@Article{IPB-2504, author = {Schreiber, T. and Prange, A. and Schäfer, P. and Iwen, T. and Grützner, R. and Marillonnet, S. and Lepage, A. and Javelle, M. and Paul, W. and Tissier, A. and}, title = {{Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR/Cas endonucleases}}, year = {2024}, journal = {Mol. Plant}, doi = {10.1016/j.molp.2024.03.013}, url = {https://doi.org/10.1016/j.molp.2024.03.013}, abstract = {In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. We identified two groups of exonucleases from the Herpes Virus and the bacteriophage T7 families that conferred an up to 38-fold increase in HDR frequencies when fused to Cas9/Cas12a in a Tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrate stable and heritable knock-ins of in wheat in 1% of the primary transformants. Our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.} } @Article{IPB-2499, author = {Frey, M. and Vahabi, K. and Cankar, K. and Lackus, N. D. and Padilla-Gonzalez, F. and Ro, D.-K. and Rieseberg, L. and Spring, O. and Tissier, A. and}, title = {{Sesquiterpene lactones – insights into biosynthesis, regulation and signalling roles}}, year = {2024}, pages = {1-27}, journal = {Crit. Rev. Plant Sci.}, doi = {10.1080/07352689.2024.2307240}, url = {https://doi.org/10.1080/07352689.2024.2307240}, abstract = {Sesquiterpene lactones (STLs) are bitter tasting plant specialized metabolites derived from farnesyl pyrophosphate (FPP) that contain a characteristic lactone ring. STLs can be found in many plant families that are distantly related to each other and outside the plant kingdom. They are especially prevalent in the plant families Apiaceae and Asteraceae, the latter being one of the largest plant families besides the Orchidaceae. The STL diversity is especially large in the Asteraceae, which made them an ideal object for chemosystematic studies in these species. Many STLs show a high bioactivity, for example as protective compounds against herbivory. STLs are also relevant for pharmaceutical applications, such as the treatment of malaria with artemisinin. Recent findings have dramatically changed our knowledge about the biosynthesis of STLs, as well as their developmental, spatial, and environmental regulation. This review intents to update the currently achieved progress in these aspects. With the advancement of genome editing tools such as CRISPR/Cas and the rapid acceleration of the speed of genome sequencing, even deeper insights into the biosynthesis, regulation, and enzyme evolution of STL can be expected in the future. Apart from their role as protective compounds, there may be a more subtle role of STL in regulatory processes of plants that will be discussed as well.} } @Article{IPB-212, author = {Wasternack, C. and Hause, B. and}, title = {{BFP1: One of 700 Arabidopsis F-box proteins mediates degradation of JA oxidases to promote plant immunity}}, year = {2024}, pages = {375-376}, journal = {Mol. Plant}, doi = {10.1016/j.molp.2024.02.008}, url = {https://doi.org/10.1016/j.molp.2024.02.008}, volume = {17}, } @Article{IPB-198, author = {Frey, M. and Bathe, U. and Meink, L. and Balcke, G. U. and Schmidt, J. and Frolov, A. and Soboleva, A. and Hassanin, A. and Davari, M. D. and Frank, O. and Schlagbauer, V. and Dawid, C. and Tissier, A. and}, title = {{Combinatorial biosynthesis in yeast leads to over 200 diterpenoids}}, year = {2024}, pages = {193-200}, journal = {Metab. Eng.}, doi = {10.1016/j.ymben.2024.02.006}, url = {https://doi.org/10.1016/j.ymben.2024.02.006}, volume = {82}, abstract = {Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyldiphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases.} } @Article{IPB-204, author = {Launhardt, L. and Uhlenberg, J. and Stellmach, H. and Schomburg, M. and Hause, B. and Heilmann, I. and Heilmann, M. and}, title = {{Association of the Arabidopsis oleoyl Δ12‐desaturase FAD2 with pre‐cis‐Golgi stacks at endoplasmic reticulum‐Golgi‐exit sites}}, year = {2024}, pages = {242-263}, journal = {Plant J.}, doi = {10.1111/tpj.16492}, url = {https://doi.org/10.1111/tpj.16492}, volume = {117}, abstract = {The unsaturation of phospholipids influences the function of membranes. In Arabidopsis thaliana, the oleoyl Δ12‐desaturase FAD2 converts oleic (18:1Δ9) to linoleic acid (18:2Δ9,12) and influences phospholipid unsaturation in different cellular membranes. Despite its importance, the precise localization of Arabidopsis FAD2 has not been unambiguously described. As FAD2 is thought to modify phospholipid‐associated fatty acids at the endoplasmic reticulum (ER), from where unsaturates are distributed to other cellular sites, we hypothesized that FAD2 locates to ER subdomains enabling trafficking of lipid intermediates through the secretory pathway. Fluorescent FAD2 fusions used to test this hypothesis were first assessed for functionality by heterologous expression in yeast (Saccharomyces cerevisiae), and in planta by Arabidopsis fad2 mutant rescue upon ectopic expression from an intrinsic FAD2 promoter fragment. Light sheet fluorescence, laser scanning confocal or spinning disc microscopy of roots, leaves, or mesophyll protoplasts showed the functional fluorescence‐tagged FAD2 variants in flattened donut‐shaped structures of ~0.5–1 μm diameter, in a pattern not resembling mere ER association. High‐resolution imaging of coexpressed organellar markers showed fluorescence‐tagged FAD2 in a ring‐shaped pattern surrounding ER‐proximal Golgi particles, colocalizing with pre‐cis‐Golgi markers. This localization required the unusual C‐terminal retention signal of FAD2, and deletion or substitutions in this protein region resulted in relaxed distribution and diffuse association with the ER. The distinct association of FAD2 with pre‐cis‐Golgi stacks in Arabidopsis root and leaf tissue is consistent with a contribution of FAD2 to membrane lipid homeostasis through the secretory pathway, as verified by an increased plasma membrane liquid phase order in the fad2 mutant.} } @Article{IPB-2497, author = {Grützner, R. and König, K. and Horn, C. and Engler, C. and Laub, A. and Vogt, T. and Marillonnet, S. and}, title = {{A transient expression tool box for anthocyanin biosynthesis in Nicotiana benthamiana}}, year = {2023}, journal = {Plant Biotechnol. J.}, doi = {10.1111/pbi.14261}, url = {https://doi.org/10.1111/pbi.14261}, abstract = {Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3‐O‐rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3‐O‐glucoside, cyanidin 3‐O‐glucoside and delphinidin 3‐O‐glucoside could be obtained in high amounts in a few days. Additionally, co‐infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5.} } @Article{IPB-306, author = {Zeng, M. and Krajinski, F. and Dam, N. M. and Hause, B. and}, title = {{Jarin-1, an inhibitor of JA-Ile biosynthesis in Arabidopsis thaliana , acts differently in other plant species}}, year = {2023}, pages = {e2273515}, journal = {Plant Signaling \& Behavior}, doi = {10.1080/15592324.2023.2273515}, url = {https://doi.org/10.1080/15592324.2023.2273515}, volume = {18}, abstract = {Jasmonates (JAs), including jasmonic acid (JA) and its biologically active derivative JA-Ile, are lipid-derived plant signaling molecules. They govern plant responses to stresses, such as wounding and insect herbivory. Wounding elicits a rapid increase of JA and JA-Ile levels as well as the expression of JAR1, coding for the enzyme involved in JA-Ile biosynthesis. Endogenous increase and application of JAs, such as MeJA, a JA methylester, result in increased defense levels, often accompanied by diminished growth. A JA-Ile biosynthesis inhibitor, jarin-1, was shown to exclusively inhibit the JA-conjugating enzyme JAR1 in Arabidopsis thaliana. To investigate whether jarin-1 does function similarly in other plants, we tested this in Medicago truncatula, Solanum lycopersicum, and Brassica nigra seedlings in a root growth inhibition assay. Application of jarin-1 alleviated the inhibition of root growth after MeJA application in M. truncatula seedlings, proving that jarin-1 is biologically active in M. truncatula. Jarin-1 did not show, however, a similar effect in S. lycopersicum and B. nigra seedlings treated with MeJA. Even JA-Ile levels were not affected by application of jarin-1 in wounded leaf disks from S. lycopersicum. Based on these results, we conclude that the effect of jarin-1 is highly species-specific. Researchers intending to use jarin-1 for studying the function of JAR1 or JA-Ile in their model plants, must test its functionality before use.} } @Article{IPB-305, author = {Zeng, M. and Dam, N. M. and Hause, B. and}, title = {{MtEIN2 affects nitrate uptake and accumulation of photosynthetic pigments under phosphate and nitrate deficiency in Medicago truncatula}}, year = {2023}, pages = {e13899}, journal = {Physiol. Plant.}, doi = {10.1111/ppl.13899}, url = {https://doi.org/10.1111/ppl.13899}, volume = {175}, abstract = {Ethylene (ET) controls many facets of plant growth and development under abiotic and biotic stresses. MtEIN2, as a critical element of the ET signaling pathway, is essential in biotic interactions. However, the role of MtEIN2 in responding to abiotic stress, such as combined nutrient deficiency, is less known. To assess the role of ethylene signaling in nutrient uptake, we manipulated nitrate (NO3−) and phosphate (Pi) availability for wild-type (WT) and the ethylene-insensitive (MtEIN2-defective) mutant, sickle, in Medicago truncatula. We measured leaf biomass and photosynthetic pigments in WT and sickle to identify conditions leading to different responses in both genotypes. Under combined NO3− and Pi deficiency, sickle plants had higher chlorophyll and carotenoid contents than WT plants. Under these conditions, nitrate content and gene expression levels of nitrate transporters were higher in the sickle mutant than in the WT. This led to the conclusion that MtEIN2 is associated with nitrate uptake and the content of photosynthetic pigments under combined Pi and NO3−deficiency in M. truncatula. We conclude that ethylene perception plays a critical role in regulating the nutrient status of plants.} } @Article{IPB-267, author = {Ninck, S. and Halder, V. and Krahn, J. H. and Beisser, D. and Resch, S. and Dodds, I. and Scholtysik, R. and Bormann, J. and Sewald, L. and Gupta, M. D. and Heilmann, G. and Bhandari, D. D. and Morimoto, K. and Buscaill, P. and Hause, B. and van der Hoorn, R. A. L. and Kaschani, F. and Kaiser, M. and}, title = {{Chemoproteomics Reveals the Pan-HER Kinase Inhibitor Neratinib To Target an Arabidopsis Epoxide Hydrolase Related to Phytohormone Signaling}}, year = {2023}, pages = {1076-1088}, journal = {ACS Chem. Biol.}, doi = {10.1021/acschembio.2c00322}, url = {https://doi.org/10.1021/acschembio.2c00322}, volume = {18}, abstract = {Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for the identification of functional SA agonists in Arabidopsis thaliana that revealed Neratinib (Ner), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that Ner covalently modifies a surface-exposed cysteine residue of Arabidopsis epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition. Physiologically, the Ner application induces jasmonate metabolism in an AtEH7-dependent manner as an early response. In addition, it modulates PATHOGENESIS RELATED 1 (PR1) expression as a hallmark of SA signaling activation as a later effect. AtEH7, however, is not the exclusive target for this physiological readout induced by Ner. Although the underlying molecular mechanisms of AtEH7-dependent modulation of jasmonate signaling and Ner-induced PR1-dependent activation of SA signaling and thus defense response regulation remain unknown, our present work illustrates the powerful combination of forward chemical genetics and chemical proteomics for identifying novel phytohormone signaling modulatory factors. It also suggests that marginally explored metabolic enzymes such as epoxide hydrolases may have further physiological roles in modulating signaling.} } @Article{IPB-259, author = {Manh, M. B. and Ost, C. and Peiter, E. and Hause, B. and Krupinska, K. and Humbeck, K. and}, title = {{WHIRLY1 acts upstream of ABA-related reprogramming of drought-induced gene expression in Barley and affects stress-related histone modifications}}, year = {2023}, pages = {6326}, journal = {Int. J. Mol. Sci.}, doi = {10.3390/ijms24076326}, url = {https://doi.org/10.3390/ijms24076326}, volume = {24}, abstract = {WHIRLY1, a small plant-specific ssDNA-binding protein, dually located in chloroplasts and the nucleus, is discussed to act as a retrograde signal transmitting a stress signal from the chloroplast to the nucleus and triggering there a stress-related gene expression. In this work, we investigated the function of WHIRLY1 in the drought stress response of barley, employing two overexpression lines (oeW1-2 and oeW1-15). The overexpression of WHIRLY1 delayed the drought-stress-related onset of senescence in primary leaves. Two abscisic acid (ABA)-dependent marker genes of drought stress, HvNCED1 and HvS40, whose expression in the wild type was induced during drought treatment, were not induced in overexpression lines. In addition, a drought-related increase in ABA concentration in the leaves was suppressed in WHIRLY1 overexpression lines. To analyze the impact of the gain-of-function of WHIRLY1 on the drought-related reprogramming of nuclear gene expression, RNAseq was performed comparing the wild type and an overexpression line. Cluster analyses revealed a set of genes highly up-regulated in response to drought in the wild type but not in the WHIRLY1 overexpression lines. Among these genes were many stress- and abscisic acid (ABA)-related ones. Another cluster comprised genes up-regulated in the oeW1 lines compared to the wild type. These were related to primary metabolism, chloroplast function and growth. Our results indicate that WHIRLY1 acts as a hub, balancing trade-off between stress-related and developmental pathways. To test whether the gain-of-function of WHIRLY1 affects the epigenetic control of stress-related gene expression, we analyzed drought-related histone modifications in different regions of the promoter and at the transcriptional start sites of HvNCED1 and HvS40. Interestingly, the level of euchromatic marks (H3K4me3 and H3K9ac) was clearly decreased in both genes in a WHIRLY1 overexpression line. Our results indicate that WHIRLY1, which is discussed to act as a retrograde signal, affects the ABA-related reprogramming of nuclear gene expression during drought via differential histone modifications.} } @Article{IPB-254, author = {Israeli, A. and Schubert, R. and Man, N. and Teboul, N. and Serrani Yarce, J. C. and Rosowski, E. E. and Wu, M.-F. and Levy, M. and Efroni, I. and Ljung, K. and Hause, B. and Reed, J. W. and Ori, N. and}, title = {{Modulating auxin response stabilizes tomato fruit set}}, year = {2023}, pages = {2336-2355}, journal = {Plant Physiol.}, doi = {10.1093/plphys/kiad205}, url = {https://doi.org/10.1093/plphys/kiad205}, volume = {192}, abstract = {Fruit formation depends on successful fertilization and is highly sensitive to weather fluctuations that affect pollination. Auxin promotes fruit initiation and growth following fertilization. Class A auxin response factors (Class A ARFs) repress transcription in the absence of auxin and activate transcription in its presence. Here, we explore how multiple members of the ARF family regulate fruit set and fruit growth in tomato (Solanum lycopersicum) and Arabidopsis thaliana, and test whether reduction of SlARF activity improves yield stability in fluctuating temperatures. We found that several tomato Slarf mutant combinations produced seedless parthenocarpic fruits, most notably mutants deficient in SlARF8A and SlARF8B genes. Arabidopsis Atarf8 mutants deficient in the orthologous gene had less complete parthenocarpy than did tomato Slarf8a Slarf8b mutants. Conversely, Atarf6 Atarf8 double mutants had reduced fruit growth after fertilization. AtARF6 and AtARF8 likely switch from repression to activation of fruit growth in response to a fertilization-induced auxin increase in gynoecia. Tomato plants with reduced SlARF8A and SlARF8B gene dosage had substantially higher yield than the wild type under controlled or ambient hot and cold growth conditions. In field trials, partial reduction in the SlARF8 dose increased yield under extreme temperature with minimal pleiotropic effects. The stable yield of the mutant plants resulted from a combination of early onset of fruit set, more fruit-bearing branches and more flowers setting fruits. Thus, ARF8 proteins mediate the control of fruit set, and relieving this control with Slarf8 mutations may be utilized in breeding to increase yield stability in tomato and other crops.} } @Article{IPB-232, author = {Darwish, E. and Ghosh, R. and Bentzer, J. and Tsardakas Renhuldt, N. and Proux‐Wera, E. and Kamal, N. and Spannagl, M. and Hause, B. and Sirijovski, N. and Van Aken, O. and}, title = {{The dynamics of touch‐responsive gene expression in cereals}}, year = {2023}, pages = {282-302}, journal = {Plant J.}, doi = {10.1111/tpj.16269}, url = {https://doi.org/10.1111/tpj.16269}, volume = {116}, abstract = {Wind, rain, herbivores, obstacles, neighbouring plants, etc. provide important mechanical cues to steerplant growth and survival. Mechanostimulation to stimulate yield and stress resistance of crops is of signifi-cant research interest, yet a molecular understanding of transcriptional responses to touch is largely absentin cereals. To address this, we performed whole-genome transcriptomics following mechanostimulation ofwheat, barley, and the recent genome-sequenced oat. The largest transcriptome changes occurred 25 minafter touching, with most of the genes being upregulated. While most genes returned to basal expressionlevel by 1–2 h in oat, many genes retained high expression even 4 h post-treatment in barley and wheat.Functional categories such as transcription factors, kinases, phytohormones, and Ca2+regulation wereaffected. In addition, cell wall-related genes involved in (hemi)cellulose, lignin, suberin, and callose biosyn-thesis were touch-responsive, providing molecular insight into mechanically induced changes in cell wallcomposition. Furthermore, several cereal-specific transcriptomic footprints were identified that were notobserved in Arabidopsis. In oat and barley, we found evidence for systemic spreading of touch-induced sig-nalling. Finally, we provide evidence that both the jasmonic acid-dependent and the jasmonic acid-independent pathways underlie touch-signalling in cereals, providing a detailed framework and markergenes for further study of (a)biotic stress responses in cereals.} } @Article{IPB-228, author = {Cankar, K. and Hakkert, J. C. and Sevenier, R. and Papastolopoulou, C. and Schipper, B. and Baixinho, J. P. and Fernández, N. and Matos, M. S. and Serra, A. T. and Santos, C. N. and Vahabi, K. and Tissier, A. and Bundock, P. and Bosch, D. and}, title = {{Lactucin synthase inactivation boosts the accumulation of anti-inflammatory 8-deoxylactucin and its derivatives in Chicory (Cichorium intybus L.)}}, year = {2023}, pages = {6061-6072}, journal = {J. Agr. Food Chem.}, doi = {10.1021/acs.jafc.2c08959}, url = {https://doi.org/10.1021/acs.jafc.2c08959}, volume = {71}, abstract = {For several sesquiterpene lactones (STLs) found in Asteraceae plants, very interesting biomedical activities have been demonstrated. Chicory roots accumulate the guaianolide STLs 8-deoxylactucin, lactucin, and lactucopicrin predominantly in oxalated forms in the latex. In this work, a supercritical fluid extract fraction of chicory STLs containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin was shown to have anti-inflammatory activity in an inflamed intestinal mucosa model. To increase the accumulation of these two compounds in chicory taproots, the lactucin synthase that takes 8-deoxylactucin as the substrate for the regiospecific hydroxylation to generate lactucin needs to be inactivated. Three candidate cytochrome P450 enzymes of the CYP71 clan were identified in chicory. Their targeted inactivation using the CRISPR/Cas9 approach identified CYP71DD33 to have lactucin synthase activity. The analysis of the terpene profile of the taproots of plants with edits in CYP71DD33 revealed a nearly complete elimination of the endogenous chicory STLs lactucin and lactucopicrin and their corresponding oxalates. Indeed, in the same lines, the interruption of biosynthesis resulted in a strong increase of 8-deoxylactucin and its derivatives. The enzyme activity of CYP71DD33 to convert 8-deoxylactucin to lactucin was additionally demonstrated in vitro using yeast microsome assays. The identified chicory lactucin synthase gene is predominantly expressed in the chicory latex, indicating that the late steps in the STL biosynthesis take place in the latex. This study contributes to further elucidation of the STL pathway in chicory and shows that root chicory can be positioned as a crop from which different health products can be extracted.} } @Article{IPB-219, author = {Ai, H. and Bellstaedt, J. and Bartusch, K. S. and Eschen‐Lippold, L. and Babben, S. and Balcke, G. U. and Tissier, A. and Hause, B. and Andersen, T. G. and Delker, C. and Quint, M. and}, title = {{Auxin‐dependent regulation of cell division rates governs root thermomorphogenesis}}, year = {2023}, pages = {e111926}, journal = {EMBO J.}, doi = {10.15252/embj.2022111926}, url = {https://doi.org/10.15252/embj.2022111926}, volume = {42}, abstract = {Roots are highly plastic organs enabling plants to adapt to a changing below-ground environment. In addition to abiotic factors like nutrients or mechanical resistance, plant roots also respond to temperature variation. Below the heat stress threshold, Arabidopsis thaliana seedlings react to elevated temperature by promoting primary root growth, possibly to reach deeper soil regions with potentially better water saturation. While above-ground thermomorphogenesis is enabled by thermo-sensitive cell elongation, it was unknown how temperature modulates root growth. We here show that roots are able to sense and respond to elevated temperature independently of shoot-derived signals. This response is mediated by a yet unknown root thermosensor that employs auxin as a messenger to relay temperature signals to the cell cycle. Growth promotion is achieved primarily by increasing cell division rates in the root apical meristem, depending on de novo local auxin biosynthesis and temperature-sensitive organization of the polar auxin transport system. Hence, the primary cellular target of elevated ambient temperature differs fundamentally between root and shoot tissues, while the messenger auxin remains the same.} } @Article{IPB-295, author = {Schindele, P. and Merker, L. and Schreiber, T. and Prange, A. and Tissier, A. and Puchta, H. and}, title = {{Enhancing gene editing and gene targeting efficiencies in Arabidopsis thaliana by using an intron‐containing version of ttLbCas12a}}, year = {2023}, pages = {457-459}, journal = {Plant Biotechnol. J.}, doi = {10.1111/pbi.13964}, url = {https://doi.org/10.1111/pbi.13964}, volume = {21}, } @Article{IPB-294, author = {Saadat, N. P. and van Aalst, M. and Brand, A. and Ebenhöh, O. and Tissier, A. and Matuszyńska, A. B. and}, title = {{Shifts in carbon partitioning by photosynthetic activity increase terpenoid synthesis in glandular trichomes}}, year = {2023}, pages = {1716-1728}, journal = {Plant J.}, doi = {10.1111/tpj.16352}, url = {https://doi.org/10.1111/tpj.16352}, volume = {115}, abstract = {Several commercially important secondary metabolites are produced and accumulated in high amounts by glandular trichomes, giving the prospect of using them as metabolic cell factories. Due to extremely high metabolic fluxes through glandular trichomes, previous research focused on how such flows are achieved. The question regarding their bioenergetics became even more interesting with the discovery of photosynthetic activity in some glandular trichomes. Despite recent advances, how primary metabolism contributes to the high metabolic fluxes in glandular trichomes is still not fully elucidated. Using computational methods and available multi-omics data, we first developed a quantitative framework to investigate the possible role of photosynthetic energy supply in terpenoid production and next tested experimentally the simulation-driven hypothesis. With this work, we provide the first reconstruction of specialised metabolism in Type-VI photosynthetic glandular trichomes of Solanum lycopersicum. Our model predicted that increasing light intensities results in a shift of carbon partitioning from catabolic to anabolic reactions driven by the energy availability of the cell. Moreover, we show the benefit of shifting between isoprenoid pathways under different light regimes, leading to a production of different classes of terpenes. Our computational predictions were confirmed in vivo, demonstrating a significant increase in production of monoterpenoids while the sesquiterpenes remained unchanged under higher light intensities. The outcomes of this research provide quantitative measures to assess the beneficial role of chloroplast in glandular trichomes for enhanced production of secondary metabolites and can guide the design of new experiments that aim at modulating terpenoid production.} } @Article{IPB-2512, author = {Schreiber, T. and Tripathee, S. and Iwen, T. and Prange, A. and Vahabi, K. and Grützner, R. and Horn, C. and Marillonnet, S. and Tissier, A. and}, title = {{DNA double strand breaks lead to de novo transcription and translation of damage-induced long RNAs in planta}}, year = {2022}, journal = {bioRxiv}, doi = {10.1101/2022.05.11.491484}, url = {https://doi.org/10.1101/2022.05.11.491484}, abstract = {DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.} } @Article{IPB-370, author = {Mittelberger, C. and Hause, B. and Janik, K. and}, title = {{The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection}}, year = {2022}, pages = {e0272467}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0272467}, url = {https://doi.org/10.1371/journal.pone.0272467}, volume = {17}, abstract = {’Candidatus Phytoplasma mali’, is a bacterial pathogen associated with the so-called apple proliferation disease in Malus × domestica. The pathogen manipulates its host with a set of effector proteins, among them SAP11CaPm, which shares similarity to SAP11AYWB from ’Candidatus Phytoplasma asteris’. SAP11AYWB interacts and destabilizes the class II CIN transcription factors of Arabidopsis thaliana, namely AtTCP4 and AtTCP13 as well as the class II CYC/TB1 transcription factor AtTCP18, also known as BRANCHED1 being an important factor for shoot branching. It has been shown that SAP11CaPm interacts with the Malus × domestica orthologues of AtTCP4 (MdTCP25) and AtTCP13 (MdTCP24), but an interaction with MdTCP16, the orthologue of AtTCP18, has never been proven. The aim of this study was to investigate this potential interaction and close a knowledge gap regarding the function of SAP11CaPm. A Yeast two-hybrid test and Bimolecular Fluorescence Complementation in planta revealed that SAP11CaPm interacts with MdTCP16. MdTCP16 is known to play a role in the control of the seasonal growth of perennial plants and an increase of MdTCP16 gene expression has been detected in apple leaves in autumn. In addition to this, MdTCP16 is highly expressed during phytoplasma infection. Binding of MdTCP16 by SAP11CaPm might lead to the induction of shoot proliferation and early bud break, both of which are characteristic symptoms of apple proliferation disease.} } @Article{IPB-369, author = {Milde, R. and Schnabel, A. and Ditfe, T. and Hoehenwarter, W. and Proksch, C. and Westermann, B. and Vogt, T. and}, title = {{Chemical synthesis of trans 8-methyl-6-nonenoyl-CoA and functional expression unravel capsaicin synthase activity encoded by the Pun1 Locus}}, year = {2022}, pages = {6878}, journal = {Molecules}, doi = {10.3390/molecules27206878}, url = {https://doi.org/10.3390/molecules27206878}, volume = {27}, abstract = {Capsaicin, produced by diverse Capsicum species, is among the world’s most popular spices and of considerable pharmaceutical relevance. Although the capsaicinoid biosynthetic pathway has been investigated for decades, several biosynthetic steps have remained partly hypothetical. Genetic evidence suggested that the decisive capsaicin synthase is encoded by the Pun1 locus. Yet, the genetic evidence of the Pun1 locus was never corroborated by functionally active capsaicin synthase that presumably catalyzes an amide bond formation between trans 8-methyl-6-nonenoyl-CoA derived from branched-chain amino acid biosynthesis and vanilloylamine derived from the phenylpropanoid pathway. In this report, we demonstrate the enzymatic activity of a recombinant capsaicin synthase encoded by Pun1, functionally expressed in Escherichia coli, and provide information on its substrate specificity and catalytic properties. Recombinant capsaicin synthase is specific for selected aliphatic CoA-esters and highly specific for vanilloylamine. Partly purified from E. coli, the recombinant active enzyme is a monomeric protein of 51 kDa that is independent of additional co-factors or associated proteins, as previously proposed. These data can now be used to design capsaicin synthase variants with different properties and alternative substrate preferences.} } @Article{IPB-350, author = {Jäckel, L. and Schnabel, A. and Stellmach, H. and Klauß, U. and Matschi, S. and Hause, G. and Vogt, T. and}, title = {{The terminal enzymatic step in piperine biosynthesis is co‐localized with the product piperine in specialized cells of black pepper (Piper nigrum L.)}}, year = {2022}, pages = {731–747}, journal = {Plant J.}, doi = {10.1111/tpj.15847}, url = {https://doi.org/10.1111/tpj.15847}, volume = {111}, abstract = {Piperine (1-piperoyl piperidine) is responsible for the pungent perception of dried black pepper (Pipernigrum) fruits and essentially contributes to the aromatic properties of this spice in combination with ablend of terpenoids. The final step in piperine biosynthesis involves piperine synthase (PS), which catalyzesthe reaction of piperoyl CoA and piperidine to the biologically active and pungent amide. Nevertheless, experimental data on the cellular localization of piperine and the complete biosynthetic pathway are missing. Not only co-localization of enzymes and products, but also potential transport of piperamides to thesink organs is a possible alternative. This work, which includes purification of the native enzyme, immunolocalization, laser microdissection, fluorescence microscopy, and electron microscopy combinedwith liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), providesexperimental evidence that piperine and PS are co-localized in specialized cells of the black pepper fruit peri-sperm. PS accumulates during early stages of fruit development and its level declines before the fruits arefully mature. The product piperine is co-localized to PS and can be monitored at the cellular level by itsstrong bluish fluorescence. Rising piperine levels during fruit maturation are consistent with the increasingnumbers of fluorescent cells within the perisperm. Signal intensities of individual laser-dissected cells whenmonitored by LC-ESI-MS/MS indicate molar concentrations of this alkaloid. Significant levels of piperineand additional piperamides were also detected in cells distributed in the cortex of black pepper roots. Insummary, the data provide comprehensive experimental evidence of and insights into cell-specific biosyn-thesis and storage of piperidine alkaloids, specific and characteristic for the Piperaceae. By a combination offluorescence microscopy and LC-MS/MS analysis we localized the major piperidine alkaloids to specific cellsof the fruit perisperm and the root cortex. Immunolocalization of native piperine and piperamide synthasesshows that enzymes are co-localized with high concentrations of products in these idioblasts.} } @Article{IPB-327, author = {El Amerany, F. and Rhazi, M. and Balcke, G. and Wahbi, S. and Meddich, A. and Taourirte, M. and Hause, B. and}, title = {{The effect of chitosan on plant physiology, wound response, and fruit quality of tomato}}, year = {2022}, pages = {5006}, journal = {Polymers}, doi = {10.3390/polym14225006}, url = {https://doi.org/10.3390/polym14225006}, volume = {14}, abstract = {In agriculture, chitosan has become popular as a metabolic enhancer; however, no deep information has been obtained yet regarding its mechanisms on vegetative tissues. This work was conducted to test the impact of chitosan applied at different plant growth stages on plant development, physiology, and response to wounding as well as fruit shape and composition. Five concentrations of chitosan were tested on tomato. The most effective chitosan doses that increased leaf number, leaf area, plant biomass, and stomatal conductance were 0.75 and 1 mg mL−1. Chitosan (1 mg mL−1) applied as foliar spray increased the levels of jasmonoyl–isoleucine and abscisic acid in wounded roots. The application of this dose at vegetative and flowering stages increased chlorophyll fluorescence (Fv/Fm) values, whereas application at the fruit maturation stage reduced the Fv/Fm values. This decline was positively correlated with fruit shape and negatively correlated with the pH and the content of soluble sugars, lycopene, total flavonoids, and nitrogen in fruits. Moreover, the levels of primary metabolites derived from glycolysis, such as inositol phosphate, lactic acid, and ascorbic acid, increased in response to treatment of plants with 1 mg mL−1- chitosan. Thus, chitosan application affects various plant processes by influencing stomata aperture, cell division and expansion, fruit maturation, mineral assimilation, and defense responses.} } @Article{IPB-411, author = {Zeng, M. and Hause, B. and van Dam, N. M. and Uthe, H. and Hoffmann, P. and Krajinski, F. and Martínez‐Medina, A. and}, title = {{The mycorrhizal symbiosis alters the plant defence strategy in a model legume plant}}, year = {2022}, pages = {3412-3428}, journal = {Plant Cell Environ.}, doi = {10.1111/pce.14421}, url = {https://doi.org/10.1111/pce.14421}, volume = {45}, abstract = {Arbuscular mycorrhizal (AM) symbiosis modulates plant‐herbivore interactions. Still, how it shapes the overall plant defence strategy and the mechanisms involved remain unclear. We investigated how AM symbiosis simultaneously modulates plant resistance and tolerance to a shoot herbivore, and explored the underlying mechanisms. Bioassays with Medicago truncatula plants were used to study the effect of the AM fungus Rhizophagus irregularis on plant resistance and tolerance to Spodoptera exigua herbivory. By performing molecular and chemical analyses, we assessed the impact of AM symbiosis on herbivore‐triggered phosphate (Pi)‐ and jasmonate (JA)‐related responses. Upon herbivory, AM symbiosis led to an increased leaf Pi content by boosting the mycorrhizal Pi‐uptake pathway. This enhanced both plant tolerance and herbivore performance. AM symbiosis counteracted the herbivore‐triggered JA burst, reducing plant resistance. To disentangle the role of the mycorrhizal Pi‐uptake pathway in the plant\'s response to herbivory, we used the mutant line ha1‐2, impaired in the H+‐ATPase gene HA1, which is essential for Pi‐uptake via the mycorrhizal pathway. We found that mycorrhiza‐triggered enhancement of herbivore performance was compromised in ha1‐2 plants. AM symbiosis thus affects the defence pattern of M. truncatula by altering resistance and tolerance simultaneously. We propose that the mycorrhizal Pi‐uptake pathway is involved in the modulation of the plant defence strategy.} } @Article{IPB-394, author = {Stellmach, H. and Hose, R. and Räde, A. and Marillonnet, S. and Hause, B. and}, title = {{A new set of Golden-Gate-Based organelle marker plasmids for colocalization studies in plants}}, year = {2022}, pages = {2620}, journal = {Plants}, doi = {10.3390/plants11192620}, url = {https://doi.org/10.3390/plants11192620}, volume = {11}, abstract = {In vivo localization of proteins using fluorescence-based approaches by fusion of the protein of interest (POI) to a fluorescent protein is a cost- and time-effective tool to gain insights into its physiological function in a plant cell. Determining the proper localization, however, requires the co-expression of defined organelle markers (OM). Several marker sets are available but, so far, the procedure requires successful co-transformation of POI and OM into the same cell and/or several cloning steps. We developed a set of vectors containing markers for basic cell organelles that enables the insertion of the gene of interest (GOI) by a single cloning step using the Golden Gate cloning approach and resulting in POI–GFP fusions. The set includes markers for plasma membrane, tonoplast, nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes, plastids, and mitochondria, all labelled with mCherry. Most of them were derived from well-established marker sets, but those localized in plasma membrane and tonoplast were improved by using different proteins. The final vectors are usable for localization studies in isolated protoplasts and for transient transformation of leaves of Nicotiana benthamiana. Their functionality is demonstrated using two enzymes involved in biosynthesis of jasmonic acid and located in either plastids or peroxisomes.} } @Article{IPB-393, author = {Soboleva, A. and Frolova, N. and Bureiko, K. and Shumilina, J. and Balcke, G. U. and Zhukov, V. A. and Tikhonovich, I. A. and Frolov, A. and}, title = {{Dynamics of Reactive Carbonyl Species in Pea Root Nodules in Response to Polyethylene Glycol (PEG)-Induced Osmotic Stress}}, year = {2022}, pages = {2726}, journal = {Int. J. Mol. Sci.}, doi = {10.3390/ijms23052726}, url = {https://doi.org/10.3390/ijms23052726}, volume = {23}, abstract = {Drought dramatically affects crop productivity worldwide. For legumes this effect is especially pronounced, as their symbiotic association with rhizobia is highly-sensitive to dehydration. This might be attributed to the oxidative stress, which ultimately accompanies plants’ response to water deficit. Indeed, enhanced formation of reactive oxygen species in root nodules might result in up-regulation of lipid peroxidation and overproduction of reactive carbonyl compounds (RCCs), which readily modify biomolecules and disrupt cell functions. Thus, the knowledge of the nodule carbonyl metabolome dynamics is critically important for understanding the drought-related losses of nitrogen fixation efficiency and plant productivity. Therefore, here we provide, to the best of our knowledge, for the first time a comprehensive overview of the pea root nodule carbonyl metabolome and address its alterations in response to polyethylene glycol-induced osmotic stress as the first step to examine the changes of RCC patterns in drought treated plants. RCCs were extracted from the nodules and derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The relative quantification of CHH-derivatives by liquid chromatography-high resolution mass spectrometry with a post-run correction for derivative stability revealed in total 194 features with intensities above 1 × 105 counts, 19 of which were down- and three were upregulated. The upregulation of glyceraldehyde could accompany non-enzymatic conversion of glyceraldehyde-3-phosphate to methylglyoxal. The accumulation of 4,5-dioxovaleric acid could be the reason for down-regulation of porphyrin metabolism, suppression of leghemoglobin synthesis, inhibition of nitrogenase and degradation of legume-rhizobial symbiosis in response to polyethylene glycol (PEG)-induced osmotic stress effect. This effect needs to be confirmed with soil-based drought models.} } @Article{IPB-389, author = {Ruberti, C. and Feitosa-Araujo, E. and Xu, Z. and Wagner, S. and Grenzi, M. and Darwish, E. and Lichtenauer, S. and Fuchs, P. and Parmagnani, A. S. and Balcerowicz, D. and Schoenaers, S. and de la Torre, C. and Mekkaoui, K. and Nunes-Nesi, A. and Wirtz, M. and Vissenberg, K. and Van Aken, O. and Hause, B. and Costa, A. and Schwarzländer, M. and}, title = {{MCU proteins dominate in vivo mitochondrial Ca2+ uptake in Arabidopsis roots}}, year = {2022}, pages = {4428-4452}, journal = {Plant Cell}, doi = {10.1093/plcell/koac242}, url = {https://doi.org/10.1093/plcell/koac242}, volume = {34}, abstract = {Abstract Ca2+ signaling is central to plant development and acclimation. While Ca2+-responsive proteins have been investigated intensely in plants, only a few Ca2+-permeable channels have been identified, and our understanding of how intracellular Ca2+ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca2+ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca2+ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca2+ transport in planta and that this mechanism is the major route for fast Ca2+ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca2+ imaging in living root tips and the stimulation of Ca2+ transients of different amplitudes, we demonstrated that mitochondrial Ca2+ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca2+ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca2+ entry route in planta and link mitochondrial Ca2+ transport with phytohormone signaling.} } @Article{IPB-324, author = {Dippe, M. and Davari, M. D. and Weigel, B. and Heinke, R. and Vogt, T. and Wessjohann, L. A. and}, title = {{Altering the regiospecificity of a catechol O‐methyltransferase through rational design: Vanilloid vs. isovanilloid motifs in the B‐ring of flavonoids}}, year = {2022}, pages = {e202200511}, journal = {ChemCatChem}, doi = {10.1002/cctc.202200511}, url = {https://doi.org/10.1002/cctc.202200511}, volume = {14}, abstract = {Rational re-design of the substrate pocket of phenylpropanoid-flavonoid O-methyltransferase (PFOMT) from Mesembryanthe-mum crystallinum, an enzyme that selectively methylates the 3’-position (= meta-position) in catechol-moieties of flavonoids to guiacol-moieties, provided the basis for the generation of variants with opposite, i. e. 4’- (para-) regioselectivity and enhanced catalytic efficiency. A double variant (Y51R/N202W) identified through a newly developed colorimetric assay efficiently modified the para-position in flavanone and flavano-nol substrates, providing access to the sweetener molecule hesperetin and other rare plant flavonoids having an isovanil-loid motif.} } @Article{IPB-321, author = {Danila, F. and Schreiber, T. and Ermakova, M. and Hua, L. and Vlad, D. and Lo, S. and Chen, Y. and Lambret‐Frotte, J. and Hermanns, A. S. and Athmer, B. and von Caemmerer, S. and Yu, S. and Hibberd, J. M. and Tissier, A. and Furbank, R. T. and Kelly, S. and Langdale, J. A. and}, title = {{A single promoter‐TALE system for tissue‐specific and tuneable expression of multiple genes in rice}}, year = {2022}, pages = {1786-1806}, journal = {Plant Biotechnol. J.}, doi = {10.1111/pbi.13864}, url = {https://doi.org/10.1111/pbi.13864}, volume = {20}, abstract = {In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of >multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems—dTALE1-STAP1 and dTALE2-STAP2—can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.} } @Article{IPB-318, author = {Cankar, K. and Hakkert, J. C. and Sevenier, R. and Campo, E. and Schipper, B. and Papastolopoulou, C. and Vahabi, K. and Tissier, A. and Bundock, P. and Bosch, D. and}, title = {{CRISPR/Cas9 targeted inactivation of the kauniolide synthase in chicory results in accumulation of costunolide and its conjugates in taproots}}, year = {2022}, pages = {940003}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2022.940003}, url = {https://doi.org/10.3389/fpls.2022.940003}, volume = {13}, abstract = {Chicory taproots accumulate sesquiterpene lactones lactucin, lactucopicrin, and 8-deoxylactucin, predominantly in their oxalated forms. The biosynthetic pathway for chicory sesquiterpene lactones has only partly been elucidated; the enzymes that convert farnesyl pyrophosphate to costunolide have been described. The next biosynthetic step of the conversion of costunolide to the tricyclic structure, guaianolide kauniolide, has so far not been elucidated in chicory. In this work three putative kauniolide synthase genes were identified in chicory named CiKLS1, CiKLS2, and CiKLS3. Their activity to convert costunolide to kauniolide was demonstrated in vitro using yeast microsome assays. Next, introduction of CRISPR/Cas9 reagents into chicory protoplasts was used to inactivate multiple chicory KLS genes and several chicory lines were successfully regenerated. The inactivation of the kauniolide synthase genes in chicory by the CRISPR/Cas9 approach resulted in interruption of the sesquiterpene lactone biosynthesis in chicory leaves and taproots. In chicory taproots, but not in leaves, accumulation of costunolide and its conjugates was observed to high levels, namely 1.5 mg/g FW. These results confirmed that all three genes contribute to STL accumulation, albeit to different extent. These observations demonstrate that three genes oriented in tandem on the chicory genome encode kauniolide synthases that initiate the conversion of costunolide toward the sesquiterpene lactones in chicory.} } @Article{IPB-317, author = {Brand, A. and Tissier, A. and}, title = {{Control of resource allocation between primary and specialized metabolism in glandular trichomes}}, year = {2022}, pages = {102172}, journal = {Curr. Opin. Plant Biol.}, doi = {10.1016/j.pbi.2022.102172}, url = {https://doi.org/10.1016/j.pbi.2022.102172}, volume = {66}, abstract = {Plant specialized metabolites are often synthesized and stored in dedicated morphological structures such as glandular trichomes, resin ducts, or laticifers where they accumulate in large concentrations. How this high productivity is achieved is still elusive, in particular, with respect to the interface between primary and specialized metabolism. Here, we focus on glandular trichomes to survey recent progress in understanding how plant metabolic cell factories manage to balance homeostasis of essential central metabolites while producing large quantities of compounds that constitute a metabolic sink. In particular, we review the role of gene duplications, transcription factors and photosynthesis.} } @Article{IPB-311, author = {Asfaw, K. G. and Liu, Q. and Eghbalian, R. and Purper, S. and Akaberi, S. and Dhakarey, R. and Münch, S. W. and Wehl, I. and Bräse, S. and Eiche, E. and Hause, B. and Bogeski, I. and Schepers, U. and Riemann, M. and Nick, P. and}, title = {{The jasmonate biosynthesis Gene OsOPR7 can mitigate salinity induced mitochondrial oxidative stress}}, year = {2022}, pages = {111156}, journal = {Plant Sci.}, doi = {10.1016/j.plantsci.2021.111156}, url = {https://doi.org/10.1016/j.plantsci.2021.111156}, volume = {316}, abstract = {Salinity poses a serious threat to global agriculture and human food security. A better understanding of plant adaptation to salt stress is, therefore, mandatory. In the non-photosynthetic cells of the root, salinity perturbs oxidative balance in mitochondria, leading to cell death. In parallel, plastids accumulate the jasmonate precursor cis (+)12-Oxo-Phyto-Dienoic Acid (OPDA) that is then translocated to peroxisomes and has been identified as promoting factor for salt-induced cell death as well. In the current study, we probed for a potential interaction between these three organelles that are primarily dealing with oxidative metabolism. We made use of two tools: (i) Rice OPDA Reductase 7 (OsOPR7), an enzyme localised in peroxisomes converting OPDA into the precursors of the stress hormone JA-Ile. (ii) A Trojan Peptoid, Plant PeptoQ, which can specifically target to mitochondria and scavenge excessive superoxide accumulating in response to salt stress. We show that overexpression of OsOPR7 as GFP fusion in tobacco (Nicotiana tabacum L. cv. Bright Yellow 2, BY-2) cells, as well as a pretreatment with Plant PeptoQ can mitigate salt stress with respect to numerous aspects including proliferation, expansion, ionic balance, redox homeostasis, and mortality. This mitigation correlates with a more robust oxidative balance, evident from a higher activity of superoxide dismutase (SOD), lower levels of superoxide and lipid peroxidation damage, and a conspicuous and specific upregulation of mitochondrial SOD transcripts. Although both, Plant PeptoQ and ectopic OsOPR7, were acting in parallel and mostly additive, there are two specific differences: (i) OsOPR7 is strictly localised to the peroxisomes, while Plant PeptoQ found in mitochondria. (ii) Plant PeptoQ activates transcripts of NAC, a factor involved in retrograde signalling from mitochondria to the nucleus, while these transcripts are suppressed significantly in the cells overexpressing OsOPR7. The fact that overexpression of a peroxisomal enzyme shifting the jasmonate pathway from the cell-death signal OPDA towards JA-Ile, a hormone linked with salt adaptation, is accompanied by more robust redox homeostasis in a different organelle, the mitochondrion, indicates that cross-talk between peroxisome and mitochondrion is a crucial factor for efficient adaptation to salt stress.} } @Article{IPB-309, author = {Abuslima, E. and Kanbar, A. and Raorane, M. L. and Eiche, E. and Junker, B. H. and Hause, B. and Riemann, M. and Nick, P. and}, title = {{Gain time to adapt: How sorghum acquires tolerance to salinity}}, year = {2022}, pages = {1008172}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2022.1008172}, url = {https://doi.org/10.3389/fpls.2022.1008172}, volume = {13}, abstract = {Salinity is a global environmental threat to agricultural production and food security around the world. To delineate salt-induced damage from adaption events we analysed a pair of sorghum genotypes which are contrasting in their response to salt stress with respect to physiological, cellular, metabolomic, and transcriptional responses. We find that the salt-tolerant genotype Della can delay the transfer of sodium from the root to the shoot, more swiftly deploy accumulation of proline and antioxidants in the leaves and transfer more sucrose to the root as compared to its susceptible counterpart Razinieh. Instead Razinieh shows metabolic indicators for a higher extent photorespiration under salt stress. Following sodium accumulation by a fluorescent dye in the different regions of the root, we find that Della can sequester sodium in the vacuoles of the distal elongation zone. The timing of the adaptive responses in Della leaves indicates a rapid systemic signal from the roots that is travelling faster than sodium itself. We arrive at a model where resistance and susceptibility are mainly a matter of temporal patterns in signalling.} } @Article{IPB-400, author = {Vendemiatti, E. and Therezan, R. and Vicente, M. and Pinto, M. and Bergau, N. and Yang, L. and Bernardi, W. and Alencar, S. and Zsögön, A. and Tissier, A. and Benedito, V. and Peres, L. and}, title = {{The genetic complexity of type-IV trichome development reveals the steps towards an insect-resistant tomato}}, year = {2022}, pages = {1309}, journal = {Plants}, doi = {10.3390/plants11101309}, url = {https://doi.org/10.3390/plants11101309}, volume = {11}, abstract = {The leaves of the wild tomato Solanum galapagense harbor type-IV glandular trichomes (GT) that produce high levels of acylsugars (AS), conferring insect resistance. Conversely, domesticated tomatoes (S. lycopersicum) lack type-IV trichomes on the leaves of mature plants, preventing high AS production, thus rendering the plants more vulnerable to insect predation. We hypothesized that cultivated tomatoes engineered to harbor type-IV trichomes on the leaves of adult plants could be insect-resistant. We introgressed the genetic determinants controlling type-IV trichome development from S. galapagense into cv. Micro-Tom (MT) and created a line named “Galapagos-enhanced trichomes” (MT-Get). Mapping-by-sequencing revealed that five chromosomal regions of S. galapagense were present in MT-Get. Further genetic mapping showed that S. galapagense alleles in chromosomes 1, 2, and 3 were sufficient for the presence of type-IV trichomes on adult organs but at lower densities. Metabolic and gene expression analyses demonstrated that type-IV trichome density was not accompanied by the AS production and exudation in MT-Get. Although the plants produce a significant amount of acylsugars, those are still not enough to make them resistant to whiteflies. We demonstrate that type-IV glandular trichome development is insufficient for high AS accumulation. The results from our study provided additional insights into the steps necessary for breeding an insect-resistant tomato.} } @INBOOK{IPB-8, author = {Sánchez, F. J. and Arciniegas, J. P. and Brand, A. and Vacca, O. and Mosquera, A. J. and Medina, A. and Chavarriaga, P. and}, title = {{Methods in Enzymology}}, year = {2022}, pages = {31-62}, chapter = {{Metabolic engineering of cassava to improve carotenoids}}, journal = {Carotenoids: Carotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology}, editor = {Eleanore T. Wurtzel}, doi = {10.1016/bs.mie.2022.03.004}, url = {https://doi.org/10.1016/bs.mie.2022.03.004}, volume = {671}, abstract = {Cassava is a staple food used in many countries around the world, despite deficiencies in micronutrients such as provitamin A carotenoids. Unfortunately, improvement of the cassava nutritional content by use of conventional breeding is slow and difficult. Therefore, there is an urgent need to develop and standardize protocols using biotechnological tools to improve cassava. The Alliance of Biodiversity International and the International Center for Tropical Agriculture (CIAT) have worked on cassava genetic transformation over the last 30 years. Here, we describe, step by step, the procedures used for genetic transformation of cassava variety TMS60444, to improve carotenoids and other traits. This protocol includes stock setup, reagents, media preparation, materials, and equipment, for the genetic transformation of embryogenic tissues. The main expected output in publishing this protocol is to provide the basis for a reproducible and reliable method to genetically modify and/or gene edit Latin American and Asian cassava varieties.} } @Article{IPB-526, author = {Zabel, S. and Brandt, W. and Porzel, A. and Athmer, B. and Bennewitz, S. and Schäfer, P. and Kortbeek, R. W. J. and Bleeker, P. M. and Tissier, A. and}, title = {{A single cytochrome P450 oxidase from Solanum habrochaites sequentially oxidizes 7-epi-zingiberene to derivatives toxic to whiteflies and various microorganisms}}, year = {2021}, pages = {1309-1325}, journal = {Plant J.}, doi = {10.1111/tpj.15113}, url = {https://doi.org/10.1111/tpj.15113}, volume = {105}, abstract = {Secretions from glandular trichomes potentially protect plants against a variety of aggressors. In the tomato clade of the Solanum genus, glandular trichomes of wild species produce a rich source of chemical diversity at the leaf surface. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we report the identification and characterisation of 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxyzingiberene (9H10epoZ), two derivatives of 7-epi-zingiberene produced in glandular trichomes of S. habrochaites LA2167. Using a combination of transcriptomics and genetics, we identified a gene coding for a cytochrome P450 oxygenase, ShCYP71D184, that is highly expressed in trichomes and co-segregates with the presence of the zingiberene derivatives. Transient expression assays in Nicotiana benthamiana showed that ShCYP71D184 carries out two successive oxidations to generate 9HZ and 9H10epoZ. Bioactivity assays showed that 9-hydroxy-10,11-epoxyzingiberene in particular exhibits substantial toxicity against B. tabaci and various microorganisms including Phytophthora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.} } @Article{IPB-525, author = {Yang, C. and Marillonnet, S. and Tissier, A. and}, title = {{The scarecrow-like transcription factor SlSCL3 regulates volatile terpene biosynthesis and glandular trichome size in tomato (Solanum lycopersicum)}}, year = {2021}, pages = {1102-1118}, journal = {Plant J.}, doi = {10.1111/tpj.15371}, url = {https://onlinelibrary.wiley.com/doi/full/10.1111/tpj.15371}, volume = {107}, abstract = {Tomato (Solanum lycopersicum L.) type VI glandular trichomes that occur on the surface of leaves, stems, young fruits and flowers produce and store a blend of volatile monoterpenes and sesquiterpenes. These compounds play important roles in the interaction with pathogens and herbivorous insects. Although the function of terpene synthases in the biosynthesis of volatile terpenes in tomato has been comprehensively investigated, the deciphering of their transcriptional regulation is only just emerging. We selected transcription factors that are over-expressed in trichomes based on existing transcriptome data and silenced them individually by virus-induced gene silencing. Of these, SlSCL3, a scarecrow-like (SCL) subfamily transcription factor, led to a significant decrease in volatile terpene content and expression of the corresponding terpene synthase genes when its transcription level was downregulated. Overexpression of SlSCL3 dramatically increased both the volatile terpene content and glandular trichome size, whereas its homozygous mutants showed reduced terpene biosynthesis. However, its heterozygous mutants also showed a significantly elevated volatile terpene content and enlarged glandular trichomes, similar to the overexpression plants. SlSCL3 modulates the expression of terpene biosynthetic pathway genes by transcriptional activation, but neither direct protein–DNA binding nor interaction with known regulators was observed. Moreover, transcript levels of the endogenous copy of SlSCL3 were decreased in the overexpression plants but increased in the heterozygous and homozygous mutants, suggesting feedback repression of its own promoter. Taken together, our results provide new insights into the role of SlSCL3 in the complex regulation of volatile terpene biosynthesis and glandular trichome development in tomato.} } @Article{IPB-519, author = {Tannert, M. and Balcke, G. U. and Tissier, A. and Köck, M. and}, title = {{At4g29530 is a phosphoethanolamine phosphatase homologous to PECP1 with a role in flowering time regulation}}, year = {2021}, pages = {1072-1083}, journal = {Plant J.}, doi = {10.1111/tpj.15367}, url = {https://doi.org/10.1111/tpj.15367}, volume = {107}, abstract = {Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in membranes. The biosynthesis of phospholipids occurs mainly via the Kennedy pathway. Recent studies have shown that through this pathway, choline (Cho) moieties are synthesized through the methylation of phosphoethanolamine (PEtn) to phosphocholine (PCho) by phospho-base N-methyltransferase. In Arabidopsis thaliana, the phosphoethanolamine/phosphocholine phosphatase1 (PECP1) is described as an enzyme that regulates the synthesis of PCho by decreasing the PEtn level during phosphate starvation to avoid the energy-consuming methylation step. By homology search, we identified a gene (At4g29530) encoding a putative PECP1 homolog from Arabidopsis with a currently unknown biological function in planta. We found that At4g29530 is not induced by phosphate starvation, and is mainly expressed in leaves and flowers. The analysis of null mutants and overexpression lines revealed that PEtn, rather than PCho, is the substrate in vivo, as in PECP1. Hydrophilic interaction chromatography-coupled mass spectrometry analysis of head group metabolites shows an increased PEtn level and decreased ethanolamine level in null mutants. At4g29530 null mutants have an early flowering phenotype, which is corroborated by a higher PC/PE ratio. Furthermore, we found an increased PCho level. The choline level was not changed, so the results corroborate that the PEtn-dependent pathway is the main route for the generation of Cho moieties. We assume that the PEtn-hydrolyzing enzyme participates in fine-tuning the metabolic pathway, and helps prevent the energy-consuming biosynthesis of PCho through the methylation pathway.} } @Article{IPB-452, author = {Grützner, R. and Martin, P. and Horn, C. and Mortensen, S. and Cram, E. J. and Lee-Parsons, C. W. and Stuttmann, J. and Marillonnet, S. and}, title = {{High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns}}, year = {2021}, pages = {100135}, journal = {Plant Communications}, doi = {10.1016/j.xplc.2020.100135}, url = {https://www.sciencedirect.com/science/article/pii/S2590346220301784}, volume = {2}, abstract = {The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana, we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.} } @Article{IPB-451, author = {Grützner, R. and Schubert, R. and Horn, C. and Yang, C. and Vogt, T. and Marillonnet, S. and}, title = {{Engineering Betalain Biosynthesis in Tomato for High Level Betanin Production in Fruits}}, year = {2021}, pages = {682443}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2021.682443}, url = {https://www.frontiersin.org/articles/10.3389/fpls.2021.682443/full}, volume = {12}, abstract = {Betalains are pigments found in plants of the Caryophyllales order, and include the red-purple betacyanins and the yellow-orange betaxanthins. The red pigment from red beets, betanin, is made from tyrosine by a biosynthetic pathway that consists of a cytochrome P450, a L-DOPA dioxygenase, and a glucosyltransferase. The entire pathway was recently reconstituted in plants that do not make betalains naturally including potato and tomato plants. The amount of betanin produced in these plants was however not as high as in red beets. It was recently shown that a plastidic arogenate dehydrogenase gene involved in biosynthesis of tyrosine in plants is duplicated in Beta vulgaris and other betalain-producing plants, and that one of the two encoded enzymes, BvADHα, has relaxed feedback inhibition by tyrosine, contributing to the high amount of betanin found in red beets. We have reconstituted the complete betanin biosynthetic pathway in tomato plants with or without a BvADHα gene, and with all genes expressed under control of a fruit-specific promoter. The plants obtained with a construct containing BvADHα produced betanin at a higher level than plants obtained with a construct lacking this gene. These results show that use of BvADHα can be useful for high level production of betalains in heterologous hosts. Unlike red beets that produce both betacyanins and betaxanthins, the transformed tomatoes produced betacyanins only, conferring a bright purple-fuschia color to the tomato juice.} } @Article{IPB-430, author = {Bittner, A. and Hause, B. and Baier, M. and}, title = {{Cold-priming causes oxylipin dampening during the early cold and light response of Arabidopsis thaliana}}, year = {2021}, pages = {7163-7179}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/erab314}, url = {https://doi.org/10.1093/jxb/erab314}, volume = {72}, abstract = {Abstract The comparison of transcriptome time-courses of the first 2 h of the cold or highlight response of 24 h cold primed and naive Arabidopsis thaliana showed that priming quickly modifies gene expression in a trigger-specific manner. It dampened up- as well as down-regulation of genes in the cold and in the light. 1/3 of the priming-regulated genes were jasmonate sensitive, including the full set of genes required for oxylipin biosynthesis. qPCR-based analysis in wildtype plants and mutants demonstrated that OPDA (12-oxo phytenoic acid) biosynthesis relative to the jasmonic acid (JA) availability controls dampening of the genes for oxylipin biosynthetic enzymes: Gene regulation in oxylipin biosynthesis mutants more strongly depended on the biosynthesis of the JA precursor OPDA than on its conversion to JA. Additionally, priming-dependent dampening during triggering was more linked to OPDA than to JA level regulation and spray application of OPDA prior to triggering counteracted gene dampening. In contrast to cold-priming induced dampening of ZAT10, priming regulation of the oxylipin hub was insensitive to priming-induced accumulation of thylakoid ascorbate peroxidase and mediated by modulation of the oxylipin sensitivity of genes for OPDA biosynthesis.} } @Article{IPB-497, author = {Püllmann, P. and Knorrscheidt, A. and Münch, J. and Palme, P. R. and Hoehenwarter, W. and Marillonnet, S. and Alcalde, M. and Westermann, B. and Weissenborn, M. J. and}, title = {{A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases}}, year = {2021}, pages = {562}, journal = {Commun. Biol.}, doi = {10.1038/s42003-021-02076-3}, url = {https://www.nature.com/articles/s42003-021-02076-3}, volume = {4}, abstract = {AbstractFungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.} } @Article{IPB-495, author = {Peters, K. and Balcke, G. and Kleinenkuhnen, N. and Treutler, H. and Neumann, S. and}, title = {{Untargeted in silico compound classification—A novel metabolomics method to assess the chemodiversity in bryophytes}}, year = {2021}, pages = {3251}, journal = {Int. J. Mol. Sci.}, doi = {10.3390/ijms22063251}, url = {https://www.mdpi.com/1422-0067/22/6/3251}, volume = {22}, abstract = {In plant ecology, biochemical analyses of bryophytes and vascular plants are often conducted on dried herbarium specimen as species typically grow in distant and inaccessible locations. Here, we present an automated in silico compound classification framework to annotate metabolites using an untargeted data independent acquisition (DIA)–LC/MS–QToF-sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH) ecometabolomics analytical method. We perform a comparative investigation of the chemical diversity at the global level and the composition of metabolite families in ten different species of bryophytes using fresh samples collected on-site and dried specimen stored in a herbarium for half a year. Shannon and Pielou’s diversity indices, hierarchical clustering analysis (HCA), sparse partial least squares discriminant analysis (sPLS-DA), distance-based redundancy analysis (dbRDA), ANOVA with post-hoc Tukey honestly significant difference (HSD) test, and the Fisher’s exact test were used to determine differences in the richness and composition of metabolite families, with regard to herbarium conditions, ecological characteristics, and species. We functionally annotated metabolite families to biochemical processes related to the structural integrity of membranes and cell walls (proto-lignin, glycerophospholipids, carbohydrates), chemical defense (polyphenols, steroids), reactive oxygen species (ROS) protection (alkaloids, amino acids, flavonoids), nutrition (nitrogen- and phosphate-containing glycerophospholipids), and photosynthesis. Changes in the composition of metabolite families also explained variance related to ecological functioning like physiological adaptations of bryophytes to dry environments (proteins, peptides, flavonoids, terpenes), light availability (flavonoids, terpenes, carbohydrates), temperature (flavonoids), and biotic interactions (steroids, terpenes). The results from this study allow to construct chemical traits that can be attributed to biogeochemistry, habitat conditions, environmental changes and biotic interactions. Our classification framework accelerates the complex annotation process in metabolomics and can be used to simplify biochemical patterns. We show that compound classification is a powerful tool that allows to explore relationships in both molecular biology by “zooming in” and in ecology by “zooming out”. The insights revealed by our framework allow to construct new research hypotheses and to enable detailed follow-up studies.} } @Article{IPB-492, author = {Ordon, J. and Martin, P. and Erickson, J. L. and Ferik, F. and Balcke, G. and Bonas, U. and Stuttmann, J. and}, title = {{Disentangling cause and consequence: genetic dissection of the DANGEROUS MIX2 risk locus, and activation of the DM2h NLR in autoimmunity}}, year = {2021}, pages = {1008-1023}, journal = {Plant J.}, doi = {10.1111/tpj.15215}, url = {https://onlinelibrary.wiley.com/doi/10.1111/tpj.15215}, volume = {106}, abstract = {Nucleotide-binding domain–leucine-rich repeat-type immune receptors (NLRs) protect plants against pathogenic microbes through intracellular detection of effector proteins. However, this comes at a cost, as NLRs can also induce detrimental autoimmunity in genetic interactions with foreign alleles. This may occur when independently evolved genomes are combined in inter- or intraspecific crosses, or when foreign alleles are introduced by mutagenesis or transgenesis. Most autoimmunity-inducing NLRs are encoded within highly variable NLR gene clusters with no known immune functions, which were termed autoimmune risk loci. Whether risk NLRs differ from sensor NLRs operating in natural pathogen resistance and how risk NLRs are activated in autoimmunity is unknown. Here, we analyzed the DANGEROUS MIX2 risk locus, a major autoimmunity hotspot in Arabidopsis thaliana. By gene editing and heterologous expression, we show that a single gene, DM2h, is necessary and sufficient for autoimmune induction in three independent cases of autoimmunity in accession Landsberg erecta. We focus on autoimmunity provoked by an EDS1-yellow fluorescent protein (YFP)NLS fusion protein to characterize DM2h functionally and determine features of EDS1-YFPNLS activating the immune receptor. Our data suggest that risk NLRs function in a manner reminiscent of sensor NLRs, while autoimmunity-inducing properties of EDS1-YFPNLS in this context are unrelated to the protein\'s functions as an immune regulator. We propose that autoimmunity, at least in some cases, may be caused by spurious, stochastic interactions of foreign alleles with coincidentally matching risk NLRs.} } @Article{IPB-483, author = {Mielke, S. and Zimmer, M. and Meena, M. K. and Dreos, R. and Stellmach, H. and Hause, B. and Voiniciuc, C. and Gasperini, D. and}, title = {{Jasmonate biosynthesis arising from altered cell walls is prompted by turgor-driven mechanical compression}}, year = {2021}, pages = {eabf0356}, journal = {Sci. Adv.}, doi = {10.1126/sciadv.abf0356}, url = {https://advances.sciencemag.org/content/7/7/eabf0356}, volume = {7}, abstract = {Despite the vital roles of jasmonoyl-isoleucine (JA-Ile) in governing plant growth and environmental acclimation, it remains unclear what intracellular processes lead to its induction. Here, we provide compelling genetic evidence that mechanical and osmotic regulation of turgor pressure represents a key elicitor of JA-Ile biosynthesis. After identifying cell wall mutant alleles in KORRIGAN1 (KOR1) with elevated JA-Ile in seedling roots, we found that ectopic JA-Ile resulted from cell nonautonomous signals deriving from enlarged cortex cells compressing inner tissues and stimulating JA-Ile production. Restoring cortex cell size by cell type–specific KOR1 complementation, by isolating a genetic kor1 suppressor, and by lowering turgor pressure with hyperosmotic treatments abolished JA-Ile signaling. Conversely, hypoosmotic treatment activated JA-Ile signaling in wild-type plants. Furthermore, constitutive JA-Ile levels guided mutant roots toward greater water availability. Collectively, these findings enhance our understanding on JA-Ile biosynthesis initiation and reveal a previously undescribed role of JA-Ile in orchestrating environmental resilience.} } @Article{IPB-516, author = {Stuttmann, J. and Barthel, K. and Martin, P. and Ordon, J. and Erickson, J. L. and Herr, R. and Ferik, F. and Kretschmer, C. and Berner, T. and Keilwagen, J. and Marillonnet, S. and Bonas, U. and}, title = {{Highly efficient multiplex editing: one‐shot generation of 8× Nicotiana benthamiana and 12× Arabidopsis mutants}}, year = {2021}, pages = {8-22}, journal = {Plant J.}, doi = {10.1111/tpj.15197}, url = {https://doi.org/10.1111/tpj.15197}, volume = {106}, abstract = {Genome editing by RNA-guided nucleases, such as SpCas9, has been used in numerous different plant species. However, to what extent multiple independent loci can be targeted simultaneously by multiplexing has not been well documented. Here, we developed a toolkit, based on a highly intron-optimized zCas9i gene, which allows assembly of nuclease constructs expressing up to 32 single guide RNAs (sgRNAs). We used this toolkit to explore the limits of multiplexing in two major model species, and report on the isolation of transgene-free octuple (8×) Nicotiana benthamiana and duodecuple (12×) Arabidopsis thaliana mutant lines in a single generation (T1 and T2, respectively). We developed novel counter-selection markers for N. benthamiana, most importantly Sl-FAST2, comparable to the well-established Arabidopsis seed fluorescence marker, and FCY-UPP, based on the production of toxic 5-fluorouracil in the presence of a precursor. Targeting eight genes with an array of nine different sgRNAs and relying on FCY-UPP for selection of non-transgenic T1, we identified N. benthamiana mutant lines with astonishingly high efficiencies: All analyzed plants carried mutations in all genes (approximately 112/116 target sites edited). Furthermore, we targeted 12 genes by an array of 24 sgRNAs in A. thaliana. Efficiency was significantly lower in A. thaliana, and our results indicate Cas9 availability is the limiting factor in such higher-order multiplexing applications. We identified a duodecuple mutant line by a combination of phenotypic screening and amplicon sequencing. The resources and results presented provide new perspectives for how multiplexing can be used to generate complex genotypes or to functionally interrogate groups of candidate genes.} } @Article{IPB-511, author = {Sheikh, A. H. and Fraz Hussain, R. M. and Tabassum, N. and Badmi, R. and Marillonnet, S. and Scheel, D. and Lee, J. and Sinha, A. and}, title = {{Possible role of WRKY transcription factors in regulating immunity in Oryza sativa ssp. indica}}, year = {2021}, pages = {101623}, journal = {Physiol. Mol. Plant Pathol.}, doi = {10.1016/j.pmpp.2021.101623}, url = {https://doi.org/10.1016/j.pmpp.2021.101623}, volume = {114}, abstract = {Plants have developed a robust transcription machinery to combat potential pathogenic organisms. One of the hallmarks of early immune responses is the activation of the WRKY transcription factors post infection. Specific WRKYs proteins from Arabidopsis are known substrates of MAPK pathway to mediate the flg22 elicited early immunity. In the current study, using the Golden Gate cloning strategy, we aim to clone the entire WRKY transcription factor family from Oryza sativa ssp. indica consisting of more than 100 members and study their MAPK interaction and subsequent role in PTI. Using a reporter LUC assay in protoplasts we investigated the early defense responses in a few interesting OsWRKY candidates. Interestingly, we observed stringent regulation of WRKY expression in cells and their transcriptional expression only under specific stress responses. The phenomenon of gene expression regulation by intron retention (IR) was prevalently observed in rice WRKY transcripts. We could show the role of WRKY8, 24, and 77 in early defense responses. It was observed that WRKY24 enhanced the expression of early defense response marker genes like NHL10 while WRKY8 and WRKY77 supressed their expression. This study highlights the complicated mechanism by which OsWRKYs expression is possibly regulated and the distinctive roles of some individual members in plant immunity. At the same time this study serves as a cautionary warning for plant researchers to be mindful of the intron retention mechanism while cloning OsWRKYs.} } @Article{IPB-507, author = {Schnabel, A. and Athmer, B. and Manke, K. and Schumacher, F. and Cotinguiba, F. and Vogt, T. and}, title = {{Identification and characterization of piperine synthase from black pepper, Piper nigrum L.}}, year = {2021}, pages = {445}, journal = {Commun. Biol.}, doi = {10.1038/s42003-021-01967-9}, url = {https://www.nature.com/articles/s42003-021-01967-9}, volume = {4}, abstract = {Black pepper (Piper nigrum L.) is the world’s most popular spice and is also used as an ingredient in traditional medicine. Its pungent perception is due to the interaction of its major compound, piperine (1-piperoyl-piperidine) with the human TRPV-1 or vanilloid receptor. We now identify the hitherto concealed enzymatic formation of piperine from piperoyl coenzyme A and piperidine based on a differential RNA-Seq approach from developing black pepper fruits. This enzyme is described as piperine synthase (piperoyl-CoA:piperidine piperoyl transferase) and is a member of the BAHD-type of acyltransferases encoded by a gene that is preferentially expressed in immature fruits. A second BAHD-type enzyme, also highly expressed in immature black pepper fruits, has a rather promiscuous substrate specificity, combining diverse CoA-esters with aliphatic and aromatic amines with similar efficiencies, and was termed piperamide synthase. Recombinant piperine and piperamide synthases are members of a small gene family in black pepper. They can be used to facilitate the microbial production of a broad range of medicinally relevant aliphatic and aromatic piperamides based on a wide array of CoA-donors and amine-derived acceptors, offering widespread applications.} } @Article{IPB-506, author = {Schnabel, A. and Cotinguiba, F. and Athmer, B. and Vogt, T. and}, title = {{Piper nigrum CYP719A37 catalyzes the decisive methylenedioxy bridge formation in piperine biosynthesis}}, year = {2021}, pages = {128}, journal = {Plants}, doi = {10.3390/plants10010128}, url = {https://doi.org/10.3390/plants10010128}, volume = {10}, abstract = {Abstract: Black pepper (Piper nigrum) is among the world’s most popular spices. Its pungent principle, piperine, has already been identified 200 years ago, yet the biosynthesis of piperine in black pepper remains largely enigmatic. In this report we analyzed the characteristic methylenedioxy bridge formation of the aromatic part of piperine by a combination of RNA-sequencing, functional expression in yeast, and LC-MS based analysis of substrate and product profiles. We identified a single cytochrome P450 transcript, specifically expressed in black pepper immature fruits. The corresponding gene was functionally expressed in yeast (Saccharomyces cerevisiae) and characterized for substrate specificity with a series of putative aromatic precursors with an aromatic vanilloid structure. Methylenedioxy bridge formation was only detected when feruperic acid (5-(4-hydroxy-3-methoxyphenyl)-2,4-pentadienoic acid) was used as a substrate, and the corresponding product was identified as piperic acid. Two alternative precursors, ferulic acid and feruperine, were not accepted. Our data provide experimental evidence that formation of the piperine methylenedioxy bridge takes place in young black pepper fruits after a currently hypothetical chain elongation of ferulic acid and before the formation of the amide bond. The partially characterized enzyme was classified as CYP719A37 and is discussed in terms of specificity, storage, and phylogenetic origin of CYP719 catalyzed reactions in magnoliids and eudicots.} } @Article{IPB-2523, author = {Zabel, S. and Brandt, W. and Porzel, A. and Athmer, B. and Kortbeek, R. W. J. and Bleeker, P. M. and Tissier, A. and}, title = {{Two novel 7-epi-zingiberene derivatives with biological activity from Solanum habrochaites are produced by a single cytochrome P450 monooxygenase}}, year = {2020}, journal = {bioRxiv}, doi = {10.1101/2020.04.21.052571}, abstract = {Secretions from glandular trichomes potentially protect the plant against a variety of aggressors. In the tomato genus, wild species constitute a rich source of chemical diversity produced at the leaf surface by glandular trichomes. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we identify two derivatives of 7-epi-zingiberene from S. habrochaites that had not been reported as yet. We identified them as 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene. Using a combination of genetics and transcriptomics we identified a single cytochrome P450 oxygenase, ShCYP71D184 that carries out two successive oxidations to generate the two sesquiterpenoids. Bioactivity assays showed that only 9-hydroxy-10,11-epoxyzingiberene exhibits substantial toxicity against B. tabaci. In addition, both 9-hydroxy-zingiberene and 9-hydroxy-10,11-epoxyzingiberene display substantial growth inhibitory activities against a range of microorganisms, including Bacillus subtilis, Phytophtora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.} } @Article{IPB-2519, author = {Püllmann, P. and Knorrscheidt, A. and Münch, J. and Palme, P. R. and Hoehenwarter, W. and Marillonnet, S. and Alcalde, M. and Westermann, B. and Weissenborn, M. J. and}, title = {{A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases}}, year = {2020}, journal = {bioRxiv}, doi = {10.1101/2020.07.22.216432}, abstract = {Fungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.} } @Article{IPB-2518, author = {Grützner, R. and Martin, P. and Horn, C. and Mortensen, S. and Cram, E. J. and Lee-Parsons, C. W. T. and Stuttmann, J. and Marillonnet, S. and}, title = {{Addition of Multiple Introns to a Cas9 Gene Results in Dramatic Improvement in Efficiency for Generation of Gene Knockouts in Plants}}, year = {2020}, journal = {bioRxiv}, doi = {10.1101/2020.04.03.023036}, abstract = {The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by stably transforming a Cas9 expression construct into the plant genome. The efficiency with which mutations are obtained in genes of interest can vary considerably depending on specific features of the constructs, including the source and nature of the promoters and terminators used for expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency with which mutations could be obtained in target genes in Arabidopsis thaliana with the Cas9 nuclease, we have investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLS) and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on Cas9 activity and that two NLSs work better than one. However, the most important impact on the efficiency of the constructs was obtained by addition of 13 introns into the Cas9 coding sequence, which dramatically improved editing efficiencies of the constructs; none of the primary transformants obtained with a Cas9 lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of primary transformants generated with intronized Cas9 displayed mutant phenotypes. The intronized Cas9 was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus.} } @Article{IPB-2516, author = {Barthel, K. and Martin, P. and Ordon, J. and Erickson, J. L. and Gantner, J. and Erickson, J. L. and Herr, R. and Kretschmer, C. and Ferik, F. and Berner, T. and Keilwagen, J. and Marillonnet, S. and Stuttmann, J. and Bonas, U. and}, title = {{One-shot generation of duodecuple (12x) mutant Arabidopsis: Highly efficient routine editing in model species}}, year = {2020}, journal = {bioRxiv}, doi = {10.1101/2020.03.31.018671}, url = {https://doi.org/10.1101/2020.03.31.018671}, abstract = {Genome editing by RNA-guided nucleases in model species is still hampered by low efficiencies, and isolation of transgene-free individuals often requires tedious PCR screening. Here, we present a toolkit that mitigates these drawbacks for Nicotiana benthamiana and Arabidopsis thaliana. The toolkit is based on an intron-optimized SpCas9-coding gene (zCas9i), which conveys dramatically enhanced editing efficiencies. The zCas9i gene is combined with remaining components of the genome editing system in recipient vectors, which lack only the user-defined guide RNA transcriptional units. Up to 32 guide RNA transcriptional units can be introduced to these recipients by a simple and PCR-free cloning strategy, with the choice of three different RNA polymerase III promoters for guide RNA expression. We developed new markers to aid transgene counter-selection in N. benthamiana, and demonstrate their efficacy for isolation of several genome-edited N. benthamiana lines. In Arabidopsis, we explore the limits of multiplexing by simultaneously targeting 12 genes by 24 sgRNAs. Perhaps surprisingly, the limiting factor in such higher order multiplexing applications is Cas9 availability, rather than recombination or silencing of repetitive sgRNA TU arrays. Through a combination of phenotypic screening and pooled amplicon sequencing, we identify transgene-free duodecuple mutant Arabidopsis plants directly in the T2 generation. This demonstrates high efficiency of the zCas9i gene, and reveals new perspectives for multiplexing to target gene families and to generate higher order mutants.} } @Article{IPB-552, author = {Grunewald, S. and Marillonnet, S. and Hause, G. and Haferkamp, I. and Neuhaus, H. E. and Veß, A. and Hollemann, T. and Vogt, T. and}, title = {{The Tapetal Major Facilitator NPF2.8 is Required for Accumulation of Flavonol Glycosides on the Pollen Surface in Arabidopsis thaliana}}, year = {2020}, pages = {1727-1748}, journal = {Plant Cell}, doi = {10.1105/tpc.19.00801}, volume = {32}, abstract = {The exine of angiosperm pollen grains is usually covered by a complex mix of metabolites including pollen-specific hydroxycinnamic acid amides (HCAAs) and flavonoid glycosides. Whereas the biosynthetic pathways resulting in the formation of HCAAs and flavonol glycosides have been characterized, it is unclear, how these compounds are transported to the pollen surface. In this report we provide several lines of evidence that AtNPF2.8, a member of the nitrate/peptide NTR/PTR family of transporters is required for accumulation and transport of pollen-specific flavonol 3-O-sophorosides, characterized by a glycosidic β-1,2-linkage, to the pollen surface of Arabidopsis. Ectopic, transient expression of this flavonol sophoroside transporter, termed AtFST1, fused to green fluorescent protein (GFP) demonstrated localization of AtFST1 at the plasmalemma in epidermal leaf cells of Nicotiana benthamiana whereas the tapetum-specific AtFST1-expression was confirmed by promAtFST1:GFP-reporter lines. In vitro characterization of AtFST1-activity was achieved by microbial uptake assays based on 14C-labeled flavonol glycosides. Finally, rescue of an fst1-line by complementation with a genomic fragment of the AtFST1 gene restored flavonol glycoside accumulation of pollen grains to wild-type levels corroborating the requirement of AtFST1 for transport of flavonol-3-O-sophorosides from the tapetum to the pollen surface.} } @Article{IPB-547, author = {El Amerany, F. and Meddich, A. and Wahbi, S. and Porzel, A. and Taourirte, M. and Rhazi, M. and Hause, B. and}, title = {{Foliar Application of Chitosan Increases Tomato Growth and Influences Mycorrhization and Expression of Endochitinase-Encoding Genes}}, year = {2020}, pages = {535}, journal = {Int. J. Mol. Sci.}, doi = {10.3390/ijms21020535}, volume = {21}, abstract = {Nowadays, applying bio-organic fertilizer (e.g., chitosan, Ch) or integrating beneficial microorganisms (e.g., arbuscular mycorrhizal fungi, AMF) are among the successful strategies to promote plant growth. Here, the effect of two application modes of Ch (foliar spray or root treatment) and Ch-derived nanoparticles (NPs) on tomato plants colonized with the AMF Rhizophagus irregularis were analyzed, thereby focusing on plant biomass, flowering and mycorrhization. An increase of shoot biomass and flower number was observed in arbuscular mycorrhizal (AM) plants sprayed with Ch. The interaction with AMF, however, was reduced as shown by decreased mycorrhization rates and AM-specific gene expression. To get insights into Ch effect on mycorrhization, levels of sugars, jasmonates, abscisic acid, and the expression of two chitinase-encoding genes were determined in mycorrhizal roots. Ch had no effect on sugar and phytohormone levels, but the reduced mycorrhization was correlated with down- and upregulated expression of Chi3 and Chi9, respectively. In contrast, application of NPs to leaves and Ch applied to the soil did not show any effect, neither on mycorrhization rate nor on growth of mycorrhizal plants. Concluding, Ch application to leaves enhanced plant growth and flowering and reduced interaction with AMF, whereas root treatment did not affect these parameters.} } @Article{IPB-545, author = {Dunker, F. and Trutzenberg, A. and Rothenpieler, J. S. and Kuhn, S. and Pröls, R. and Schreiber, T. and Tissier, A. and Kemen, A. and Kemen, E. and Hückelhoven, R. and Weiberg, A. and}, title = {{Oomycete small RNAs bind to the plant RNA-induced silencing complex for virulence}}, year = {2020}, pages = {e56096}, journal = {eLife}, doi = {10.7554/eLife.56096}, volume = {9}, abstract = {The exchange of small RNAs (sRNAs) between hosts and pathogens can lead to gene silencing in the recipient organism, a mechanism termed cross-kingdom RNAi (ck-RNAi). While fungal sRNAs promoting virulence are established, the significance of ck-RNAi in distinct plant pathogens is not clear. Here, we describe that sRNAs of the pathogen Hyaloperonospora arabidopsidis, which represents the kingdom of oomycetes and is phylogenetically distant from fungi, employ the host plant\'s Argonaute (AGO)/RNA-induced silencing complex for virulence. To demonstrate H. arabidopsidis sRNA (HpasRNA) functionality in ck-RNAi, we designed a novel CRISPR endoribonuclease Csy4/GUS reporter that enabled in situ visualization of HpasRNA-induced target suppression in Arabidopsis. The significant role of HpasRNAs together with AtAGO1 in virulence was revealed in plant atago1 mutants and by transgenic Arabidopsis expressing a short-tandem-target-mimic to block HpasRNAs, that both exhibited enhanced resistance. HpasRNA-targeted plant genes contributed to host immunity, as Arabidopsis gene knockout mutants displayed quantitative enhanced susceptibility.} } @Article{IPB-534, author = {Asfaw, K. G. and Liu, Q. and Xu, X. and Manz, C. and Purper, S. and Eghbalian, R. and Münch, S. W. and Wehl, I. and Bräse, S. and Eiche, E. and Hause, B. and Bogeski, I. and Schepers, U. and Riemann, M. and Nick, P. and}, title = {{A mitochondria-targeted coenzyme Q peptoid induces superoxide dismutase and alleviates salinity stress in plant cells}}, year = {2020}, pages = {11563}, journal = {Sci. Rep.}, doi = {10.1038/s41598-020-68491-4}, volume = {10}, abstract = {Salinity is a serious challenge to global agriculture and threatens human food security. Plant cells can respond to salt stress either by activation of adaptive responses, or by programmed cell death. The mechanisms deciding the respective response are far from understood, but seem to depend on the degree, to which mitochondria can maintain oxidative homeostasis. Using plant PeptoQ, a Trojan Peptoid, as vehicle, it is possible to transport a coenzyme Q10 (CoQ10) derivative into plant mitochondria. We show that salinity stress in tobacco BY-2 cells (Nicotiana tabacum L. cv Bright Yellow-2) can be mitigated by pretreatment with plant PeptoQ with respect to numerous aspects including proliferation, expansion, redox homeostasis, and programmed cell death. We tested the salinity response for transcripts from nine salt-stress related-genes representing different adaptive responses. While most did not show any significant response, the salt response of the transcription factor NtNAC, probably involved in mitochondrial retrograde signaling, was significantly modulated by the plant PeptoQ. Most strikingly, transcripts for the mitochondrial, Mn-dependent Superoxide Dismutase were rapidly and drastically upregulated in presence of the peptoid, and this response was disappearing in presence of salt. The same pattern, albeit at lower amplitude, was seen for the sodium exporter SOS1. The findings are discussed by a model, where plant PeptoQ modulates retrograde signalling to the nucleus leading to a strong expression of mitochondrial SOD, what renders mitochondria more resilient to perturbations of oxidative balance, such that cells escape salt induced cell death and remain viable.} } @Article{IPB-595, author = {Schuurink, R. and Tissier, A. and}, title = {{Glandular trichomes: micro‐organs with model status?}}, year = {2020}, pages = {2251-2266}, journal = {New Phytol.}, doi = {10.1111/nph.16283}, volume = {225}, abstract = {Glandular trichomes are epidermal outgrowths that are the site of biosynthesis and storage of large quantities of specialized metabolites. Besides their role in the protection of plants against biotic and abiotic stresses, they have attracted interest owing to the importance of the compounds they produce for human use; for example, as pharmaceuticals, flavor and fragrance ingredients, or pesticides. Here, we review what novel concepts investigations on glandular trichomes have brought to the field of specialized metabolism, particularly with respect to chemical and enzymatic diversity. Furthermore, the next challenges in the field are understanding the metabolic network underlying the high productivity of glandular trichomes and the transport and storage of metabolites. Another emerging area is the development of glandular trichomes. Studies in some model species, essentially tomato, tobacco, and Artemisia, are now providing the first molecular clues, but many open questions remain: How is the distribution and density of different trichome types on the leaf surface controlled? When is the decision for an epidermal cell to differentiate into one type of trichome or another taken? Recent advances in gene editing make it now possible to address these questions and promise exciting discoveries in the near future.} } @Article{IPB-593, author = {Schubert, R. and Werner, S. and Cirka, H. and Rödel, P. and Tandron Moya, Y. and Mock, H.-P. and Hutter, I. and Kunze, G. and Hause, B. and}, title = {{Effects of Arbuscular Mycorrhization on fruit quality in industrialized tomato production}}, year = {2020}, pages = {7029}, journal = {Int. J. Mol. Sci.}, doi = {10.3390/ijms21197029}, url = {https://doi.org/10.3390/ijms21197029}, volume = {21}, abstract = {Industrialized tomato production faces a decrease in flavors and nutritional value due to conventional breeding. Moreover, tomato production heavily relies on nitrogen and phosphate fertilization. Phosphate uptake and improvement of fruit quality by arbuscular mycorrhizal (AM) fungi are well-studied. We addressed the question of whether commercially used tomato cultivars grown in a hydroponic system can be mycorrhizal, leading to improved fruit quality. Tomato plants inoculated with Rhizophagus irregularis were grown under different phosphate concentrations and in substrates used in industrial tomato production. Changes in fruit gene expression and metabolite levels were checked by RNAseq and metabolite determination, respectively. The tests revealed that reduction of phosphate to 80% and use of mixed substrate allow AM establishment without affecting yield. By comparing green fruits from non-mycorrhizal and mycorrhizal plants, differentially expressed genes (DEGs) were found to possibly be involved in processes regulating fruit maturation and nutrition. Red fruits from mycorrhizal plants showed a trend of higher BRIX values and increased levels of carotenoids in comparison to those from non-mycorrhizal plants. Free amino acids exhibited up to four times higher levels in red fruits due to AM, showing the potential of mycorrhization to increase the nutritional value of tomatoes in industrialized production.} } @Article{IPB-592, author = {Schnabel, A. and Cotinguiba, F. and Athmer, B. and Yang, C. and Westermann, B. and Schaks, A. and Porzel, A. and Brandt, W. and Schumacher, F. and Vogt, T. and}, title = {{A piperic acid CoA ligase produces a putative precursor of piperine, the pungent principle from black pepper fruits}}, year = {2020}, pages = {569-581}, journal = {Plant J.}, doi = {10.1111/tpj.14652}, volume = {102}, abstract = {Black pepper (Piper nigrum L.) is known for the high content of piperine, a cinnamoyl amide derivative regarded as largely responsible for the pungent taste of this widely used spice. Despite its long history and worldwide use, the biosynthesis of piperine and related amides has been enigmatic up to now. In this report we describe a specific piperic acid CoA ligase from immature green fruits of P. nigrum. The corresponding enzyme was cloned and functionally expressed in E. coli. The recombinant enzyme displays a high specificity for piperic acid and does not accept the structurally related feruperic acid characterized by a similar C‐2 extension of the general C6‐C3 phenylpropanoid structure. The enzyme is also inactive with the standard set of hydroxycinnamic acids tested including caffeic acid, 4‐coumaric acid, ferulic acid, and sinapic acid. Substrate specificity is corroborated by in silico modeling which suggests a perfect fit of the substrate piperic acid to the active site of the piperic acid CoA ligase. The CoA ligase gene shows highest expression levels in immature green fruits, is also expressed in leaves and flowers, but not in roots. Virus‐induced gene silencing provided some preliminary indications that the production of piperoyl‐CoA is required for the biosynthesis of piperine in black pepper fruits.} } @Article{IPB-581, author = {Ordon, J. and Bressan, M. and Kretschmer, C. and Dall’Osto, L. and Marillonnet, S. and Bassi, R. and Stuttmann, J. and}, title = {{Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation}}, year = {2020}, pages = {151-162}, journal = {Funct. Integr. Genomics}, doi = {10.1007/s10142-019-00665-4}, volume = {20}, abstract = {Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25–30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.} } @Article{IPB-571, author = {Marillonnet, S. and Grützner, R. and}, title = {{Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline}}, year = {2020}, pages = {e115}, journal = {Curr. Protoc. Mol. Biol.}, doi = {10.1002/cpmb.115}, volume = {130}, abstract = {Methods that enable the construction of recombinant DNA molecules are essential tools for biological research and biotechnology. Golden Gate cloning is used for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one‐pot assembly procedure. Golden Gate cloning is based on the use of a type IIS restriction enzyme for digestion of the DNA fragments and vector. Because restriction sites for the type IIS enzyme used for assembly must be present at the ends of the DNA fragments and vector but absent from all internal sequences, special care must be taken to prepare DNA fragments and the recipient vector with a structure suitable for assembly by Golden Gate cloning. In this article, protocols are presented for preparation of DNA fragments, modules, and vectors suitable for Golden Gate assembly cloning. Additional protocols are presented for assembly of defined parts in a transcription unit, as well as the stitching together of multiple transcription units into multigene constructs by the modular cloning (MoClo) pipeline.} } @Article{IPB-568, author = {Leonova, T. and Popova, V. and Tsarev, A. and Henning, C. and Antonova, K. and Rogovskaya, N. and Vikhnina, M. and Baldensperger, T. and Soboleva, A. and Dinastia, E. and Dorn, M. and Shiroglasova, O. and Grishina, T. and Balcke, G. U. and Ihling, C. and Smolikova, G. and Medvedev, S. and Zhukov, V. A. and Babakov, V. and Tikhonovich, I. A. and Glomb, M. A. and Bilova, T. and Frolov, A. and}, title = {{Does Protein Glycation Impact on the Drought-Related Changes in Metabolism and Nutritional Properties of Mature Pea (Pisum sativum L.) Seeds?}}, year = {2020}, pages = {567}, journal = {Int. J. Mol. Sci.}, doi = {10.3390/ijms21020567}, volume = {21}, abstract = {Protein glycation is usually referred to as an array of non-enzymatic post-translational modifications formed by reducing sugars and carbonyl products of their degradation. The resulting advanced glycation end products (AGEs) represent a heterogeneous group of covalent adducts, known for their pro-inflammatory effects in mammals, and impacting on pathogenesis of metabolic diseases and ageing. In plants, AGEs are the markers of tissue ageing and response to environmental stressors, the most prominent of which is drought. Although water deficit enhances protein glycation in leaves, its effect on seed glycation profiles is still unknown. Moreover, the effect of drought on biological activities of seed protein in mammalian systems is still unstudied with respect to glycation. Therefore, here we address the effects of a short-term drought on the patterns of seed protein-bound AGEs and accompanying alterations in pro-inflammatory properties of seed protein in the context of seed metabolome dynamics. A short-term drought, simulated as polyethylene glycol-induced osmotic stress and applied at the stage of seed filling, resulted in the dramatic suppression of primary seed metabolism, although the secondary metabolome was minimally affected. This was accompanied with significant suppression of NF-kB activation in human SH-SY5Y neuroblastoma cells after a treatment with protein hydrolyzates, isolated from the mature seeds of drought-treated plants. This effect could not be attributed to formation of known AGEs. Most likely, the prospective anti-inflammatory effect of short-term drought is related to antioxidant effect of unknown secondary metabolite protein adducts, or down-regulation of unknown plant-specific AGEs due to suppression of energy metabolism during seed filling.} } @Article{IPB-566, author = {Kusstatscher, P. and Wicaksono, W. A. and Bergna, A. and Cernava, T. and Bergau, N. and Tissier, A. and Hause, B. and Berg, G. and}, title = {{Trichomes form genotype-specific microbial hotspots in the phyllosphere of tomato}}, year = {2020}, pages = {17}, journal = {Environ. Microbiome}, doi = {10.1186/s40793-020-00364-9}, url = {https://doi.org/10.1186/s40793-020-00364-9}, volume = {15}, abstract = {Background: The plant phyllosphere is a well-studied habitat characterized by low nutrient availability and high community dynamics. In contrast, plant trichomes, known for their production of a large number of metabolites, are a yet unexplored habitat for microbes. We analyzed the phyllosphere as well as trichomes of two tomato genotypes (Solanum lycopersicum LA4024, S. habrochaites LA1777) by targeting bacterial 16S rRNA gene fragments. Results: Leaves, leaves without trichomes, and trichomes alone harbored similar abundances of bacteria (108–109 16S rRNA gene copy numbers per gram of sample). In contrast, bacterial diversity was found significantly increased in trichome samples (Shannon index: 4.4 vs. 2.5). Moreover, the community composition was significantly different when assessed with beta diversity analysis and corresponding statistical tests. At the bacterial class level, Alphaproteobacteria (23.6%) were significantly increased, whereas Bacilli (8.6%) were decreased in trichomes. The bacterial family Sphingomonadacea (8.4%) was identified as the most prominent, trichome-specific feature; Burkholderiaceae and Actinobacteriaceae showed similar patterns. Moreover, Sphingomonas was identified as a central element in the core microbiome of trichome samples, while distinct low-abundant bacterial families including Hymenobacteraceae and Alicyclobacillaceae were exclusively found in trichome samples. Niche preferences were statistically significant for both genotypes and genotype-specific enrichments were further observed. Conclusion: Our results provide first evidence of a highly specific trichome microbiome in tomato and show the importance of micro-niches for the structure of bacterial communities on leaves. These findings provide further clues for breeding, plant pathology and protection as well as so far unexplored natural pathogen defense strategies. } } @Article{IPB-608, author = {Tang, G. and Ma, J. and Hause, B. and Nick, P. and Riemann, M. and}, title = {{Jasmonate is required for the response to osmotic stress in rice}}, year = {2020}, pages = {104047}, journal = {Environ. Exp. Bot.}, doi = {10.1016/j.envexpbot.2020.104047}, volume = {175}, abstract = {Plants have the ability to alleviate the harmful effects caused by abiotic and biotic stress. Phytohormones play a very important role in the acclimation to these stresses. To study the role of jasmonate in the acclimation to osmotic stress, an ALLENE OXIDE CYCLASE (AOC) mutant of rice (cpm2), disrupted in the biosynthesis of jasmonic acid (JA), and its wild type (WT) background were employed to investigate their responses to osmotic stress caused by treatment with polyethylene glycol (PEG) 6000. WT showed tolerance to osmotic stress, correlated with a fast transient increase of JA and JA-isoleucine (JA-Ile) in the shoots prior to an increase in abscisic acid (ABA), followed by a second increase in jasmonates when exposing to osmotic stress during 24 h. In roots, the pattern of hormonal increase was similar, but the response appeared to be faster, and remained transient, also with respect to low levels of jasmonates upon continuing osmotic stress. The mutant, which was containing extremely low levels of jasmonates, was hypersensitive to the stress. However, ABA accumulated in both, shoots and roots of cpm2, to similar (but not equal) levels as those seen in the WT, demonstrating that the biosynthesis or catabolism of ABA in response to osmotic stress is at least partially independent of JA, but can be modulated by JA. Our results suggest that jasmonates operate in parallel, presumably synergistically, to ABA, and are indispensable for osmotic stress tolerance in rice.} } @Article{IPB-607, author = {Tabassum, N. and Eschen-Lippold, L. and Athmer, B. and Baruah, M. and Brode, M. and Maldonado-Bonilla, L. D. and Hoehenwarter, W. and Hause, G. and Scheel, D. and Lee, J. and}, title = {{Phosphorylation‐dependent control of an RNA granule‐localized protein that fine‐tunes defence gene expression at a post‐transcriptional level}}, year = {2020}, pages = {1023-1039}, journal = {Plant J.}, doi = {10.1111/tpj.14573}, volume = {101}, abstract = {Mitogen‐activated protein kinase (MAPK) cascades are key signalling modules of plant defence responses to pathogen‐associated molecular patterns (PAMPs, e.g. bacterial flg22 peptide). The Tandem Zinc Finger Protein 9 (TZF9) is an RNA‐binding protein that is phosphorylated by two PAMP‐responsive MAPKs, MPK3 and MPK6. We mapped the major phosphosites in TZF9 and showed their importance for controlling in vitro RNA‐binding activity, in vivo flg22‐induced rapid disappearance of TZF9‐labelled processing body‐like structures and TZF9 protein turnover. Microarray analysis showed a strong discordance between transcriptome (total mRNA) and translatome (polysome‐associated mRNA) in the tzf9 mutant, with more mRNAs associated to ribosomes in the absence of TZF9. This suggests that TZF9 may sequester and inhibit translation of subsets of mRNAs. Fittingly, TZF9 physically interacts with poly(A)‐binding protein 2 (PAB2), a hallmark constituent of stress granules – a site for stress‐induced translational stalling/arrest. TZF9 even promotes stress granule assembly in the absence of stress. Hence, MAPKs may control defence gene expression post‐transcriptionally through release from translation arrest within TZF9‐PAB2‐containing RNA granules or perturbing PAB2 functions in translation control (e.g. in the mRNA closed‐loop model of translation).} } @INBOOK{IPB-19, author = {Marillonnet, S. and Werner, S. and}, title = {{DNA Cloning and Assembly}}, year = {2020}, pages = {125-141}, chapter = {{Assembly of Multigene Constructs Using the Modular Cloning System MoClo}}, journal = {Methods Mol. Biol.}, editor = {In: Chandran S., George K.}, doi = {10.1007/978-1-0716-0908-8_8}, volume = {2205}, abstract = {Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for complex pathway engineering. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the Modular Cloning system MoClo. Making constructs using the MoClo system requires users to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.} } @INBOOK{IPB-17, author = {Hause, B. and Requena, N. and}, title = {{Arbuscular Mycorrhizal Fungi}}, year = {2020}, pages = {185-196}, chapter = {{Detection of Arbuscular Mycorrhizal Fungal Gene Expression by In Situ Hybridization}}, journal = {Methods Mol. Biol.}, editor = {Ferrol, N. \& Lanfranco, L., eds.}, doi = {10.1007/978-1-0716-0603-2_14}, volume = {2146}, abstract = {The complexity of the obligate symbiotic interaction of arbuscular mycorrhizal (AM) fungi and their host roots requires sophisticated molecular methods. In particular, to capture the dynamic of the interaction, cell-specific methods for gene expression analysis are required. In situ hybridization is a technique that allows to determine the location of transcript accumulation within tissues, being of special interest for these fungi that cannot be genetically modified. The method requires proper fixation and embedding methods as well as specific probes for the hybridization allowing detection of specific transcripts. In this chapter, we present a method to prepare roots, which have established a symbiosis with an arbuscular mycorrhizal fungus for the detection of fungal transcripts. This includes chemical fixation, subsequent embedding in a suitable medium, sectioning and pretreatment of sections, the hybridization procedure itself, as well as the immunological detection of RNA-RNA hybrids.} } @INBOOK{IPB-16, author = {Grützner, R. and Marillonnet, S. and}, title = {{DNA Cloning and Assembly}}, year = {2020}, pages = {107-123}, chapter = {{Generation of MoClo Standard Parts Using Golden Gate Cloning}}, journal = {Methods Mol. Biol.}, editor = {In: Chandran S., George K.}, doi = {10.1007/978-1-0716-0908-8_7}, url = {https://experiments.springernature.com/articles/10.1007/978-1-0716-0908-8_7}, volume = {2205}, abstract = {Availability of efficient DNA assembly methods is a basic requirement for synthetic biology. A variety of modular cloning systems have been developed, based on Golden Gate cloning for DNA assembly, to enable users to assemble multigene constructs from libraries of standard parts using a series of successive one-pot assembly reactions. Standard parts contain the DNA sequence coding for a genetic element of interest such as a promoter, coding sequence or terminator. Standard parts for the modular cloning system MoClo must be flanked by two BsaI restriction sites and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of basic parts. This protocol requires the following steps (1) defining the type of basic part that needs to be cloned, (2) designing primers for amplification, (3) performing PCR amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large basic parts, it is preferable to first clone subparts as intermediate level −1 constructs. These subparts are sequenced individually and are then further assembled to make the final level 0 module.} } @Article{IPB-2525, author = {Dunker, F. and Trutzenberg, A. and Rothenpieler, J. S. and Kuhn, S. and Pröls, R. and Schreiber, T. and Tissier, A. and Hückelhoven, R. and Weiberg, A. and}, title = {{Oomycete small RNAs invade the plant RNA-induced silencing complex for virulence}}, year = {2019}, journal = {bioRxiv}, doi = {10.1101/689190}, abstract = {Fungal small RNAs (sRNAs) hijack the plant RNA silencing pathway to manipulate host gene expression, named cross-kingdom RNA interference (ckRNAi). It is currently unknown how conserved and significant ckRNAi is for microbial virulence. Here, we found for the first time that sRNAs of a pathogen representing the oomycete kingdom invade the host plant’s Argonaute (AGO)/RNA-induced silencing complex. To demonstrate the functionality of the plant-invading oomycete Hyaloperonospora arabidopsidis sRNAs (HpasRNAs), we designed a novel CRISPR endoribonuclease Csy4/GUS repressor reporter to visualize in situ pathogen-induced target suppression in Arabidopsis thaliana host plant. By using 5’ RACE-PCR we demonstrated HpasRNAs-directed cleavage of plant mRNAs. The significant role of HpasRNAs together with AtAGO1 in virulence was demonstrated by plant atago1 mutants and by transgenic Arabidopsis expressing a target mimic to block HpasRNAs, that both exhibited enhanced resistance. Individual HpasRNA plant targets contributed to host immunity, as Arabidopsis gene knockout or HpasRNA-resistant gene versions exhibited quantitative enhanced or reduced susceptibility, respectively. Together with previous reports, we found that ckRNAi is conserved among oomycete and fungal pathogens.} } @Article{IPB-672, author = {Hausner, J. and Jordan, M. and Otten, C. and Marillonnet, S. and Büttner, D. and}, title = {{Modular Cloning of the Type III Secretion Gene Cluster from the Plant-Pathogenic Bacterium Xanthomonas euvesicatoria}}, year = {2019}, pages = {532-547}, journal = {ACS Synth. Biol.}, doi = {10.1021/acssynbio.8b00434}, volume = {8}, abstract = {Type III secretion (T3S) systems are essential pathogenicity factors of most Gram-negative bacteria and translocate effector proteins into plant or animal cells. T3S systems can, therefore, be used as tools for protein delivery into eukaryotic cells, for instance after transfer of the T3S gene cluster into nonpathogenic recipient strains. Here, we report the modular cloning of the T3S gene cluster from the plant-pathogenic bacterium Xanthomonas euvesicatoria. The resulting multigene construct encoded a functional T3S system and delivered effector proteins into plant cells. The modular design of the T3S gene cluster allowed the efficient replacement and rearrangement of single genes or operons and the insertion of reporter genes for functional studies. In the present study, we used the modular T3S system to analyze the assembly of a fluorescent fusion of the predicted cytoplasmic ring protein HrcQ. Our studies demonstrate the use of the modular T3S gene cluster for functional analyses and mutant approaches in X. euvesicatoria. A potential application of the modular T3S system as protein delivery tool is discussed.} } @Article{IPB-671, author = {Hause, B. and}, title = {{Elevated CO2‐induced improvement of mycorrhization – which players lie in‐between?}}, year = {2019}, pages = {5-7}, journal = {New Phytol.}, doi = {10.1111/nph.16023}, volume = {224}, abstract = {This article is a Commentary on Zhou et al. 224: 106–116.} } @Article{IPB-651, author = {Dreher, D. and Baldermann, S. and Schreiner, M. and Hause, B. and}, title = {{An arbuscular mycorrhizal fungus and a root pathogen induce different volatiles emitted by Medicago truncatula roots}}, year = {2019}, pages = {85-90}, journal = {J. Adv. Res.}, doi = {10.1016/j.jare.2019.03.002}, volume = {19}, abstract = {Plants are in permanent contact with various microorganisms and are always impacted by them. To better understand the first steps of a plant’s recognition of soil-borne microorganisms, the early release of volatile organic compounds (VOCs) emitted from roots of Medicago truncatula in response to the symbiont Rhizophagus irregularis or the pathogenic oomycete Aphanomyces euteiches was analysed. More than 90 compounds were released from roots as detected by an untargeted gas chromatography-mass spectrometry approach. Principal component analyses clearly distinguished untreated roots from roots treated with either R. irregularis or A. euteiches. Several VOCs were found to be emitted specifically in response to each of the microorganisms. Limonene was specifically emitted from wild-type roots after contact with R. irregularis spores but not from roots of the mycorrhiza-deficient mutant does not make infections3. The application of limonene to mycorrhizal roots, however, did not affect the mycorrhization rate. Inoculation of roots with A. euteiches zoospores resulted in the specific emission of several sesquiterpenes, such as nerolidol, viridiflorol and nerolidol-epoxyacetate but application of nerolidol to zoospores of A. euteiches did not affect their vitality. Therefore, plants discriminate between different microorganisms at early stages of their interaction and respond differently to the level of root-emitted volatiles.} } @Article{IPB-634, author = {Bergau, N. and Maul, S. and Rujescu, D. and Simm, A. and Navarrete Santos, A. and}, title = {{Reduction of Glycolysis Intermediate Concentrations in the Cerebrospinal Fluid of Alzheimer’s Disease Patients}}, year = {2019}, pages = {871}, journal = {Front. Neurosci.}, doi = {10.3389/fnins.2019.00871}, volume = {13}, abstract = {The profile of 122 metabolites in the cerebrospinal fluid (CSF) of patients suffering from Alzheimer’s disease (AD) and controls was studied. Among the 122 metabolites analyzed, 61 could be detected. Statistically significant differences between the AD and control group were only detected for metabolites of the glycolysis. Thus, accurate quantification of 11 glycolytic metabolites was done. We detected a significant reduction of five of them, namely phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate, pyruvate and dihydroxyacetone phosphate in the AD CSF compared to controls. These results correlate with the known reduction of glucose metabolism in the brain of patients with AD and indicate that metabolic analysis of the central carbon metabolism can be a potential tool in AD diagnostic. Although the Receiver operating characteristic (ROC) analyses of the metabolites do not reach the level of the diagnostic informativity of AD biomarkers, the combination of specific glycolysis metabolites with the established biomarkers may lead to an improvement in sensitivity and specificity.} } @Article{IPB-700, author = {Mittelberger, C. and Stellmach, H. and Hause, B. and Kerschbamer, C. and Schlink, K. and Letschka, T. and Janik, K. and}, title = {{A Novel Effector Protein of Apple Proliferation Phytoplasma Disrupts Cell Integrity of Nicotiana spp. Protoplasts}}, year = {2019}, pages = {4613}, journal = {Int. J. Mol. Sci.}, doi = {10.3390/ijms20184613}, volume = {20}, abstract = {Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by ‘Candidatus Phytoplasma mali’ (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called “Protein in Malus Expressed 2” (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.} } @Article{IPB-630, author = {Bathe, U. and Tissier, A. and}, title = {{Cytochrome P450 enzymes: A driving force of plant diterpene diversity}}, year = {2019}, pages = {149-162}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2018.12.003}, volume = {161}, abstract = {In plant terpene biosynthesis, oxidation of the hydrocarbon backbone produced by terpene synthases is typically carried out by cytochrome P450 oxygenases (CYPs). The modifications introduced by CYPs include hydroxylations, sequential oxidations at one position and ring rearrangements and closures. These reactions significantly expand the structural diversity of terpenoids, but also provide anchoring points for further decorations by various transferases. In recent years, there has been a significant increase in reports of CYPs involved in plant terpene pathways. Plant diterpenes represent an important class of metabolites that includes hormones and a number of industrially relevant compounds such as pharmaceutical, aroma or food ingredients. In this review, we provide a comprehensive survey on CYPs reported to be involved in plant diterpene biosynthesis to date. A phylogenetic analysis showed that only few CYP clans are represented in diterpene biosynthesis, namely CYP71, CYP85 and CYP72. Remarkably few CYP families and subfamilies within those clans are involved, indicating specific expansion of these clades in plant diterpene biosynthesis. Nonetheless, the evolutionary trajectory of CYPs of specialized diterpene biosynthesis is diverse. Some are recently derived from gibberellin biosynthesis, while others have a more ancient history with recent expansions in specific plant families. Among diterpenoids, labdane-related diterpenoids represent a dominant class. The availability of CYPs from diverse plant species able to catalyze oxidations in specific regions of the labdane-related backbones provides opportunities for combinatorial biosynthesis to produce novel diterpene compounds that can be screened for biological activities of interest.} } @Article{IPB-629, author = {Bathe, U. and Frolov, A. and Porzel, A. and Tissier, A. and}, title = {{CYP76 Oxidation Network of Abietane Diterpenes in Lamiaceae Reconstituted in Yeast}}, year = {2019}, pages = {13437-13450}, journal = {J. Agr. Food Chem.}, doi = {10.1021/acs.jafc.9b00714}, volume = {67}, abstract = {Rosemary and sage species from Lamiaceae contain high amounts of structurally related but diverse abietane diterpenes. A number of substances from this compound family have potential pharmacological activities and are used in the food and cosmetic industry. This has raised interest in their biosynthesis. Investigations in Rosmarinus officinalis and some sage species have uncovered two main groups of cytochrome P450 oxygenases that are involved in the oxidation of the precursor abietatriene. CYP76AHs produce ferruginol and 11-hydroxyferruginol, while CYP76AKs catalyze oxidations at the C20 position. Using a modular Golden-Gate-compatible assembly system for yeast expression, these enzymes were systematically tested either alone or in combination. A total of 14 abietane diterpenes could be detected, 8 of which have not been reported thus far. We demonstrate here that yeast is a valid system for engineering and reconstituting the abietane diterpene network, allowing for the discovery of novel compounds with potential bioactivity.} } @Article{IPB-712, author = {Püllmann, P. and Ulpinnis, C. and Marillonnet, S. and Gruetzner, R. and Neumann, S. and Weissenborn, M. J. and}, title = {{Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design}}, year = {2019}, pages = {10932}, journal = {Sci. Rep.}, doi = {10.1038/s41598-019-47376-1}, volume = {9}, abstract = {Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Mutagenesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.} } @Article{IPB-752, author = {Yadav, H. and Dreher, D. and Athmer, B. and Porzel, A. and Gavrin, A. and Baldermann, S. and Tissier, A. and Hause, B. and}, title = {{Medicago TERPENE SYNTHASE 10 Is Involved in Defense Against an Oomycete Root Pathogen}}, year = {2019}, pages = {1598-1613}, journal = {Plant Physiol.}, doi = {10.1104/pp.19.00278}, volume = {180}, abstract = {In nature, plants interact with numerous beneficial or pathogenic soil-borne microorganisms. Plants have developed various defense strategies to expel pathogenic microbes, some of which function soon after pathogen infection. We used Medicago truncatula and its oomycete pathogen Aphanomyces euteiches to elucidate early responses of the infected root. A. euteiches causes root rot disease in legumes and is a limiting factor in legume production. Transcript profiling of seedlings and adult plant roots inoculated with A. euteiches zoospores for 2 h revealed specific upregulation of a gene encoding a putative sesquiterpene synthase (M. truncatula TERPENE SYNTHASE 10 [MtTPS10]) in both developmental stages. MtTPS10 was specifically expressed in roots upon oomycete infection. Heterologous expression of MtTPS10 in yeast led to production of a blend of sesquiterpenes and sesquiterpene alcohols, with NMR identifying a major peak corresponding to himalachol. Moreover, plants carrying a tobacco (Nicotiana tabacum) retrotransposon Tnt1 insertion in MtTPS10 lacked the emission of sesquiterpenes upon A. euteiches infection, supporting the assumption that the identified gene encodes a multiproduct sesquiterpene synthase. Mttps10 plants and plants with reduced MtTPS10 transcript levels created by expression of an MtTPS10-artificial microRNA in roots were more susceptible to A. euteiches infection than were the corresponding wild-type plants and roots transformed with the empty vector, respectively. Sesquiterpenes produced by expression of MtTPS10 in yeast also inhibited mycelial growth and A. euteiches zoospore germination. These data suggest that sesquiterpene production in roots by MtTPS10 plays a previously unrecognized role in the defense response of M. truncatula against A. euteiches.} } @Article{IPB-749, author = {Wasternack, C. and Hause, B. and}, title = {{The missing link in jasmonic acid biosynthesis}}, year = {2019}, pages = {776-777}, journal = {Nat. Plants}, doi = {10.1038/s41477-019-0492-y}, volume = {5}, abstract = {Jasmonic acid biosynthesis starts in chloroplasts and is finalized in peroxisomes. The required export of a crucial intermediate out of the chloroplast is now shown to be mediated by a protein from the outer envelope called JASSY.} } @Article{IPB-745, author = {Wasternack, C. and Hause, B. and Abel, S. and}, title = {{Benno Parthier (1932–2019)}}, year = {2019}, pages = {519-520}, journal = {Plant Mol. Biol.}, doi = {10.1007/s11103-019-00927-6}, volume = {101}, } @Article{IPB-731, author = {Schulze, A. and Zimmer, M. and Mielke, S. and Stellmach, H. and Melnyk, C. W. and Hause, B. and Gasperini, D. and}, title = {{Wound-Induced Shoot-to-Root Relocation of JA-Ile Precursors Coordinates Arabidopsis Growth}}, year = {2019}, pages = {1383-1394}, journal = {Mol. Plant}, doi = {10.1016/j.molp.2019.05.013}, volume = {12}, abstract = {Multicellular organisms rely on the movement of signaling molecules across cells, tissues, and organs to communicate among distal sites. In plants, localized leaf damage activates jasmonic acid (JA)-dependent transcriptional reprogramming in both harmed and unharmed tissues. Although it has been indicated that JA species can translocate from damaged into distal sites, the identity of the mobile compound(s), the tissues through which they translocate, and the effect of their relocation remain unknown. Here, we found that following shoot wounding, the relocation of endogenous jasmonates through the phloem is essential to initiate JA signaling and stunt growth in unharmed roots of Arabidopsis thaliana. By employing grafting experiments and hormone profiling, we uncovered that the hormone precursor cis-12-oxo-phytodienoic acid (OPDA) and its derivatives, but not the bioactive JA-Ile conjugate, translocate from wounded shoots into undamaged roots. Upon root relocation, the mobile precursors cooperatively regulated JA responses through their conversion into JA-Ile and JA signaling activation. Collectively, our findings demonstrate the existence of long-distance translocation of endogenous OPDA and its derivatives, which serve as mobile molecules to coordinate shoot-to-root responses, and highlight the importance of a controlled redistribution of hormone precursors among organs during plant stress acclimation.} } @Article{IPB-730, author = {Schubert, R. and Dobritzsch, S. and Gruber, C. and Hause, G. and Athmer, B. and Schreiber, T. and Marillonnet, S. and Okabe, Y. and Ezura, H. and Acosta, I. F. and Tarkowská, D. and Hause, B. and}, title = {{Tomato MYB21 Acts in Ovules to Mediate Jasmonate-Regulated Fertility}}, year = {2019}, pages = {1043-1062}, journal = {Plant Cell}, doi = {10.1105/tpc.18.00978}, volume = {31}, abstract = {The function of the plant hormone jasmonic acid (JA) in the development of tomato (Solanum lycopersicum) flowers was analyzed with a mutant defective in JA perception (jasmonate-insensitive1-1, jai1-1). In contrast with Arabidopsis (Arabidopsis thaliana) JA-insensitive plants, which are male sterile, the tomato jai1-1 mutant is female sterile, with major defects in female development. To identify putative JA-dependent regulatory components, we performed transcriptomics on ovules from flowers at three developmental stages from wild type and jai1-1 mutants. One of the strongly downregulated genes in jai1-1 encodes the MYB transcription factor SlMYB21. Its Arabidopsis ortholog plays a crucial role in JA-regulated stamen development. SlMYB21 was shown here to exhibit transcription factor activity in yeast, to interact with SlJAZ9 in yeast and in planta, and to complement Arabidopsis myb21-5. To analyze SlMYB21 function, we generated clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9 (Cas9) mutants and identified a mutant by Targeting Induced Local Lesions in Genomes (TILLING). These mutants showed female sterility, corroborating a function of MYB21 in tomato ovule development. Transcriptomics analysis of wild type, jai1-1, and myb21-2 carpels revealed processes that might be controlled by SlMYB21. The data suggest positive regulation of JA biosynthesis by SlMYB21, but negative regulation of auxin and gibberellins. The results demonstrate that SlMYB21 mediates at least partially the action of JA and might control the flower-to-fruit transition.} } @Article{IPB-729, author = {Schubert, R. and Grunewald, S. and von Sivers, L. and Hause, B. and}, title = {{Effects of Jasmonate on Ethylene Function during the Development of Tomato Stamens}}, year = {2019}, pages = {277}, journal = {Plants}, doi = {10.3390/plants8080277}, volume = {8}, abstract = {The phenotype of the tomato mutant jasmonate-insensitive1-1 (jai1-1) mutated in the JA-Ile co-receptor COI1 demonstrates JA function in flower development, since it is female-sterile. In addition, jai1-1 exhibits a premature anther dehydration and pollen release, being in contrast to a delayed anther dehiscence in the JA-insensitive Arabidopsis mutant coi1-1. The double mutant jai1-1 Never ripe (jai1-1 Nr), which is in addition insensitive to ethylene (ET), showed a rescue of the jai1-1 phenotype regarding pollen release. This suggests that JA inhibits a premature rise in ET to prevent premature stamen desiccation. To elucidate the interplay of JA and ET in more detail, stamen development in jai1-1 Nr was compared to wild type, jai1-1 and Nr regarding water content, pollen vitality, hormone levels, and accumulation of phenylpropanoids and transcripts encoding known JA- and ET-regulated genes. For the latter, RT-qPCR based on nanofluidic arrays was employed. The data showed that additional prominent phenotypic features of jai1-1, such as diminished water content and pollen vitality, and accumulation of phenylpropanoids were at least partially rescued by the ET-insensitivity. Hormone levels and accumulation of transcripts were not affected. The data revealed that strictly JA-regulated processes cannot be rescued by ET-insensitivity, thereby emphasizing a rather minor role of ET in JA-regulated stamen development.} } @Article{IPB-728, author = {Schreiber, T. and Prange, A. and Hoppe, T. and Tissier, A. and}, title = {{Split-TALE: A TALE-Based Two-Component System for Synthetic Biology Applications in Planta}}, year = {2019}, pages = {1001-1012}, journal = {Plant Physiol.}, doi = {10.1104/pp.18.01218}, volume = {179}, abstract = {Transcription activator-like effectors (TALEs) are bacterial Type-III effector proteins from phytopathogenic Xanthomonas species that act as transcription factors in plants. The modular DNA-binding domain of TALEs can be reprogrammed to target nearly any DNA sequence. Here, we designed and optimized a two-component AND-gate system for synthetic circuits in plants based on TALEs. In this system, named split-TALE (sTALE), the TALE DNA binding domain and the transcription activation domain are separated and each fused to protein interacting domains. Physical interaction of interacting domains leads to TALE-reconstitution and can be monitored by reporter gene induction. This setup was used for optimization of the sTALE scaffolds, which result in an AND-gate system with an improved signal-to-noise ratio. We also provide a toolkit of ready-to-use vectors and single modules compatible with Golden Gate cloning and MoClo syntax. In addition to its implementation in synthetic regulatory circuits, the sTALE system allows the analysis of protein-protein interactions in planta.} } @Article{IPB-726, author = {Sarkar, D. and Rovenich, H. and Jeena, G. and Nizam, S. and Tissier, A. and Balcke, G. U. and Mahdi, L. K. and Bonkowski, M. and Langen, G. and Zuccaro, A. and}, title = {{The inconspicuous gatekeeper: endophytic Serendipita vermifera acts as extended plant protection barrier in the rhizosphere}}, year = {2019}, pages = {886-901}, journal = {New Phytol.}, doi = {10.1111/nph.15904}, volume = {224}, abstract = {In nature, beneficial and pathogenic fungi often simultaneously colonise plants. Despite substantial efforts to understand the composition of natural plant−microbe communities, the mechanisms driving such multipartite interactions remain largely unknown.Here we address how the interaction between the beneficial root endophyte Serendipita vermifera and the pathogen Bipolaris sorokiniana affects fungal behaviour and determines barley host responses using a gnotobiotic soil‐based split‐root system.Fungal confrontation in soil resulted in induction of B. sorokiniana genes involved in secondary metabolism and a significant repression of genes encoding putative effectors. In S. vermifera, genes encoding hydrolytic enzymes were strongly induced. This antagonistic response was not activated during the tripartite interaction in barley roots. Instead, we observed a specific induction of S. vermifera genes involved in detoxification and redox homeostasis. Pathogen infection but not endophyte colonisation resulted in substantial host transcriptional reprogramming and activation of defence. In the presence of S. vermifera, pathogen infection and disease symptoms were significantly reduced despite no marked alterations of the plant transcriptional response.The activation of stress response genes and concomitant repression of putative effector gene expression in B. sorokiniana during confrontation with the endophyte suggest a reduction of the pathogen\'s virulence potential before host plant infection.} } @Article{IPB-722, author = {Ronzan, M. and Piacentini, D. and Fattorini, L. and Federica, D. R. and Caboni, E. and Eiche, E. and Ziegler, J. and Hause, B. and Riemann, M. and Betti, C. and Altamura, M. M. and Falasca, G. and}, title = {{Auxin-jasmonate crosstalk in Oryza sativa L. root system formation after cadmium and/or arsenic exposure}}, year = {2019}, pages = {59-69}, journal = {Environ. Exp. Bot.}, doi = {10.1016/j.envexpbot.2019.05.013}, volume = {165}, abstract = {Soil pollutants may affect root growth through interactions among phytohormones like auxin and jasmonates. Rice is frequently grown in paddy fields contaminated by cadmium and arsenic, but the effects of these pollutants on jasmonates/auxin crosstalk during adventitious and lateral roots formation are widely unknown. Therefore, seedlings of Oryza sativa cv. Nihonmasari and of the jasmonate-biosynthetic mutant coleoptile photomorphogenesis2 were exposed to cadmium and/or arsenic, and/or jasmonic acid methyl ester, and then analysed through morphological, histochemical, biochemical and molecular approaches.In both genotypes, arsenic and cadmium accumulated in roots more than shoots. In the roots, arsenic levels were more than twice higher than cadmium levels, either when arsenic was applied alone, or combined with cadmium. Pollutants reduced lateral root density in the wild -type in every treatment condition, but jasmonic acid methyl ester increased it when combined with each pollutant. Interestingly, exposure to cadmium and/or arsenic did not change lateral root density in the mutant. The transcript levels of OsASA2 and OsYUCCA2, auxin biosynthetic genes, increased in the wild-type and mutant roots when pollutants and jasmonic acid methyl ester were applied alone. Auxin (indole-3-acetic acid) levels transiently increased in the roots with cadmium and/or arsenic in the wild-type more than in the mutant. Arsenic and cadmium, when applied alone, induced fluctuations in bioactive jasmonate contents in wild-type roots, but not in the mutant. Auxin distribution was evaluated in roots of OsDR5::GUS seedlings exposed or not to jasmonic acid methyl ester added or not with cadmium and/or arsenic. The DR5::GUS signal in lateral roots was reduced by arsenic, cadmium, and jasmonic acid methyl ester. Lipid peroxidation, evaluated as malondialdehyde levels, was higher in the mutant than in the wild-type, and increased particularly in As presence, in both genotypes.Altogether, the results show that an auxin/jasmonate interaction affects rice root system development in the presence of cadmium and/or arsenic, even if exogenous jasmonic acid methyl ester only slightly mitigates pollutants toxicity.} } @INBOOK{IPB-28, author = {Marillonnet, S. and Werner, S. and}, title = {{Microbial Metabolic Engineering}}, year = {2019}, pages = {93-109}, chapter = {{Assembly of Complex Pathways Using Type IIs Restriction Enzymes}}, journal = {Methods Mol. Biol.}, editor = {Santos, C. N. S. \& Ajikumar, P. K., eds.}, doi = {10.1007/978-1-4939-9142-6_7}, volume = {1927}, abstract = {Efficient DNA assembly methods are essential tools for synthetic biology and metabolic engineering. Among several recently developed methods that allow assembly of multiple DNA fragments in a single step, DNA assembly using type IIS enzymes provides many advantages for complex pathway engineering. In particular, it provides the ability for the user to quickly assemble multigene constructs using a series of simple one-pot assembly steps starting from libraries of cloned and sequenced parts. We describe here a protocol for assembly of multigene constructs using the modular cloning system (MoClo). Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. Basic parts that are not yet available need to be made. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.} } @INBOOK{IPB-27, author = {Janik, K. and Stellmach, H. and Mittelberger, C. and Hause, B. and}, title = {{Phytoplasmas}}, year = {2019}, pages = {321-331}, chapter = {{Characterization of Phytoplasmal Effector Protein Interaction with Proteinaceous Plant Host Targets Using Bimolecular Fluorescence Complementation (BiFC)}}, journal = {Methods Mol. Biol.}, editor = {Musetti, R. \& Pagliari, L., eds.}, doi = {10.1007/978-1-4939-8837-2_24}, volume = {1875}, abstract = {Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves and how to transform these protoplasts with BiFC expression vectors containing the phytoplasma effector and the host interaction partner, respectively. If an interaction occurs, a fluorescent YFP-complex is reconstituted in the protoplast, which can be visualized using fluorescence microscopy.} } @INBOOK{IPB-24, author = {Hause, B. and Yadav, H. and}, title = {{The Model Legume Medicago truncatula}}, year = {2019}, pages = {1179-1184}, chapter = {{Creation of composite plants – transformation of Medicago truncatula roots}}, editor = {de Bruijn, F., ed.}, doi = {10.1002/9781119409144.ch152}, abstract = {Medicago truncatula, owing to its small diploid genome (∼500 Mbp), short life cycle, and high natural diversity makes it a good model plant and has opened the door of opportunities for scientists interested in studying legume biology. But over the years, challenges are also being faced for genetic manipulation of this plant. Many genetic manipulation protocols have been published involving Agrobacterium tumefaciens, a pathogen causing tumor disease in plants. These protocols apart from being difficult to achieve, are also time consuming. Nowadays, an easy, less time consuming and highly reproducible Agrobacterium rhizogenes based method is in use by many research groups. This method generates composite plants having transformed roots on a wild‐type shoot. Here, stable transformed lines that can be propagated over time are not achieved by this method, but for root‐development or root–microbe interaction studies this method has proven to be a useful tool for the community. In addition, transformed roots can be propagated by root organ cultures (ROCs), wherein transformed roots are propagated on sucrose containing media without any shoot part. Occasionally, even stable transgenic plants can be regenerated from transgenic roots. In this chapter, developments and improvements of various transformation protocols are discussed. The suitability of composite plants is highlighted by a study on mycorrhization of transformed and non‐transformed roots, which did not show differences in the mycorrhization rate and developmental stages of the arbuscular mycorrhizal (AM) fungus inside the roots as well as in transcript accumulation and metabolite levels of roots. Finally, applications of the A. rhizogenes based transformation method are discussed.} } @Article{IPB-2537, author = {Püllmann, P. and Ulpinnis, C. and Marillonnet, S. and Gruetzner, R. and Neumann, S. and Weissenborn, M. J. and}, title = {{Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design}}, year = {2018}, journal = {bioRxiv}, doi = {10.1101/453621}, abstract = {Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Muta-genesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.} } @Article{IPB-2534, author = {Ordon, J. and Bressan, M. and Kretschmer, C. and Dall'Osto, L. and Marillonnet, S. and Bassi, R. and Stuttmann, J. and}, title = {{Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation}}, year = {2018}, journal = {bioRxiv}, doi = {10.1101/393439}, abstract = {Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.} } @Article{IPB-769, author = {Dhakarey, R. and Raorane, M. L. and Treumann, A. and Peethambaran, P. K. and Schendel, R. R. and Sahi, V. P. and Hause, B. and Bunzel, M. and Henry, A. and Kohli, A. and Riemann, M. and}, title = {{Corrigendum: Physiological and Proteomic Analysis of the Rice Mutant cpm2 Suggests a Negative Regulatory Role of Jasmonic Acid in Drought Tolerance}}, year = {2018}, pages = {465}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2018.00465}, volume = {9}, } @Article{IPB-758, author = {Bennewitz, S. and Bergau, N. and Tissier, A. and}, title = {{QTL Mapping of the Shape of Type VI Glandular Trichomes in Tomato}}, year = {2018}, pages = {1421}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2018.01421}, volume = {9}, abstract = {Glandular trichomes contribute to the high resistance of wild tomato species against insect pests not only thanks to the metabolites they produce but also because of morphological and developmental features which support the high production of these defense compounds. In Solanum habrochaites, type VI trichomes have a distinct spherical shape and a large intercellular storage cavity where metabolites can accumulate and are released upon breaking off of the glandular cells. In contrast, the type VI trichomes of S. lycopersicum have a four-leaf clover shape corresponding to the four glandular cells and a small internal cavity with limited capacity for storage of compounds. To better characterize the genetic factors underlying these trichome morphological differences we created a back-cross population of 116 individuals between S. habrochaites LA1777 and S. lycopersicum var. cerasiforme WVa106. A trichome score that reflects the shape of the type VI trichomes allowing the quantification of this trait was designed. The scores were distributed normally across the population, which was mapped with a total of 192 markers. This resulted in the identification of six quantitative trait locus (QTLs) on chromosomes I, VII, VII, and XI. The QTL on chromosome I with the highest LOD score was confirmed and narrowed down to a 500 gene interval in an advanced population derived from one of the back-cross lines. Our results provide the foundation for the genetic dissection of type VI trichome morphology and the introgression of these trichome traits into cultivated tomato lines for increased insect resistance.} } @Article{IPB-755, author = {Akaberi, S. and Wang, H. and Claudel, P. and Riemann, M. and Hause, B. and Hugueney, P. and Nick, P. and}, title = {{Grapevine fatty acid hydroperoxide lyase generates actin-disrupting volatiles and promotes defence-related cell death}}, year = {2018}, pages = {2883-2896}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/ery133}, volume = {69}, abstract = {Fatty acid hydroperoxides can generate short-chained volatile aldehydes that may participate in plant defence. A grapevine hydroperoxide lyase (VvHPL1) clustering to the CYP74B class was functionally characterized with respect to a role in defence. In grapevine leaves, transcripts of this gene accumulated rapidly to high abundance in response to wounding. Cellular functions of VvHPL1 were investigated upon heterologous expression in tobacco BY-2 cells. A C-terminal green fluorescent protein (GFP) fusion of VvHPL1 was located in plastids. The overexpression lines were found to respond to salinity stress or the bacterial elicitor harpin by increasing cell death. This signal-dependent mortality response was mitigated either by addition of exogenous jasmonic acid or by treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases. By feeding different substrates to recombinantly expressed enzyme, VvHPL1 could also be functionally classified as true 13-HPL. The cognate products generated by this 13-HPL were cis-3-hexenal and trans-2-hexenal. Using a GFP-tagged actin marker line, one of these isomeric products, cis-3-hexenal, was found specifically to elicit a rapid disintegration of actin filaments. This response was not only observed in the heterologous system (tobacco BY-2), but also in a grapevine cell strain expressing this marker, as well as in leaf discs from an actin marker grape used as a homologous system. These results are discussed in the context of a role for VvHPL1 in a lipoxygenase-dependent signalling pathway triggering cell death-related defence that bifurcates from jasmonate-dependent basal immunity.} } @Article{IPB-853, author = {Xu, H. and Lybrand, D. and Bennewitz, S. and Tissier, A. and Last, R. L. and Pichersky, E. and}, title = {{Production of trans-chrysanthemic acid, the monoterpene acid moiety of natural pyrethrin insecticides, in tomato fruit}}, year = {2018}, pages = {271-278}, journal = {Metab. Eng.}, doi = {10.1016/j.ymben.2018.04.004}, volume = {47}, abstract = {The pyrethrum plant, Tanacetum cinerariifolium (Asteraceae) synthesizes a class of compounds called pyrethrins that have strong insecticidal properties but are safe to humans. Class I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of three jasmonic-acid derived alcohols. We reconstructed the trans-chrysanthemic acid biosynthetic pathway in tomato fruits, which naturally produce high levels of the tetraterpene pigment lycopene, an isoprenoid which shares a common precursor, dimethylallyl diphosphate (DMAPP), with trans-chrysanthemic acid. trans-Chrysanthemic acid biosynthesis in tomato fruit was achieved by expressing the chrysanthemyl diphosphate synthase gene from T. cinerariifolium, encoding the enzyme that uses DMAPP to make trans-chrysanthemol, under the control of the fruit specific promoter PG, as well as an alcohol dehydrogenease (ADH) gene and aldehyde dehydrogenase (ALDH) gene from a wild tomato species, also under the control of the PG promoter. Tomato fruits expressing all three genes had a concentration of trans-chrysanthemic acid that was about 1.7-fold higher (by weight) than the levels of lycopene present in non-transgenic fruit, while the level of lycopene in the transgenic plants was reduced by 68%. Ninety seven percent of the diverted DMAPP was converted to trans-chrysanthemic acid, but 62% of this acid was further glycosylated. We conclude that the tomato fruit is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.} } @Article{IPB-849, author = {Wasternack, C. and Hause, B. and}, title = {{A Bypass in Jasmonate Biosynthesis – the OPR3-independent Formation}}, year = {2018}, pages = {276-279}, journal = {Trends Plant Sci.}, doi = {10.1016/j.tplants.2018.02.011}, volume = {23}, abstract = {For the first time in 25 years, a new pathway for biosynthesis of jasmonic acid (JA) has been identified. JA production takes place via 12-oxo-phytodienoic acid (OPDA) including reduction by OPDA reductases (OPRs). A loss-of-function allele, opr3-3, revealed an OPR3-independent pathway converting OPDA to JA.} } @Article{IPB-844, author = {Vogt, T. and}, title = {{Unusual spermine-conjugated hydroxycinnamic acids on pollen: function and evolutionary advantage}}, year = {2018}, pages = {5311-5315}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/ery359}, volume = {69}, abstract = {Conjugates between polyamines and hydroxycinnamic acids are found on the pollen surface of all higher plants, both mono- and dicots. But we don’t know why they are there. Delporte et al. (2018) have now shown that in the tapetum of the Asteraceae (sunflower family) a new type of BAHD-acyltransferase is expressed, able to transfer coenzyme A-activated coumaric acid to all four primary and secondary amine groups of the polyamine spermine. In the case of chicory this sequential addition results in a fully substituted tetracoumaroyl–spermine conjugate and points to an evolutionary advantage of these functionally enigmatic compounds.} } @Article{IPB-841, author = {van Aubel, G. and Serderidis, S. and Ivens, J. and Clinkemaillie, A. and Legrève, A. and Hause, B. and Van Cutsem, P. and}, title = {{Oligosaccharides successfully thwart hijacking of the salicylic acid pathway by Phytophthora infestans in potato leaves}}, year = {2018}, pages = {1901-1911}, journal = {Plant Pathol.}, doi = {10.1111/ppa.12908}, volume = {67}, abstract = {Potato growing is severely threatened by the late blight agent Phytophthora infestans, which is usually controlled by massive amounts of fungicides. While variety resistance is often bypassed by the pathogen, the plant innate immunity opens the way to new biological plant protection tools e.g. the COS‐OGA elicitor. This oligosaccharide composition mimics the interaction between plants and fungal pathogens as it combines chitosan oligomers (COS) with pectin‐derived oligogalacturonides (OGA).Two different COS‐OGA elicitors were evaluated against potato late blight: FytoSave® mainly efficient against powdery mildews and FytoSol, a new composition still under development. Next to the evaluation of their protective effect, a comparative study of plant defense induction was performed focusing on the effect of repeated sprayings as well as on the stimulation of salicylic acid (SA), jasmonic acid and ethylene‐related pathways during the biotrophic and the necrotrophic growth stages of the pathogen.The FytoSave® elicitor strongly increased the SA content but failed to induce a sufficient protection against late blight while FytoSol maintained or even decreased the free SA content in presence of P. infestans and was completely efficient. Surprisingly, the necrotrophic development of P. infestans occurred along with a strong leaf accumulation of free SA and SA‐related transcripts. It may represent an attempt by P. infestans to divert plant defenses for its own benefit. Preventive sprayings with FytoSol but not FytoSave® completely impeded this hijacking. FytoSol seemed to keep the SA pathway under control, thereby preventing its diversion by P. infestans.} } @Article{IPB-838, author = {Tissier, A. and}, title = {{Plant secretory structures: more than just reaction bags}}, year = {2018}, pages = {73-79}, journal = {Curr. Opin. Biotech.}, doi = {10.1016/j.copbio.2017.08.003}, volume = {49}, abstract = {Plants have a remarkable capacity for the production of a wide range of metabolites. Much has been reported and reviewed on the diversity of these metabolites and how it is achieved, for example through the evolution of enzyme families. In comparison, relatively little is known on the extraordinary metabolic productivity of dedicated organs where many of these metabolites are synthesized and accumulate. Plant glandular trichomes are such specialized metabolite factories, for which recent omics analyses have shed new light on the adaptive metabolic strategies that support high metabolic fluxes. In photosynthetic trichomes such as those of the Solanaceae, these include CO2 refixation and possibly C4-like metabolism which contribute to the high productivity of these sink organs.} } @Article{IPB-836, author = {Tannert, M. and May, A. and Ditfe, D. and Berger, S. and Balcke, G. U. and Tissier, A. and Köck, M. and}, title = {{Pi starvation-dependent regulation of ethanolamine metabolism by phosphoethanolamine phosphatase PECP1 in Arabidopsis roots}}, year = {2018}, pages = {467-481}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/erx408}, volume = {69}, abstract = {A universal plant response to phosphorus deprivation is the up-regulation of a diverse array of phosphatases. As reported recently, the AtPECP1 gene encodes a phosphatase with in vitro substrate specificity for phosphoethanolamine and phosphocholine. The putative substrates suggested that AtPECP1 is related to phospholipid metabolism; however, the biological function of AtPECP1 is as yet not understood. In addition, whereas lipid remodelling processes as part of the phosphorus starvation response have been extensively studied, knowledge of the polar head group metabolism and its regulation is lacking. We found that AtPECP1 is expressed in the cytosol and exerts by far its strongest activity in roots of phosphate-starved plants. We established a novel LC-MS/MS-based method for the quantitative and simultaneous measurement of the head group metabolites. The analysis of Atpecp1 null mutants and overexpression lines revealed that phosphoethanolamine, but not phosphocholine is the substrate of AtPECP1 in vivo. The impact on head group metabolite levels is greatest in roots of both loss-of-function and gain-of-function transgenic lines, indicating that the biological role of AtPECP1 is mainly restricted to roots. We suggest that phosphoethanolamine hydrolysis by AtPECP1 during Pi starvation is required to down-regulate the energy-consuming biosynthesis of phosphocholine through the methylation pathway.} } @Article{IPB-833, author = {Stauder, R. and Welsch, R. and Camagna, M. and Kohlen, W. and Balcke, G. U. and Tissier, A. and Walter, M. H. and}, title = {{Strigolactone Levels in Dicot Roots Are Determined by an Ancestral Symbiosis-Regulated Clade of the PHYTOENE SYNTHASE Gene Family}}, year = {2018}, pages = {255}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2018.00255}, volume = {9}, abstract = {Strigolactones (SLs) are apocarotenoid phytohormones synthesized from carotenoid precursors. They are produced most abundantly in roots for exudation into the rhizosphere to cope with mineral nutrient starvation through support of root symbionts. Abscisic acid (ABA) is another apocarotenoid phytohormone synthesized in roots, which is involved in responses to abiotic stress. Typically low carotenoid levels in roots raise the issue of precursor supply for the biosynthesis of these two apocarotenoids in this organ. Increased ABA levels upon abiotic stress in Poaceae roots are known to be supported by a particular isoform of phytoene synthase (PSY), catalyzing the rate-limiting step in carotenogenesis. Here we report on novel PSY3 isogenes from Medicago truncatula (MtPSY3) and Solanum lycopersicum (SlPSY3) strongly expressed exclusively upon root interaction with symbiotic arbuscular mycorrhizal (AM) fungi and moderately in response to phosphate starvation. They belong to a widespread clade of conserved PSYs restricted to dicots (dPSY3) distinct from the Poaceae-PSY3s involved in ABA formation. An ancient origin of dPSY3s and a potential co-evolution with the AM symbiosis is discussed in the context of PSY evolution. Knockdown of MtPSY3 in hairy roots of M. truncatula strongly reduced SL and AM-induced C13 α-ionol/C14 mycorradicin apocarotenoids. Inhibition of the reaction subsequent to phytoene synthesis revealed strongly elevated levels of phytoene indicating induced flux through the carotenoid pathway in roots upon mycorrhization. dPSY3 isogenes are coregulated with upstream isogenes and downstream carotenoid cleavage steps toward SLs (D27, CCD7, CCD8) suggesting a combined carotenoid/apocarotenoid pathway, which provides “just in time”-delivery of precursors for apocarotenoid formation.} } @Article{IPB-797, author = {Kowarschik, K. and Hoehenwarter, W. and Marillonnet, S. and Trujillo, M. and}, title = {{UbiGate: a synthetic biology toolbox to analyse ubiquitination}}, year = {2018}, pages = {1749-1763}, journal = {New Phytol.}, doi = {10.1111/nph.14900}, volume = {217}, abstract = {Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post‐translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process.Here, we present UbiGate − a synthetic biology toolbox, together with an inducible bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by ‘Golden Gate’ cloning.This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression.We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time‐ and cost‐effective manner.} } @Article{IPB-786, author = {Gelová, Z. and ten Hoopen, P. and Novák, O. and Motyka, V. and Pernisová, M. and Dabravolski, S. and Didi, V. and Tillack, I. and Oklešťková, J. and Strnad, M. and Hause, B. and Haruštiaková, D. and Conrad, U. and Janda, L. and Hejátko, J. and}, title = {{Antibody-mediated modulation of cytokinins in tobacco: organ-specific changes in cytokinin homeostasis}}, year = {2018}, pages = {441-454}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/erx426}, volume = {69}, abstract = {Cytokinins comprise a group of phytohormones with an organ-specific mode of action. Although the mechanisms controlling the complex networks of cytokinin metabolism are partially known, the role of individual cytokinin types in the maintenance of cytokinin homeostasis remains unclear. Utilizing the overproduction of single-chain Fv antibodies selected for their ability to bind trans-zeatin riboside and targeted to the endoplasmic reticulum, we post-synthetically modulated cytokinin ribosides, the proposed transport forms of cytokinins. We observed asymmetric activity of cytokinin biosynthetic genes and cytokinin distribution in wild-type tobacco seedlings with higher cytokinin abundance in the root than in the shoot. Antibody-mediated modulation of cytokinin ribosides further enhanced the relative cytokinin abundance in the roots and induced cytokinin-related phenotypes in an organ-specific manner. The activity of cytokinin oxidase/dehydrogenase in the roots was strongly up-regulated in response to antibody-mediated formation of the cytokinin pool in the endoplasmic reticulum. However, we only detected a slight decrease in the root cytokinin levels. In contrast, a significant decrease of cytokinins occurred in the shoot. We suggest the roots as the main site of cytokinin biosynthesis in tobacco seedlings. Conversely, cytokinin levels in the shoot seem to depend largely on long-range transport of cytokinin} } @Article{IPB-784, author = {Gantner, J. and Ordon, J. and Ilse, T. and Kretschmer, C. and Gruetzner, R. and Löfke, C. and Dagdas, Y. and Bürstenbinder, K. and Marillonnet, S. and Stuttmann, J. and}, title = {{Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system}}, year = {2018}, pages = {e0197185}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0197185}, volume = {13}, abstract = {Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.} } @Article{IPB-782, author = {Frolov, A. and Mamontova, T. and Ihling, C. and Lukasheva, E. and Bankin, M. and Chantseva, V. and Vikhnina, M. and Soboleva, A. and Shumilina, J. and Mavropolo-Stolyarenko, G. and Grishina, T. and Osmolovskaya, N. and Zhukov, V. and Hoehenwarter, W. and Sinz, A. and Tikhononovich, I. and Wessjohann, L. and Bilova, T. and Smolikova, G. and Medvedev, S. and}, title = {{Mining seed proteome: from protein dynamics to modification profiles}}, year = {2018}, pages = {43-58}, journal = {Biol. Commun.}, doi = {10.21638/spbu03.2018.106}, volume = {63}, abstract = {In the modern world, crop plants represent a major source of daily consumed foods. Among them, cereals and legumes — i.e. the crops accumulating oils, carbohydrates and proteins in their seeds — dominate in European agriculture, tremendously impacting global protein consumption and biodiesel production. Therefore, the seeds of crop plants attract the special attention of biologists, biochemists, nutritional physiologists and food chemists. Seed development and germination, as well as age- and stress-related changes in their viability and nutritional properties, can be addressed by a variety of physiological and biochemical methods. In this context, the methods of functional genomics can be applied to address characteristic changes in seed metabolism, which can give access to stress-resistant genotypes. Among these methods, proteomics is one of the most effective tools, allowing mining metabolism changes on the protein level. Here we discuss the main methodological approaches of seed proteomics in the context of physiological changes related to environmental stress and ageing. We provide a comprehensive comparison of gel- and chromatographybased approaches with a special emphasis on advantages and disadvantages of both strategies in characterization of the seed proteome.} } @Article{IPB-779, author = {Ferlian, O. and Biere, A. and Bonfante, P. and Buscot, F. and Eisenhauer, N. and Fernandez, I. and Hause, B. and Herrmann, S. and Krajinski-Barth, F. and Meier, I. C. and Pozo, M. J. and Rasmann, S. and Rillig, M. C. and Tarkka, M. T. and van Dam, N. M. and Wagg, C. and Martinez-Medina, A. and}, title = {{Growing Research Networks on Mycorrhizae for Mutual Benefits}}, year = {2018}, pages = {975-984}, journal = {Trends Plant Sci.}, doi = {10.1016/j.tplants.2018.08.008}, volume = {23}, abstract = {Research on mycorrhizal interactions has traditionally developed into separate disciplines addressing different organizational levels. This separation has led to an incomplete understanding of mycorrhizal functioning. Integration of mycorrhiza research at different scales is needed to understand the mechanisms underlying the context dependency of mycorrhizal associations, and to use mycorrhizae for solving environmental issues. Here, we provide a road map for the integration of mycorrhiza research into a unique framework that spans genes to ecosystems. Using two key topics, we identify parallels in mycorrhiza research at different organizational levels. Based on two current projects, we show how scientific integration creates synergies, and discuss future directions. Only by overcoming disciplinary boundaries, we will achieve a more comprehensive understanding of the functioning of mycorrhizal associations.} } @Article{IPB-777, author = {Fattorini, L. and Hause, B. and Gutierrez, L. and Veloccia, A. and Della Rovere, F. and Piacentini, D. and Falasca, G. and Altamura, M. M. and}, title = {{Jasmonate promotes auxin-induced adventitious rooting in dark-grown Arabidopsis thaliana seedlings and stem thin cell layers by a cross-talk with ethylene signalling and a modulation of xylogenesis}}, year = {2018}, pages = {182}, journal = {BMC Plant Biol.}, doi = {10.1186/s12870-018-1392-4}, volume = {18}, abstract = {BackgroundAdventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA \+ Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA \+ Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored.ResultsMeJA, at 0.01 μM, enhances AR-formation, when combined with IBA \+ Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression.ConclusionsThe JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species.} } @Article{IPB-819, author = {Peters, K. and Worrich, A. and Weinhold, A. and Alka, O. and Balcke, G. and Birkemeyer, C. and Bruelheide, H. and Calf, O. W. and Dietz, S. and Dührkop, K. and Gaquerel, E. and Heinig, U. and Kücklich, M. and Macel, M. and Müller, C. and Poeschl, Y. and Pohnert, G. and Ristok, C. and Rodríguez, V. M. and Ruttkies, C. and Schuman, M. and Schweiger, R. and Shahaf, N. and Steinbeck, C. and Tortosa, M. and Treutler, H. and Ueberschaar, N. and Velasco, P. and Weiß, B. M. and Widdig, A. and Neumann, S. and van Dam, N. M. and}, title = {{Current Challenges in Plant Eco-Metabolomics}}, year = {2018}, pages = {1385}, journal = {Int. J. Mol. Sci.}, doi = {10.3390/ijms19051385}, volume = {19}, abstract = {The relatively new research discipline of Eco-Metabolomics is the application of metabolomics techniques to ecology with the aim to characterise biochemical interactions of organisms across different spatial and temporal scales. Metabolomics is an untargeted biochemical approach to measure many thousands of metabolites in different species, including plants and animals. Changes in metabolite concentrations can provide mechanistic evidence for biochemical processes that are relevant at ecological scales. These include physiological, phenotypic and morphological responses of plants and communities to environmental changes and also interactions with other organisms. Traditionally, research in biochemistry and ecology comes from two different directions and is performed at distinct spatiotemporal scales. Biochemical studies most often focus on intrinsic processes in individuals at physiological and cellular scales. Generally, they take a bottom-up approach scaling up cellular processes from spatiotemporally fine to coarser scales. Ecological studies usually focus on extrinsic processes acting upon organisms at population and community scales and typically study top-down and bottom-up processes in combination. Eco-Metabolomics is a transdisciplinary research discipline that links biochemistry and ecology and connects the distinct spatiotemporal scales. In this review, we focus on approaches to study chemical and biochemical interactions of plants at various ecological levels, mainly plant–organismal interactions, and discuss related examples from other domains. We present recent developments and highlight advancements in Eco-Metabolomics over the last decade from various angles. We further address the five key challenges: (1) complex experimental designs and large variation of metabolite profiles; (2) feature extraction; (3) metabolite identification; (4) statistical analyses; and (5) bioinformatics software tools and workflows. The presented solutions to these challenges will advance connecting the distinct spatiotemporal scales and bridging biochemistry and ecology} } @INBOOK{IPB-37, author = {Tissier, A. and}, title = {{Molecular Pharming: Applications, Challenges, and Emerging Areas}}, year = {2018}, pages = {353-382}, chapter = {{Harnessing Plant Trichome Biochemistry for the Production of Useful Compounds}}, editor = {Kermode, A. R. \& Jiang, L., eds.}, doi = {10.1002/9781118801512.ch14}, abstract = {Plant glandular trichomes are epidermal differentiations that are dedicated to the production of specialized metabolites, which constitute a first line of defense against pathogens and herbivores. The secretions of these metabolic factories are chemically very diverse, including of terpenoid, fatty acid, or phenylpropanoid origins. They find uses in various industrial areas, for example as pharmaceutical, flavor, or fragrance ingredients or as insecticides. Recent progress in the elucidation of biosynthesis pathways for these compounds has opened up novel opportunities for metabolic engineering in microorganisms as well as in plants.} } @INBOOK{IPB-36, author = {Schreiber, T. and Tissier, A. and}, title = {{Molecular Pharming: Applications, Challenges, and Emerging Areas}}, year = {2018}, pages = {25-42}, chapter = {{Synthetic Transcription Activator-Like Effector-Activated Promoters for Coordinated Orthogonal Gene Expression in Plants}}, editor = {Kermode, A. R. \& Jiang, L., eds.}, doi = {10.1002/9781118801512.ch2}, abstract = {Transcription activator‐like effectors (TALEs) can be programmed to bind specific DNA sequences. This property was used to construct libraries of synthetic TALE‐activated promoters (STAPs), which drive varying levels of gene expression. After a brief description of these promoters, we explore how these STAPs can be used for various applications in plant synthetic biology, in particular for the coordinated expression of multiple genes for metabolic engineering and in the design and implementation of gene regulatory networks.} } @Article{IPB-2541, author = {Gantner, J. and Ilse, T. and Ordon, J. and Kretschmer, C. and Gruetzner, R. and Löfke, C. and Dagdas, Y. and Bürstenbinder, K. and Marillonnet, S. and Stuttmann, J. and}, title = {{Peripheral infrastructure vectors and an extended set of plant parts for the modular cloning system}}, year = {2017}, journal = {bioRxiv}, doi = {10.1101/237768}, abstract = {Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.} } @Article{IPB-873, author = {Dreher, D. and Yadav, H. and Zander, S. and Hause, B. and}, title = {{Is there genetic variation in mycorrhization of Medicago truncatula?}}, year = {2017}, pages = {e3713}, journal = {PeerJ}, doi = {10.7717/peerj.3713}, volume = {5}, abstract = {Differences in the plant’s response among ecotypes or accessions are often used to identify molecular markers for the respective process. In order to analyze genetic diversity of Medicago truncatula in respect to interaction with the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, mycorrhizal colonization was evaluated in 32 lines of the nested core collection representing the genetic diversity of the SARDI collection. All studied lines and the reference line Jemalong A17 were inoculated with R. irregularis and the mycorrhization rate was determined at three time points after inoculation. There were, however, no reliable and consistent differences in mycorrhization rates among all lines. To circumvent possible overlay of potential differences by use of the highly effective inoculum, native sandy soil was used in an independent experiment. Here, significant differences in mycorrhization rates among few of the lines were detectable, but the overall high variability in the mycorrhization rate hindered clear conclusions. To narrow down the number of lines to be tested in more detail, root system architecture (RSA) of in vitro-grown seedlings of all lines under two different phosphate (Pi) supply condition was determined in terms of primary root length and number of lateral roots. Under high Pi supply (100 µM), only minor differences were observed, whereas in response to Pi-limitation (3 µM) several lines exhibited a drastically changed number of lateral roots. Five lines showing the highest alterations or deviations in RSA were selected and inoculated with R. irregularis using two different Pi-fertilization regimes with either 13 mM or 3 mM Pi. Mycorrhization rate of these lines was checked in detail by molecular markers, such as transcript levels of RiTubulin and MtPT4. Under high phosphate supply, the ecotypes L000368 and L000555 exhibited slightly increased fungal colonization and more functional arbuscules, respectively. To address the question, whether capability for mycorrhizal colonization might be correlated to general invasion by microorganisms, selected lines were checked for infection by the root rot causing pathogen, Aphanoymces euteiches. The mycorrhizal colonization phenotype, however, did not correlate with the resistance phenotype upon infection with two strains of A. euteiches as L000368 showed partial resistance and L000555 exhibited high susceptibility as determined by quantification of A. euteiches rRNA within infected roots. Although there is genetic diversity in respect to pathogen infection, genetic diversity in mycorrhizal colonization of M. truncatula is rather low and it will be rather difficult to use it as a trait to access genetic markers.} } @Article{IPB-871, author = {Dhakarey, R. and Raorane, M. L. and Treumann, A. and Peethambaran, P. K. and Schendel, R. R. and Sahi, V. P. and Hause, B. and Bunzel, M. and Henry, A. and Kohli, A. and Riemann, M. and}, title = {{Physiological and Proteomic Analysis of the Rice Mutant cpm2 Suggests a Negative Regulatory Role of Jasmonic Acid in Drought Tolerance}}, year = {2017}, pages = {1903}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2017.01903}, volume = {8}, abstract = {It is widely known that numerous adaptive responses of drought-stressed plants are stimulated by chemical messengers known as phytohormones. Jasmonic acid (JA) is one such phytohormone. But there are very few reports revealing its direct implication in drought related responses or its cross-talk with other phytohormones. In this study, we compared the morpho-physiological traits and the root proteome of a wild type (WT) rice plant with its JA biosynthesis mutant coleoptile photomorphogenesis 2 (cpm2), disrupted in the allene oxide cyclase (AOC) gene, for insights into the role of JA under drought. The mutant had higher stomatal conductance, higher water use efficiency and higher shoot ABA levels under severe drought as compared to the WT. Notably, roots of cpm2 were better developed compared to the WT under both, control and drought stress conditions. Root proteome was analyzed using the Tandem Mass Tag strategy to better understand this difference at the molecular level. Expectedly, AOC was unique but notably highly abundant under drought in the WT. Identification of other differentially abundant proteins (DAPs) suggested increased energy metabolism (i.e., increased mobilization of resources) and reactive oxygen species scavenging in cpm2 under drought. Additionally, various proteins involved in secondary metabolism, cell growth and cell wall synthesis were also more abundant in cpm2 roots. Proteome-guided transcript, metabolite, and histological analyses provided further insights into the favorable adaptations and responses, most likely orchestrated by the lack of JA, in the cpm2 roots. Our results in cpm2 are discussed in the light of JA crosstalk to other phytohormones. These results together pave the path for understanding the precise role of JA during drought stress in rice.} } @Article{IPB-866, author = {Blüher, D. and Laha, D. and Thieme, S. and Hofer, A. and Eschen-Lippold, L. and Masch, A. and Balcke, G. and Pavlovic, I. and Nagel, O. and Schonsky, A. and Hinkelmann, R. and Wörner, J. and Parvin, N. and Greiner, R. and Weber, S. and Tissier, A. and Schutkowski, M. and Lee, J. and Jessen, H. and Schaaf, G. and Bonas, U. and}, title = {{A 1-phytase type III effector interferes with plant hormone signaling}}, year = {2017}, pages = {2159}, journal = {Nat. Commun.}, doi = {10.1038/s41467-017-02195-8}, volume = {8}, abstract = {Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen’s benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP6), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP6 at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP6 to InsP5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana.} } @Article{IPB-865, author = {Bjornson, M. and Balcke, G. U. and Xiao, Y. and de Souza, A. and Wang, J.-Z. and Zhabinskaya, D. and Tagkopoulos, I. and Tissier, A. and Dehesh, K. and}, title = {{Integrated omics analyses of retrograde signaling mutant delineate interrelated stress-response strata}}, year = {2017}, pages = {70-84}, journal = {Plant J.}, doi = {10.1111/tpj.13547}, volume = {91}, abstract = {To maintain homeostasis in the face of intrinsic and extrinsic insults, cells have evolved elaborate quality control networks to resolve damage at multiple levels. Interorganellar communication is a key requirement for this maintenance, however the underlying mechanisms of this communication have remained an enigma. Here we integrate the outcome of transcriptomic, proteomic, and metabolomics analyses of genotypes including ceh1, a mutant with constitutively elevated levels of both the stress‐specific plastidial retrograde signaling metabolite methyl‐erythritol cyclodiphosphate (MEcPP) and the defense hormone salicylic acid (SA), as well as the high MEcPP but SA deficient genotype ceh1/eds16, along with corresponding controls. Integration of multi‐omic analyses enabled us to delineate the function of MEcPP from SA, and expose the compartmentalized role of this retrograde signaling metabolite in induction of distinct but interdependent signaling cascades instrumental in adaptive responses. Specifically, here we identify strata of MEcPP‐sensitive stress‐response cascades, among which we focus on selected pathways including organelle‐specific regulation of jasmonate biosynthesis; simultaneous induction of synthesis and breakdown of SA; and MEcPP‐mediated alteration of cellular redox status in particular glutathione redox balance. Collectively, these integrated multi‐omic analyses provided a vehicle to gain an in‐depth knowledge of genome‐metabolism interactions, and to further probe the extent of these interactions and delineate their functional contributions. Through this approach we were able to pinpoint stress‐mediated transcriptional and metabolic signatures and identify the downstream processes modulated by the independent or overlapping functions of MEcPP and SA in adaptive responses.} } @Article{IPB-864, author = {Bilova, T. and Paudel, G. and Shilyaev, N. and Schmidt, R. and Brauch, D. and Tarakhovskaya, E. and Milrud, S. and Smolikova, G. and Tissier, A. and Vogt, T. and Sinz, A. and Brandt, W. and Birkemeyer, C. and Wessjohann, L. A. and Frolov, A. and}, title = {{Global proteomic analysis of advanced glycation end products in the Arabidopsis proteome provides evidence for age-related glycation hot spots}}, year = {2017}, pages = {15758-15776}, journal = {J. Biol. Chem.}, doi = {10.1074/jbc.M117.794537}, volume = {292}, abstract = {Glycation is a post-translational modification resulting from the interaction of protein amino and guanidino groups with carbonyl compounds. Initially, amino groups react with reducing carbohydrates, yielding Amadori and Heyns compounds. Their further degradation results in formation of advanced glycation end products (AGEs), also originating from α-dicarbonyl products of monosaccharide autoxidation and primary metabolism. In mammals, AGEs are continuously formed during the life of the organism, accumulate in tissues, are well-known markers of aging, and impact age-related tissue stiffening and atherosclerotic changes. However, the role of AGEs in age-related molecular alterations in plants is still unknown. To fill this gap, we present here a comprehensive study of the age-related changes in the Arabidopsis thaliana glycated proteome, including the proteins affected and specific glycation sites therein. We also consider the qualitative and quantitative changes in glycation patterns in terms of the general metabolic background, pathways of AGE formation, and the status of plant anti-oxidative/anti-glycative defense. Although the patterns of glycated proteins were only minimally influenced by plant age, the abundance of 96 AGE sites in 71 proteins was significantly affected in an age-dependent manner and clearly indicated the existence of age-related glycation hot spots in the plant proteome. Homology modeling revealed glutamyl and aspartyl residues in close proximity (less than 5 Å) to these sites in three aging-specific and eight differentially glycated proteins, four of which were modified in catalytic domains. Thus, the sites of glycation hot spots might be defined by protein structure that indicates, at least partly, site-specific character of glycation.} } @Article{IPB-861, author = {Balcke, G. U. and Bennewitz, S. and Bergau, N. and Athmer, B. and Henning, A. and Majovsky, P. and Jiménez-Gómez, J. M. and Hoehenwarter, W. and Tissier, A. and}, title = {{Multi-Omics of Tomato Glandular Trichomes Reveals Distinct Features of Central Carbon Metabolism Supporting High Productivity of Specialized Metabolites}}, year = {2017}, pages = {960-983}, journal = {Plant Cell}, doi = {10.1105/tpc.17.00060}, volume = {29}, abstract = {Glandular trichomes are metabolic cell factories with the capacity to produce large quantities of secondary metabolites. Little is known about the connection between central carbon metabolism and metabolic productivity for secondary metabolites in glandular trichomes. To address this gap in our knowledge, we performed comparative metabolomics, transcriptomics, proteomics, and 13C-labeling of type VI glandular trichomes and leaves from a cultivated (Solanum lycopersicum LA4024) and a wild (Solanum habrochaites LA1777) tomato accession. Specific features of glandular trichomes that drive the formation of secondary metabolites could be identified. Tomato type VI trichomes are photosynthetic but acquire their carbon essentially from leaf sucrose. The energy and reducing power from photosynthesis are used to support the biosynthesis of secondary metabolites, while the comparatively reduced Calvin-Benson-Bassham cycle activity may be involved in recycling metabolic CO2. Glandular trichomes cope with oxidative stress by producing high levels of polyunsaturated fatty acids, oxylipins, and glutathione. Finally, distinct mechanisms are present in glandular trichomes to increase the supply of precursors for the isoprenoid pathways. Particularly, the citrate-malate shuttle supplies cytosolic acetyl-CoA and plastidic glycolysis and malic enzyme support the formation of plastidic pyruvate. A model is proposed on how glandular trichomes achieve high metabolic productivity.} } @Article{IPB-948, author = {Wang, J.-Z. and Li, B. and Xiao, Y. and Ni, Y. and Ke, H. and Yang, P. and de Souza, A. and Bjornson, M. and He, X. and Shen, Z. and Balcke, G. U. and Briggs, S. P. and Tissier, A. and Kliebenstein, D. J. and Dehesh, K. and}, title = {{Initiation of ER Body Formation and Indole Glucosinolate Metabolism by the Plastidial Retrograde Signaling Metabolite, MEcPP}}, year = {2017}, pages = {1400-1416}, journal = {Mol. Plant}, doi = {10.1016/j.molp.2017.09.012}, volume = {10}, abstract = {Plants have evolved tightly regulated signaling networks to respond and adapt to environmental perturbations, but the nature of the signaling hub(s) involved have remained an enigma. We have previously established that methylerythritol cyclodiphosphate (MEcPP), a precursor of plastidial isoprenoids and a stress-specific retrograde signaling metabolite, enables cellular readjustments for high-order adaptive functions. Here, we specifically show that MEcPP promotes two Brassicaceae-specific traits, namely endoplasmic reticulum (ER) body formation and induction of indole glucosinolate (IGs) metabolism selectively, via transcriptional regulation of key regulators NAI1 for ER body formation and MYB51/122 for IGs biosynthesis). The specificity of MEcPP is further confirmed by the lack of induction of wound-inducible ER body genes as well as IGs by other altered methylerythritol phosphate pathway enzymes. Genetic analyses revealed MEcPP-mediated COI1-dependent induction of these traits. Moreover, MEcPP signaling integrates the biosynthesis and hydrolysis of IGs through induction of nitrile-specifier protein1 and reduction of the suppressor, ESM1, and production of simple nitriles as the bioactive end product. The findings position the plastidial metabolite, MEcPP, as the initiation hub, transducing signals to adjust the activity of hard-wired gene circuitry to expand phytochemical diversity and alter the associated subcellular structure required for functionality of the secondary metabolites, thereby tailoring plant stress responses.} } @Article{IPB-944, author = {Tissier, A. and Morgan, J. A. and Dudareva, N. and}, title = {{Plant Volatiles: Going ‘In’ but not ‘Out’ of Trichome Cavities}}, year = {2017}, pages = {930-938}, journal = {Trends Plant Sci.}, doi = {10.1016/j.tplants.2017.09.001}, volume = {22}, abstract = {Plant glandular trichomes are able to secrete and store large amounts of volatile organic compounds (VOCs). VOCs typically accumulate in dedicated extracellular spaces, which can be either subcuticular, as in the Lamiaceae or Asteraceae, or intercellular, as in the Solanaceae. Volatiles are retained at high concentrations in these storage cavities with limited release into the atmosphere and without re-entering the secretory cells, where they would be toxic. This implies the existence of mechanisms allowing transport of VOCs to the cavity but preventing their diffusion out once they have been delivered. The cuticle and cell wall lining the cavity are likely to have key roles in retaining volatiles, but their exact composition and the potential molecular players involved are largely unknown.} } @Article{IPB-932, author = {Scheibner, F. and Marillonnet, S. and Büttner, D. and}, title = {{The TAL Effector AvrBs3 from Xanthomonas campestris pv. vesicatoria Contains Multiple Export Signals and Can Enter Plant Cells in the Absence of the Type III Secretion Translocon}}, year = {2017}, pages = {2180}, journal = {Front. Microbiol.}, doi = {10.3389/fmicb.2017.02180}, volume = {8}, abstract = {Pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. Effector protein delivery is controlled by the T3S chaperone HpaB, which presumably escorts effector proteins to the secretion apparatus. One intensively studied effector is the transcription activator-like (TAL) effector AvrBs3, which binds to promoter sequences of plant target genes and activates plant gene expression. It was previously reported that type III-dependent delivery of AvrBs3 depends on the N-terminal protein region. The signals that control T3S and translocation of AvrBs3, however, have not yet been characterized. In the present study, we show that T3S and translocation of AvrBs3 depend on the N-terminal 10 and 50 amino acids, respectively. Furthermore, we provide experimental evidence that additional signals in the N-terminal 30 amino acids and the region between amino acids 64 and 152 promote translocation of AvrBs3 in the absence of HpaB. Unexpectedly, in vivo translocation assays revealed that AvrBs3 is delivered into plant cells even in the absence of HrpF, which is the predicted channel-forming component of the T3S translocon in the plant plasma membrane. The presence of HpaB- and HrpF-independent transport routes suggests that the delivery of AvrBs3 is initiated during early stages of the infection process, presumably before the activation of HpaB or the insertion of the translocon into the plant plasma membrane.} } @Article{IPB-894, author = {Janik, K. and Mithöfer, A. and Raffeiner, M. and Stellmach, H. and Hause, B. and Schlink, K. and}, title = {{An effector of apple proliferation phytoplasma targets TCP transcription factors—a generalized virulence strategy of phytoplasma?}}, year = {2017}, pages = {435-442}, journal = {Mol. Plant Pathol.}, doi = {10.1111/mpp.12409}, volume = {18}, abstract = {The plant pathogen Candidatus Phytoplasma mali (P. mali) is the causative agent of apple proliferation, a disease of increasing importance in apple‐growing areas within Europe. Despite its economic importance, little is known about the molecular mechanisms of disease manifestation within apple trees. In this study, we identified two TCP (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR) transcription factors of Malus x domestica as binding partners of the P. mali SAP11‐like effector ATP\_00189. Phytohormone analyses revealed an effect of P. mali infection on jasmonates, salicylic acid and abscisic acid levels, showing that P. mali affects phytohormonal levels in apple trees, which is in line with the functions of the effector assumed from its binding to TCP transcription factors. To our knowledge, this is the first characterization of the molecular targets of a P. mali effector and thus provides the basis to better understand symptom development and disease progress during apple proliferation. As SAP11 homologues are found in several Phytoplasma species infecting a broad range of different plants, SAP11‐like proteins seem to be key players in phytoplasmal infection.} } @Article{IPB-882, author = {Frolov, A. and Bilova, T. and Paudel, G. and Berger, R. and Balcke, G. U. and Birkemeyer, C. and Wessjohann, L. A. and}, title = {{Early responses of mature Arabidopsis thaliana plants to reduced water potential in the agar-based polyethylene glycol infusion drought model}}, year = {2017}, pages = {70-83}, journal = {J. Plant Physiol.}, doi = {10.1016/j.jplph.2016.09.013}, volume = {208}, abstract = {Drought is one of the most important environmental stressors resulting in increasing losses of crop plant productivity all over the world. Therefore, development of new approaches to increase the stress tolerance of crop plants is strongly desired. This requires precise and adequate modeling of drought stress. As this type of stress manifests itself as a steady decrease in the substrate water potential (ψw), agar plates infused with polyethylene glycol (PEG) are the perfect experimental tool: they are easy in preparation and provide a constantly reduced ψw, which is not possible in soil models. However, currently, this model is applicable only to seedlings and cannot be used for evaluation of stress responses in mature plants, which are obviously the most appropriate objects for drought tolerance research. To overcome this limitation, here we introduce a PEG-based agar infusion model suitable for 6–8-week-old A. thaliana plants, and characterize, to the best of our knowledge for the first time, the early drought stress responses of adult plants grown on PEG-infused agar. We describe essential alterations in the primary metabolome (sugars and related compounds, amino acids and polyamines) accompanied by qualitative and quantitative changes in protein patterns: up to 87 unique stress-related proteins were annotated under drought stress conditions, whereas further 84 proteins showed a change in abundance. The obtained proteome patterns differed slightly from those reported for seedlings and soil-based models.} } @INBOOK{IPB-43, author = {Schreiber, T. and Tissier, A. and}, title = {{Plant Gene Regulatory Networks}}, year = {2017}, pages = {185-204}, chapter = {{Generation of dTALEs and Libraries of Synthetic TALE-Activated Promoters for Engineering of Gene Regulatory Networks in Plants}}, journal = {Methods Mol. Biol.}, editor = {Kaufmann, K. \& Mueller-Roeber, B., eds.}, doi = {10.1007/978-1-4939-7125-1_13}, volume = {1629}, abstract = {Transcription factors with programmable DNA-binding specificity constitute valuable tools for the design of orthogonal gene regulatory networks for synthetic biology. Transcription activator-like effectors (TALEs), as natural transcription regulators, were used to design, build, and test libraries of synthetic TALE-activated promoters (STAPs) that show a broad range of expression levels in plants. In this chapter, we present protocols for the construction of artificial TALEs and corresponding STAPs.} } @Article{IPB-1008, author = {Jud, W. and Fischer, L. and Canaval, E. and Wohlfahrt, G. and Tissier, A. and Hansel, A. and}, title = {{Plant surface reactions: an opportunistic ozone defence mechanism impacting atmospheric chemistry}}, year = {2016}, pages = {277-292}, journal = {Atmos. Chem. Phys.}, doi = {10.5194/acp-16-277-2016}, volume = {16}, abstract = {Elevated tropospheric ozone concentrations are considered a toxic threat to plants, responsible for global crop losses with associated economic costs of several billion dollars per year. Plant injuries have been linked to the uptake of ozone through stomatal pores and oxidative damage of the internal leaf tissue. But a striking question remains: can surface reactions limit the stomatal uptake of ozone and therefore reduce its detrimental effects to plants?In this laboratory study we could show that semi-volatile organic compounds exuded by the glandular trichomes of different Nicotiana tabacum varieties are an efficient ozone sink at the plant surface. In our experiments, different diterpenoid compounds were responsible for a strongly variety-dependent ozone uptake of plants under dark conditions, when stomatal pores are almost closed. Surface reactions of ozone were accompanied by a prompt release of oxygenated volatile organic compounds, which could be linked to the corresponding precursor compounds: ozonolysis cis-abienol (C20H34O) – a diterpenoid with two exocyclic double bonds – caused emissions of formaldehyde (HCHO) and methyl vinyl ketone (C4H6O). The ring-structured cembratrien-diols (C20H34O2) with three endocyclic double bonds need at least two ozonolysis steps to form volatile carbonyls such as 4-oxopentanal (C5H8O2), which we could observe in the gas phase, too.Fluid dynamic calculations were used to model ozone distribution in the diffusion-limited leaf boundary layer under daylight conditions. In the case of an ozone-reactive leaf surface, ozone gradients in the vicinity of stomatal pores are changed in such a way that the ozone flux through the open stomata is strongly reduced.Our results show that unsaturated semi-volatile compounds at the plant surface should be considered as a source of oxygenated volatile organic compounds, impacting gas phase chemistry, as well as efficient ozone sink improving the ozone tolerance of plants.} } @Article{IPB-1002, author = {Hettwer, K. and Böttcher, C. and Frolov, A. and Mittasch, J. and Albert, A. and von Roepenack-Lahaye, E. and Strack, D. and Milkowski, C. and}, title = {{Dynamic metabolic changes in seeds and seedlings of Brassica napus (oilseed rape) suppressing UGT84A9 reveal plasticity and molecular regulation of the phenylpropanoid pathway}}, year = {2016}, pages = {46-57}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2016.01.014}, volume = {124}, abstract = {In Brassica napus, suppression of the key biosynthetic enzyme UDP-glucose:sinapic acid glucosyltransferase (UGT84A9) inhibits the biosynthesis of sinapine (sinapoylcholine), the major phenolic component of seeds. Based on the accumulation kinetics of a total of 158 compounds (110 secondary and 48 primary metabolites), we investigated how suppression of the major sink pathway of sinapic acid impacts the metabolome of developing seeds and seedlings. In UGT84A9-suppressing (UGT84A9i) lines massive alterations became evident in late stages of seed development affecting the accumulation levels of 58 secondary and 7 primary metabolites. UGT84A9i seeds were characterized by decreased amounts of various hydroxycinnamic acid (HCA) esters, and increased formation of sinapic and syringic acid glycosides. This indicates glycosylation and β-oxidation as metabolic detoxification strategies to bypass intracellular accumulation of sinapic acid. In addition, a net loss of sinapic acid upon UGT84A9 suppression may point to a feedback regulation of HCA biosynthesis. Surprisingly, suppression of UGT84A9 under control of the seed-specific NAPINC promoter was maintained in cotyledons during the first two weeks of seedling development and associated with a reduced and delayed transformation of sinapine into sinapoylmalate. The lack of sinapoylmalate did not interfere with plant fitness under UV-B stress. Increased UV-B radiation triggered the accumulation of quercetin conjugates whereas the sinapoylmalate level was not affected.} } @Article{IPB-1001, author = {Hazman, M. and Hause, B. and Eiche, E. and Riemann, M. and Nick, P. and}, title = {{Different forms of osmotic stress evoke qualitatively different responses in rice}}, year = {2016}, pages = {45-56}, journal = {J. Plant Physiol.}, doi = {10.1016/j.jplph.2016.05.027}, volume = {202}, abstract = {Drought, salinity and alkalinity are distinct forms of osmotic stress with serious impacts on rice productivity. We investigated, for a salt-sensitive rice cultivar, the response to osmotically equivalent doses of these stresses. Drought, experimentally mimicked by mannitol (single factor: osmotic stress), salinity (two factors: osmotic stress and ion toxicity), and alkalinity (three factors: osmotic stress, ion toxicity, and depletion of nutrients and protons) produced different profiles of adaptive and damage responses, both locally (in the root) as well as systemically (in the shoot). The combination of several stress factors was not necessarily additive, and we even observed cases of mitigation, when two (salinity), or three stressors (alkalinity) were compared to the single stressor (drought). The response to combinations of individual stress factors is therefore not a mere addition of the partial stress responses, but rather represents a new quality of response. We interpret this finding in a model, where the output to signaling molecules is not determined by their abundance per se, but qualitatively depends on their adequate integration into an adaptive signaling network. This output generates a systemic signal that will determine the quality of the shoot response to local concentrations of ions.} } @Article{IPB-980, author = {Dobritzsch, M. and Lübken, T. and Eschen-Lippold, L. and Gorzolka, K. and Blum, E. and Matern, A. and Marillonnet, S. and Böttcher, C. and Dräger, B. and Rosahl, S. and}, title = {{MATE Transporter-Dependent Export of Hydroxycinnamic Acid Amides}}, year = {2016}, pages = {583-596}, journal = {Plant Cell}, doi = {10.1105/tpc.15.00706}, volume = {28}, abstract = {The ability of Arabidopsis thaliana to successfully prevent colonization by Phytophthora infestans, the causal agent of late blight disease of potato (Solanum tuberosum), depends on multilayered defense responses. To address the role of surface-localized secondary metabolites for entry control, droplets of a P. infestans zoospore suspension, incubated on Arabidopsis leaves, were subjected to untargeted metabolite profiling. The hydroxycinnamic acid amide coumaroylagmatine was among the metabolites secreted into the inoculum. In vitro assays revealed an inhibitory activity of coumaroylagmatine on P. infestans spore germination. Mutant analyses suggested a requirement of the p-coumaroyl-CoA:agmatine N4-p-coumaroyl transferase ACT for the biosynthesis and of the MATE transporter DTX18 for the extracellular accumulation of coumaroylagmatine. The host plant potato is not able to efficiently secrete coumaroylagmatine. This inability is overcome in transgenic potato plants expressing the two Arabidopsis genes ACT and DTX18. These plants secrete agmatine and putrescine conjugates to high levels, indicating that DTX18 is a hydroxycinnamic acid amide transporter with a distinct specificity. The export of hydroxycinnamic acid amides correlates with a decreased ability of P. infestans spores to germinate, suggesting a contribution of secreted antimicrobial compounds to pathogen defense at the leaf surface.} } @Article{IPB-972, author = {Bilova, T. and Lukasheva, E. and Brauch, D. and Greifenhagen, U. and Paudel, G. and Tarakhovskaya, E. and Frolova, N. and Mittasch, J. and Balcke, G. U. and Tissier, A. and Osmolovskaya, N. and Vogt, T. and Wessjohann, L. A. and Birkemeyer, C. and Milkowski, C. and Frolov, A. and}, title = {{A Snapshot of the Plant Glycated Proteome: STRUCTURAL, FUNCTIONAL, AND MECHANISTIC ASPECTS}}, year = {2016}, pages = {7621-7636}, journal = {J. Biol. Chem.}, doi = {10.1074/jbc.M115.678581}, volume = {291}, abstract = {Glycation is the reaction of carbonyl compounds (reducing sugars and α-dicarbonyls) with amino acids, lipids, and proteins, yielding early and advanced glycation end products (AGEs). The AGEs can be formed via degradation of early glycation intermediates (glycoxidation) and by interaction with the products of monosaccharide autoxidation (autoxidative glycosylation). Although formation of these potentially deleterious compounds is well characterized in animal systems and thermally treated foods, only a little information about advanced glycation in plants is available. Thus, the knowledge of the plant AGE patterns and the underlying pathways of their formation are completely missing. To fill this gap, we describe the AGE-modified proteome of Brassica napus and characterize individual sites of advanced glycation by the methods of liquid chromatography-based bottom-up proteomics. The modification patterns were complex but reproducible: 789 AGE-modified peptides in 772 proteins were detected in two independent experiments. In contrast, only 168 polypeptides contained early glycated lysines, which did not resemble the sites of advanced glycation. Similar observations were made with Arabidopsis thaliana. The absence of the early glycated precursors of the AGE-modified protein residues indicated autoxidative glycosylation, but not glycoxidation, as the major pathway of AGE formation. To prove this assumption and to identify the potential modifying agents, we estimated the reactivity and glycative potential of plant-derived sugars using a model peptide approach and liquid chromatography-mass spectrometry-based techniques. Evaluation of these data sets together with the assessed tissue carbohydrate contents revealed dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, ribulose, erythrose, and sucrose as potential precursors of plant AGEs.} } @Article{IPB-970, author = {Bergau, N. and Navarette Santos, A. and Henning, A. and Balcke, G. U. and Tissier, A. and}, title = {{Autofluorescence as a Signal to Sort Developing Glandular Trichomes by Flow Cytometry}}, year = {2016}, pages = {949}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2016.00949}, volume = {7}, abstract = {The industrial relevance of a number of metabolites produced in plant glandular trichomes (GTs) has spurred research on these specialized organs for a number of years. Most of the research, however, has focused on the elucidation of secondary metabolite pathways and comparatively little has been undertaken on the development and differentiation of GTs. One way to gain insight into these developmental processes is to generate stage-specific transcriptome and metabolome data. The difficulty for this resides in the isolation of early stages of development of the GTs. Here we describe a method for the separation and isolation of intact young and mature type VI trichomes from the wild tomato species Solanum habrochaites. The final and key step of the method uses cell sorting based on distinct autofluorescence signals of the young and mature trichomes. We demonstrate that sorting by flow cytometry allows recovering pure fractions of young and mature trichomes. Furthermore, we show that the sorted trichomes can be used for transcript and metabolite analyses. Because many plant tissues or cells have distinct autofluorescence components, the principles of this method can be generally applicable for the isolation of specific cell types without prior labeling.} } @Article{IPB-967, author = {Arnold, M. D. and Gruber, C. and Floková, K. and Miersch, O. and Strnad, M. and Novák, O. and Wasternack, C. and Hause, B. and}, title = {{The Recently Identified Isoleucine Conjugate of cis-12-Oxo-Phytodienoic Acid Is Partially Active in cis-12-Oxo-Phytodienoic Acid-Specific Gene Expression of Arabidopsis thaliana}}, year = {2016}, pages = {e0162829}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0162829}, volume = {11}, abstract = {Oxylipins of the jasmonate family are active as signals in plant responses to biotic and abiotic stresses as well as in development. Jasmonic acid (JA), its precursor cis-12-oxo-phytodienoic acid (OPDA) and the isoleucine conjugate of JA (JA-Ile) are the most prominent members. OPDA and JA-Ile have individual signalling properties in several processes and differ in their pattern of gene expression. JA-Ile, but not OPDA, is perceived by the SCFCOI1-JAZ co-receptor complex. There are, however, numerous processes and genes specifically induced by OPDA. The recently identified OPDA-Ile suggests that OPDA specific responses might be mediated upon formation of OPDA-Ile. Here, we tested OPDA-Ile-induced gene expression in wild type and JA-deficient, JA-insensitive and JA-Ile-deficient mutant background. Tests on putative conversion of OPDA-Ile during treatments revealed only negligible conversion. Expression of two OPDA-inducible genes, GRX480 and ZAT10, by OPDA-Ile could be detected in a JA-independent manner in Arabidopsis seedlings but less in flowering plants. The data suggest a bioactivity in planta of OPDA-Ile.} } @Article{IPB-1062, author = {Wasternack, C. and Hause, B. and}, title = {{OPDA-Ile – a new JA-Ile-independent signal?}}, year = {2016}, pages = {e1253646}, journal = {Plant Signal Behav.}, doi = {10.1080/15592324.2016.1253646}, volume = {11}, abstract = {Expression takes place for most of the jasmonic acid (JA)-induced genes in a COI1-dependent manner via perception of its conjugate JA-Ile in the SCFCOI1-JAZ co-receptor complex. There are, however, numerous genes and processes, which are preferentially induced COI1-independently by the precursor of JA, 12-oxo-phytodienoic acid (OPDA). After recent identification of the Ile-conjugate of OPDA, OPDA-Ile, biological activity of this compound could be unequivocally proven in terms of gene expression. Any interference of OPDA, JA, or JA-Ile in OPDA-Ile-induced gene expression could be excluded by using different genetic background. The data suggest individual signaling properties of OPDA-Ile. Future studies for analysis of an SCFCOI1-JAZ co-receptor-independent route of signaling are proposed.} } @Article{IPB-1055, author = {Treutler, H. and Tsugawa, H. and Porzel, A. and Gorzolka, K. and Tissier, A. and Neumann, S. and Balcke, G. U. and}, title = {{Discovering Regulated Metabolite Families in Untargeted Metabolomics Studies}}, year = {2016}, pages = {8082-8090}, journal = {Anal. Chem.}, doi = {10.1021/acs.analchem.6b01569}, volume = {88}, abstract = {The identification of metabolites by mass spectrometry constitutes a major bottleneck which considerably limits the throughput of metabolomics studies in biomedical or plant research. Here, we present a novel approach to analyze metabolomics data from untargeted, data-independent LC-MS/MS measurements. By integrated analysis of MS1 abundances and MS/MS spectra, the identification of regulated metabolite families is achieved. This approach offers a global view on metabolic regulation in comparative metabolomics. We implemented our approach in the web application “MetFamily”, which is freely available at http://msbi.ipb-halle.de/MetFamily/. MetFamily provides a dynamic link between the patterns based on MS1-signal intensity and the corresponding structural similarity at the MS/MS level. Structurally related metabolites are annotated as metabolite families based on a hierarchical cluster analysis of measured MS/MS spectra. Joint examination with principal component analysis of MS1 patterns, where this annotation is preserved in the loadings, facilitates the interpretation of comparative metabolomics data at the level of metabolite families. As a proof of concept, we identified two trichome-specific metabolite families from wild-type tomato Solanum habrochaites LA1777 in a fully unsupervised manner and validated our findings based on earlier publications and with NMR.} } @Article{IPB-1053, author = {Taylor, I. and Wang, Y. and Seitz, K. and Baer, J. and Bennewitz, S. and Mooney, B. P. and Walker, J. C. and}, title = {{Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission}}, year = {2016}, pages = {e0147203}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0147203}, volume = {11}, abstract = {Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity.} } @Article{IPB-1043, author = {Scheler, U. and Brandt, W. and Porzel, A. and Rothe, K. and Manzano, D. and Božić, D. and Papaefthimiou, D. and Balcke, G. U. and Henning, A. and Lohse, S. and Marillonnet, S. and Kanellis, A. K. and Ferrer, A. and Tissier, A. and}, title = {{Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast}}, year = {2016}, pages = {12942}, journal = {Nat. Commun.}, doi = {10.1038/ncomms12942}, volume = {7}, abstract = {Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.} } @Article{IPB-1042, author = {Scheibner, F. and Schulz, S. and Hausner, J. and Marillonnet, S. and Büttner, D. and}, title = {{Type III-Dependent Translocation of HrpB2 by a Nonpathogenic hpaABC Mutant of the Plant-Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria}}, year = {2016}, pages = {3331-3347}, journal = {Appl. Environ. Microbiol.}, doi = {10.1128/AEM.00537-16}, volume = {82}, abstract = {The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate effector proteins into plant cells. The T3S apparatus spans both bacterial membranes and is associated with an extracellular pilus and a channel-like translocon in the host plasma membrane. T3S is controlled by the switch protein HpaC, which suppresses secretion and translocation of the predicted inner rod protein HrpB2 and promotes secretion of translocon and effector proteins. We previously reported that HrpB2 interacts with HpaC and the cytoplasmic domain of the inner membrane protein HrcU (C. Lorenz, S. Schulz, T. Wolsch, O. Rossier, U. Bonas, and D. Büttner, PLoS Pathog 4:e1000094, 2008, http://dx.doi.org/10.1371/journal.ppat.1000094). However, the molecular mechanisms underlying the control of HrpB2 secretion are not yet understood. Here, we located a T3S and translocation signal in the N-terminal 40 amino acids of HrpB2. The results of complementation experiments with HrpB2 deletion derivatives revealed that the T3S signal of HrpB2 is essential for protein function. Furthermore, interaction studies showed that the N-terminal region of HrpB2 interacts with the cytoplasmic domain of HrcU, suggesting that the T3S signal of HrpB2 contributes to substrate docking. Translocation of HrpB2 is suppressed not only by HpaC but also by the T3S chaperone HpaB and its secreted regulator, HpaA. Deletion of hpaA, hpaB, and hpaC leads to a loss of pathogenicity but allows the translocation of fusion proteins between the HrpB2 T3S signal and effector proteins into leaves of host and non-host plants.IMPORTANCE The T3S system of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is essential for pathogenicity and delivers effector proteins into plant cells. T3S depends on HrpB2, which is a component of the predicted periplasmic inner rod structure of the secretion apparatus. HrpB2 is secreted during the early stages of the secretion process and interacts with the cytoplasmic domain of the inner membrane protein HrcU. Here, we localized the secretion and translocation signal of HrpB2 in the N-terminal 40 amino acids and show that this region is sufficient for the interaction with the cytoplasmic domain of HrcU. Our results suggest that the T3S signal of HrpB2 is required for the docking of HrpB2 to the secretion apparatus. Furthermore, we provide experimental evidence that the N-terminal region of HrpB2 is sufficient to target effector proteins for translocation in a nonpathogenic X. campestris pv. vesicatoria strain.} } @Article{IPB-1034, author = {Pedranzani, H. and Rodríguez-Rivera, M. and Gutiérrez, M. and Porcel, R. and Hause, B. and Ruiz-Lozano, J. M. and}, title = {{Arbuscular mycorrhizal symbiosis regulates physiology and performance of Digitaria eriantha plants subjected to abiotic stresses by modulating antioxidant and jasmonate levels}}, year = {2016}, pages = {141-152}, journal = {Mycorrhiza}, doi = {10.1007/s00572-015-0653-4}, volume = {26}, abstract = {This study evaluates antioxidant responses and jasmonate regulation in Digitaria eriantha cv. Sudafricana plants inoculated (AM) and non-inoculated (non-AM) with Rhizophagus irregularis and subjected to drought, cold, or salinity. Stomatal conductance, photosynthetic efficiency, biomass production, hydrogen peroxide accumulation, lipid peroxidation, antioxidants enzymes activities, and jasmonate levels were determined. Stomatal conductance and photosynthetic efficiency decreased in AM and non-AM plants under all stress conditions. However, AM plants subjected to drought, salinity, or non-stress conditions showed significantly higher stomatal conductance values. AM plants subjected to drought or non-stress conditions increased their shoot/root biomass ratios, whereas salinity and cold caused a decrease in these ratios. Hydrogen peroxide accumulation, which was high in non-AM plant roots under all treatments, increased significantly in non-AM plant shoots under cold stress and in AM plants under non-stress and drought conditions. Lipid peroxidation increased in the roots of all plants under drought conditions. In shoots, although lipid peroxidation decreased in AM plants under non-stress and cold conditions, it increased under drought and salinity. AM plants consistently showed high catalase (CAT) and ascorbate peroxidase (APX) activity under all treatments. By contrast, the glutathione reductase (GR) and superoxide dismutase (SOD) activity of AM roots was lower than that of non-AM plants and increased in shoots. The endogenous levels of cis-12-oxophytodienoc acid (OPDA), jasmonic acid (JA), and 12-OH-JA showed a significant increase in AM plants as compared to non-AM plants. 11-OH-JA content only increased in AM plants subjected to drought. Results show that D. eriantha is sensitive to drought, salinity, and cold stresses and that inoculation with AM fungi regulates its physiology and performance under such conditions, with antioxidants and jasmonates being involved in this process.} } @Article{IPB-1033, author = {Paudel, G. and Bilova, T. and Schmidt, R. and Greifenhagen, U. and Berger, R. and Tarakhovskaya, E. and Stöckhardt, S. and Balcke, G. U. and Humbeck, K. and Brandt, W. and Sinz, A. and Vogt, T. and Birkemeyer, C. and Wessjohann, L. and Frolov, A. and}, title = {{Osmotic stress is accompanied by protein glycation in Arabidopsis thaliana}}, year = {2016}, pages = {6283-6295}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/erw395}, volume = {67}, abstract = {Among the environmental alterations accompanying oncoming climate changes, drought is the most important factor influencing crop plant productivity. In plants, water deficit ultimately results in the development of oxidative stress and accumulation of osmolytes (e.g. amino acids and carbohydrates) in all tissues. Up-regulation of sugar biosynthesis in parallel to the increasing overproduction of reactive oxygen species (ROS) might enhance protein glycation, i.e. interaction of carbonyl compounds, reducing sugars and α-dicarbonyls with lysyl and arginyl side-chains yielding early (Amadori and Heyns compounds) and advanced glycation end-products (AGEs). Although the constitutive plant protein glycation patterns were characterized recently, the effects of environmental stress on AGE formation are unknown so far. To fill this gap, we present here a comprehensive in-depth study of the changes in Arabidopsis thaliana advanced glycated proteome related to osmotic stress. A 3 d application of osmotic stress revealed 31 stress-specifically and 12 differentially AGE-modified proteins, representing altogether 56 advanced glycation sites. Based on proteomic and metabolomic results, in combination with biochemical, enzymatic and gene expression analysis, we propose monosaccharide autoxidation as the main stress-related glycation mechanism, and glyoxal as the major glycation agent in plants subjected to drought.} } @Article{IPB-1027, author = {Otto, M. and Naumann, C. and Brandt, W. and Wasternack, C. and Hause, B. and}, title = {{Activity Regulation by Heteromerization of Arabidopsis Allene Oxide Cyclase Family Members}}, year = {2016}, pages = {3}, journal = {Plants}, doi = {10.3390/plants5010003}, volume = {5}, abstract = {Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 \+ AOC1 and AOC4 \+ AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles.} } @Article{IPB-1010, author = {Klopotek, Y. and Franken, P. and Klaering, H.-P. and Fischer, K. and Hause, B. and Hajirezaei, M.-R. and Druege, U. and}, title = {{A higher sink competitiveness of the rooting zone and invertases are involved in dark stimulation of adventitious root formation in Petunia hybrida cuttings}}, year = {2016}, pages = {10-22}, journal = {Plant Sci.}, doi = {10.1016/j.plantsci.2015.11.001}, volume = {243}, abstract = {The contribution of carbon assimilation and allocation and of invertases to the stimulation of adventitious root formation in response to a dark pre-exposure of petunia cuttings was investigated, considering the rooting zone (stem base) and the shoot apex as competing sinks. Dark exposure had no effect on photosynthesis and dark respiration during the subsequent light period, but promoted dry matter partitioning to the roots. Under darkness, higher activities of cytosolic and vacuolar invertases were maintained in both tissues when compared to cuttings under light. This was partially associated with higher RNA levels of respective genes. However, activity of cell wall invertases and transcript levels of one cell wall invertase isogene increased specifically in the stem base during the first two days after cutting excision under both light and darkness. During five days after excision, RNA accumulation of four invertase genes indicated preferential expression in the stem base compared to the apex. Darkness shifted the balance of expression of one cytosolic and two vacuolar invertase genes towards the stem base. The results indicate that dark exposure before planting enhances the carbon sink competitiveness of the rooting zone and that expression and activity of invertases contribute to the shift in carbon allocation.} } @INBOOK{IPB-54, author = {Schreiber, T. and Tissier, A. and}, title = {{Synthetic Biology and Metabolic Engineering in Plants and Microbes Part B: Metabolism in Plants}}, year = {2016}, pages = {361-378}, chapter = {{Libraries of Synthetic TALE-Activated Promoters: Methods and Applications}}, journal = {Methods Enzymol.}, editor = {O\'Connor, S. E., ed.}, doi = {10.1016/bs.mie.2016.03.004}, volume = {576}, abstract = {The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits.} } @INBOOK{IPB-46, author = {Bilova, T. and Greifenhagen, U. and Paudel, G. and Lukasheva, E. and Brauch, D. and Osmolovskaya, N. and Tarakhovskaya, E. and Balcke, G. U. and Tissier, A. and Vogt, T. and Milkowski, C. and Birkemeyer, C. and Wessjohann, L. and Frolov, A. and}, title = {{Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives}}, year = {2016}, pages = {295-316}, chapter = {{Glycation of Plant Proteins under Environmental Stress — Methodological Approaches, Potential Mechanisms and Biological Role}}, editor = {Shanker, A. K. \& Shanker, C., eds.}, doi = {10.5772/61860}, abstract = {Environmental stress is one of the major factors reducing crop productivity. Due to the oncoming climate changes, the effects of drought and high light on plants play an increasing role in modern agriculture. These changes are accompanied with a progressing contamination of soils with heavy metals. Independent of their nature, environmental alterations result in development of oxidative stress, i.e. increase of reactive oxygen species (ROS) contents, and metabolic adjustment, i.e. accumulation of soluble primary metabolites (amino acids and sugars). However, a simultaneous increase of ROS and sugar concentrations ultimately results in protein glycation, i.e. non-enzymatic interaction of reducing sugars or their degradation products (α-dicarbonyls) with proteins. The eventually resulting advanced glycation end-products (AGEs) are known to be toxic and pro-inflammatory in mammals. Recently, their presence was unambiguously demonstrated in vivo in stressed Arabidopsis thaliana plants. Currently, information on protein targets, modification sites therein, mediators and mechanisms of plant glycation are being intensively studied. In this chapter, we comprehensively review the methodological approaches for plant glycation research and discuss potential mechanisms of AGE formation under stress conditions. On the basis of these patterns and additional in vitro experiments, the pathways and mechanisms of plant glycation can be proposed.} } @Article{IPB-1073, author = {Bergau, N. and Bennewitz, S. and Syrowatka, F. and Hause, G. and Tissier, A. and}, title = {{The development of type VI glandular trichomes in the cultivated tomato Solanum lycopersicum and a related wild species S. habrochaites}}, year = {2015}, pages = {289}, journal = {BMC Plant Biol.}, doi = {10.1186/s12870-015-0678-z}, volume = {15}, abstract = {BackgroundType VI glandular trichomes represent the most abundant trichome type on leaves and stems of tomato plants and significantly contribute to herbivore resistance, particularly in the wild species. Despite this, their development has been poorly studied so far. The goal of this study is to fill this gap. Using a variety of cell imaging techniques, a detailed record of the anatomy and developmental stages of type VI trichomes in the cultivated tomato (Solanum lycopersicum) and in a related wild species (S. habrochaites) is provided.ResultsIn both species, the development of these structures follows a highly reproducible cell division pattern. The two species differ in the shape of the trichome head which is round in S. habrochaites and like a four-leaf clover in S. lycopersicum, correlating with the presence of a large intercellular cavity in S. habrochaites where the produced metabolites accumulate. In both species, the junction between the intermediate cell and the four glandular cells constitute a breaking point facilitating the decapitation of the trichome and thereby the quick release of the metabolites. A strongly auto-fluorescent compound transiently accumulates in the early stages of development suggesting a potential role in the differentiation process. Finally, immuno-labelling with antibodies recognizing specific cell wall components indicate a key role of pectin and arabinogalactan components in the differentiation of type VI trichomes.ConclusionsOur observations explain the adaptive morphologies of type VI trichomes for metabolite storage and release and provide a framework for further studies of these important metabolic cellular factories. This is required to better exploit their potential, in particular for the breeding of pest resistance in tomato.} } @Article{IPB-1109, author = {Hazman, M. and Hause, B. and Eiche, E. and Nick, P. and Riemann, M. and}, title = {{Increased tolerance to salt stress in OPDA-deficient rice ALLENE OXIDE CYCLASE mutants is linked to an increased ROS-scavenging activity}}, year = {2015}, pages = {3339-3352}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/erv142}, volume = {66}, abstract = {Salinity stress represents a global constraint for rice, the most important staple food worldwide. Therefore the role of the central stress signal jasmonate for the salt response was analysed in rice comparing the responses to salt stress for two jasmonic acid (JA) biosynthesis rice mutants (cpm2 and hebiba) impaired in the function of ALLENE OXIDE CYCLASE (AOC) and their wild type. The aoc mutants were less sensitive to salt stress. Interestingly, both mutants accumulated smaller amounts of Na\+ ions in their leaves, and showed better scavenging of reactive oxygen species (ROS) under salt stress. Leaves of the wild type and JA mutants accumulated similar levels of abscisic acid (ABA) under stress conditions, and the levels of JA and its amino acid conjugate, JA–isoleucine (JA-Ile), showed only subtle alterations in the wild type. In contrast, the wild type responded to salt stress by strong induction of the JA precursor 12-oxophytodienoic acid (OPDA), which was not observed in the mutants. Transcript levels of representative salinity-induced genes were induced less in the JA mutants. The absence of 12-OPDA in the mutants correlated not only with a generally increased ROS-scavenging activity, but also with the higher activity of specific enzymes in the antioxidative pathway, such as glutathione S-transferase, and fewer symptoms of damage as, for example, indicated by lower levels of malondialdehyde. The data are interpreted in a model where the absence of OPDA enhanced the antioxidative power in mutant leaves.} } @Article{IPB-1100, author = {Fellenberg, C. and Vogt, T. and}, title = {{Evolutionarily conserved phenylpropanoid pattern on angiosperm pollen}}, year = {2015}, pages = {212-218}, journal = {Trends Plant Sci.}, doi = {10.1016/j.tplants.2015.01.011}, volume = {20}, abstract = {The male gametophyte of higher plants appears as a solid box containing the essentials to transmit genetic material to the next generation. These consist of haploid generative cells that are required for reproduction, and an invasive vegetative cell producing the pollen tube, both mechanically protected by a rigid polymer, the pollen wall, and surrounded by a hydrophobic pollen coat. This coat mediates the direct contact to the biotic and abiotic environments. It contains a mixture of compounds required not only for fertilization but also for protection against biotic and abiotic stressors. Among its metabolites, the structural characteristics of two types of phenylpropanoids, hydroxycinnamic acid amides and flavonol glycosides, are highly conserved in Angiosperm pollen. Structural and functional aspects of these compounds will be discussed.} } @Article{IPB-1089, author = {Dobritzsch, S. and Weyhe, M. and Schubert, R. and Dindas, J. and Hause, G. and Kopka, J. and Hause, B. and}, title = {{Dissection of jasmonate functions in tomato stamen development by transcriptome and metabolome analyses}}, year = {2015}, pages = {28}, journal = {BMC Biol.}, doi = {10.1186/s12915-015-0135-3}, volume = {13}, abstract = {BackgroundJasmonates are well known plant signaling components required for stress responses and development. A prominent feature of jasmonate biosynthesis or signaling mutants is the loss of fertility. In contrast to the male sterile phenotype of Arabidopsis mutants, the tomato mutant jai1-1 exhibits female sterility with additional severe effects on stamen and pollen development. Its senescence phenotype suggests a function of jasmonates in regulation of processes known to be mediated by ethylene. To test the hypothesis that ethylene involved in tomato stamen development is regulated by jasmonates, a temporal profiling of hormone content, transcriptome and metabolome of tomato stamens was performed using wild type and jai1-1.ResultsWild type stamens showed a transient increase of jasmonates that is absent in jai1-1. Comparative transcriptome analyses revealed a diminished expression of genes involved in pollen nutrition at early developmental stages of jai1-1 stamens, but an enhanced expression of ethylene-related genes at late developmental stages. This finding coincides with an early increase of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) in jai1-1 and a premature pollen release from stamens, a phenotype similarly visible in an ethylene overproducing mutant. Application of jasmonates to flowers of transgenic plants affected in jasmonate biosynthesis diminished expression of ethylene-related genes, whereas the double mutant jai1-1 NeverRipe (ethylene insensitive) showed a complementation of jai1-1 phenotype in terms of dehiscence and pollen release.ConclusionsOur data suggest an essential role of jasmonates in the temporal inhibition of ethylene production to prevent premature desiccation of stamens and to ensure proper timing in flower development.} } @Article{IPB-1082, author = {Brückner, K. and Schäfer, P. and Weber, E. and Grützner, R. and Marillonnet, S. and Tissier, A. and}, title = {{A library of synthetic transcription activator-like effector-activated promoters for coordinated orthogonal gene expression in plants}}, year = {2015}, pages = {707-716}, journal = {Plant J.}, doi = {10.1111/tpj.12843}, volume = {82}, abstract = {A library of synthetic promoters containing the binding site of a single designer transcription activator‐like effector (dTALE) was constructed. The promoters contain a constant sequence, consisting of an 18‐base long dTALE‐binding site and a TATA box, flanked by degenerate sequences of 49 bases downstream and 19 bases upstream. Forty‐three of these promoters were sequenced and tested in transient assays in Nicotiana benthamiana using a GUS reporter gene. The strength of expression of the promoters ranged from around 5% to almost 100% of the viral 35S promoter activity. We then demonstrated the utility of these promoters for metabolic engineering by transiently expressing three genes for the production of a plant diterpenoid in N. benthamiana. The simplicity of the promoter structure shows great promise for the development of genetic circuits, with wide potential applications in plant synthetic biology and metabolic engineering.} } @Article{IPB-1080, author = {Brandt, W. and Manke, K. and Vogt, T. and}, title = {{A catalytic triad – Lys-Asn-Asp – Is essential for the catalysis of the methyl transfer in plant cation-dependent O-methyltransferases}}, year = {2015}, pages = {130-139}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2014.12.018}, volume = {113}, abstract = {Crystal structure data of cation-dependent catechol O-methyltransferases (COMTs) from mammals and related caffeoyl coenzyme A OMTs (CCoAOMTs) from plants have suggested operative molecular mechanisms. These include bivalent cations that facilitate deprotonation of vicinal aromatic dihydroxy systems and illustrate a conserved arrangement of hydroxyl and carboxyl ligands consistent with the requirements of a metal-activated catalytic mechanism. The general concept of metal-dependent deprotonation via a complexed aspartate is only one part of a more pronounced proton relay, as shown by semiempirical and DFT quantum mechanical calculations and experimental validations. A previously undetected catalytic triad, consisting of Lys157-Asn181-Asp228 residues is required for complete methyl transfer in case of a cation-dependent phenylpropanoid and flavonoid OMT, as described in this report. This triad appears essential for efficient methyl transfer to catechol-like hydroxyl group in phenolics. The observation is consistent with a catalytic lysine in the case of mammalian COMTs, but jettisons existing assumptions on the initial abstraction of the meta-hydroxyl proton to the metal stabilizing Asp154 (PFOMT) or comparable Asp-carboxyl groups in type of cation-dependent enzymes in plants. The triad is conserved among all characterized plant CCoAOMT-like enzymes, which are required not only for methylation of soluble phenylpropanoids like coumarins or monolignol monomers, but is also present in the similar microbial and mammalian cation-dependent enzymes which methylate a comparable set of substrates.} } @Article{IPB-1078, author = {Božić, D. and Papaefthimiou, D. and Brückner, K. and de Vos, R. C. H. and Tsoleridis, C. A. and Katsarou, D. and Papanikolaou, A. and Pateraki, I. and Chatzopoulou, F. M. and Dimitriadou, E. and Kostas, S. and Manzano, D. and Scheler, U. and Ferrer, A. and Tissier, A. and Makris, A. M. and Kampranis, S. C. and Kanellis, A. K. and}, title = {{Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis}}, year = {2015}, pages = {e0124106}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0124106}, volume = {10}, abstract = {Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.} } @Article{IPB-1142, author = {Patron, N. J. and Orzaez, D. and Marillonnet, S. and Warzecha, H. and Matthewman, C. and Youles, M. and Raitskin, O. and Leveau, A. and Farré, G. and Rogers, C. and Smith, A. and Hibberd, J. and Webb, A. A. R. and Locke, J. and Schornack, S. and Ajioka, J. and Baulcombe, D. C. and Zipfel, C. and Kamoun, S. and Jones, J. D. G. and Kuhn, H. and Robatzek, S. and Van Esse, H. P. and Sanders, D. and Oldroyd, G. and Martin, C. and Field, R. and O'Connor, S. and Fox, S. and Wulff, B. and Miller, B. and Breakspear, A. and Radhakrishnan, G. and Delaux, P.-M. and Loqué, D. and Granell, A. and Tissier, A. and Shih, P. and Brutnell, T. P. and Quick, W. P. and Rischer, H. and Fraser, P. D. and Aharoni, A. and Raines, C. and South, P. F. and Ané, J.-M. and Hamberger, B. R. and Langdale, J. and Stougaard, J. and Bouwmeester, H. and Udvardi, M. and Murray, J. A. H. and Ntoukakis, V. and Schäfer, P. and Denby, K. and Edwards, K. J. and Osbourn, A. and Haseloff, J. and}, title = {{Standards for plant synthetic biology: a common syntax for exchange of DNA parts}}, year = {2015}, pages = {13-19}, journal = {New Phytol.}, doi = {10.1111/nph.13532}, volume = {208}, abstract = {Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease‐mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.} } @Article{IPB-1137, author = {Nagel, R. and Bernholz, C. and Vranová, E. and Košuth, J. and Bergau, N. and Ludwig, S. and Wessjohann, L. and Gershenzon, J. and Tissier, A. and Schmidt, A. and}, title = {{Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate}}, year = {2015}, pages = {847-859}, journal = {Plant J.}, doi = {10.1111/tpj.13064}, volume = {84}, abstract = {Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C10), farnesyl diphosphate (FDP, C15) or geranylgeranyl diphosphate (GGDP, C20). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C25) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C25 versus C20 product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C20) as a product and a larger R group (Met) resulting in GFDP (C25).} } @Article{IPB-1130, author = {Matschi, S. and Hake, K. and Herde, M. and Hause, B. and Romeis, T. and}, title = {{The Calcium-Dependent Protein Kinase CPK28 Regulates Development by Inducing Growth Phase-Specific, Spatially Restricted Alterations in Jasmonic Acid Levels Independent of Defense Responses in Arabidopsis}}, year = {2015}, pages = {591-606}, journal = {Plant Cell}, doi = {10.1105/tpc.15.00024}, volume = {27}, abstract = {Phytohormones play an important role in development and stress adaptations in plants, and several interacting hormonal pathways have been suggested to accomplish fine-tuning of stress responses at the expense of growth. This work describes the role played by the CALCIUM-DEPENDENT PROTEIN KINASE CPK28 in balancing phytohormone-mediated development in Arabidopsis thaliana, specifically during generative growth. cpk28 mutants exhibit growth reduction solely as adult plants, coinciding with altered balance of the phytohormones jasmonic acid (JA) and gibberellic acid (GA). JA-dependent gene expression and the levels of several JA metabolites were elevated in a growth phase-dependent manner in cpk28, and accumulation of JA metabolites was confined locally to the central rosette tissue. No elevated resistance toward herbivores or necrotrophic pathogens was detected for cpk28 plants, either on the whole-plant level or specifically within the tissue displaying elevated JA levels. Abolishment of JA biosynthesis or JA signaling led to a full reversion of the cpk28 growth phenotype, while modification of GA signaling did not. Our data identify CPK28 as a growth phase-dependent key negative regulator of distinct processes: While in seedlings, CPK28 regulates reactive oxygen species-mediated defense signaling; in adult plants, CPK28 confers developmental processes by the tissue-specific balance of JA and GA without affecting JA-mediated defense responses.} } @Article{IPB-1125, author = {Lischweski, S. and Muchow, A. and Guthörl, D. and Hause, B. and}, title = {{Jasmonates act positively in adventitious root formation in petunia cuttings}}, year = {2015}, pages = {229}, journal = {BMC Plant Biol.}, doi = {10.1186/s12870-015-0615-1}, volume = {15}, abstract = {BackgroundPetunia is a model to study the process of adventitious root (AR) formation on leafy cuttings. Excision of cuttings leads to a transient increase in jasmonates, which is regarded as an early, transient and critical event for rooting. Here, the role of jasmonates in AR formation on petunia cuttings has been studied by a reverse genetic approach.ResultsTo reduce the endogenous levels of jasmonates, transgenic plants were generated expressing a Petunia hybrida ALLENE OXIDE CYCLASE (PhAOC)-RNAi construct. The transgenic plants exhibited strongly reduced PhAOC transcript and protein levels as well as diminished accumulation of cis-12-oxo-phytodienoic acid, jasmonic acid and jasmonoyl-isoleucine after wounding in comparison to wild type and empty vector expressing plants. Reduced levels of endogenous jasmonates resulted in formation of lower numbers of ARs. However, this effect was not accompanied by altered levels of auxin and aminocyclopropane carboxylate (ACC, precursor of ethylene) or by impaired auxin and ethylene-induced gene expression. Neither activity of cell-wall invertases nor accumulation of soluble sugars was altered by jasmonate deficiency.ConclusionsDiminished numbers of AR in JA-deficient cuttings suggest that jasmonates act as positive regulators of AR formation in petunia wild type. However, wound-induced rise in jasmonate levels in petunia wild type cuttings seems not to be causal for increased auxin and ethylene levels and for sink establishment.} } @Article{IPB-1169, author = {Walter, M. H. and Stauder, R. and Tissier, A. and}, title = {{Evolution of root-specific carotenoid precursor pathways for apocarotenoid signal biogenesis}}, year = {2015}, pages = {1-10}, journal = {Plant Sci.}, doi = {10.1016/j.plantsci.2014.12.017}, volume = {233}, abstract = {Various cleavage products of C40 carotenoid substrates are formed preferentially or exclusively in roots. Such apocarotenoid signaling or regulatory compounds differentially induced in roots during environmental stress responses including root colonization by arbuscular mycorrhizal fungi include ABA, strigolactones and C13 α-ionol/C14 mycorradicin derivatives. The low carotenoid levels in roots raise the question of whether there is a regulated precursor supply channeled into apocarotenoid formation distinct from default carotenoid pathways. This review describes root-specific isogene components of carotenoid pathways toward apocarotenoid formation, highlighting a new PSY3 class of phytoene synthase genes in dicots. It is clearly distinct from the monocot PSY3 class co-regulated with ABA formation. At least two members of the exclusive dicot PSY3s are regulated by nutrient stress and mycorrhization. This newly recognized dicot PSY3 (dPSY3 vs. mPSY3 from monocots) class probably represents an ancestral branch in the evolution of the plant phytoene synthase family. The evolutionary history of PSY genes is compared with the evolution of MEP pathway isogenes encoding 1-deoxy-d-xylulose 5-phosphate synthases (DXS), particularly DXS2, which is co-regulated with dPSY3s in mycorrhizal roots. Such stress-inducible isoforms for rate-limiting steps in root carotenogenesis might be components of multi-enzyme complexes committed to apocarotenoid rather than to carotenoid formation.} } @Article{IPB-1110, author = {Heinze, M. and Brandt, W. and Marillonnet, S. and Roos, W. and}, title = {{“Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints}}, year = {2015}, pages = {448-462}, journal = {Plant Cell}, doi = {10.1105/tpc.114.135343}, volume = {27}, abstract = {The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins.} } @INBOOK{IPB-62, author = {Tissier, A. and Ziegler, J. and Vogt, T. and}, title = {{Ecological Biochemistry: Environmental and Interspecies Interactions}}, year = {2015}, pages = {14-37}, chapter = {{Specialized Plant Metabolites: Diversity and Biosynthesis}}, editor = {Krauss, G.-J. \& Nies, D. H., eds.}, doi = {10.1002/9783527686063.ch2}, abstract = {Plant secondary metabolites, also termed specialized plant metabolites, currently comprise more than 200 000 natural products that are all based on a few biosynthetic pathways and key primary metabolites. Some pathways like flavonoid and terpenoid biosynthesis are universally distributed in the plant kingdom, whereas others like alkaloid or cyanogenic glycoside biosynthesis are restricted to a limited set of taxa. Diversification is achieved by an array of mechanisms at the genetic and enzymatic level including gene duplications, substrate promiscuity of enzymes, cell‐specific regulatory systems, together with modularity and combinatorial aspects. Specialized metabolites reflect adaptations to a specific environment. The observed diversity illustrates the heterogeneity and multitude of ecological habitats and niches that plants have colonized so far and constitutes a reservoir of potential new metabolites that may provide adaptive advantage in the face of environmental changes. The code that connects the observed chemical diversity to this ecological diversity is largely unknown. One way to apprehend this diversity is to realize its tremendous plasticity and evolutionary potential. This chapter presents an overview of the most widespread and popular secondary metabolites, which provide a definite advantage to adapt to or to colonize a particular environment, making the boundary between the “primary” and the “secondary” old fashioned and blurry.} } @INBOOK{IPB-60, author = {Marillonnet, S. and Werner, S. and}, title = {{Glyco-Engineering}}, year = {2015}, pages = {269-284}, chapter = {{Assembly of Multigene Constructs Using Golden Gate Cloning}}, journal = {Methods Mol. Biol.}, editor = {Castilho, A., ed.}, doi = {10.1007/978-1-4939-2760-9_19}, volume = {1321}, abstract = {Efficient DNA assembly methods are required for synthetic biology. Standardization of DNA parts is an essential element that not only facilitates reuse of the same parts for various constructs but also allows standardization of the assembly strategy. We provide here a protocol for assembly of multigene constructs from standard biological parts using the modular cloning system MoClo. Making constructs using this system requires to first define the structure of the final construct and to identify all basic parts and vectors required for the construction strategy. The cloning strategy is in large part determined by the structure of the final construct, which is then made using a series of one-pot Golden Gate cloning reactions.} } @INBOOK{IPB-59, author = {Hause, B. and Hause, G. and}, title = {{Ecological Biochemistry: Environmental and Interspecies Interactions}}, year = {2015}, pages = {366-382}, chapter = {{Microscope Techniques and Single Cell Analysis}}, editor = {Krauss, G.-J. \& Nies, D. H., eds.}, doi = {10.1002/9783527686063.ch19}, abstract = {For centuries, progress in biological research has been connected to the development of tools and equipment that allow new insights into the living matter. The invention of and improvements in optical systems were very important because exceeding the limits of the optical resolution of the human eye delivered new insights into tissues, cells, and subcellular compartments on the one hand and cellular processes on the other. Even the very first light microscopes, developed at the beginning of the seventeenth century, enabled the discovery of “Cells as little boxes” by Robert Hooke, and of bacteria by Antoni van Leeuwenhoek. Since then, many aspects of microscopes have been improved and new illumination, staining, and detection methods have been developed in order to increase the optical resolution. In this chapter, we describe the principles and possibilities of the use of microscopes in biology, as well as specific methods of preparing biological materials in order to obtain optimum microscopic images with an appropriate scientific message. Further, emphasis is given on staining techniques used for biological materials including transgenic approaches that use the wide variance of fluorescent proteins.} } @Article{IPB-1276, author = {Weier, D. and Thiel, J. and Kohl, S. and Tarkowská, D. and Strnad, M. and Schaarschmidt, S. and Weschke, W. and Weber, H. and Hause, B. and}, title = {{Gibberellin-to-abscisic acid balances govern development and differentiation of the nucellar projection of barley grains}}, year = {2014}, pages = {5291-5304}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/eru289}, volume = {65}, abstract = {In cereal grains, the maternal nucellar projection (NP) constitutes the link to the filial organs, forming a transfer path for assimilates and signals towards the endosperm. At transition to the storage phase, the NP of barley (Hordeum vulgare) undergoes dynamic and regulated differentiation forming a characteristic pattern of proliferating, elongating, and disintegrating cells. Immunolocalization revealed that abscisic acid (ABA) is abundant in early non-elongated but not in differentiated NP cells. In the maternally affected shrunken-endosperm mutant seg8, NP cells did not elongate and ABA remained abundant. The amounts of the bioactive forms of gibberellins (GAs) as well as their biosynthetic precursors were strongly and transiently increased in wild-type caryopses during the transition and early storage phases. In seg8, this increase was delayed and less pronounced together with deregulated gene expression of specific ABA and GA biosynthetic genes. We concluded that differentiation of the barley NP is driven by a distinct and specific shift from lower to higher GA:ABA ratios and that the spatial–temporal change of GA:ABA balances is required to form the differentiation gradient, which is a prerequisite for ordered transfer processes through the NP. Deregulated ABA:GA balances in seg8 impair the differentiation of the NP and potentially compromise transfer of signals and assimilates, resulting in aberrant endosperm growth. These results highlight the impact of hormonal balances on the proper release of assimilates from maternal to filial organs and provide new insights into maternal effects on endosperm differentiation and growth of barley grains.} } @Article{IPB-1274, author = {Wasternack, C. and Hause, B. and}, title = {{Blütenduft, Abwehr, Entwicklung: Jasmonsäure - ein universelles Pflanzenhormon}}, year = {2014}, pages = {164-171}, journal = {Biologie in unserer Zeit}, doi = {10.1002/biuz.201410535}, volume = {44}, abstract = {Pflanzen müssen gegen vielfältige biotische und abiotische Umwelteinflusse eine Abwehr aufbauen. Aber gleichzeitig müssen sie wachsen und sich vermehren. Jasmonate sind neben anderen Hormonen ein zentrales Signal bei der Etablierung von Abwehrmechanismen, aber auch Signal von Entwicklungsprozessen wie Blüten‐ und Trichombildung, sowie der Hemmung von Wachstum. Biosynthese und essentielle Komponenten der Signaltransduktion von JA und seinem biologisch aktiven Konjugat JA‐Ile sind gut untersucht. Der Rezeptor ist ein Proteinkomplex, der “JA‐Ile‐Wahrnehmung” mit proteasomalem Abbau von Repressorproteinen verbindet. Dadurch können positiv agierende Transkriptionsfaktoren wirksam werden und vielfältige Genexpressionsänderungen auslösen. Dies betrifft die Bildung von Abwehrproteinen, Enzymen der JA‐Biosynthese und Sekundärstoffbildung, und Proteinen von Signalketten und Entwicklungsprozessen. Die Kenntnisse zur JA‐Ile‐Wirkung werden in Landwirtschaft und Biotechnologie genutzt.} } @Article{IPB-1267, author = {Staniek, A. and Bouwmeester, H. and Fraser, P. D. and Kayser, O. and Martens, S. and Tissier, A. and van der Krol, S. and Wessjohann, L. and Warzecha, H. and}, title = {{Natural products - learning chemistry from plants}}, year = {2014}, pages = {326-336}, journal = {Biotechnol. J.}, doi = {10.1002/biot.201300059}, volume = {9}, abstract = {Plant natural products (PNPs) are unique in that they represent a vast array of different structural features, ranging from relatively simple molecules to very complex ones. Given the fact that many plant secondary metabolites exhibit profound biological activity, they are frequently used as fragrances and flavors, medicines, as well as industrial chemicals. As the intricate structures of PNPs often cannot be mimicked by chemical synthesis, the original plant providers constitute the sole source for their industrial, large‐scale production. However, sufficient supply is not guaranteed for all molecules of interest, making the development of alternative production systems a priority. Modern techniques, such as genome mining and thorough biochemical analysis, have helped us gain preliminary understanding of the enzymatic formation of the valuable ingredients in planta. Herein, we review recent advances in the application of biocatalytical processes, facilitating generation of complex PNPs through utilization of plant‐derived specific enzymes and combinatorial biochemistry. We further evaluate the options of employing heterologous organisms harboring PNP biosynthetic pathways for the production of secondary metabolites of interest.} } @Article{IPB-1259, author = {Schneider, J. D. and Marillonnet, S. and Castilho, A. and Gruber, C. and Werner, S. and Mach, L. and Klimyuk, V. and Mor, T. S. and Steinkellner, H. and}, title = {{Oligomerization status influences subcellular deposition and glycosylation of recombinant butyrylcholinesterase in Nicotiana benthamiana}}, year = {2014}, pages = {832-839}, journal = {Plant Biotechnol. J.}, doi = {10.1111/pbi.12184}, volume = {12}, abstract = {Plants have a proven track record for the expression of biopharmaceutically interesting proteins. Importantly, plants and mammals share a highly conserved secretory pathway that allows similar folding, assembly and posttranslational modifications of proteins. Human butyrylcholinesterase (BChE) is a highly sialylated, tetrameric serum protein, investigated as a bioscavenger for organophosphorous nerve agents. Expression of recombinant BChE (rBChE) in Nicotiana benthamiana results in accumulation of both monomers as well as assembled oligomers. In particular, we show here that co‐expression of BChE with a novel gene‐stacking vector, carrying six mammalian genes necessary for in planta protein sialylation, resulted in the generation of rBChE decorated with sialylated N‐glycans. The N‐glycosylation profile of monomeric rBChE secreted to the apoplast largely resembles the plasma‐derived orthologue. In contrast, rBChE purified from total soluble protein extracts was decorated with a significant portion of ER‐typical oligomannosidic structures. Biochemical analyses and live‐cell imaging experiments indicated that impaired N‐glycan processing is due to aberrant deposition of rBChE oligomers in the endoplasmic reticulum or endoplasmic‐reticulum‐derived compartments. In summary, we show the assembly of rBChE multimers, however, also points to the need for in‐depth studies to explain the unexpected subcellular targeting of oligomeric BChE in plants.} } @Article{IPB-1245, author = {Phan, H. T. and Hause, B. and Hause, G. and Arcalis, E. and Stoger, E. and Maresch, D. and Altmann, F. and Joensuu, J. and Conrad, U. and}, title = {{Influence of Elastin-Like Polypeptide and Hydrophobin on Recombinant Hemagglutinin Accumulations in Transgenic Tobacco Plants}}, year = {2014}, pages = {e99347}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0099347}, volume = {9}, abstract = {Fusion protein strategies are useful tools to enhance expression and to support the development of purification technologies. The capacity of fusion protein strategies to enhance expression was explored in tobacco leaves and seeds. C-terminal fusion of elastin-like polypeptides (ELP) to influenza hemagglutinin under the control of either the constitutive CaMV 35S or the seed-specific USP promoter resulted in increased accumulation in both leaves and seeds compared to the unfused hemagglutinin. The addition of a hydrophobin to the C-terminal end of hemagglutinin did not significantly increase the expression level. We show here that, depending on the target protein, both hydrophobin fusion and ELPylation combined with endoplasmic reticulum (ER) targeting induced protein bodies in leaves as well as in seeds. The N-glycosylation pattern indicated that KDEL sequence-mediated retention of leaf-derived hemagglutinins and hemagglutinin-hydrophobin fusions were not completely retained in the ER. In contrast, hemagglutinin-ELP from leaves contained only the oligomannose form, suggesting complete ER retention. In seeds, ER retention seems to be nearly complete for all three constructs. An easy and scalable purification method for ELPylated proteins using membrane-based inverse transition cycling could be applied to both leaf- and seed-expressed hemagglutinins.} } @Article{IPB-1236, author = {Meesters, C. and Mönig, T. and Oeljeklaus, J. and Krahn, D. and Westfall, C. S. and Hause, B. and Jez, J. M. and Kaiser, M. and Kombrink, E. and}, title = {{A chemical inhibitor of jasmonate signaling targets JAR1 in Arabidopsis thaliana}}, year = {2014}, pages = {830-836}, journal = {Nat. Chem. Biol.}, doi = {10.1038/nchembio.1591}, volume = {10}, abstract = {Jasmonates are lipid-derived plant hormones that regulate plant defenses and numerous developmental processes. Although the biosynthesis and molecular function of the most active form of the hormone, (\+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), have been unraveled, it remains poorly understood how the diversity of bioactive jasmonates regulates such a multitude of plant responses. Bioactive analogs have been used as chemical tools to interrogate the diverse and dynamic processes of jasmonate action. By contrast, small molecules impairing jasmonate functions are currently unknown. Here, we report on jarin-1 as what is to our knowledge the first small-molecule inhibitor of jasmonate responses that was identified in a chemical screen using Arabidopsis thaliana. Jarin-1 impairs the activity of JA-Ile synthetase, thereby preventing the synthesis of the active hormone, JA-Ile, whereas closely related enzymes are not affected. Thus, jarin-1 may serve as a useful chemical tool in search for missing regulatory components and further dissection of the complex jasmonate signaling networks.} } @Article{IPB-1204, author = {Engler, C. and Youles, M. and Gruetzner, R. and Ehnert, T.-M. and Werner, S. and Jones, J. D. G. and Patron, N. J. and Marillonnet, S. and}, title = {{A Golden Gate Modular Cloning Toolbox for Plants}}, year = {2014}, pages = {839-843}, journal = {ACS Synth. Biol.}, doi = {10.1021/sb4001504}, volume = {3}, abstract = {Plant Synthetic Biology requires robust and efficient methods for assembling multigene constructs. Golden Gate cloning provides a precision module-based cloning technique for facile assembly of multiple genes in one construct. We present here a versatile resource for plant biologists comprising a set of cloning vectors and 96 standardized parts to enable Golden Gate construction of multigene constructs for plant transformation. Parts include promoters, untranslated sequences, reporters, antigenic tags, localization signals, selectable markers, and terminators. The comparative performance of parts in the model plant Nicotiana benthamiana is discussed.} } @Article{IPB-1202, author = {Druege, U. and Franken, P. and Lischewski, S. and Ahkami, A. H. and Zerche, S. and Hause, B. and Hajirezaei, M. R. and}, title = {{Transcriptomic analysis reveals ethylene as stimulator and auxin as regulator of adventitious root formation in petunia cuttings}}, year = {2014}, pages = {494}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2014.00494}, volume = {5}, abstract = {Adventitious root (AR) formation in the stem base (SB) of cuttings is the basis for propagation of many plant species and petunia is used as model to study this developmental process. Following AR formation from 2 to 192 hours post-excision (hpe) of cuttings, transcriptome analysis by microarray revealed a change of the character of the rooting zone from SB to root identity. The greatest shift in the number of differentially expressed genes was observed between 24 and 72 hpe, when the categories storage, mineral nutrient acquisition, anti-oxidative and secondary metabolism, and biotic stimuli showed a notable high number of induced genes. Analyses of phytohormone-related genes disclosed multifaceted changes of the auxin transport system, auxin conjugation and the auxin signal perception machinery indicating a reduction in auxin sensitivity and phase-specific responses of particular auxin-regulated genes. Genes involved in ethylene biosynthesis and action showed a more uniform pattern as a high number of respective genes were generally induced during the whole process of AR formation. The important role of ethylene for stimulating AR formation was demonstrated by the application of inhibitors of ethylene biosynthesis and perception as well as of the precursor aminocyclopropane-1-carboxylic acid, all changing the number and length of AR. A model is proposed showing the putative role of polar auxin transport and resulting auxin accumulation in initiation of subsequent changes in auxin homeostasis and signal perception with a particular role of Aux/IAA expression. These changes might in turn guide the entrance into the different phases of AR formation. Ethylene biosynthesis, which is stimulated by wounding and does probably also respond to other stresses and auxin, acts as important stimulator of AR formation probably via the expression of ethylene responsive transcription factor genes, whereas the timing of different phases seems to be controlled by auxin.} } @Article{IPB-1200, author = {Dey, S. and Wenig, M. and Langen, G. and Sharma, S. and Kugler, K. G. and Knappe, C. and Hause, B. and Bichlmeier, M. and Babaeizad, V. and Imani, J. and Janzik, I. and Stempfl, T. and Hückelhoven, R. and Kogel, K.-H. and Mayer, K. F. X. and Vlot, A. C. and}, title = {{Bacteria-Triggered Systemic Immunity in Barley Is Associated with WRKY and ETHYLENE RESPONSIVE FACTORs But Not with Salicylic Acid}}, year = {2014}, pages = {2133-2151}, journal = {Plant Physiol.}, doi = {10.1104/pp.114.249276}, volume = {166}, abstract = {Leaf-to-leaf systemic immune signaling known as systemic acquired resistance is poorly understood in monocotyledonous plants. Here, we characterize systemic immunity in barley (Hordeum vulgare) triggered after primary leaf infection with either Pseudomonas syringae pathovar japonica (Psj) or Xanthomonas translucens pathovar cerealis (Xtc). Both pathogens induced resistance in systemic, uninfected leaves against a subsequent challenge infection with Xtc. In contrast to systemic acquired resistance in Arabidopsis (Arabidopsis thaliana), systemic immunity in barley was not associated with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 or the local or systemic accumulation of salicylic acid. Instead, we documented a moderate local but not systemic induction of abscisic acid after infection of leaves with Psj. In contrast to salicylic acid or its functional analog benzothiadiazole, local applications of the jasmonic acid methyl ester or abscisic acid triggered systemic immunity to Xtc. RNA sequencing analysis of local and systemic transcript accumulation revealed unique gene expression changes in response to both Psj and Xtc and a clear separation of local from systemic responses. The systemic response appeared relatively modest, and quantitative reverse transcription-polymerase chain reaction associated systemic immunity with the local and systemic induction of two WRKY and two ETHYLENE RESPONSIVE FACTOR (ERF)-like transcription factors. Systemic immunity against Xtc was further associated with transcriptional changes after a secondary/systemic Xtc challenge infection; these changes were dependent on the primary treatment. Taken together, bacteria-induced systemic immunity in barley may be mediated in part by WRKY and ERF-like transcription factors, possibly facilitating transcriptional reprogramming to potentiate immunity.} } @Article{IPB-1192, author = {Bucher, M. and Hause, B. and Krajinski, F. and Küster, H. and}, title = {{Through the doors of perception to function in arbuscular mycorrhizal symbioses}}, year = {2014}, pages = {833-840}, journal = {New Phytol.}, doi = {10.1111/nph.12862}, volume = {204}, abstract = {The formation of an arbuscular mycorrhizal (AM) symbiosis is initiated by the bidirectional exchange of diffusible molecules. While strigolactone hormones, secreted from plant roots, stimulate hyphal branching and fungal metabolism, fungal short‐chain chitin oligomers as well as sulfated and nonsulfated lipochitooligosaccharides (s/nsMyc‐LCOs) elicit pre‐symbiosis responses in the host. Fungal LCO signals are structurally related to rhizobial Nod‐factor LCOs. Genome‐wide expression studies demonstrated that defined sets of genes were induced by Nod‐, sMyc‐ and nsMyc‐LCOs, indicating LCO‐specific perception in the pre‐symbiosis phase. During hyphopodium formation and the subsequent root colonization, cross‐talk between plant roots and AM fungi also involves phytohormones. Notably, gibberellins control arbuscule formation via DELLA proteins, which themselves serve as positive regulators of arbuscule formation. The establishment of arbuscules is accompanied by a substantial transcriptional and post‐transcriptional reprogramming of host roots, ultimately defining the unique protein composition of arbuscule‐containing cells. Based on cellular expression profiles, key checkpoints of AM development as well as candidate genes encoding transcriptional regulators and regulatory microRNAs were identified. Detailed functional analyses of promoters specified short motifs sufficient for cell‐autonomous gene regulation in cells harboring arbuscules, and suggested simultaneous, multi‐level regulation of the mycorrhizal phosphate uptake pathway by integrating AM symbiosis and phosphate starvation response signaling.} } @Article{IPB-1191, author = {Brückner, K. and Božić, D. and Manzano, D. and Papaefthimiou, D. and Pateraki, I. and Scheler, U. and Ferrer, A. and de Vos, R. C. and Kanellis, A. K. and Tissier, A. and}, title = {{Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes}}, year = {2014}, pages = {52-64}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2014.01.021}, volume = {101}, abstract = {Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.} } @Article{IPB-1187, author = {Bosch, M. and Wright, L. P. and Gershenzon, J. and Wasternack, C. and Hause, B. and Schaller, A. and Stintzi, A. and}, title = {{Jasmonic Acid and Its Precursor 12-Oxophytodienoic Acid Control Different Aspects of Constitutive and Induced Herbivore Defenses in Tomato}}, year = {2014}, pages = {396-410}, journal = {Plant Physiol.}, doi = {10.1104/pp.114.237388}, volume = {166}, abstract = {The jasmonate family of growth regulators includes the isoleucine (Ile) conjugate of jasmonic acid (JA-Ile) and its biosynthetic precursor 12-oxophytodienoic acid (OPDA) as signaling molecules. To assess the relative contribution of JA/JA-Ile and OPDA to insect resistance in tomato (Solanum lycopersicum), we silenced the expression of OPDA reductase3 (OPR3) by RNA interference (RNAi). Consistent with a block in the biosynthetic pathway downstream of OPDA, OPR3-RNAi plants contained wild-type levels of OPDA but failed to accumulate JA or JA-Ile after wounding. JA/JA-Ile deficiency in OPR3-RNAi plants resulted in reduced trichome formation and impaired monoterpene and sesquiterpene production. The loss of these JA/JA-Ile -dependent defense traits rendered them more attractive to the specialist herbivore Manduca sexta with respect to feeding and oviposition. Oviposition preference resulted from reduced levels of repellant monoterpenes and sesquiterpenes. Feeding preference, on the other hand, was caused by increased production of cis-3-hexenal acting as a feeding stimulant for M. sexta larvae in OPR3-RNAi plants. Despite impaired constitutive defenses and increased palatability of OPR3-RNAi leaves, larval development was indistinguishable on OPR3-RNAi and wild-type plants, and was much delayed compared with development on the jasmonic acid-insensitive1 (jai1) mutant. Apparently, signaling through JAI1, the tomato ortholog of the ubiquitin ligase CORONATINE INSENSITIVE1 in Arabidopsis (Arabidopsis thaliana), is required for defense, whereas the conversion of OPDA to JA/JA-Ile is not. Comparing the signaling activities of OPDA and JA/JA-Ile, we found that OPDA can substitute for JA/JA-Ile in the local induction of defense gene expression, but the production of JA/JA-Ile is required for a systemic response.} } @Article{IPB-1232, author = {López-Ráez, J. A. and Fernandez, I. and García, J. M. and Berrio, E. and Bonfante, P. and Walter, M. H. and Pozo, M. J. and}, title = {{Differential spatio-temporal expression of carotenoid cleavage dioxygenases regulates apocarotenoid fluxes during AM symbiosis}}, year = {2014}, pages = {59-69}, journal = {Plant Sci.}, doi = {10.1016/j.plantsci.2014.10.010}, volume = {230}, abstract = {Apocarotenoids are a class of compounds that play important roles in nature. In recent years, a prominent role for these compounds in arbuscular mycorrhizal (AM) symbiosis has been shown. They are derived from carotenoids by the action of the carotenoid cleavage dioxygenase (CCD) enzyme family. In the present study, using tomato as a model, the spatio-temporal expression pattern of the CCD genes during AM symbiosis establishment and functioning was investigated. In addition, the levels of the apocarotenoids strigolactones (SLs), C13 α-ionol and C14 mycorradicin (C13/C14) derivatives were analyzed. The results suggest an increase in SLs promoted by the presence of the AM fungus at the early stages of the interaction, which correlated with an induction of the SL biosynthesis gene SlCCD7. At later stages, induction of SlCCD7 and SlCCD1 expression in arbusculated cells promoted the production of C13/C14 apocarotenoid derivatives. We show here that the biosynthesis of apocarotenoids during AM symbiosis is finely regulated throughout the entire process at the gene expression level, and that CCD7 constitutes a key player in this regulation. Once the symbiosis is established, apocarotenoid flux would be turned towards the production of C13/C14 derivatives, thus reducing SL biosynthesis and maintaining a functional symbiosis.} } @Article{IPB-1226, author = {Krajinski, F. and Courty, P.-E. and Sieh, D. and Franken, P. and Zhang, H. and Bucher, M. and Gerlach, N. and Kryvoruchko, I. and Zoeller, D. and Udvardi, M. and Hause, B. and}, title = {{The H\+-ATPase HA1 of Medicago truncatula Is Essential for Phosphate Transport and Plant Growth during Arbuscular Mycorrhizal Symbiosis}}, year = {2014}, pages = {1808-1817}, journal = {Plant Cell}, doi = {10.1105/tpc.113.120436}, volume = {26}, abstract = {A key feature of arbuscular mycorrhizal symbiosis is improved phosphorus nutrition of the host plant via the mycorrhizal pathway, i.e., the fungal uptake of Pi from the soil and its release from arbuscules within root cells. Efficient transport of Pi from the fungus to plant cells is thought to require a proton gradient across the periarbuscular membrane (PAM) that separates fungal arbuscules from the host cell cytoplasm. Previous studies showed that the H\+-ATPase gene HA1 is expressed specifically in arbuscule-containing root cells of Medicago truncatula. We isolated a ha1-2 mutant of M. truncatula and found it to be impaired in the development of arbuscules but not in root colonization by Rhizophagus irregularis hyphae. Artificial microRNA silencing of HA1 recapitulated this phenotype, resulting in small and truncated arbuscules. Unlike the wild type, the ha1-2 mutant failed to show a positive growth response to mycorrhizal colonization under Pi-limiting conditions. Uptake experiments confirmed that ha1-2 mutants are unable to take up phosphate via the mycorrhizal pathway. Increased pH in the apoplast of abnormal arbuscule-containing cells of the ha1-2 mutant compared with the wild type suggests that HA1 is crucial for building a proton gradient across the PAM and therefore is indispensible for the transfer of Pi from the fungus to the plant.} } @Article{IPB-1223, author = {Hilou, A. and Zhang, H. and Franken, P. and Hause, B. and}, title = {{Do jasmonates play a role in arbuscular mycorrhiza-induced local bioprotection of Medicago truncatula against root rot disease caused by Aphanomyces euteiches?}}, year = {2014}, pages = {45-54}, journal = {Mycorrhiza}, doi = {10.1007/s00572-013-0513-z}, volume = {24}, abstract = {Bioprotective effects of mycorrhization with two different arbuscular mycorrhizal (AM) fungi, Funneliformis mosseae and Rhizophagus irregularis, against Aphanomyces euteiches, the causal agent of root rot in legumes, were studied in Medicago truncatula using phenotypic and molecular markers. Previous inoculation with an AM-fungus reduced disease symptoms as well as the amount of pathogen within roots, as determined by the levels of A. euteiches rRNA or transcripts of the gene sterol C24 reductase. Inoculation with R. irregularis was as efficient as that with F. mosseae. To study whether jasmonates play a regulatory role in bioprotection of M. truncatula by the AM fungi, composite plants harboring transgenic roots were used to modulate the expression level of the gene encoding M. truncatula allene oxide cyclase 1, a key enzyme in jasmonic acid biosynthesis. Neither an increase nor a reduction in allene oxide cyclase levels resulted in altered bioprotection by the AM fungi against root infection by A. euteiches. These data suggest that jasmonates do not play a major role in the local bioprotective effect of AM fungi against the pathogen A. euteiches in M. truncatula roots.} } @INBOOK{IPB-73, author = {Thieme, F. and Marillonnet, S. and}, title = {{DNA Cloning and Assembly Methods}}, year = {2014}, pages = {37-48}, chapter = {{Quick and Clean Cloning}}, journal = {Methods Mol. Biol.}, editor = {Valla, S. \& Lale, R., eds.}, doi = {10.1007/978-1-62703-764-8_3}, volume = {1116}, abstract = {Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.} } @INBOOK{IPB-70, author = {Engler, C. and Marillonnet, S. and}, title = {{DNA Cloning and Assembly Methods}}, year = {2014}, pages = {119-131}, chapter = {{Golden Gate Cloning}}, journal = {Methods Mol. Biol.}, editor = {Valla, S. \& Lale, R., eds.}, doi = {10.1007/978-1-62703-764-8_9}, volume = {1116}, abstract = {DNA assembly methods are essential tools for biological research and biotechnology. Therefore various methods have been developed to clone DNA fragments of interest. Conventional methods usually require several cloning steps to generate a construct of interest. At each step, a single DNA fragment is transferred from a donor plasmid or PCR product to a recipient vector. In the past few years, a number of methods have been developed to facilitate and speed up this process. One of these methods, Golden Gate cloning, allows assembling up to nine fragments at a time in a recipient plasmid. Cloning is performed by pipetting in a single tube all plasmid donors, the recipient vector, a type IIS restriction enzyme and ligase, and incubating the mix in a thermal cycler. Despite the simplicity of the cloning procedure, the majority of clones obtained after transformation contain the expected construct. Using Golden Gate cloning however requires the use of carefully designed donor and recipient plasmids. We provide here a protocol describing how to design these plasmids and also describe the conditions necessary to perform the assembly reaction.} } @INBOOK{IPB-66, author = {Balcke, G. U. and Bennewitz, S. and Zabel, S. and Tissier, A. and}, title = {{Plant Isoprenoids}}, year = {2014}, pages = {189-202}, chapter = {{Isoprenoid and Metabolite Profiling of Plant Trichomes}}, journal = {Methods Mol. Biol.}, editor = {Rodríguez-Concepción, M., ed.}, doi = {10.1007/978-1-4939-0606-2_13}, volume = {1153}, abstract = {Plant glandular trichomes are specialized secretory structures located on the surface of the aerial parts of plants with large biosynthetic capacity, often with terpenoids as output molecules. The collection of plant trichomes requires a method to separate trichomes from leaf epidermal tissues. For metabolite profiling, trichome tissue needs to be rapidly quenched in order to maintain the indigenous state of intracellular intermediates. Appropriate extraction and chromatographic separation methods must be available, which address the wide-ranging polarity of metabolites. In this chapter, a protocol for trichome harvest using a frozen paint brush is presented. A work flow for broad-range metabolite profiling using LC-MS2 analysis is described, which is applicable to assess very hydrophilic isoprenoid precursors as well as more hydrophobic metabolites from trichomes and other plant tissues.} } @Article{IPB-1364, author = {Wils, C. R. and Brandt, W. and Manke, K. and Vogt, T. and}, title = {{A single amino acid determines position specificity of an Arabidopsis thaliana CCoAOMT-like O-methyltransferase}}, year = {2013}, pages = {683-689}, journal = {FEBS Lett.}, doi = {10.1016/j.febslet.2013.01.040}, volume = {587}, abstract = {Caffeoyl‐coenzyme A O‐methyltransferase (CCoAOMT)‐like proteins from plants display a conserved position specificity towards the meta‐position of aromatic vicinal dihydroxy groups, consistent with the methylation pattern observed in vivo. A CCoAOMT‐like enzyme identified from Arabidopsis thaliana encoded by the gene At4g26220 shows a strong preference for methylating the para position of flavanones and dihydroflavonols, whereas flavones and flavonols are methylated in the meta‐position. Sequence alignments and homology modelling identified several unique amino acids compared to motifs of other CCoAOMT‐like enzymes. Mutation of a single glycine, G46 towards a tyrosine was sufficient for a reversal of the unusual para‐ back to meta‐O‐methylation of flavanones and dihydroflavonols.} } @Article{IPB-1359, author = {Wasternack, C. and Hause, B. and}, title = {{Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany}}, year = {2013}, pages = {1021-1058}, journal = {Ann. Bot.}, doi = {10.1093/aob/mct067}, volume = {111}, abstract = {BackgroundJasmonates are important regulators in plant responses to biotic and abiotic stresses as well as in development. Synthesized from lipid-constituents, the initially formed jasmonic acid is converted to different metabolites including the conjugate with isoleucine. Important new components of jasmonate signalling including its receptor were identified, providing deeper insight into the role of jasmonate signalling pathways in stress responses and development.ScopeThe present review is an update of the review on jasmonates published in this journal in 2007. New data of the last five years are described with emphasis on metabolites of jasmonates, on jasmonate perception and signalling, on cross-talk to other plant hormones and on jasmonate signalling in response to herbivores and pathogens, in symbiotic interactions, in flower development, in root growth and in light perception.ConclusionsThe last few years have seen breakthroughs in the identification of JASMONATE ZIM DOMAIN (JAZ) proteins and their interactors such as transcription factors and co-repressors, and the crystallization of the jasmonate receptor as well as of the enzyme conjugating jasmonate to amino acids. Now, the complex nature of networks of jasmonate signalling in stress responses and development including hormone cross-talk can be addressed.} } @Article{IPB-1358, author = {Wasternack, C. and Forner, S. and Strnad, M. and Hause, B. and}, title = {{Jasmonates in flower and seed development}}, year = {2013}, pages = {79-85}, journal = {Biochimie}, doi = {10.1016/j.biochi.2012.06.005}, volume = {95}, abstract = {Jasmonates are ubiquitously occurring lipid-derived signaling compounds active in plant development and plant responses to biotic and abiotic stresses. Upon environmental stimuli jasmonates are formed and accumulate transiently. During flower and seed development, jasmonic acid (JA) and a remarkable number of different metabolites accumulate organ- and tissue specifically. The accumulation is accompanied with expression of jasmonate-inducible genes. Among these genes there are defense genes and developmentally regulated genes. The profile of jasmonate compounds in flowers and seeds covers active signaling molecules such as JA, its precursor 12-oxophytodienoic acid (OPDA) and amino acid conjugates such as JA-Ile, but also inactive signaling molecules occur such as 12-hydroxy-JA and its sulfated derivative. These latter compounds can occur at several orders of magnitude higher level than JA. Metabolic conversion of JA and JA-Ile to hydroxylated compounds seems to inactivate JA signaling, but also specific functions of jasmonates in flower and seed development were detected. In tomato OPDA is involved in embryo development. Occurrence of jasmonates, expression of JA-inducible genes and JA-dependent processes in flower and seed development will be discussed.} } @Article{IPB-1350, author = {Staniek, A. and Bouwmeester, H. and Fraser, P. D. and Kayser, O. and Martens, S. and Tissier, A. and van der Krol, S. and Wessjohann, L. and Warzecha, H. and}, title = {{Natural products - modifying metabolite pathways in plants}}, year = {2013}, pages = {1159-1171}, journal = {Biotechnol. J.}, doi = {10.1002/biot.201300224}, volume = {8}, abstract = {The diversity of plant natural product (PNP) molecular structures is reflected in the variety of biochemical and genetic pathways that lead to their formation and accumulation. Plant secondary metabolites are important commodities, and include fragrances, colorants, and medicines. Increasing the extractable amount of PNP through plant breeding, or more recently by means of metabolic engineering, is a priority. The prerequisite for any attempt at metabolic engineering is a detailed knowledge of the underlying biosynthetic and regulatory pathways in plants. Over the past few decades, an enormous body of information about the biochemistry and genetics of biosynthetic pathways involved in PNPs production has been generated. In this review, we focus on the three large classes of plant secondary metabolites: terpenoids (or isoprenoids), phenylpropanoids, and alkaloids. All three provide excellent examples of the tremendous efforts undertaken to boost our understanding of biosynthetic pathways, resulting in the first successes in plant metabolic engineering. We further consider what essential information is still missing, and how future research directions could help achieve the rational design of plants as chemical factories for high‐value products.} } @Article{IPB-1342, author = {Schaarschmidt, S. and Gresshoff, P. M. and Hause, B. and}, title = {{Analyzing the soybean transcriptome during autoregulation of mycorrhization identifies the transcription factors GmNF-YA1a/b as positive regulators of arbuscular mycorrhization}}, year = {2013}, pages = {R62}, journal = {Genome Biol.}, doi = {10.1186/gb-2013-14-6-r62}, volume = {14}, abstract = {BackgroundSimilarly to the legume-rhizobia symbiosis, the arbuscular mycorrhiza interaction is controlled by autoregulation representing a feedback inhibition involving the CLAVATA1-like receptor kinase NARK in shoots. However, little is known about signals and targets down-stream of NARK. To find NARK-related transcriptional changes in mycorrhizal soybean (Glycine max) plants, we analyzed wild-type and two nark mutant lines interacting with the arbuscular mycorrhiza fungus Rhizophagus irregularis.ResultsAffymetrix GeneChip analysis of non-inoculated and partially inoculated plants in a split-root system identified genes with potential regulation by arbuscular mycorrhiza or NARK. Most transcriptional changes occur locally during arbuscular mycorrhiza symbiosis and independently of NARK. RT-qPCR analysis verified nine genes as NARK-dependently regulated. Most of them have lower expression in roots or shoots of wild type compared to nark mutants, including genes encoding the receptor kinase GmSIK1, proteins with putative function as ornithine acetyl transferase, and a DEAD box RNA helicase. A predicted annexin named GmAnnx1a is differentially regulated by NARK and arbuscular mycorrhiza in distinct plant organs. Two putative CCAAT-binding transcription factor genes named GmNF-YA1a and GmNF-YA1b are down-regulated NARK-dependently in non-infected roots of mycorrhizal wild-type plants and functional gene analysis confirmed a positive role for these genes in the development of an arbuscular mycorrhiza symbiosis.ConclusionsOur results indicate GmNF-YA1a/b as positive regulators in arbuscular mycorrhiza establishment, whose expression is down-regulated by NARK in the autoregulated root tissue thereby diminishing subsequent infections. Genes regulated independently of arbuscular mycorrhization by NARK support an additional function of NARK in symbioses-independent mechanisms.} } @Article{IPB-1332, author = {Nahar, K. and Kyndt, T. and Hause, B. and Höfte, M. and Gheysen, G. and}, title = {{Brassinosteroids Suppress Rice Defense Against Root-Knot Nematodes Through Antagonism With the Jasmonate Pathway}}, year = {2013}, pages = {106-115}, journal = {Mol. Plant Microbe Interact.}, doi = {10.1094/MPMI-05-12-0108-FI}, volume = {26}, abstract = {The importance of phytohormone balance is increasingly recognized as central to the outcome of plant–pathogen interactions. Next to their well-known developmental role, brassinosteroids (BR) were recently found to be involved in plant innate immunity. In this study, we examined the role of BR in rice (Oryza sativa) innate immunity during infection with the root-knot nematode Meloidogyne graminicola, and we studied the inter-relationship with the jasmonate (JA) pathway. Exogenous epibrassinolide (BL) supply at low concentrations induced susceptibility in the roots whereas high concentrations of BL enforced systemic defense against this nematode. Upon high exogenous BL supply on the shoot, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) confirmed a strong feedback inhibitory effect, leading to reduced BR biosynthesis in the root. Moreover, we demonstrate that the immune suppressive effect of BR is at least partly due to negative cross-talk with the JA pathway. Mutants in the BR biosynthesis or signaling pathway accumulate slightly higher levels of the immediate JA-precursor 12-oxo-phytodienoic acid, and qRT-PCR data showed that the BR and JA pathway are mutually antagonistic in rice roots. Collectively, these results suggest that the balance between the BR and JA pathway is an effective regulator of the outcome of the rice–M. graminicola interaction.} } @Article{IPB-1329, author = {Matsuba, Y. and Nguyen, T. T. and Wiegert, K. and Falara, V. and Gonzales-Vigil, E. and Leong, B. and Schäfer, P. and Kudrna, D. and Wing, R. A. and Bolger, A. M. and Usadel, B. and Tissier, A. and Fernie, A. R. and Barry, C. S. and Pichersky, E. and}, title = {{Evolution of a Complex Locus for Terpene Biosynthesis in Solanum}}, year = {2013}, pages = {2022-2036}, journal = {Plant Cell}, doi = {10.1105/tpc.113.111013}, volume = {25}, abstract = {Functional gene clusters, containing two or more genes encoding different enzymes for the same pathway, are sometimes observed in plant genomes, most often when the genes specify the synthesis of specialized defensive metabolites. Here, we show that a cluster of genes in tomato (Solanum lycopersicum; Solanaceae) contains genes for terpene synthases (TPSs) that specify the synthesis of monoterpenes and diterpenes from cis-prenyl diphosphates, substrates that are synthesized by enzymes encoded by cis-prenyl transferase (CPT) genes also located within the same cluster. The monoterpene synthase genes in the cluster likely evolved from a diterpene synthase gene in the cluster by duplication and divergence. In the orthologous cluster in Solanum habrochaites, a new sesquiterpene synthase gene was created by a duplication event of a monoterpene synthase followed by a localized gene conversion event directed by a diterpene synthase gene. The TPS genes in the Solanum cluster encoding cis-prenyl diphosphate–utilizing enzymes are closely related to a tobacco (Nicotiana tabacum; Solanaceae) diterpene synthase encoding Z-abienol synthase (Nt-ABS). Nt-ABS uses the substrate copal-8-ol diphosphate, which is made from the all-trans geranylgeranyl diphosphate by copal-8-ol diphosphate synthase (Nt-CPS2). The Solanum gene cluster also contains an ortholog of Nt-CPS2, but it appears to encode a nonfunctional protein. Thus, the Solanum functional gene cluster evolved by duplication and divergence of TPS genes, together with alterations in substrate specificity to utilize cis-prenyl diphosphates and through the acquisition of CPT genes.} } @Article{IPB-1306, author = {Frolov, A. and Henning, A. and Böttcher, C. and Tissier, A. and Strack, D. and}, title = {{An UPLC-MS/MS Method for the Simultaneous Identification and Quantitation of Cell Wall Phenolics in Brassica napus Seeds}}, year = {2013}, pages = {1219-1227}, journal = {J. Agr. Food Chem.}, doi = {10.1021/jf3042648}, volume = {61}, abstract = {The seed residues left after pressing of rapeseed oil are rich in proteins and could be used for human nutrition and animal feeding. These press cakes contain, however, antinutritives, with fiber being the most abundant one. The analysis of fiber phenolic component (localized to seed coat cell walls) is, therefore, important in breeding and food quality control. However, correct structure and content assignments of cell wall-bound phenolics are challenging due to their low stability during sample preparation. Here, a novel LC-MS/MS-based method for the simultaneous identification and quantitation of 66 cell wall-bound phenolics and their derivatives is described. The method was internally standardized, corrected for degradation effects during sample preparation, and cross-validated with a well-established UV-based procedure. This approach was successfully applied to the analysis of cell wall phenolic patterns in different B. napus cultivars and proved to be suitable for marker compound search as well as assay development.} } @Article{IPB-1294, author = {Brückner, K. and Tissier, A. and}, title = {{High-level diterpene production by transient expression in Nicotiana benthamiana}}, year = {2013}, pages = {46}, journal = {Plant Meth.}, doi = {10.1186/1746-4811-9-46}, volume = {9}, abstract = {BackgroundCharacterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.ResultsAgrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.ConclusionWith this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.} } @Article{IPB-1292, author = {Bogdanović, M. D. and Dragićević, M. B. and Tanić, N. T. and Todorović, S. I. and Mišić, D. M. and Živković, S. T. and Tissier, A. and Simonović, A. D. and}, title = {{Reverse Transcription of 18S rRNA with Poly(dT)18 and Other Homopolymers}}, year = {2013}, pages = {55-63}, journal = {Plant Mol. Biol. Rep.}, doi = {10.1007/s11105-012-0474-y}, volume = {31}, abstract = {Ribosomal 18S RNA is widely used as a housekeeping gene in expression studies, including end-point PCR, Northern analysis, and real-time experiments. However, there are two disadvantages and two points of error introduction in using 18S rRNA as a reference gene. First, 18S has no poly(A) tail, so it is commonly reverse transcribed with specific primers or random hexamers, independently from poly(dT)-primed transcripts. Secondly, due to its abundance, the 18S cDNA must be extensively diluted to be comparable to the tested genes. In this study, 18S rRNA from five taxonomically diverse plant species, including Physcomitrella patens, Adiantum capillus-veneris, Centaurium erythraea, Arabidopsis thaliana, and Zea mays, was successfully reverse transcribed (RT) using poly(dT)18. As all other homopolymers, including poly(dA)18, poly(dC)18, and poly(dG)18, could serve as RT primers, it was concluded that homopolymers anneal by mispriming at the sites of complementary homopolymeric runs or segments rich in complementary base. Poly(dC)18 was the most efficient as RT primer, and the only one which interfered with subsequent PCR, giving species-specific pattern of products. Poly(dT)-primed RT reactions were less efficient in comparison to specific primer or random hexamer-primed reactions. Homopolymeric priming of 18S in RT reactions is general in terms of RNA origin and the method of RNA isolation and is possibly applicable to other tailless housekeeping genes.} } @Article{IPB-1322, author = {Kathagen, A. and Schulte, A. and Balcke, G. and Phillips, H. S. and Martens, T. and Matschke, J. and Günther, H. S. and Soriano, R. and Modrusan, Z. and Sandmann, T. and Kuhl, C. and Tissier, A. and Holz, M. and Krawinkel, L. A. and Glatzel, M. and Westphal, M. and Lamszus, K. and}, title = {{Hypoxia and oxygenation induce a metabolic switch between pentose phosphate pathway and glycolysis in glioma stem-like cells}}, year = {2013}, pages = {763-780}, journal = {Acta Neuropathol.}, doi = {10.1007/s00401-013-1173-y}, volume = {126}, abstract = {Fluctuations in oxygen tension during tissue remodeling impose a major metabolic challenge in human tumors. Stem-like tumor cells in glioblastoma, the most common malignant brain tumor, possess extraordinary metabolic flexibility, enabling them to initiate growth even under non-permissive conditions. We identified a reciprocal metabolic switch between the pentose phosphate pathway (PPP) and glycolysis in glioblastoma stem-like (GS) cells. Expression of PPP enzymes is upregulated by acute oxygenation but downregulated by hypoxia, whereas glycolysis enzymes, particularly those of the preparatory phase, are regulated inversely. Glucose flux through the PPP is reduced under hypoxia in favor of flux through glycolysis. PPP enzyme expression is elevated in human glioblastomas compared to normal brain, especially in highly proliferative tumor regions, whereas expression of parallel preparatory phase glycolysis enzymes is reduced in glioblastomas, except for strong upregulation in severely hypoxic regions. Hypoxia stimulates GS cell migration but reduces proliferation, whereas oxygenation has opposite effects, linking the metabolic switch to the “go or grow” potential of the cells. Our findings extend Warburg’s observation that tumor cells predominantly utilize glycolysis for energy production, by suggesting that PPP activity is elevated in rapidly proliferating tumor cells but suppressed by acute severe hypoxic stress, favoring glycolysis and migration to protect cells against hypoxic cell damage.} } @Article{IPB-1319, author = {Ischebeck, T. and Werner, S. and Krishnamoorthy, P. and Lerche, J. and Meijon, M. and Stenzel, I. and Löfke, C. and Wiessner, T. and Im, Y. J. and Perera, I. Y. and Iven, T. and Feussner, I. and Busch, W. and Boss, W. F. and Teichmann, T. and Hause, B. and Persson, S. and Heilmann, I. and}, title = {{Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis}}, year = {2013}, pages = {4894-4911}, journal = {Plant Cell}, doi = {10.1105/tpc.113.116582}, volume = {25}, abstract = {The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.} } @INBOOK{IPB-85, author = {Wasternack, C. and Hause, B. and}, title = {{Festkolloquium der Leopoldina anlässlich des 80. Geburtstages von Herrn Altpräsidenten Benno Parthier}}, year = {2013}, pages = {29-38}, chapter = {{Benno Parthier und die Jasmonatforschung in Halle}}, journal = {Nova Acta Leopoldina}, editor = {Hacker, J., ed.}, url = {https://www.leopoldina.org/publikationen/detailansicht/publication/festkolloquium-der-leopoldina-anlaesslich-des-80-geburtstages-von-herrn-altpraesidenten-benno-parthie/}, volume = {Supplementum Nr. 28}, } @INBOOK{IPB-84, author = {Walter, M. H. and}, title = {{Molecular Microbial Ecology of the Rhizosphere}}, year = {2013}, pages = {513-524}, chapter = {{Role of Carotenoid Metabolism in the Arbuscular Mycorrhizal Symbiosis}}, editor = {de Bruijn, F. J., ed.}, doi = {10.1002/9781118297674.ch48}, abstract = {Cleavage products of carotenoids (apocarotenoids) exert a variety of often poorly characterized functions in roots and in rhizospheric interactions of plants with both symbionts and parasites. They are generated by regiospecific cleavage enzymes (CCDs, NCEDs) that act in a single or sequential way on C40 carotenoids. Among such apocarotenoids are one well‐known phytohormone controlling drought stress response networks (abscisic acid, ABA) and a newly discovered class of growth regulators involved in adaptive reactions to nutrient stress (strigolactones, SL). A third class of apocarotenoids consists of derivatives of a cyclic cyclohexenone (CH) and a linear mycorradicin (MR) type. They accumulate abundantly as part of a so‐called yellow pigment complex in roots colonized by arbuscular mycorrhizal (AM) fungi. Mycorrhizal phenotypes of pathway knockdown and loss‐of‐function mutants are reviewed in order to clarify the role of the three apocarotenoid classes for the AM symbiosis. One case of pathway interconnection between SL and CH/MR biogenesis through CCD7 is discussed along with other implications for interplay between pathways. SLs appear to preferentially affect early steps of root colonization by AM fungi and thus colonization levels. In contrast, accumulation of CH/MR derivatives is associated with arbuscule formation. Arbuscules are transient structures, which undergo constant degradation and reformation (turnover). Colocalization of CH/MR derivatives with degrading arbuscules and other observations suggest a phytoalexin‐like function in a plant‐controlled degradation of degenerating or poorly functional arbuscules. A model is presented, which proposes maintenance of high levels of functional arbuscules delivering phosphate through plant management of their rapid turnover.} } @INBOOK{IPB-79, author = {Hause, B. and Mielke, K. and Forner, S. and}, title = {{Jasmonate Signaling}}, year = {2013}, pages = {135-144}, chapter = {{Cell-Specific Detection of Jasmonates by Means of an Immunocytological Approach}}, journal = {Methods Mol. Biol.}, editor = {Goossens, A. \& Pauwels, L., eds.}, doi = {10.1007/978-1-62703-414-2_11}, volume = {1011}, abstract = {To determine the location of specific molecules within tissues or cells, immunological techniques are frequently used. However, immunolocalization of small molecules, such as jasmonic acid (JA) and its bioactive amino acid conjugate, JA-isoleucine, requires proper fixation and embedding methods as well as specific antibodies. In this chapter, we present a method to prepare plant tissues for the detection of jasmonates, including the chemical fixation to immobilize JA within the tissue, the subsequent embedding in a suitable medium, and the immunolabeling procedure itself.} } @INBOOK{IPB-78, author = {Engler, C. and Marillonnet, S. and}, title = {{Synthetic Biology}}, year = {2013}, pages = {141-156}, chapter = {{Combinatorial DNA Assembly Using Golden Gate Cloning}}, journal = {Methods Mol. Biol.}, editor = {Polizzi, K. M. \& Kontoravdi, C., eds.}, doi = {10.1007/978-1-62703-625-2_12}, volume = {1073}, abstract = {A basic requirement for synthetic biology is the availability of efficient DNA assembly methods. We have previously reported the development of Golden Gate cloning, a method that allows parallel assembly of multiple DNA fragments in a one-tube reaction. Golden Gate cloning can be used for different levels of construct assembly: from gene fragments to complete gene coding sequences, from basic genetic elements to full transcription units, and finally from transcription units to multigene constructs. We provide here a protocol for DNA assembly using Golden Gate cloning, taking as an example the level of assembly of gene fragments to complete coding sequences, a level of cloning that can be used to perform DNA shuffling. Such protocol requires the following steps: (1) selecting fusion sites within parental sequences (sites at which parental sequences will be recombined), (2) amplifying all DNA fragments by PCR to add flanking restriction sites, (3) cloning the amplified fragments in intermediate constructs, and (4) assembling all or selected sets of intermediate constructs in a compatible recipient vector using a one-pot restriction-ligation.} } @Article{IPB-1461, author = {Wessjohann, L. and Vogt, T. and Kufka, J. and Klein, R. and}, title = {{Prenyl- und Methyltransferasen in Natur und Synthese}}, year = {2012}, pages = {22-25}, journal = {BIOspektrum}, doi = {10.1007/s12268-012-0137-4}, volume = {18}, abstract = {Late stage enzymatic prenylation and methylation are means to diversify (natural) compounds and to specify their functions. In eukaryotes and microbes, these steps are performed by large enzyme families, the prenyl and methyl transferases, which modify various types of small molecules, like isoprenoids, phenolics or alkaloids, but also DNA and proteins. We investigate the theoretical basis of these processes and possible commercial applications in synthetic chemistry.} } @Article{IPB-1460, author = {Werner, S. and Engler, C. and Weber, E. and Gruetzner, R. and Marillonnet, S. and}, title = {{Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system}}, year = {2012}, pages = {38-43}, journal = {Bioengineered}, doi = {10.4161/bbug.3.1.18223}, volume = {3}, abstract = {Recent progress in the field of synthetic biology has led to the creation of cells containing synthetic genomes. Although these first synthetic organisms contained copies of natural genomes, future work will be directed toward engineering of organisms with modified genomes and novel phenotypes. Much work, however, remains to be done to be able to routinely engineer novel biological functions. As a tool that will be useful for such purpose, we have recently developed a modular cloning system (MoClo) that allows high throughput assembly of multiple genetic elements. We present here new features of this cloning system that allow to increase the speed of assembly of multigene constructs. As an example, 68 DNA fragments encoding basic genetic elements were assembled using three one-pot cloning steps, resulting in a 50 kb construct containing 17 eukaryotic transcription units. This cloning system should be useful for generating the multiple construct variants that will be required for developing gene networks encoding novel functions, and fine-tuning the expression levels of the various genes involved.} } @Article{IPB-1454, author = {Wasternack, C. and Goetz, S. and Hellwege, A. and Forner, S. and Strnad, M. and Hause, B. and}, title = {{Another JA/COI1-independent role of OPDA detected in tomato embryo development}}, year = {2012}, pages = {1349-1353}, journal = {Plant Signal Behav.}, doi = {10.4161/psb.21551}, volume = {7}, abstract = {Jasmonates (JAs) are ubiquitously occurring signaling compounds in plants formed in response to biotic and abiotic stress as well as in development. (\+)-7-iso-jasmonoyl isoleucine, the bioactive JA, is involved in most JA-dependent processes mediated by the F-box protein COI1 in a proteasome-dependent manner. However, there is an increasing number of examples, where the precursor of JA biosynthesis, cis-(\+)-12-oxophytodienoic acid (OPDA) is active in a JA/COI1-independent manner. Here, we discuss those OPDA-dependent processes, thereby giving emphasis on tomato embryo development. Recent data on seed coat-generated OPDA and its role in embryo development is discussed based on biochemical and genetic evidences.} } @Article{IPB-1450, author = {Vadassery, J. and Reichelt, M. and Hause, B. and Gershenzon, J. and Boland, W. and Mithöfer, A. and}, title = {{CML42-Mediated Calcium Signaling Coordinates Responses to Spodoptera Herbivory and Abiotic Stresses in Arabidopsis}}, year = {2012}, pages = {1159-1175}, journal = {Plant Physiol.}, doi = {10.1104/pp.112.198150}, volume = {159}, abstract = {In the interaction between Arabidopsis (Arabidopsis thaliana) and the generalist herbivorous insect Spodoptera littoralis, little is known about early events in defense signaling and their link to downstream phytohormone pathways. S. littoralis oral secretions induced both Ca2\+ and phytohormone elevation in Arabidopsis. Plant gene expression induced by oral secretions revealed up-regulation of a gene encoding a calmodulin-like protein, CML42. Functional analysis of cml42 plants revealed more resistance to herbivory than in the wild type, because caterpillars gain less weight on the mutant, indicating that CML42 negatively regulates plant defense; cml42 also showed increased aliphatic glucosinolate content and hyperactivated transcript accumulation of the jasmonic acid (JA)-responsive genes VSP2 and Thi2.1 upon herbivory, which might contribute to increased resistance. CML42 up-regulation is negatively regulated by the jasmonate receptor Coronatine Insensitive1 (COI1), as loss of functional COI1 resulted in prolonged CML42 activation. CML42 thus acts as a negative regulator of plant defense by decreasing COI1-mediated JA sensitivity and the expression of JA-responsive genes and is independent of herbivory-induced JA biosynthesis. JA-induced Ca2\+ elevation and root growth inhibition were more sensitive in cml42, also indicating higher JA perception. Our results indicate that CML42 acts as a crucial signaling component connecting Ca2\+ and JA signaling. CML42 is localized to cytosol and nucleus. CML42 is also involved in abiotic stress responses, as kaempferol glycosides were down-regulated in cml42, and impaired in ultraviolet B resistance. Under drought stress, the level of abscisic acid accumulation was higher in cml42 plants. Thus, CML42 might serve as a Ca2\+ sensor having multiple functions in insect herbivory defense and abiotic stress responses.} } @Article{IPB-1449, author = {Tissier, A. and}, title = {{Glandular trichomes: what comes after expressed sequence tags?}}, year = {2012}, pages = {51-68}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2012.04913.x}, volume = {70}, abstract = {Glandular trichomes cover the surface of many plant species. They exhibit tremendous diversity, be it in their shape or the compounds they secrete. This diversity is expressed between species but also within species or even individual plants. The industrial uses of some trichome secretions and their potential as a defense barrier, for example against arthropod pests, has spurred research into the biosynthesis pathways that lead to these specialized metabolites. Because complete biosynthesis pathways take place in the secretory cells, the establishment of trichome‐specific expressed sequence tag libraries has greatly accelerated their elucidation. Glandular trichomes also have an important metabolic capacity and may be considered as true cell factories. To fully exploit the potential of glandular trichomes as breeding or engineering objects, several research areas will have to be further investigated, such as development, patterning, metabolic fluxes and transcription regulation. The purpose of this review is to provide an update on the methods and technologies which have been used to investigate glandular trichomes and to propose new avenues of research to deepen our understanding of these specialized structures.} } @Article{IPB-1444, author = {Stenzel, I. and Otto, M. and Delker, C. and Kirmse, N. and Schmidt, D. and Miersch, O. and Hause, B. and Wasternack, C. and}, title = {{ALLENE OXIDE CYCLASE (AOC) gene family members of Arabidopsis thaliana: tissue- and organ-specific promoter activities and in vivo heteromerization}}, year = {2012}, pages = {6125-6138}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/ers261}, volume = {63}, abstract = {Jasmonates are important signals in plant stress responses and plant development. An essential step in the biosynthesis of jasmonic acid (JA) is catalysed by ALLENE OXIDE CYCLASE (AOC) which establishes the naturally occurring enantiomeric structure of jasmonates. In Arabidopsis thaliana, four genes encode four functional AOC polypeptides (AOC1, AOC2, AOC3, and AOC4) raising the question of functional redundancy or diversification. Analysis of transcript accumulation revealed an organ-specific expression pattern, whereas detailed inspection of transgenic lines expressing the GUS reporter gene under the control of individual AOC promoters showed partially redundant promoter activities during development: (i) In fully developed leaves, promoter activities of AOC1, AOC2, and AOC3 appeared throughout all leaf tissue, but AOC4 promoter activity was vascular bundle-specific; (ii) only AOC3 and AOC4 showed promoter activities in roots; and (iii) partially specific promoter activities were found for AOC1 and AOC4 in flower development. In situ hybridization of flower stalks confirmed the GUS activity data. Characterization of single and double AOC loss-of-function mutants further corroborates the hypothesis of functional redundancies among individual AOCs due to a lack of phenotypes indicative of JA deficiency (e.g. male sterility). To elucidate whether redundant AOC expression might contribute to regulation on AOC activity level, protein interaction studies using bimolecular fluorescence complementation (BiFC) were performed and showed that all AOCs can interact among each other. The data suggest a putative regulatory mechanism of temporal and spatial fine-tuning in JA formation by differential expression and via possible heteromerization of the four AOCs.} } @Article{IPB-1436, author = {Sallaud, C. and Giacalone, C. and Töpfer, R. and Goepfert, S. and Bakaher, N. and Rösti, S. and Tissier, A. and}, title = {{Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes}}, year = {2012}, pages = {1-17}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2012.05068.x}, volume = {72}, abstract = {Leaves of tobacco (Nicotiana tabacum) are covered with glandular trichomes that produce sucrose esters and diterpenoids in varying quantities, depending on cultivar type. The bicyclic diterpene Z‐abienol is the major labdanoid present in some oriental tobacco cultivars, where it constitutes a precursor of important flavours and aromas. We describe here the identification and characterization of two genes governing the biosynthesis of Z‐abienol in N. tabacum. As for other angiosperm labdanoid diterpenes, the biosynthesis of Z‐abienol proceeds in two steps. NtCPS2 encodes a class‐II terpene synthase that synthesizes 8‐hydroxy‐copalyl diphosphate, and NtABS encodes a kaurene synthase‐like (KSL) protein that uses 8‐hydroxy‐copalyl diphosphate to produce Z‐abienol. Phylogenetic analysis indicates that NtABS belongs to a distinct clade of KSL proteins that comprises the recently identified tomato (Solanum habrochaites) santalene and bergamotene synthase. RT‐PCR results show that both genes are preferentially expressed in trichomes. Moreover, microscopy of NtCPS2 promoter‐GUS fusion transgenics demonstrated a high specificity of expression to trichome glandular cells. Ectopic expression of both genes, but not of either one alone, driven by a trichome‐specific promoter in transgenic Nicotiana sylvestris conferred Z‐abienol formation to this species, which does not normally produce it. Furthermore, sequence analysis of over 100 tobacco cultivars revealed polymorphisms in NtCPS2 that lead to a prematurely truncated protein in cultivars lacking Z‐abienol, thus establishing NtCPS2 as a major gene controlling Z‐abienol biosynthesis in tobacco. These results offer new perspectives for tobacco breeding and the metabolic engineering of labdanoid diterpenes, as well as for structure–function relationship studies of terpene synthases.} } @Article{IPB-1419, author = {Landgraf, R. and Schaarschmidt, S. and Hause, B. and}, title = {{Repeated leaf wounding alters the colonization of Medicago truncatula roots by beneficial and pathogenic microorganisms}}, year = {2012}, pages = {1344-1357}, journal = {Plant Cell Environ.}, doi = {10.1111/j.1365-3040.2012.02495.x}, volume = {35}, abstract = {In nature, plants are subject to various stresses that are often accompanied by wounding of the aboveground tissues. As wounding affects plants locally and systemically, we investigated the impact of leaf wounding on interactions of Medicago truncatula with root‐colonizing microorganisms, such as the arbuscular mycorrhizal (AM) fungus Glomus intraradices, the pathogenic oomycete Aphanomyces euteiches and the nitrogen‐fixing bacterium Sinorhizobium meliloti. To obtain a long‐lasting wound response, repeated wounding was performed and resulted in locally and systemically increased jasmonic acid (JA) levels accompanied by the expression of jasmonate‐induced genes, among them the genes encoding allene oxide cyclase 1 (MtAOC1) and a putative cell wall‐bound invertase (cwINV). After repeated wounding, colonization with the AM fungus was increased, suggesting a role of jasmonates as positive regulators of mycorrhization, whereas the interaction with the rhizobacterium was not affected. In contrast, wounded plants appeared to be less susceptible to pathogens which might be caused by JA‐induced defence mechanisms. The effects of wounding on mycorrhization and pathogen infection could be partially mimicked by foliar application of JA. In addition to JA itself, the positive effect on mycorrhization might be mediated by systemically induced cwINV, which was previously shown to exhibit a regulatory function on interaction with AM fungi.} } @Article{IPB-1381, author = {Bleeker, P. M. and Mirabella, R. and Diergaarde, P. J. and VanDoorn, A. and Tissier, A. and Kant, M. R. and Prins, M. and de Vos, M. and Haring, M. A. and Schuurink, R. C. and}, title = {{Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative}}, year = {2012}, pages = {20124-20129}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, doi = {10.1073/pnas.1208756109}, volume = {109}, abstract = {Tomato breeding has been tremendously efficient in increasing fruit quality and quantity but did not focus on improving herbivore resistance. The biosynthetic pathway for the production of 7-epizingiberene in a wild tomato was introduced into a cultivated greenhouse variety with the aim to obtain herbivore resistance. 7-Epizingiberene is a specific sesquiterpene with toxic and repellent properties that is produced and stored in glandular trichomes. We identified 7-epizingiberene synthase (ShZIS) that belongs to a new class of sesquiterpene synthases, exclusively using Z-Z-farnesyl-diphosphate (zFPP) in plastids, probably arisen through neo-functionalization of a common ancestor. Expression of the ShZIS and zFPP synthases in the glandular trichomes of cultivated tomato resulted in the production of 7-epizingiberene. These tomatoes gained resistance to several herbivores that are pests of tomato. Hence, introduction of this sesquiterpene biosynthetic pathway into cultivated tomatoes resulted in improved herbivore resistance.} } @Article{IPB-1377, author = {Bektas, I. and Fellenberg, C. and Paulsen, H. and}, title = {{Water-soluble chlorophyll protein (WSCP) of Arabidopsis is expressed in the gynoecium and developing silique}}, year = {2012}, pages = {251-259}, journal = {Planta}, doi = {10.1007/s00425-012-1609-y}, volume = {236}, abstract = {Water-soluble chlorophyll protein (WSCP) has been found in many Brassicaceae, most often in leaves. In many cases, its expression is stress-induced, therefore, it is thought to be involved in some stress response. In this work, recombinant WSCP from Arabidopsis thaliana (AtWSCP) is found to form chlorophyll-protein complexes in vitro that share many properties with recombinant or native WSCP from Brassica oleracea, BoWSCP, including an unusual heat resistance up to 100°C in aqueous solution. A polyclonal antibody raised against the recombinant apoprotein is used to identify plant tissues expressing AtWSCP. The only plant organs containing significant amounts of AtWSCP are the gynoecium in open flowers and the septum of developing siliques, specifically the transmission tract. In fully grown but still green siliques, the protein has almost disappeared. Possible implications for AtWSCP functions are discussed.} } @Article{IPB-1376, author = {Balcke, G. U. and Handrick, V. and Bergau, N. and Fichtner, M. and Henning, A. and Stellmach, H. and Tissier, A. and Hause, B. and Frolov, A. and}, title = {{An UPLC-MS/MS method for highly sensitive high-throughput analysis of phytohormones in plant tissues}}, year = {2012}, pages = {47}, journal = {Plant Meth.}, doi = {10.1186/1746-4811-8-47}, volume = {8}, abstract = {BackgroundPhytohormones are the key metabolites participating in the regulation of multiple functions of plant organism. Among them, jasmonates, as well as abscisic and salicylic acids are responsible for triggering and modulating plant reactions targeted against pathogens and herbivores, as well as resistance to abiotic stress (drought, UV-irradiation and mechanical wounding). These factors induce dramatic changes in phytohormone biosynthesis and transport leading to rapid local and systemic stress responses. Understanding of underlying mechanisms is of principle interest for scientists working in various areas of plant biology. However, highly sensitive, precise and high-throughput methods for quantification of these phytohormones in small samples of plant tissues are still missing.ResultsHere we present an LC-MS/MS method for fast and highly sensitive determination of jasmonates, abscisic and salicylic acids. A single-step sample preparation procedure based on mixed-mode solid phase extraction was efficiently combined with essential improvements in mobile phase composition yielding higher efficiency of chromatographic separation and MS-sensitivity. This strategy resulted in dramatic increase in overall sensitivity, allowing successful determination of phytohormones in small (less than 50 mg of fresh weight) tissue samples. The method was completely validated in terms of analyte recovery, sensitivity, linearity and precision. Additionally, it was cross-validated with a well-established GC-MS-based procedure and its applicability to a variety of plant species and organs was verified.ConclusionThe method can be applied for the analyses of target phytohormones in small tissue samples obtained from any plant species and/or plant part relying on any commercially available (even less sensitive) tandem mass spectrometry instrumentation.} } @Article{IPB-1407, author = {Goetz, S. and Hellwege, A. and Stenzel, I. and Kutter, C. and Hauptmann, V. and Forner, S. and McCaig, B. and Hause, G. and Miersch, O. and Wasternack, C. and Hause, B. and}, title = {{Role of cis-12-Oxo-Phytodienoic Acid in Tomato Embryo Development}}, year = {2012}, pages = {1715-1727}, journal = {Plant Physiol.}, doi = {10.1104/pp.111.192658}, volume = {158}, abstract = {Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.} } @Article{IPB-1406, author = {Gaupels, F. and Sarioglu, H. and Beckmann, M. and Hause, B. and Spannagl, M. and Draper, J. and Lindermayr, C. and Durner, J. and}, title = {{Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling}}, year = {2012}, pages = {2285-2299}, journal = {Plant Physiol.}, doi = {10.1104/pp.112.205336}, volume = {160}, abstract = {In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling.} } @Article{IPB-1401, author = {Fellenberg, C. and van Ohlen, M. and Handrick, V. and Vogt, T. and}, title = {{The role of CCoAOMT1 and COMT1 in Arabidopsis anthers}}, year = {2012}, pages = {51-61}, journal = {Planta}, doi = {10.1007/s00425-011-1586-6}, volume = {236}, abstract = {Arabidopsis caffeoyl coenzyme A dependent O-methyltransferase 1 (CCoAOMT1) and caffeic acid O-methyltransferase 1 (COMT1) display a similar substrate profile although with distinct substrate preferences and are considered the key methyltransferases (OMTs) in the biosynthesis of lignin monomers, coniferyl and sinapoylalcohol. Whereas CCoAOMT1 displays a strong preference for caffeoyl coenzyme A, COMT1 preferentially methylates 5-hydroxyferuloyl CoA derivatives and also performs methylation of flavonols with vicinal aromatic dihydroxy groups, such as quercetin. Based on different knockout lines, phenolic profiling, and immunohistochemistry, we present evidence that both enzymes fulfil distinct, yet different tasks in Arabidopsis anthers. CCoAOMT1 besides its role in vascular tissues can be localized to the tapetum of young stamens, contributing to the biosynthesis of spermidine phenylpropanoid conjugates. COMT1, although present in the same organ, is not localized in the tapetum, but in two directly adjacent cells layers, the endothecium and the epidermal layer of stamens. In vivo localization and phenolic profiling of comt1 plants provide evidence that COMT1 neither contributes to the accumulation of spermidine phenylpropanoid conjugates nor to the flavonol glycoside pattern of pollen grains.} } @Article{IPB-1400, author = {Fellenberg, C. and Ziegler, J. and Handrick, V. and Vogt, T. and}, title = {{Polyamine homeostasis in wild type and phenolamide deficient Arabidopsis thaliana stamens}}, year = {2012}, pages = {180}, journal = {Front. Plant Sci.}, doi = {10.3389/fpls.2012.00180}, volume = {3}, abstract = {Polyamines (PAs) like putrescine, spermidine, and spermine are ubiquitous polycationic molecules that occur in all living cells and have a role in a wide variety of biological processes. High amounts of spermidine conjugated to hydroxycinnamic acids are detected in the tryphine of Arabidopsis thaliana pollen grains. Tapetum localized spermidine hydroxycinnamic acid transferase (SHT) is essential for the biosynthesis of these anther specific tris-conjugated spermidine derivatives. Sht knockout lines show a strong reduction of hydroxycinnamic acid amides (HCAAs). The effect of HCAA-deficient anthers on the level of free PAs was measured by a new sensitive and reproducible method using 9-fluorenylmethyl chloroformate (FMOC) and fluorescence detection by HPLC. PA concentrations can be accurately determined even when very limited amounts of plant material, as in the case of A. thaliana stamens, are available. Analysis of free PAs in wild type stamens compared to sht deficient mutants and transcript levels of key PA biosynthetic genes revealed a highly controlled regulation of PA homeostasis in A. thaliana anthers.} } @INBOOK{IPB-90, author = {Walter, M. H. and Floss, D. S. and Paetzold, H. and Manke, K. and Vollrath, J. and Brandt, W. and Strack, D. and}, title = {{Isoprenoid Synthesis in Plants and Microorganisms}}, year = {2012}, pages = {251-270}, chapter = {{Control of Plastidial Isoprenoid Precursor Supply: Divergent 1-Deoxy-D-Xylulose 5-Phosphate Synthase (DXS) Isogenes Regulate the Allocation to Primary or Secondary Metabolism}}, editor = {Bach, T. J. \& Rohmer, M., eds.}, doi = {10.1007/978-1-4614-4063-5_17}, abstract = {Following the description of two separate pathways for isoprenoid precursor biosynthesis in plants, a new level of complexity has been introduced by the discovery of two divergent gene classes encoding the first enzyme of the plastidial methylerythritol phosphate (MEP) pathway. These nonredundant 1-deoxy-d-xylulose 5-phosphate synthase (DXS) isogenes are differentially expressed in such a way that DXS1 appears to serve housekeeping functions, whereas DXS2 is associated with the production of specialized (secondary) isoprenoids involved in ecological functions. Examples of the latter are apocarotenoid formation in roots colonized by arbuscular mycorrhizal fungi and mono- or diterpenoid biosynthesis in trichomes. Knockdown of DXS2 genes can specifically suppress secondary isoprenoid formation without affecting basic plant functions. Analyzing DXS isogenes along the progression of land plant evolution shows separation in structure and complementary expression already at the level of gymnosperms, which is maintained in all angiosperms except Arabidopsis.} } @INBOOK{IPB-88, author = {Tissier, A. and}, title = {{Transgenic Plants - Advances and Limitations}}, year = {2012}, pages = {353-378}, chapter = {{Trichome Specific Expression: Promoters and Their Applications}}, editor = {Çiftçi, Y. O., ed.}, doi = {10.5772/32101}, } @INBOOK{IPB-87, author = {Tissier, A. and Sallaud, C. and Rontein, D. and}, title = {{Isoprenoid Synthesis in Plants and Microorganisms}}, year = {2012}, pages = {271-283}, chapter = {{Tobacco Trichomes as a Platform for Terpenoid Biosynthesis Engineering}}, editor = {Bach, T. J. \& Rohmer, M., eds.}, doi = {10.1007/978-1-4614-4063-5_18}, abstract = {Many plant species have evolved specialized organs dedicated to the production of a restricted number of secondary metabolites. These organs have secretory tissues which can lead to very significant accumulations of products, in the range of mg per g of fresh weight. These natural cell factories are therefore interesting targets for metabolic engineering. Plant glandular trichomes in particular have attracted interest because of the relative ease to isolate them and to analyse the compounds they produce because they are secreted onto the leaf surface. Depending on the species, trichomes can produce a variety of metabolites. Terpenoids however are particularly well represented and have been used by humans in a variety of industries, including as aroma, fragrance and pharmaceutical ingredients. Tobacco trichomes produce diterpenoids in large amounts and were therefore chosen as a model system for engineering the biosynthesis of this important class of compounds. We present here our strategy and first results, which bode well for the future of glandular trichomes as engineered natural cellular factories.} } @Article{IPB-1504, author = {Mielke, K. and Forner, S. and Kramell, R. and Conrad, U. and Hause, B. and}, title = {{Cell-specific visualization of jasmonates in wounded tomato and Arabidopsis leaves using jasmonate-specific antibodies}}, year = {2011}, pages = {1069-1080}, journal = {New Phytol.}, doi = {10.1111/j.1469-8137.2010.03638.x}, volume = {190}, abstract = {Jasmonates are well‐characterized signals in the development of plants and their response to abiotic and biotic stresses, such as touch and wounding by herbivores. A gap in our knowledge on jasmonate‐induced processes, however, is the cellular localization of jasmonates.Here, a novel antibody‐based approach was developed to visualize jasmonates in cross‐sections of plant material. Antibodies raised in rabbits against BSA‐coupled jasmonic acid (JA) are specific for JA, its methyl ester and isoleucine conjugate. They do not bind to 12‐oxophytodienoic acid, 12‐hydoxy‐JA or coronatine. These antibodies were used in combination with newly established fixation and embedding methods.Jasmonates were rapidly and uniformly distributed within all cells near the site of damage of a mechanically wounded tomato (Solanum lycopersicum) leaf. Leaf tissue distally located to the wound site exhibited identical distribution, but had a lower signal intensity. The occurrence of jasmonates in all cell types of a wounded leaf was accompanied by transcript accumulation of early JA‐induced genes visualized by in situ hybridization.With these new antibodies, a powerful tool is available to detect cell‐specifically the occurrence of jasmonates in any jasmonate‐dependent stress response or developmental process of plants.} } @Article{IPB-1492, author = {Helber, N. and Wippel, K. and Sauer, N. and Schaarschmidt, S. and Hause, B. and Requena, N. and}, title = {{A Versatile Monosaccharide Transporter That Operates in the Arbuscular Mycorrhizal Fungus Glomus sp Is Crucial for the Symbiotic Relationship with Plants}}, year = {2011}, pages = {3812-3823}, journal = {Plant Cell}, doi = {10.1105/tpc.111.089813}, volume = {23}, abstract = {For more than 400 million years, plants have maintained a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi. This evolutionary success can be traced to the role of these fungi in providing plants with mineral nutrients, particularly phosphate. In return, photosynthates are given to the fungus, which support its obligate biotrophic lifestyle. Although the mechanisms involved in phosphate transfer have been extensively studied, less is known about the reciprocal transfer of carbon. Here, we present the high-affinity Monosaccharide Transporter2 (MST2) from Glomus sp with a broad substrate spectrum that functions at several symbiotic root locations. Plant cell wall sugars can efficiently outcompete the Glc uptake capacity of MST2, suggesting they can serve as alternative carbon sources. MST2 expression closely correlates with that of the mycorrhiza-specific PhosphateTransporter4 (PT4). Furthermore, reduction of MST2 expression using host-induced gene silencing resulted in impaired mycorrhiza formation, malformed arbuscules, and reduced PT4 expression. These findings highlight the symbiotic role of MST2 and support the hypothesis that the exchange of carbon for phosphate is tightly linked. Unexpectedly, we found that the external mycelium of AM fungi is able to take up sugars in a proton-dependent manner. These results imply that the sugar uptake system operating in this symbiosis is more complex than previously anticipated.} } @Article{IPB-1483, author = {Escalante-Pérez, M. and Krol, E. and Stange, A. and Geiger, D. and Al-Rasheid, K. A. S. and Hause, B. and Neher, E. and Hedrich, R. and}, title = {{A special pair of phytohormones controls excitability, slow closure, and external stomach formation in the Venus flytrap}}, year = {2011}, pages = {15492-15497}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, doi = {10.1073/pnas.1112535108}, volume = {108}, abstract = {Venus flytrap\'s leaves can catch an insect in a fraction of a second. Since the time of Charles Darwin, scientists have struggled to understand the sensory biology and biomechanics of this plant, Dionaea muscipula. Here we show that insect-capture of Dionaea traps is modulated by the phytohormone abscisic acid (ABA) and jasmonates. Water-stressed Dionaea, as well as those exposed to the drought-stress hormone ABA, are less sensitive to mechanical stimulation. In contrast, application of 12-oxo-phytodienoic acid (OPDA), a precursor of the phytohormone jasmonic acid (JA), the methyl ester of JA (Me-JA), and coronatine (COR), the molecular mimic of the isoleucine conjugate of JA (JA-Ile), triggers secretion of digestive enzymes without any preceding mechanical stimulus. Such secretion is accompanied by slow trap closure. Under physiological conditions, insect-capture is associated with Ca2\+ signaling and a rise in OPDA, Apparently, jasmonates bypass hapto-electric processes associated with trap closure. However, ABA does not affect OPDA-dependent gland activity. Therefore, signals for trap movement and secretion seem to involve separate pathways. Jasmonates are systemically active because application to a single trap induces secretion and slow closure not only in the given trap but also in all others. Furthermore, formerly touch-insensitive trap sectors are converted into mechanosensitive ones. These findings demonstrate that prey-catching Dionaea combines plant-specific signaling pathways, involving OPDA and ABA with a rapidly acting trigger, which uses ion channels, action potentials, and Ca2\+ signals.} } @Article{IPB-1529, author = {Zdyb, A. and Demchenko, K. and Heumann, J. and Mrosk, C. and Grzeganek, P. and Göbel, C. and Feussner, I. and Pawlowski, K. and Hause, B. and}, title = {{Jasmonate biosynthesis in legume and actinorhizal nodules}}, year = {2011}, pages = {568-579}, journal = {New Phytol.}, doi = {10.1111/j.1469-8137.2010.03504.x}, volume = {189}, abstract = {Jasmonic acid (JA) is a plant signalling compound that has been implicated in the regulation of mutualistic symbioses. In order to understand the spatial distribution of JA biosynthetic capacity in nodules of two actinorhizal species, Casaurina glauca and Datisca glomerata, and one legume, Medicago truncatula, we determined the localization of allene oxide cyclase (AOC) which catalyses a committed step in JA biosynthesis. In all nodule types analysed, AOC was detected exclusively in uninfected cells.The levels of JA were compared in the roots and nodules of the three plant species. The nodules and noninoculated roots of the two actinorhizal species, and the root systems of M. truncatula, noninoculated or nodulated with wild‐type Sinorhizobium meliloti or with mutants unable to fix nitrogen, did not show significant differences in JA levels. However, JA levels in all plant organs examined increased significantly on mechanical disturbance.To study whether JA played a regulatory role in the nodules of M. truncatula, composite plants containing roots expressing an MtAOC1‐sense or MtAOC1‐RNAi construct were inoculated with S. meliloti. Neither an increase nor reduction in AOC levels resulted in altered nodule formation.These data suggest that jasmonates are not involved in the development and function of root nodules.} } @Article{IPB-1525, author = {Wirsing, L. and Naumann, K. and Vogt, T. and}, title = {{Arabidopsis methyltransferase fingerprints by affinity-based protein profiling}}, year = {2011}, pages = {220-225}, journal = {Anal. Biochem.}, doi = {10.1016/j.ab.2010.09.029}, volume = {408}, abstract = {Precise annotation of time and spatial distribution of enzymes involved in plant secondary metabolism by gel electrophoresis are usually difficult due to their low abundance. Therefore, effective methods to enrich these enzymes are required to correlate available transcript and metabolite data with the actual presence of active enzymes in wild-type and mutant plants or to monitor variations of these enzymes under various types of biotic and abiotic stress conditions. S-Adenosyl-L-methionine-dependent O-methyltransferases play important roles in the modification of natural products such as phenylpropanoids or alkaloids. In plants they occur as small superfamilies with defined roles for each of its members in different organs and tissues. We explored the use of S-adenosyl-L-homocysteine as a selectivity function in affinity-based protein profiling supported by capture compound mass spectrometry. Due to their high affinity to this ligand it was possible to identify developmental changes of flower-specific patterns of plant natural product O-methyltransferases and corroborate the absence of individual O-methyltransferases in the corresponding Arabidopsis knockout lines. Developmental changes in the OMT pattern were correlated with transcript data obtained by qPCR.} } @Article{IPB-1523, author = {Weber, E. and Gruetzner, R. and Werner, S. and Engler, C. and Marillonnet, S. and}, title = {{Assembly of Designer TAL Effectors by Golden Gate Cloning}}, year = {2011}, pages = {e19722}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0019722}, volume = {6}, abstract = {Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.} } @Article{IPB-1522, author = {Weber, E. and Engler, C. and Gruetzner, R. and Werner, S. and Marillonnet, S. and}, title = {{A Modular Cloning System for Standardized Assembly of Multigene Constructs}}, year = {2011}, pages = {e16765}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0016765}, volume = {6}, abstract = {The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo) system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.} } @Article{IPB-1521, author = {Walter, M. H. and Strack, D. and}, title = {{Carotenoids and their cleavage products: Biosynthesis and functions}}, year = {2011}, pages = {663-692}, journal = {Nat. Prod. Rep.}, doi = {10.1039/C0NP00036A}, volume = {28}, abstract = {Covering: up to mid-2010This review focuses on plant carotenoids, but it also includes progress made on microbial and animal carotenoid metabolism to better understand the functions and the evolution of these structurally diverse compounds with a common backbone. Plants have evolved isogenes for specific key steps of carotenoid biosynthesis with differential expression profiles, whose characteristic features will be compared. Perhaps the most exciting progress has been made in studies of carotenoid cleavage products (apocarotenoids) with an ever-expanding variety of novel functions being discovered. This review therefore covers structural, molecular genetic and functional aspects of carotenoids and apocarotenoids alike. Apocarotenoids are specifically tailored from carotenoids by a family of oxidative cleavage enzymes, but whether there are contributions to their generation from chemical oxidation, photooxidation or other mechanisms is largely unknown. Control of carotenoid homeostasis is discussed in the context of biosynthetic and degradative reactions but also in the context of subcellular environments for deposition and sequestration within and outside of plastids. Other aspects of carotenoid research, including metabolic engineering and synthetic biology approaches, will only be covered briefly.} } @Article{IPB-1520, author = {Thieme, F. and Engler, C. and Kandzia, R. and Marillonnet, S. and}, title = {{Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes}}, year = {2011}, pages = {e20556}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0020556}, volume = {6}, abstract = {We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.} } @INBOOK{IPB-96, author = {Engler, C. and Marillonnet, S. and}, title = {{cDNA Libraries}}, year = {2011}, pages = {167-181}, chapter = {{Generation of Families of Construct Variants Using Golden Gate Shuffling}}, journal = {Methods Mol. Biol.}, editor = {Lu, C. et al., eds.}, doi = {10.1007/978-1-61779-065-2_11}, volume = {729}, abstract = {Current standard cloning methods based on the use of restriction enzymes and ligase are very versatile, but are not well suited for high-throughput cloning projects or for assembly of many DNA fragments from several parental plasmids in a single step. We have previously reported the development of an efficient cloning method based on the use of type IIs restriction enzymes and restriction–ligation. Such method allows seamless assembly of multiple fragments from several parental plasmids with high efficiency, and also allows performing DNA shuffling if fragments prepared from several homologous genes are assembled together in a single restriction–ligation. Such protocol, called Golden Gate shuffling, requires performing the following steps: (1) sequences from several homologous genes are aligned, and recombination sites defined on conserved sequences; (2) modules defined by the position of these recombination sites are amplified by PCR with primers designed to equip them with flanking BsaI sites; (3) the amplified fragments are cloned as intermediate constructs and sequenced; and (4) finally, the intermediate modules are assembled together in a compatible recipient vector in a one-pot restriction–ligation. Depending on the needs of the user, and because of the high cloning efficiency, the resulting constructs can either be screened and analyzed individually, or, if required in larger numbers, directly used in functional screens to detect improved protein variants.} } @Article{IPB-1600, author = {Wolfram, K. and Schmidt, J. and Wray, V. and Milkowski, C. and Schliemann, W. and Strack, D. and}, title = {{Profiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus)}}, year = {2010}, pages = {1076-1084}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2010.04.007}, volume = {71}, abstract = {A dsRNAi approach silencing a key enzyme of sinapate ester biosynthesis (UDP-glucose:sinapate glucosyltransferase, encoded by the UGT84A9 gene) in oilseed rape (Brassica napus) seeds was performed to reduce the anti-nutritive properties of the seeds by lowering the content of the major seed component sinapine (sinapoylcholine) and various minor sinapate esters. The transgenic seeds have been produced so far to the T6 generation and revealed a steady suppression of sinapate ester accumulation. HPLC analysis of the wild-type and transgenic seeds revealed, as in the previous generations, marked alterations of the sinapate ester pattern of the transformed seeds. Besides strong reduction of the amount of the known sinapate esters, HPLC analysis revealed unexpectedly the appearance of several minor hitherto unknown rapeseed constituents. These compounds were isolated and identified by mass spectrometric and NMR spectroscopic analyses. Structures of 11 components were elucidated to be 4-O-glucosides of syringate, caffeyl alcohol and its 7,8-dihydro derivative as well as of sinapate and sinapine, along with sinapoylated kaempferol glycosides, a hexoside of a cyclic spermidine alkaloid and a sinapine derivative with an ether-bridge to a C6–C3-unit. These results indicate a strong impact of the transgenic approach on the metabolic network of phenylpropanoids in B. napus seeds. Silencing of UGT84A9 gene expression disrupt the metabolic flow through sinapoylglucose and alters the amounts and nature of the phenylpropanoid endproducts.} } @Article{IPB-1595, author = {Weichert, N. and Saalbach, I. and Weichert, H. and Kohl, S. and Erban, A. and Kopka, J. and Hause, B. and Varshney, A. and Sreenivasulu, N. and Strickert, M. and Kumlehn, J. and Weschke, W. and Weber, H. and}, title = {{Increasing Sucrose Uptake Capacity of Wheat Grains Stimulates Storage Protein Synthesis}}, year = {2010}, pages = {698-710}, journal = {Plant Physiol.}, doi = {10.1104/pp.109.150854}, volume = {152}, abstract = {Increasing grain sink strength by improving assimilate uptake capacity could be a promising approach toward getting higher yield. The barley (Hordeum vulgare) sucrose transporter HvSUT1 (SUT) was expressed under control of the endosperm-specific Hordein B1 promoter (HO). Compared with the wild type, transgenic HOSUT grains take up more sucrose (Suc) in vitro, showing that the transgene is functional. Grain Suc levels are not altered, indicating that Suc fluxes are influenced rather than steady-state levels. HOSUT grains have increased percentages of total nitrogen and prolamins, which is reflected in increased levels of phenylalanine, tyrosine, tryptophan, isoleucine, and leucine at late grain development. Transcript profiling indicates specific stimulation of prolamin gene expression at the onset of storage phase. Changes in gene expression and metabolite levels related to carbon metabolism and amino acid biosynthesis suggest deregulated carbon-nitrogen balance, which together indicate carbon sufficiency and relative depletion of nitrogen. Genes, deregulated together with prolamin genes, might represent candidates, which respond positively to assimilate supply and are related to sugar-starch metabolism, cytokinin and brassinosteroid functions, cell proliferation, and sugar/abscisic acid signaling. Genes showing inverse expression patterns represent potential negative regulators. It is concluded that HvSUT1 overexpression increases grain protein content but also deregulates the metabolic status of wheat (Triticum aestivum) grains, accompanied by up-regulated gene expression of positive and negative regulators related to sugar signaling and assimilate supply. In HOSUT grains, alternating stimulation of positive and negative regulators causes oscillatory patterns of gene expression and highlights the capacity and great flexibility to adjust wheat grain storage metabolism in response to metabolic alterations.} } @Article{IPB-1592, author = {Walter, M. H. and Floss, D. S. and Strack, D. and}, title = {{Apocarotenoids: hormones, mycorrhizal metabolites and aroma volatiles}}, year = {2010}, pages = {1-17}, journal = {Planta}, doi = {10.1007/s00425-010-1156-3}, volume = {232}, abstract = {Apocarotenoids are tailored from carotenoids by oxidative enzymes [carotenoid cleavage oxygenases (CCOs)], cleaving specific double bonds of the polyene chain. The cleavage products can act as hormones, signaling compounds, chromophores and scent/aroma constituents. Recent advances were the identification of strigolactones as apocarotenoids and the description of their novel role as shoot branching inhibitor hormones. Strigolactones are also involved in plant signaling to both harmful (parasitic weeds) and beneficial [arbuscular mycorrhizal (AM) fungi] rhizosphere residents. This review describes the progress in the characterization of CCOs, termed CCDs and NCEDs, in plants. It highlights the importance of sequential cleavage reactions of C40 carotenoid precursors, the apocarotenoid cleavage oxygenase (ACO) nature of several CCOs and the topic of compartmentation. Work on the biosynthesis of abundant C13 cyclohexenone and C14 mycorradicin apocarotenoids in mycorrhizal roots has revealed a new role of CCD1 as an ACO of C27 apocarotenoid intermediates, following their predicted export from plastid to cytosol. Manipulation of the AM-induced apocarotenoid pathway further suggests novel roles of C13 apocarotenoids in controlling arbuscule turnover in the AM symbiosis. CCD7 has been established as a biosynthetic crosspoint, controlling both strigolactone and AM-induced C13 apocarotenoid biosynthesis. Interdependence of the two apocarotenoid pathways may thus play a role in AM-mediated reduction of parasitic weed infestations. Potential scenarios of C13 scent/aroma volatile biogenesis are discussed, including the novel mechanism revealed from mycorrhizal roots. The recent progress in apocarotenoid research opens up new perspectives for fundamental work, but has also great application potential for the horticulture, food and fragrance industries.} } @Article{IPB-1591, author = {Vogt, T. and}, title = {{Phenylpropanoid Biosynthesis}}, year = {2010}, pages = {2-20}, journal = {Mol. Plant}, doi = {10.1093/mp/ssp106}, volume = {3}, abstract = {The general phenylpropanoid metabolism generates an enormous array of secondary metabolites based on the few intermediates of the shikimate pathway as the core unit. The resulting hydroxycinnamic acids and esters are amplified in several cascades by a combination of reductases, oxygenases, and transferases to result in an organ and developmentally specific pattern of metabolites, characteristic for each plant species. During the last decade, methodology driven targeted and non-targeted approaches in several plant species have enabled the identification of the participating enzymes of this complex biosynthetic machinery, and revealed numerous genes, enzymes, and metabolites essential for regulation and compartmentation. Considerable success in structural and computational biology, combined with the analytical sensitivity to detect even trace compounds and smallest changes in the metabolite, transcript, or enzyme pattern, has facilitated progress towards a comprehensive view of the plant response to its biotic and abiotic environment. Transgenic approaches have been used to reveal insights into an apparently redundant gene and enzyme pattern required for functional integrity and plasticity of the various phenylpropanoid biosynthetic pathways. Nevertheless, the function and impact of all members of a gene family remain to be completely established. This review aims to give an update on the various facets of the general phenylpropanoid pathway, which is not only restricted to common lignin or flavonoid biosynthesis, but feeds into a variety of other aromatic metabolites like coumarins, phenolic volatiles, or hydrolyzable tannins.} } @Article{IPB-1589, author = {Vogel, J. T. and Walter, M. H. and Giavalisco, P. and Lytovchenko, A. and Kohlen, W. and Charnikhova, T. and Simkin, A. J. and Goulet, C. and Strack, D. and Bouwmeester, H. J. and Fernie, A. R. and Klee, H. J. and}, title = {{SlCCD7 controls strigolactone biosynthesis, shoot branching and mycorrhiza-induced apocarotenoid formation in tomato}}, year = {2010}, pages = {300-311}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2009.04056.x}, volume = {61}, abstract = {The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch‐inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C13 cyclohexenone and C14 mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source–sink interactions and production of arbuscular mycorrhiza‐induced apocarotenoids.} } @Article{IPB-1585, author = {Teutschbein, J. and Gross, W. and Nimtz, M. and Milkowski, C. and Hause, B. and Strack, D. and}, title = {{Identification and Localization of a Lipase-like Acyltransferase in Phenylpropanoid Metabolism of Tomato (Solanum lycopersicum)}}, year = {2010}, pages = {38374-38381}, journal = {J. Biol. Chem.}, doi = {10.1074/jbc.M110.171637}, volume = {285}, abstract = {We have isolated an enzyme classified as chlorogenate: glucarate caffeoyltransferase (CGT) from seedlings of tomato (Solanum lycopersicum) that catalyzes the formation of caffeoylglucarate and caffeoylgalactarate using chlorogenate (5-O-caffeoylquinate) as acyl donor. Peptide sequences obtained by trypsin digestion and spectrometric sequencing were used to isolate the SlCGT cDNA encoding a protein of 380 amino acids with a putative targeting signal of 24 amino acids indicating an entry of the SlCGT into the secretory pathway. Immunogold electron microscopy revealed the localization of the enzyme in the apoplastic space of tomato leaves. Southern blot analysis of genomic cDNA suggests that SlCGT is encoded by a single-copy gene. The SlCGT cDNA was functionally expressed in Nicotiana benthamiana leaves and proved to confer chlorogenate-dependent caffeoyltransferase activity in the presence of glucarate. Sequence comparison of the deduced amino acid sequence identified the protein unexpectedly as a GDSL lipase-like protein, representing a new member of the SGNH protein superfamily. Lipases of this family employ a catalytic triad of Ser-Asp-His with Ser as nucleophile of the GDSL motif. Site-directed mutagenesis of each residue of the assumed respective SlCGT catalytic triad, however, indicated that the catalytic triad of the GDSL lipase is not essential for SlCGT enzymatic activity. SlCGT is therefore the first example of a GDSL lipase-like protein that lost hydrolytic activity and has acquired a completely new function in plant metabolism, functioning in secondary metabolism as acyltransferase in synthesis of hydroxycinnamate esters by employing amino acid residues different from the lipase catalytic triad.} } @Article{IPB-1584, author = {Stumpe, M. and Göbel, C. and Faltin, B. and Beike, A. K. and Hause, B. and Himmelsbach, K. and Bode, J. and Kramell, R. and Wasternack, C. and Frank, W. and Reski, R. and Feussner, I. and}, title = {{The moss Physcomitrella patens contains cyclopentenones but no jasmonates: mutations in allene oxide cyclase lead to reduced fertility and altered sporophyte morphology}}, year = {2010}, pages = {740-749}, journal = {New Phytol.}, doi = {10.1111/j.1469-8137.2010.03406.x}, volume = {188}, abstract = {Two cDNAs encoding allene oxide cyclases (PpAOC1, PpAOC2), key enzymes in the formation of jasmonic acid (JA) and its precursor (9S,13S)‐12‐oxo‐phytodienoic acid (cis‐(\+)‐OPDA), were isolated from the moss Physcomitrella patens.Recombinant PpAOC1 and PpAOC2 show substrate specificity against the allene oxide derived from 13‐hydroperoxy linolenic acid (13‐HPOTE); PpAOC2 also shows substrate specificity against the allene oxide derived from 12‐hydroperoxy arachidonic acid (12‐HPETE).In protonema and gametophores the occurrence of cis‐(\+)‐OPDA, but neither JA nor the isoleucine conjugate of JA nor that of cis‐(\+)‐OPDA was detected.Targeted knockout mutants for PpAOC1 and for PpAOC2 were generated, while double mutants could not be obtained. The ΔPpAOC1 and ΔPpAOC2 mutants showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis.} } @Article{IPB-1538, author = {Breuillin, F. and Schramm, J. and Hajirezaei, M. and Ahkami, A. and Favre, P. and Druege, U. and Hause, B. and Bucher, M. and Kretzschmar, T. and Bossolini, E. and Kuhlemeier, C. and Martinoia, E. and Franken, P. and Scholz, U. and Reinhardt, D. and}, title = {{Phosphate systemically inhibits development of arbuscular mycorrhiza in Petunia hybrida and represses genes involved in mycorrhizal functioning}}, year = {2010}, pages = {1002-1017}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2010.04385.x}, volume = {64}, abstract = {Most terrestrial plants form arbuscular mycorrhiza (AM), mutualistic associations with soil fungi of the order Glomeromycota. The obligate biotrophic fungi trade mineral nutrients, mainly phosphate (Pi), for carbohydrates from the plants. Under conditions of high exogenous phosphate supply, when the plant can meet its own P requirements without the fungus, AM are suppressed, an effect which could be interpreted as an active strategy of the plant to limit carbohydrate consumption of the fungus by inhibiting its proliferation in the roots. However, the mechanisms involved in fungal inhibition are poorly understood. Here, we employ a transcriptomic approach to get insight into potential shifts in metabolic activity and symbiotic signalling, and in the defence status of plants exposed to high Pi levels. We show that in mycorrhizal roots of petunia, a similar set of symbiosis‐related genes is expressed as in mycorrhizal roots of Medicago, Lotus and rice. Pi acts systemically to repress symbiotic gene expression and AM colonization in the root. In established mycorrhizal roots, Pi repressed symbiotic gene expression rapidly, whereas the inhibition of colonization followed with a lag of more than a week. Taken together, these results suggest that Pi acts by repressing essential symbiotic genes, in particular genes encoding enzymes of carotenoid and strigolactone biosynthesis, and symbiosis‐associated phosphate transporters. The role of these effects in the suppression of symbiosis under high Pi conditions is discussed.} } @Article{IPB-1573, author = {Paetzold, H. and Garms, S. and Bartram, S. and Wieczorek, J. and Urós-Gracia, E.-M. and Rodríguez-Concepción, M. and Boland, W. and Strack, D. and Hause, B. and Walter, M. H. and}, title = {{The Isogene 1-Deoxy-D-Xylulose 5-Phosphate Synthase 2 Controls Isoprenoid Profiles, Precursor Pathway Allocation, and Density of Tomato Trichomes}}, year = {2010}, pages = {904-916}, journal = {Mol. Plant}, doi = {10.1093/mp/ssq032}, volume = {3}, abstract = {Plant isoprenoids are formed from precursors synthesized by the mevalonate (MVA) pathway in the cytosol or by the methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms (DXS1, DXS2). In tomato (Solanum lycopersicum), the SlDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SlDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SlDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SlDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of 13C to 12C natural isotope ratios. A compensatory up-regulation of SlDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.} } @Article{IPB-1570, author = {Mittasch, J. and Mikolajewski, S. and Breuer, F. and Strack, D. and Milkowski, C. and}, title = {{Genomic microstructure and differential expression of the genes encoding UDP-glucose:sinapate glucosyltransferase (UGT84A9) in oilseed rape (Brassica napus)}}, year = {2010}, pages = {1485-1500}, journal = {Theor. Appl. Genet.}, doi = {10.1007/s00122-010-1270-4}, volume = {120}, abstract = {In oilseed rape (Brassica napus), the glucosyltransferase UGT84A9 catalyzes the formation of 1-O-sinapoyl-β-glucose, which feeds as acyl donor into a broad range of accumulating sinapate esters, including the major antinutritive seed component sinapoylcholine (sinapine). Since down-regulation of UGT84A9 was highly efficient in decreasing the sinapate ester content, the genes encoding this enzyme were considered as potential targets for molecular breeding of low sinapine oilseed rape. B. napus harbors two distinguishable sequence types of the UGT84A9 gene designated as UGT84A9-1 and UGT84A9-2. UGT84A9-1 is the predominantly expressed variant, which is significantly up-regulated during the seed filling phase, when sinapate ester biosynthesis exhibits strongest activity. In the allotetraploid genome of B. napus, UGT84A9-1 is represented by two loci, one derived from the Brassica C-genome (UGT84A9a) and one from the Brassica A-genome (UGT84A9b). Likewise, for UGT84A9-2 two loci were identified in B. napus originating from both diploid ancestor genomes (UGT84A9c, Brassica C-genome; UGT84A9d, Brassica A-genome). The distinct UGT84A9 loci were genetically mapped to linkage groups N15 (UGT84A9a), N05 (UGT84A9b), N11 (UGT84A9c) and N01 (UGT84A9d). All four UGT84A9 genomic loci from B. napus display a remarkably low micro-collinearity with the homologous genomic region of Arabidopsis thaliana chromosome III, but exhibit a high density of transposon-derived sequence elements. Expression patterns indicate that the orthologous genes UGT84A9a and UGT84A9b should be considered for mutagenesis inactivation to introduce the low sinapine trait into oilseed rape.} } @Article{IPB-1569, author = {Milkowski, C. and Strack, D. and}, title = {{Sinapate esters in brassicaceous plants: biochemistry, molecular biology, evolution and metabolic engineering}}, year = {2010}, pages = {19-35}, journal = {Planta}, doi = {10.1007/s00425-010-1168-z}, volume = {232}, abstract = {Brassicaceous plants are characterized by a pronounced metabolic flux toward sinapate, produced by the shikimate/phenylpropanoid pathway, which is converted into a broad spectrum of O-ester conjugates. The abundant sinapate esters in Brassica napus and Arabidopsis thaliana reflect a well-known metabolic network, including UDP-glucose:sinapate glucosyltransferase (SGT), sinapoylglucose:choline sinapoyltransferase (SCT), sinapoylglucose:l-malate sinapoyltransferase (SMT) and sinapoylcholine (sinapine) esterase (SCE). 1-O-Sinapoylglucose, produced by SGT during seed development, is converted to sinapine by SCT and hydrolyzed by SCE in germinating seeds. The released sinapate feeds via sinapoylglucose into the biosynthesis of sinapoylmalate in the seedlings catalyzed by SMT. Sinapoylmalate is involved in protecting the leaves against the deleterious effects of UV-B radiation. Sinapine might function as storage vehicle for ready supply of choline for phosphatidylcholine biosynthesis in young seedlings. The antinutritive character of sinapine and related sinapate esters hamper the use of the valuable seed protein of the oilseed crop B. napus for animal feed and human nutrition. Due to limited variation in seed sinapine content within the assortment of B. napus cultivars, low sinapine lines cannot be generated by conventional breeding giving rise to genetic engineering of sinapate ester metabolism as a promising means. In this article we review the progress made throughout the last decade in identification of genes involved in sinapate ester metabolism and characterization of the encoded enzymes. Based on gene structures and enzyme recruitment, evolution of sinapate ester metabolism is discussed. Strategies of targeted metabolic engineering, designed to generate low-sinapate ester lines of B. napus, are evaluated.} } @Article{IPB-1568, author = {Mallona, I. and Lischewski, S. and Weiss, J. and Hause, B. and Egea-Cortines, M. and}, title = {{Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida}}, year = {2010}, pages = {4}, journal = {BMC Plant Biol.}, doi = {10.1186/1471-2229-10-4}, volume = {10}, abstract = {BackgroundIdentification of genes with invariant levels of gene expression is a prerequisite for validating transcriptomic changes accompanying development. Ideally expression of these genes should be independent of the morphogenetic process or environmental condition tested as well as the methods used for RNA purification and analysis.ResultsIn an effort to identify endogenous genes meeting these criteria nine reference genes (RG) were tested in two Petunia lines (Mitchell and V30). Growth conditions differed in Mitchell and V30, and different methods were used for RNA isolation and analysis. Four different software tools were employed to analyze the data. We merged the four outputs by means of a non-weighted unsupervised rank aggregation method. The genes identified as optimal for transcriptomic analysis of Mitchell and V30 were EF1α in Mitchell and CYP in V30, whereas the least suitable gene was GAPDH in both lines.ConclusionsThe least adequate gene turned out to be GAPDH indicating that it should be rejected as reference gene in Petunia. The absence of correspondence of the best-suited genes suggests that assessing reference gene stability is needed when performing normalization of data from transcriptomic analysis of flower and leaf development.} } @Article{IPB-1565, author = {Leitner, M. and Kaiser, R. and Hause, B. and Boland, W. and Mithöfer, A. and}, title = {{Does mycorrhization influence herbivore-induced volatile emission in Medicago truncatula?}}, year = {2010}, pages = {89-101}, journal = {Mycorrhiza}, doi = {10.1007/s00572-009-0264-z}, volume = {20}, abstract = {Symbiosis with mycorrhizal fungi substantially impacts secondary metabolism and defensive traits of colonised plants. In the present study, we investigated the influence of mycorrhization (Glomus intraradices) on inducible indirect defences against herbivores using the model legume Medicago truncatula. Volatile emission by mycorrhizal and non-mycorrhizal plants was measured in reaction to damage inflicted by Spodoptera spp. and compared to the basal levels of volatile emission by plants of two different cultivars. Emitted volatiles were recorded using closed-loop stripping and gas chromatography/mass spectrometry. The documented volatile patterns were evaluated using multidimensional scaling to visualise patterns and stepwise linear discriminant analysis to distinguish volatile blends of plants with distinct physiological status and genetic background. Volatile blends emitted by different cultivars of M. truncatula prove to be clearly distinct, whereas mycorrhization only slightly influenced herbivore-induced volatile emissions. Still, the observed differences were sufficient to create classification rules to distinguish mycorrhizal and non-mycorrhizal plants by the volatiles emitted. Moreover, the effect of mycorrhization turned out to be opposed in the two cultivars examined. Root symbionts thus seem to alter indirect inducible defences of M. truncatula against insect herbivores. The impact of this effect strongly depends on the genetic background of the plant and, hence, in part explains the highly contradictory results on tripartite interactions gathered to date.} } @Article{IPB-1564, author = {Kopertekh, L. and Schulze, K. and Frolov, A. and Strack, D. and Broer, I. and Schiemann, J. and}, title = {{Cre-mediated seed-specific transgene excision in tobacco}}, year = {2010}, pages = {597-605}, journal = {Plant Mol. Biol.}, doi = {10.1007/s11103-009-9595-6}, volume = {72}, abstract = {Here we report the production of marker-free transgenic plants expressing phenolic compounds with high pharmacological value. Our strategy consisted in simultaneous delivery of lox-target and cre-containing constructs into the plant genome by cotransformation. In the Cre-vector, the cre recombinase gene was controlled by a seed-specific napin promoter. In the lox-target construct the selectable bar gene was placed between two lox sites in direct orientation, while a napin promoter driven vstI gene was inserted outside of the lox sites. Upon seed-specific cre induction the bar expression cassette was excised from the tobacco genome. Genetic and molecular analysis of T1 progeny plants indicated DNA excision in all 10 transgenic lines tested. RP-HPLC analysis demonstrated that the expression of the vstI gene resulted in accumulation of trans-resveratrol and its glycosylated derivative piceid in seeds of all marker free lines. These findings indicate that the seed-specific marker gene excision did not interfere with the expression of the gene of interest. Our data demonstrated the feasi of a developmentally controlled cre gene to mediate site-specific excision in tobacco very efficiently.} } @Article{IPB-1563, author = {Klopotek, Y. and Haensch, K.-T. and Hause, B. and Hajirezaei, M.-R. and Druege, U. and}, title = {{Dark exposure of petunia cuttings strongly improves adventitious root formation and enhances carbohydrate availability during rooting in the light}}, year = {2010}, pages = {547-554}, journal = {J. Plant Physiol.}, doi = {10.1016/j.jplph.2009.11.008}, volume = {167}, abstract = {The effect of temporary dark exposure on adventitious root formation (ARF) in Petunia×hybrida ‘Mitchell’ cuttings was investigated. Histological and metabolic changes in the cuttings during the dark treatment and subsequent rooting in the light were recorded. Excised cuttings were exposed to the dark for seven days at 10°C followed by a nine-day rooting period in perlite or were rooted immediately for 16 days in a climate chamber at 22/20°C (day/night) and a photosynthetic photon flux density (PPFD) of 100 μmol m−2 s−1. Dark exposure prior to rooting increased, accelerated and synchronized ARF. The rooting period was reduced from 16 days (non-treated cuttings) to 9 days (treated cuttings). Under optimum conditions, despite the reduced rooting period, dark-exposed cuttings produced a higher number and length of roots than non-treated cuttings. An increase in temperature to 20 °C during the dark treatment or extending the cold dark exposure to 14 days caused a similar enhancement of root development compared to non-treated cuttings. Root meristem formation had already started during the dark treatment and was enhanced during the subsequent rooting period. Levels of soluble sugars (glucose, fructose and sucrose) and starch in leaf and basal stem tissues significantly decreased during the seven days of dark exposure. This depletion was, however, compensated during rooting after 6 and 24h for soluble sugars in leaves and the basal stem, respectively, whereas the sucrose level in the basal stem was already increased at 6 h. The association of higher carbohydrate levels with improved rooting in previously dark-exposed versus non-treated cuttings indicates that increased post-darkness carbohydrate availability and allocation towards the stem base contribute to ARF under the influence of dark treatment and provide energy for cell growth subject to a rising sink intensity in the base of the cutting.} } @Article{IPB-1555, author = {Handrick, V. and Vogt, T. and Frolov, A. and}, title = {{Profiling of hydroxycinnamic acid amides in Arabidopsis thaliana pollen by tandem mass spectrometry}}, year = {2010}, pages = {2789-2801}, journal = {Anal. Bioanal. Chem.}, doi = {10.1007/s00216-010-4129-2}, volume = {398}, abstract = {Phenylpropanoid polyamine conjugates are widespread in plant species. Their presence has been established in seeds, flower buds, and pollen grains. A biosynthetic pathway proposed for hydroxycinnamoyl spermidine conjugates has been suggested for the model plant Arabidopsis thaliana with a central acyl transfer reaction performed by a BAHD-like hydroxycinnamoyl transferase. A detailed liquid chromatography (LC)–electrospray ionization–mass spectrometry- and tandem-mass-spectrometry (MS/MS)-based survey of wild-type and spermidine hydroxycinnamoyl transferase (SHT) mutants identified more than 30 different bis- and tris-substituted spermidine conjugates, five of which were glycosylated, in the methanol-soluble fraction of the pollen exine. On the basis of characterized fragmentation patterns, a high-throughput LC–MS/MS method for highly sensitive HCAA relative quantification (targeted profiling) was developed. Only minor qualitative and quantitative differences in the pattern of bis-acyl spermidine conjugates in the SHT mutant compared to wild-type plants provide strong evidence for the presence of multiple BAHD-like acyl transferases and suggest a much more complex array of enzymatic steps in the biosynthesis of these conjugates than previously anticipated.} } @Article{IPB-1552, author = {Frolov, A. and Hoffmann, R. and}, title = {{Identification and relative quantification of specific glycation sites in human serum albumin}}, year = {2010}, pages = {2349-2356}, journal = {Anal. Bioanal. Chem.}, doi = {10.1007/s00216-010-3810-9}, volume = {397}, abstract = {Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys549 (KAm(Glc)QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.} } @Article{IPB-1551, author = {Fedorova, M. and Frolov, A. and Hoffmann, R. and}, title = {{Fragmentation behavior of Amadori-peptides obtained by non-enzymatic glycosylation of lysine residues with ADP-ribose in tandem mass spectrometry}}, year = {2010}, pages = {664-669}, journal = {J. Mass Spectrom.}, doi = {10.1002/jms.1758}, volume = {45}, abstract = {Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses.} } @Article{IPB-1548, author = {Ehrlich, H. and Hanke, T. and Simon, P. and Born, R. and Fischer, C. and Frolov, A. and Langrock, T. and Hoffmann, R. and Schwarzenbolz, U. and Henle, T. and Bazhenov, V. V. and Worch, H. and}, title = {{Carboxymethylation of the fibrillar collagen with respect to formation of hydroxyapatite}}, year = {2010}, pages = {542-551}, journal = {J. Biomed. Mater. Res. B}, doi = {10.1002/jbm.b.31551}, volume = {92B}, abstract = {Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Highly acidic macromolecules are postulated intermediates in tissue mineralization, because they sequester many calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. A prerequisite for biomineralization is the ability of cations like calcium to bind to proteins and to result in concert with appropriate anions like phosphates or carbonates in composite materials with bone‐like properties. For this mineralization process the proteins have to be modified with respect to acidification. In this study we modified the protein collagen by carboxymethylation using glucuronic acid. Our experiments showed unambigously, that Nε‐carboxymethyllysine is the major product of the in vitro nonenzymatic glycation reaction between glucuronic acid and collagen. We hypothesized that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions so that a critical nucleus of ions can be formed, leading to the formation of the mineral. Thus, the self‐organization of HAP nanocrystals on and within collagen fibrils was intensified by carboxymethylation.} } @Article{IPB-1673, author = {Zurbriggen, M. D. and Carrillo, N. and Tognetti, V. B. and Melzer, M. and Peisker, M. and Hause, B. and Hajirezaei, M.-R. and}, title = {{Chloroplast-generated reactive oxygen species play a major role in localized cell death during the non-host interaction between tobacco and Xanthomonas campestris pv. vesicatoria}}, year = {2009}, pages = {962-973}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2009.04010.x}, volume = {60}, abstract = {Attempted infection of plants by pathogens elicits a complex defensive response. In many non‐host and incompatible host interactions it includes the induction of defence‐associated genes and a form of localized cell death (LCD), purportedly designed to restrict pathogen advance, collectively known as the hypersensitive response (HR). It is preceded by an oxidative burst, generating reactive oxygen species (ROS) that are proposed to cue subsequent deployment of the HR, although neither the origin nor the precise role played by ROS in the execution of this response are completely understood. We used tobacco plants expressing cyanobacterial flavodoxin to address these questions. Flavodoxin is an electron shuttle present in prokaryotes and algae that, when expressed in chloroplasts, specifically prevents ROS formation in plastids during abiotic stress episodes. Infiltration of tobacco wild‐type leaves with high titres of Xanthomonas campestris pv. vesicatoria (Xcv ), a non‐host pathogen, resulted in ROS accumulation in chloroplasts, followed by the appearance of localized lesions typical of the HR. In contrast, chloroplast ROS build‐up and LCD were significantly reduced in Xcv ‐inoculated plants expressing plastid‐targeted flavodoxin. Metabolic routes normally inhibited by pathogens were protected in the transformants, whereas other aspects of the HR, including the induction of defence‐associated genes and synthesis of salicylic and jasmonic acid, proceeded as in inoculated wild‐type plants. Therefore, ROS generated in chloroplasts during this non‐host interaction are essential for the progress of LCD, but do not contribute to the induction of pathogenesis‐related genes or other signalling components of the response.} } @Article{IPB-1666, author = {Wasternack, C. and Hause, B. and}, title = {{Emerging complexity: jasmonate-induced volatiles affect parasitoid choice}}, year = {2009}, pages = {2451-2453}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/erp197}, volume = {60}, } @Article{IPB-1665, author = {Walter, M. H. and Floß, D. S. and Strack, D. and}, title = {{Die facettenreiche Welt der Apocarotinoide. Farben, Düfte, Aromen und Hormone}}, year = {2009}, pages = {336-344}, journal = {Biologie in unserer Zeit}, doi = {10.1002/biuz.200910402}, volume = {39}, abstract = {Apocarotinoide werden durch hochspezifische Spaltungsreaktionen oxidativer Enzyme an den Doppelbindungen von Carotinoiden maßgeschneidert. Es können neue Chromophore entstehen, die zusätzliche Nuancen des gelb‐roten Farbspektrums eröffnen. Farblose C13‐Apocarotinoide können potente Duft‐ und Aromastoffe sein. Viele Apocarotinoidfunktionen mit Hormoncharakter sind lange bekannt (Abszisinsäure in Pflanzen, Trisporsäure in Pilzen, Retinsäure in Säugern). Eine neue Klasse von Apocarotinoid‐Pflanzenhormonen, die die Sprossverzweigung der Pflanzen mitbestimmen, wurde kürzlich als Strigolactone identifiziert. In ihrer Biosynthese wie auch in der von mykorrhizainduzierten C13/C14‐Apocarotinoiden treten mehrstufige aufeinanderfolgende Carotinoidspaltungsreaktionen auf. Das Wissen über Synthesewege und Funktionen von Apocarotinoiden eröffnet neue Perspektiven für Anwendungen im Zierpflanzenbau, bei der Bekämpfung parasitischer Unkräuter und in der Beeinflussung von Blütendüften und Fruchtaromen.} } @Article{IPB-1664, author = {Vandenborre, G. and Miersch, O. and Hause, B. and Smagghe, G. and Wasternack, C. and Van Damme, E. J. and}, title = {{Spodoptera littoralis-Induced Lectin Expression in Tobacco}}, year = {2009}, pages = {1142-1155}, journal = {Plant Cell Physiol.}, doi = {10.1093/pcp/pcp065}, volume = {50}, abstract = {The induced defense response in plants towards herbivores is mainly regulated by jasmonates and leads to the accumulation of so-called jasmonate-induced proteins. Recently, a jasmonate (JA) inducible lectin called Nicotiana tabacum agglutinin or NICTABA was discovered in tobacco (N. tabacum cv Samsun) leaves. Tobacco plants also accumulate the lectin after insect attack by caterpillars. To study the functional role of NICTABA, the accumulation of the JA precursor 12-oxophytodienoic acid (OPDA), JA as well as different JA metabolites were analyzed in tobacco leaves after herbivory by larvae of the cotton leafworm (Spodoptera littoralis) and correlated with NICTABA accumulation. It was shown that OPDA, JA as well as its methyl ester can trigger NICTABA accumulation. However, hydroxylation of JA and its subsequent sulfation and glucosylation results in inactive compounds that have lost the capacity to induce NICTABA gene expression. The expression profile of NICTABA after caterpillar feeding was recorded in local as well as in systemic leaves, and compared to the expression of several genes encoding defense proteins, and genes encoding a tobacco systemin and the allene oxide cyclase, an enzyme in JA biosynthesis. Furthermore, the accumulation of NICTABA was quanti-fied after S. littoralis herbivory and immunofluorescence microscopy was used to study the localization of NICTABA in the tobacco leaf.} } @Article{IPB-1661, author = {Thiyam, U. and Claudia, P. and Jan, U. and Alfred, B. and}, title = {{De-oiled rapeseed and a protein isolate: characterization of sinapic acid derivatives by HPLC–DAD and LC–MS}}, year = {2009}, pages = {825-831}, journal = {Eur. Food Res. Technol.}, doi = {10.1007/s00217-009-1122-0}, volume = {229}, abstract = {De-oiled rapeseed is a rich source of proteins and phenolic compounds. The phenolic compounds, namely sinapic acid derivatives (SAD), could occur as free sinapic acid, esterified (as sinapine, the choline ester of sinapic acid) and decarboxylated (as canolol) forms. Rapeseed protein preparations containing very low phenolic compounds have been the focus of our ongoing research. A precipitated rapeseed protein isolate is investigated for SAD such as sinapine, sinapoyl glucose, canolol using HPLC–DAD and LC–MS. Profile of the phenolic compounds of de-oiled rapeseed, press cakes and the precipitated protein isolate are compared. HPLC–DAD analysis indicated SAD; particularly sinapine is the main phenolic compound of all the substrates. The protein derivation process did not remarkably alter the profile of the investigated protein isolate.} } @Article{IPB-1659, author = {Stehle, F. and Brandt, W. and Stubbs, M. T. and Milkowski, C. and Strack, D. and}, title = {{Sinapoyltransferases in the light of molecular evolution}}, year = {2009}, pages = {1652-1662}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2009.07.023}, volume = {70}, abstract = {Acylation is a prevalent chemical modification that to a significant extent accounts for the tremendous diversity of plant metabolites. To catalyze acyl transfer reactions, higher plants have evolved acyltransferases that accept β-acetal esters, typically 1-O-glucose esters, as an alternative to the ubiquitously occurring CoA-thioester-dependent enzymes. Shared homology indicates that the β-acetal ester-dependent acyltransferases are derived from a common hydrolytic ancestor of the Serine CarboxyPeptidase (SCP) type, giving rise to the name Serine CarboxyPeptidase-Like (SCPL) acyltransferases. We have analyzed structure–function relationships, reaction mechanism and sequence evolution of Arabidopsis 1-O-sinapoyl-β-glucose:l-malate sinapoyltransferase (AtSMT) and related enzymes to investigate molecular changes required to impart acyltransferase activity to hydrolytic enzymes. AtSMT has maintained the catalytic triad of the hydrolytic ancestor as well as part of the H-bond network for substrate recognition to bind the acyl acceptor l-malate. A Glu/Asp substitution at the amino acid position preceding the catalytic Ser supports binding of the acyl donor 1-O-sinapoyl-β-glucose and was found highly conserved among SCPL acyltransferases. The AtSMT-catalyzed acyl transfer reaction follows a random sequential bi-bi mechanism that requires both substrates 1-O-sinapoyl-β-glucose and l-malate bound in an enzyme donor–acceptor complex to initiate acyl transfer. Together with the strong fixation of the acyl acceptor l-malate, the acquisition of this reaction mechanism favours transacylation over hydrolysis in AtSMT catalysis. The model structure and enzymatic side activities reveal that the AtSMT-mediated acyl transfer proceeds via a short-lived acyl enzyme complex. With regard to evolution, the SCPL acyltransferase clade most likely represents a recent development. The encoding genes are organized in a tandem-arranged cluster with partly overlapping functions. With other enzymes encoded by the respective gene cluster on Arabidopsis chromosome 2, AtSMT shares the enzymatic side activity to disproportionate 1-O-sinapoyl-β-glucoses to produce 1,2-di-O-sinapoyl-β-glucose. In the absence of the acyl acceptor l-malate, a residual esterase activity became obvious as a remnant of the hydrolytic ancestor. With regard to the evolution of Arabidopsis SCPL acyltransferases, our results suggest early neofunctionalization of the hydrolytic ancestor toward acyltransferase activity and acyl donor specificity for 1-O-sinapoyl-β-glucose followed by subfunctionalization to recognize different acyl acceptors.} } @Article{IPB-1656, author = {Schaarschmidt, S. and Hause, B. and Strack, D. and}, title = {{Wege zur Endomykorrhiza. Einladung ans Buffet}}, year = {2009}, pages = {102-113}, journal = {Biologie in unserer Zeit}, doi = {10.1002/biuz.200610385}, volume = {39}, abstract = {Die Lebensgemeinschaft mit Mykorrhizapilzen stellt Pflanzen mineralische Nährstoffe und Wasser zur Verfügung und gilt daher als evolutionäre Grundlage für die Entwicklung der Landpflanzen. Die heute weit verbreitete arbuskuläre Mykorrhiza (AM) ist insbesondere unter widrigen Bedingungen (Nährstoffmangel, Trocken‐, Salz‐ oder Schwermetallstress sowie Pathogenbefall) für die Pflanze von Nutzen. Der pilzliche AM‐Partner, der obligat auf die Interaktion angewiesen ist, wird im Gegenzug mit Kohlenhydraten versorgt. Der Artikel beschreibt den aktuellen Stand der Forschung bezüglich der Etablierung und Regulation der AM durch die Pflanze. Es werden die frühen Erkennungssignale und die nachfolgende Wegbereitung der Pflanze für den eindringenden Pilz, die Kohlenhydratversorgung des AM‐Pilzes, wie auch die Limitierung der pilzlichen Infektionen mittels Autoregulation und die Rolle der Phytohormone für eine funktionelle und ausgeglichene Symbiose behandelt.} } @Article{IPB-1647, author = {Mrosk, C. and Forner, S. and Hause, G. and Küster, H. and Kopka, J. and Hause, B. and}, title = {{Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices}}, year = {2009}, pages = {3797-3807}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/erp220}, volume = {60}, abstract = {Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects.} } @Article{IPB-1643, author = {Lukačin, R. and Matern, U. and Hehmann, M. and Specker, S. and Vogt, T. and}, title = {{Corrigendum to “Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus” [FEBS Lett. 577 (2004) 367-370]}}, year = {2009}, pages = {855-855}, journal = {FEBS Lett.}, doi = {10.1016/j.febslet.2009.01.050}, volume = {583}, } @Article{IPB-1635, author = {Hohnjec, N. and Lenz, F. and Fehlberg, V. and Vieweg, M. F. and Baier, M. C. and Hause, B. and Küster, H. and}, title = {{The Signal Peptide of the Medicago truncatula Modular Nodulin MtNOD25 Operates as an Address Label for the Specific Targeting of Proteins to Nitrogen-Fixing Symbiosomes}}, year = {2009}, pages = {63-72}, journal = {Mol. Plant Microbe Interact.}, doi = {10.1094/MPMI-22-1-0063}, volume = {22}, abstract = {The nodule-specific MtNOD25 gene of the model legume Medicago truncatula encodes a modular nodulin composed of different repetitive modules flanked by distinct N- and C-termini. Although similarities are low with respect to all repetitive modules, both the N-terminal signal peptide (SP) and the C-terminus are highly conserved in modular nodulins from different legumes. On the cellular level, MtNOD25 is only transcribed in the infected cells of root nodules, and this activation is mediated by a 299-bp minimal promoter containing an organ-specific element. By expressing mGFP6 translational fusions in transgenic nodules, we show that MtNOD25 proteins are exclusively translocated to the symbiosomes of infected cells. This specific targeting only requires an N-terminal MtNOD25 SP that is highly conserved across a family of legume-specific symbiosome proteins. Our finding sheds light on one possible mechanism for the delivery of host proteins to the symbiosomes of infected root nodule cells and, in addition, defines a short molecular address label of only 24 amino acids whose N-terminal presence is sufficient to translocate proteins across the peribacteroid membrane.} } @Article{IPB-1634, author = {Hause, B. and Schaarschmidt, S. and}, title = {{The role of jasmonates in mutualistic symbioses between plants and soil-born microorganisms}}, year = {2009}, pages = {1589-1599}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2009.07.003}, volume = {70}, abstract = {Many plants are able to develop mutualistic interactions with arbuscular mycorrhizal fungi and/or nitrogen-fixing bacteria. Whereas the former is widely distributed among most of the land plants, the latter is restricted to species of ten plant families, including the legumes. The establishment of both associations is based on mutual recognition and a high degree of coordination at the morphological and physiological level. This requires the activity of a number of signals, including jasmonates. Here, recent knowledge on the putative roles of jasmonates in both mutualistic symbioses will be reviewed. Firstly, the action of jasmonates will be discussed in terms of the initial signal exchange between symbionts and in the resulting plant signaling cascade common for nodulation and mycorrhization. Secondly, the putative role of jasmonates in the autoregulation of the endosymbioses will be outlined. Finally, aspects of function of jasmonates in the fully established symbioses will be presented. Various processes will be discussed that are possibly mediated by jasmonates, including the redox status of nodules and the carbohydrate partitioning of mycorrhizal roots.} } @Article{IPB-1633, author = {Hause, B. and Wasternack, C. and Strack, D. and}, title = {{Jasmonates in stress responses and development}}, year = {2009}, pages = {1483-1484}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2009.07.004}, volume = {70}, } @Article{IPB-1630, author = {Grunwald, U. and Guo, W. and Fischer, K. and Isayenkov, S. and Ludwig-Müller, J. and Hause, B. and Yan, X. and Küster, H. and Franken, P. and}, title = {{Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal, Pi-fertilised and phytohormone-treated Medicago truncatula roots}}, year = {2009}, pages = {1023-1034}, journal = {Planta}, doi = {10.1007/s00425-008-0877-z}, volume = {229}, abstract = {A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by Pi fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with Pi or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of Pi concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher Pi levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices.} } @Article{IPB-1610, author = {Brakhage, A. and Gierl, A. and Hartmann, T. and Strack, D. and}, title = {{Evolution of metabolic diversity}}, year = {2009}, pages = {1619-1620}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2009.07.007}, volume = {70}, } @Article{IPB-1601, author = {Ahkami, A. H. and Lischewski, S. and Haensch, K.-T. and Porfirova, S. and Hofmann, J. and Rolletschek, H. and Melzer, M. and Franken, P. and Hause, B. and Druege, U. and Hajirezaei, M. R. and}, title = {{Molecular physiology of adventitious root formation in Petunia hybrida cuttings: involvement of wound response and primary metabolism}}, year = {2009}, pages = {613-625}, journal = {New Phytol.}, doi = {10.1111/j.1469-8137.2008.02704.x}, volume = {181}, abstract = {Adventitious root formation (ARF) in the model plant Petunia hybrida cv. Mitchell has been analysed in terms of anatomy, gene expression, enzymatic activities and levels of metabolites. This study focuses on the involvement of wound response and primary metabolism.Microscopic techniques were complemented with targeted transcript, enzyme and metabolite profiling using real time polymerase chain reaction (PCR), Northern blot, enzymatic assays, chromatography and mass spectrometry.Three days after severance from the stock plants, first meristematic cells appeared which further developed into root primordia and finally adventitious roots. Excision of cuttings led to a fast and transient increase in the wound‐hormone jasmonic acid, followed by the expression of jasmonate‐regulated genes such as cell wall invertase. Analysis of soluble and insoluble carbohydrates showed a continuous accumulation during ARF. A broad metabolite profiling revealed a strong increase in organic acids and resynthesis of essential amino acids.Substantial changes in enzyme activities and metabolite levels indicate that specific enzymes and metabolites might play a crucial role during ARF. Three metabolic phases could be defined: (i) sink establishment phase characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates; (ii) recovery phase; and (iii) maintenance phase, in which a symplastic unloading occurs.} } @Article{IPB-1626, author = {Floss, D. S. and Walter, M. H. and}, title = {{Role of carotenoid cleavage dioxygenase 1 (CCD1) in apocarotenoid biogenesis revisited}}, year = {2009}, pages = {172-175}, journal = {Plant Signal Behav.}, doi = {10.4161/psb.4.3.7840}, volume = {4}, abstract = {Oxidative tailoring of C40 carotenoids by double bond-specific cleavage enzymes (carotenoid cleavage dioxygenases, CCDs) gives rise to various apocarotenoids. AtCCD1 generating C13 and C14 apocarotenoids and orthologous enzymes in other plants are the only CCDs acting in the cytosol, while the hitherto presumed C40 substrate is localized in the plastid. A new model for CCD1 action arising from a RNAi-mediated CCD1 gene silencing study in mycorrhizal hairy roots of Medicago truncatula may solve this contradiction. This approach unexpectedly resulted in the accumulation of C27 apocarotenoids but not C40 carotenoids suggesting C27 as the main substrates for CCD1 in planta. It further implies a consecutive two-step cleavage process, in which another CCD performs the primary cleavage of C40 to C27 in the plastid followed by C27 export and further cleavage by CCD1 in the cytosol. We compare the specificities and subcellular locations of the various CCDs and propose the plastidial CCD7 to be the first player in mycorrhizal apocarotenoid biogenesis.} } @Article{IPB-1624, author = {Fellenberg, C. and Böttcher, C. and Vogt, T. and}, title = {{Phenylpropanoid polyamine conjugate biosynthesis in Arabidopsis thaliana flower buds}}, year = {2009}, pages = {1392-1400}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2009.08.010}, volume = {70}, abstract = {Phenylpropanoid polyamine conjugates have been identified in flowers of many plant species. Their presence in Arabidopsis thaliana has only been recently established in flower buds and pollen grains. Annotation and location of a cation-dependent O-methyltransferase AtTSM1 specifically to the tapetum of young flower buds enabled the subsequent identification of several genes with a putative role in phenylpropanoid polyamine conjugate biosynthesis. Based on the analysis of several A. thaliana knockout mutants, a biosynthetic pathway of these conjugates is proposed, which involves two methylation steps catalyzed by different cation-dependent O-methyltransferases, a cytochrome P450 (CYP98A8) catalyzed hydroxylation, and a conjugating acyl transfer performed by a BAHD-like, hydroxycinnamoyl (HC)-transferase. LC/MS based metabolite profiling of the cyp98A8 knockout line identified new feruloyl- and 4-coumaroylspermidine conjugates in the corresponding flowers consistent with a role of this gene in the hydroxylation of these conjugates. A pattern of minor amounts of bis- and tris-acylspermidine conjugates, likely the products of additional HC-transferases were identified in wild type as well as in the mutant lines. Transcript suppression of the genes early in the pathway was observed in knockout or RNAi-lines of the genes encoding late enzymatic steps. The implication of these findings for spermidine conjugate biosynthesis in flower buds of A. thaliana is discussed.} } @Article{IPB-1622, author = {Engler, C. and Gruetzner, R. and Kandzia, R. and Marillonnet, S. and}, title = {{Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes}}, year = {2009}, pages = {e5553}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0005553}, volume = {4}, abstract = {We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.} } @Article{IPB-1620, author = {Ehrlich, H. and Hanke, T. and Frolov, A. and Langrock, T. and Hoffmann, R. and Fischer, C. and Schwarzenbolz, U. and Henle, T. and Born, R. and Worch, H. and}, title = {{Modification of collagen in vitro with respect to formation of Nɛ-carboxymethyllysine}}, year = {2009}, pages = {51-56}, journal = {Int. J. Biol. Macromol.}, doi = {10.1016/j.ijbiomac.2008.10.001}, volume = {44}, abstract = {Developing new biopolymer-based materials with bio-identical properties is a significant challenge in modern science. One interesting route to this goal involves the biomineralization of collagen, a pre-structured and widely available protein, into a material with interesting properties. A prerequisite for biomineralization is the ability of cations (e.g., calcium) to bind to the protein and to result in concert with appropriate anions (e.g., phosphate) in composite material with e.g., bone-like properties. In order to increase the number of binding sites it is necessary to modify the protein prior to mineralization. For this glucuronic acid (GA) was used due to its carbonyl and carboxyl groups to derivatize proteinogenic amino groups transferring them into negatively charged carboxyl groups. Our experiments showed for the first time, that Nɛ-carboxymethyllysine is the major product of in vitro non-enzymatic glycosylation of collagen by glucuronic acid. For an unequivocal determination of the reaction products, the lysine residues of collagen and of the model peptide were carboxymethylated through a reductive alkylation with glyoxalic acid and compared to the glucuronic acid derivatives. Beside their identical mass spectra the common structure elements could be confirmed with FTIR. Thus, in the context of matrix engineering, by producing Nɛ-carboxymethyllysine, glucuronic acid offers a convenient way of introducing additional stable acidic groups into protein matrices.} } @Article{IPB-1619, author = {Ehrlich, H. and Hanke, T. and Born, R. and Fischer, C. and Frolov, A. and Langrock, T. and Hoffmann, R. and Schwarzenbolz, U. and Henle, T. and Simon, P. and Geiger, D. and Bazhenov, V. V. and Worch, H. and}, title = {{Mineralization of biomimetically carboxymethylated collagen fibrils in a model dual membrane diffusion system}}, year = {2009}, pages = {254-259}, journal = {J. Membr. Sci.}, doi = {10.1016/j.memsci.2008.10.003}, volume = {326}, abstract = {In the present work, we show for the first time, that Nɛ-carboxymethyllysine is the major product of the in vitro non-enzymatic glycation reaction between fibrillar collagen and glucuronic acid. Dual diffusion membrane system was effectively used for oriented crystal growth of octacalcium phosphate/hydroxyapatite on the biomimetically carboxymethylated collagen fibrils. We hypothesize that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions in such a way that a critical nucleus of ions can be formed, leading to the formation of the mineral under specific micro-environment conditions achieved by using diffusion membrane system.} } @INBOOK{IPB-104, author = {Frolov, A. and Singer, D. and Zauner, T. and Hoffmann, R. and}, title = {{Peptides for Youth}}, year = {2009}, pages = {423-424}, chapter = {{Solid Phase Synthesis and Analysis of Amadori Peptides}}, journal = {Adv. Exp. Med. Biol.}, doi = {10.1007/978-0-387-73657-0_182}, volume = {611}, } @INBOOK{IPB-103, author = {Dorka, R. and Miersch, O. and Hause, B. and Weik, P. and Wasternack, C. and}, title = {{Die Mistel in der Tumortherapie 2. Aktueller Stand der Forschung und klinische Anwendung}}, year = {2009}, pages = {49-66}, chapter = {{Chronobiologische Phänomene und Jasmonatgehalt bei Viscum album L.}}, } @Article{IPB-1711, author = {Kopycki, J. G. and Stubbs, M. T. and Brandt, W. and Hagemann, M. and Porzel, A. and Schmidt, J. and Schliemann, W. and Zenk, M. H. and Vogt, T. and}, title = {{Functional and Structural Characterization of a Cation-dependent O-Methyltransferase from the Cyanobacterium Synechocystis sp. Strain PCC 6803}}, year = {2008}, pages = {20888-20896}, journal = {J. Biol. Chem.}, doi = {10.1074/jbc.M801943200}, volume = {283}, abstract = {The coding sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 slr0095 gene was cloned and functionally expressed in Escherichia coli. The corresponding enzyme was classified as a cation- and S-adenosyl-l-methionine-dependent O-methyltransferase (SynOMT), consistent with considerable amino acid sequence identities to eukaryotic O-methyltransferases (OMTs). The substrate specificity of SynOMT was similar with those of plant and mammalian CCoAOMT-like proteins accepting a variety of hydroxycinnamic acids and flavonoids as substrates. In contrast to the known mammalian and plant enzymes, which exclusively methylate the meta-hydroxyl position of aromatic di- and trihydroxy systems, Syn-OMT also methylates the para-position of hydroxycinnamic acids like 5-hydroxyferulic and 3,4,5-trihydroxycinnamic acid, resulting in the formation of novel compounds. The x-ray structure of SynOMT indicates that the active site allows for two alternative orientations of the hydroxylated substrates in comparison to the active sites of animal and plant enzymes, consistent with the observed preferred para-methylation and position promiscuity. Lys3 close to the N terminus of the recombinant protein appears to play a key role in the activity of the enzyme. The possible implications of these results with respect to modifications of precursors of polymers like lignin are discussed.} } @Article{IPB-1710, author = {Kopycki, J. G. and Rauh, D. and Chumanevich, A. A. and Neumann, P. and Vogt, T. and Stubbs, M. T. and}, title = {{Biochemical and Structural Analysis of Substrate Promiscuity in Plant Mg2\+-Dependent O-Methyltransferases}}, year = {2008}, pages = {154-164}, journal = {J. Mol. Biol.}, doi = {10.1016/j.jmb.2008.02.019}, volume = {378}, abstract = {Plant S-adenosyl-l-methionine-dependent class I natural product O-methyltransferases (OMTs), related to animal catechol OMTs, are dependent on bivalent cations and strictly specific for the meta position of aromatic vicinal dihydroxy groups. While the primary activity of these class I enzymes is methylation of caffeoyl coenzyme A OMTs, a distinct subset is able to methylate a wider range of substrates, characterized by the promiscuous phenylpropanoid and flavonoid OMT. The observed broad substrate specificity resides in two regions: the N-terminus and a variable insertion loop near the C-terminus, which displays the lowest degree of sequence conservation between the two subfamilies. Structural and biochemical data, based on site-directed mutagenesis and domain exchange between the two enzyme types, present evidence that only small topological changes among otherwise highly conserved 3-D structures are sufficient to differentiate between an enzymatic generalist and an enzymatic specialist in plant natural product methylation.} } @Article{IPB-1697, author = {Floss, D. S. and Schliemann, W. and Schmidt, J. and Strack, D. and Walter, M. H. and}, title = {{RNA Interference-Mediated Repression of MtCCD1 in Mycorrhizal Roots of Medicago truncatula Causes Accumulation of C27 Apocarotenoids, Shedding Light on the Functional Role of CCD1}}, year = {2008}, pages = {1267-1282}, journal = {Plant Physiol.}, doi = {10.1104/pp.108.125062}, volume = {148}, abstract = {Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C40 carotenoid substrates at 9,10 and 9′,10′ positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C13 cyclohexenone and C14 mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C27 apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C27 apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C27 apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C27 → C14 \+ C13), while the first step (C40 → C27 \+ C13) may be catalyzed by CCD7 and/or CCD4 inside plastids.} } @Article{IPB-1696, author = {Floß, D. S. and Hause, B. and Lange, P. R. and Küster, H. and Strack, D. and Walter, M. H. and}, title = {{Knock-down of the MEP pathway isogene 1-deoxy-d-xylulose 5-phosphate synthase 2 inhibits formation of arbuscular mycorrhiza-induced apocarotenoids, and abolishes normal expression of mycorrhiza-specific plant marker genes}}, year = {2008}, pages = {86-100}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2008.03575.x}, volume = {56}, abstract = {The first step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by two isoforms of 1‐deoxy‐d‐ xylulose 5‐phosphate synthase (DXS1 and DXS2). In Medicago truncatula , MtDXS1 and MtDXS2 genes exhibit completely different expression patterns. Most prominently, colonization by arbuscular mycorrhizal (AM) fungi induces the accumulation of certain apocarotenoids (cyclohexenone and mycorradicin derivatives) correlated with the expression of MtDXS2 but not of MtDXS1. To prove a distinct function of DXS2, a selective RNAi approach on MtDXS2 expression was performed in transgenic hairy roots of M. truncatula. Repression of MtDXS2 consistently led to reduced transcript levels in mycorrhizal roots, and to a concomitant reduction of AM‐induced apocarotenoid accumulation. The transcript levels of MtDXS1 remained unaltered in RNAi plants, and no phenotypical changes in non‐AM plants were observed. Late stages of the AM symbiosis were adversely affected, but only upon strong repression with residual MtDXS2‐1 transcript levels remaining below approximately 10%. This condition resulted in a strong decrease in the transcript levels of MtPT4 , an AM‐specific plant phosphate transporter gene, and in a multitude of other AM‐induced plant marker genes, as shown by transcriptome analysis. This was accompanied by an increased proportion of degenerating and dead arbuscules at the expense of mature ones. The data reveal a requirement for DXS2‐dependent MEP pathway‐based isoprenoid products to sustain mycorrhizal functionality at later stages of the symbiosis. They further validate the concept of a distinct role for DXS2 in secondary metabolism, and offer a novel tool to selectively manipulate the levels of secondary isoprenoids by targeting their precursor supply.} } @Article{IPB-1695, author = {Fellenberg, C. and Milkowski, C. and Hause, B. and Lange, P.-R. and Böttcher, C. and Schmidt, J. and Vogt, T. and}, title = {{Tapetum-specific location of a cation-dependent O-methyltransferase in Arabidopsis thaliana}}, year = {2008}, pages = {132-145}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2008.03576.x}, volume = {56}, abstract = {Cation‐ and S ‐adenosyl‐l ‐methionine (AdoMet)‐dependent plant natural product methyltransferases are referred to as CCoAOMTs because of their preferred substrate, caffeoyl coenzyme A (CCoA). The enzymes are encoded by a small family of genes, some of which with a proven role in lignin monomer biosynthesis. In Arabidopsis thaliana individual members of this gene family are temporally and spatially regulated. The gene At1g67990 is specifically expressed in flower buds, and is not detected in any other organ, such as roots, leaves or stems. Several lines of evidence indicate that the At1g67990 transcript is located in the flower buds, whereas the corresponding CCoAOMT‐like protein, termed AtTSM1, is located exclusively in the tapetum of developing stamen. Flowers of At1g67990 RNAi‐suppressed plants are characterized by a distinct flower chemotype with severely reduced levels of the N  ′,N  ′′‐ bis‐(5‐hydroxyferuloyl)‐N  ′′′‐sinapoylspermidine compensated for by N1 ,N5 ,N10 ‐tris‐(5‐hydroxyferuloyl)spermidine derivative, which is characterized by the lack of a single methyl group in the sinapoyl moiety. This severe change is consistent with the observed product profile of AtTSM1 for aromatic phenylpropanoids. Heterologous expression of the recombinant protein shows the highest activity towards a series of caffeic acid esters, but 5‐hydroxyferuloyl spermidine conjugates are also accepted substrates. The in vitro substrate specificity and the in vivo RNAi‐mediated suppression data of the corresponding gene suggest a role of this cation‐dependent CCoAOMT‐like protein in the stamen/pollen development of A. thaliana .} } @Article{IPB-1694, author = {Felker, P. and Stintzing, F. and Müssig, E. and Leitenberger, M. and Carle, R. and Vogt, T. and Bunch, R. and}, title = {{Colour inheritance in cactus pear (Opuntia ficus-indica) fruits}}, year = {2008}, pages = {307-318}, journal = {Ann. Appl. Biol.}, doi = {10.1111/j.1744-7348.2008.00222.x}, volume = {152}, abstract = {The pigments of Opuntia ficus‐indica fruits, which are derived from the betalain rather than anthocyanin pathway, have an extraordinary range in colour from lime green, orange, red to purple. This is a result from varying concentrations and proportions of about half a dozen betaxanthins and betacyanins. The yellow‐orange betaxanthins are derived from spontaneous condensation of betalamic acid with amines or amino acids. The reddish‐purple betacyanins are enzymatically formed from betalamic acid and cyclo ‐dihydroxyphenylalanine (DOPA) yielding betanidin and further glycosylated on either of the two hydroxyls of the cyclo ‐DOPA moiety. In the present work, degenerated primers were used to obtain partial genomic sequences of two major genes in the biosynthetic pathway for betalains, that is the 4,5‐extradiol dioxygenase which forms the betalamic acid responsible for the yellow colour and a putative 5‐O ‐glucosyltransferase which glycosylates betanidin in Dorotheanthus bellidiformis and may be responsible for the red colour. Differences in the genomic DNA between coloured versus non‐coloured varieties were not found. Regulatory mechanisms seem to independently control pigmentation of O. ficus‐indica fruit tissues for inner core, peel and epidermis. Core pigmentation occurs first and well before fruit maturity and peel pigmentation. Peel pigmentation is fully developed at maturity, presumably related to maximum soluble solids. Epidermal pigmentation appears to be independent of core and peel pigmentation, perhaps because of light stimulation. Similar control mechanisms exist through transcription factors for the major enzyme regulating anthocyanin production in grapes.} } @Article{IPB-1693, author = {Engler, C. and Kandzia, R. and Marillonnet, S. and}, title = {{A One Pot, One Step, Precision Cloning Method with High Throughput Capability}}, year = {2008}, pages = {e3647}, journal = {PLOS ONE}, doi = {10.1371/journal.pone.0003647}, volume = {3}, abstract = {Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.} } @Article{IPB-1689, author = {Clauß, K. and Baumert, A. and Nimtz, M. and Milkowski, C. and Strack, D. and}, title = {{Role of a GDSL lipase-like protein as sinapine esterase in Brassicaceae}}, year = {2008}, pages = {802-813}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2007.03374.x}, volume = {53}, abstract = {The seeds of most members of the Brassicaceae accumulate high amounts of sinapine (sinapoylcholine) that is rapidly hydrolyzed during early stages of seed germination. One of three isoforms of sinapine esterase activity (BnSCE3) has been isolated from Brassica napus seedlings and subjected to trypsin digestion and spectrometric sequencing. The peptide sequences were used to isolate BnSCE3 cDNA, which was shown to contain an open reading frame of 1170 bp encoding a protein of 389 amino acids, including a leader peptide of 25 amino acids. Sequence comparison identified the protein as the recently cloned BnLIP2, i.e. a GDSL lipase‐like protein, which displays high sequence identity to a large number of corresponding plant proteins, including four related Arabidopsis lipases. The enzymes belong to the SGNH protein family, which use a catalytic triad of Ser‐Asp‐His, with serine as the nucleophile of the GDSL motif. The corresponding B. napus and Arabidopsis genes were heterologously expressed in Nicotiana benthamiana leaves and proved to confer sinapine esterase activity. In addition to sinapine esterase activity, the native B. napus protein (BnSCE3/BnLIP2) showed broad substrate specificity towards various other choline esters, including phosphatidylcholine. This exceptionally broad substrate specificity, which is common to a large number of other GDSL lipases in plants, hampers their functional analysis. However, the data presented here indicate a role for the GDSL lipase‐like BnSCE3/BnLIP2 as a sinapine esterase in members of the Brassicaceae, catalyzing hydrolysis of sinapine during seed germination, leading, via 1‐O ‐sinapoyl‐β‐glucose, to sinapoyl‐l ‐malate in the seedlings.} } @Article{IPB-1753, author = {Weier, D. and Mittasch, J. and Strack, D. and Milkowski, C. and}, title = {{The genes BnSCT1 and BnSCT2 from Brassica napus encoding the final enzyme of sinapine biosynthesis: molecular characterization and suppression}}, year = {2008}, pages = {375-385}, journal = {Planta}, doi = {10.1007/s00425-007-0624-x}, volume = {227}, abstract = {This study describes the molecular characterization of the genes BnSCT1 and BnSCT2 from oilseed rape (Brassica napus) encoding the enzyme 1-O-sinapoyl-β-glucose:choline sinapoyltransferase (SCT; EC 2.3.1.91). SCT catalyzes the 1-O-β-acetal ester-dependent biosynthesis of sinapoylcholine (sinapine), the most abundant phenolic compound in seeds of B. napus. GUS fusion experiments indicated that seed specificity of BnSCT1 expression is caused by an inducible promoter confining transcription to embryo tissues and the aleurone layer. A dsRNAi construct designed to silence seed-specifically the BnSCT1 gene was effective in reducing the sinapine content of Arabidopsis seeds thus defining SCT genes as targets for molecular breeding of low sinapine cultivars of B. napus. Sequence analyses revealed that in the allotetraploid genome of B. napus the gene BnSCT1 represents the C genome homologue from the B. oleracea progenitor whereas BnSCT2 was derived from the Brassica A genome of B. rapa. The BnSCT1 and BnSCT2 loci showed colinearity with the homologous Arabidopsis SNG2 gene locus although the genomic microstructure revealed the deletion of a cluster of three genes and several coding regions in the B. napus genome.} } @Article{IPB-1750, author = {Wadenbäck, J. and von Arnold, S. and Egertsdotter, U. and Walter, M. H. and Grima-Pettenati, J. and Goffner, D. and Gellerstedt, G. and Gullion, T. and Clapham, D. and}, title = {{Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR)}}, year = {2008}, pages = {379-392}, journal = {Transgenic Res.}, doi = {10.1007/s11248-007-9113-z}, volume = {17}, abstract = {An attractive objective in tree breeding is to reduce the content of lignin or alter its composition, in order to facilitate delignification in pulping. This has been achieved in transgenic angiosperm tree species. In this study we show for the first time that changes in lignin content and composition can be achieved in a conifer by taking a transgenic approach. Lignin content and composition have been altered in five-year-old transgenic plants of Norway spruce (Picea abies [L.] Karst) expressing the Norway spruce gene encoding cinnamoyl CoA reductase (CCR) in antisense orientation. The asCCR plants had a normal phenotype but smaller stem widths compared to the transformed control plants. The transcript abundance of the sense CCR gene was reduced up to 35% relative to the transformed control. The corresponding reduction in lignin content was up to 8%, which is at the lower limit of the 90–99% confidence intervals reported for natural variation. The contribution of H-lignin to the non-condensed fraction of lignin, as judged by thioacidolysis, was reduced up to 34%. The H-lignin content was strongly correlated with the total lignin content. Furthermore, the kappa number of small-scale Kraft pulps from one of the most down-regulated lines was reduced 3.5%. The transcript abundances of the various lignin biosynthetic genes were down-regulated indicating co-regulation of the biosynthetic pathway.} } @Article{IPB-1747, author = {Tretner, C. and Huth, U. and Hause, B. and}, title = {{Mechanostimulation of Medicago truncatula leads to enhanced levels of jasmonic acid}}, year = {2008}, pages = {2847-2856}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/ern145}, volume = {59}, abstract = {Wounding of plants leads to endogenous rise of jasmonic acid (JA) accompanied with the expression of a distinct set of genes. Among them are those coding for the allene oxide cyclase (AOC) that catalyses a regulatory step in JA biosynthesis, and for 1-deoxy-D-xylulose 5-phosphate synthase 2 (DXS2), an enzyme involved in isoprenoid biosynthesis. To address the question how roots and shoots of Medicago truncatula respond to mechanostimulation and wounding, M. truncatula plants were analysed in respect to JA levels as well as MtAOC1 and MtDXS2-1 transcript accumulation. Harvest-caused mechanostimulation resulted in a strong, but transient increase in JA level in roots and shoots followed by a transient increase in MtAOC1 transcript accumulation. Additional wounding of either shoots or roots led to further increased JA and MtAOC1 transcript levels in shoots, but not in roots. In situ hybridization revealed a cell-specific transcript accumulation of MtAOC1 after mechanostimulation in companion cells of the vascular tissue of the stem. AOC protein, however, was found to occur constitutively in vascular bundles. Further, transcript accumulation of MtDXS2-1 was similar to that of MtAOC1 in shoots, but its transcript levels were not enhanced in roots. Repeated touching of shoots increased MtAOC1 transcript levels and led to significantly shorter shoots and increased biomass. In conclusion, M. truncatula plants respond very sensitively to mechanostimulation with enhanced JA levels and altered transcript accumulation, which might contribute to the altered phenotype after repeated touching of plants.} } @Article{IPB-1746, author = {Tiedemann, J. and Rutten, T. and Mönke, G. and Vorwieger, A. and Rolletschek, H. and Meissner, D. and Milkowski, C. and Petereck, S. and Mock, H.-P. and Zank, T. and Bäumlein, H. and}, title = {{Dissection of a complex seed phenotype: Novel insights of FUSCA3 regulated developmental processes}}, year = {2008}, pages = {1-12}, journal = {Dev. Biol.}, doi = {10.1016/j.ydbio.2008.01.034}, volume = {317}, abstract = {A T-DNA insertion mutant of FUSCA3 (fus3-T) in Arabidopsis thaliana exhibits several of the expected deleterious effects on seed development, but not the formation of brown seeds, a colouration which results from the accumulation of large amounts of anthocyanin. A detailed phenotypic comparison between fus3-T and a known splice point mutant (fus3-3) revealed that the seeds from both mutants do not enter dormancy and can be rescued at an immature stage. Without rescue, mature fus3-3 seeds are non-viable, whereas those of fus3-T suffer only a slight loss in their germinability. A series of comparisons between the two mutants uncovered differences with respect to conditional lethality, in histological and sub-cellular features, and in the relative amounts of various storage compounds and metabolites present, leading to a further dissection of developmental processes in seeds and a partial reinterpretation of the complex seed phenotype. FUS3 function is now known to be restricted to the acquisition of embryo-dependent seed dormancy, the determination of cotyledonary cell identity, and the synthesis and accumulation of storage compounds. Based on DNA binding studies, a model is presented which can explain the differences between the mutant alleles. The fus3-T lesion is responsible for loss of function only, while the fus3-3 mutation induces various pleiotropic effects conditioned by a truncation gene product causing severe mis-differentiation.} } @Article{IPB-1741, author = {Sun, Z. and Hans, J. and Walter, M. H. and Matusova, R. and Beekwilder, J. and Verstappen, F. W. A. and Ming, Z. and van Echtelt, E. and Strack, D. and Bisseling, T. and Bouwmeester, H. J. and}, title = {{Cloning and characterisation of a maize carotenoid cleavage dioxygenase (ZmCCD1) and its involvement in the biosynthesis of apocarotenoids with various roles in mutualistic and parasitic interactions}}, year = {2008}, pages = {789-801}, journal = {Planta}, doi = {10.1007/s00425-008-0781-6}, volume = {228}, abstract = {Colonisation of maize roots by arbuscular mycorrhizal (AM) fungi leads to the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives). Other root apocarotenoids (strigolactones) are involved in signalling during early steps of the AM symbiosis but also in stimulation of germination of parasitic plant seeds. Both apocarotenoid classes are predicted to originate from cleavage of a carotenoid substrate by a carotenoid cleavage dioxygenase (CCD), but the precursors and cleavage enzymes are unknown. A Zea mays CCD (ZmCCD1) was cloned by RT-PCR and characterised by expression in carotenoid accumulating E. coli strains and analysis of cleavage products using GC–MS. ZmCCD1 efficiently cleaves carotenoids at the 9, 10 position and displays 78% amino acid identity to Arabidopsis thaliana CCD1 having similar properties. ZmCCD1 transcript levels were shown to be elevated upon root colonisation by AM fungi. Mycorrhization led to a decrease in seed germination of the parasitic plant Striga hermonthica as examined in a bioassay. ZmCCD1 is proposed to be involved in cyclohexenone and mycorradicin formation in mycorrhizal maize roots but not in strigolactone formation.} } @Article{IPB-1740, author = {Stenzel, I. and Ischebeck, T. and König, S. and Hołubowska, A. and Sporysz, M. and Hause, B. and Heilmann, I. and}, title = {{The Type B Phosphatidylinositol-4-Phosphate 5-Kinase 3 Is Essential for Root Hair Formation in Arabidopsis thaliana}}, year = {2008}, pages = {124-141}, journal = {Plant Cell}, doi = {10.1105/tpc.107.052852}, volume = {20}, abstract = {Root hairs are extensions of root epidermal cells and a model system for directional tip growth of plant cells. A previously uncharacterized Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K3) was identified and found to be expressed in the root cortex, epidermal cells, and root hairs. Recombinant PIP5K3 protein was catalytically active and converted phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Arabidopsis mutant plants homozygous for T-DNA–disrupted PIP5K3 alleles were compromised in root hair formation, a phenotype complemented by expression of wild-type PIP5K3 cDNA under the control of a 1500-bp PIP5K3 promoter fragment. Root hair–specific PIP5K3 overexpression resulted in root hair deformation and loss of cell polarity with increasing accumulation of PIP5K3 transcript. Using reestablishment of root hair formation in T-DNA mutants as a bioassay for physiological functionality of engineered PIP5K3 variants, catalytic activity was found to be essential for physiological function, indicating that PtdIns(4,5)P2 formation is required for root hair development. An N-terminal domain containing membrane occupation and recognition nexus repeats, which is not required for catalytic activity, was found to be essential for the establishment of root hair growth. Fluorescence-tagged PIP5K3 localized to the periphery of the apical region of root hair cells, possibly associating with the plasma membrane and/or exocytotic vesicles. Transient heterologous expression of full-length PIP5K3 in tobacco (Nicotiana tabacum) pollen tubes increased plasma membrane association of a PtdIns(4,5)P2-specific reporter in these tip-growing cells. The data demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P2 production in the apical region of root hair cells.} } @Article{IPB-1739, author = {Stenzel, I. and Hause, B. and Proels, R. and Miersch, O. and Oka, M. and Roitsch, T. and Wasternack, C. and}, title = {{The AOC promoter of tomato is regulated by developmental and environmental stimuli}}, year = {2008}, pages = {1859-1869}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2008.03.007}, volume = {69}, abstract = {The allene oxide cyclase (AOC) catalyzes the formation of cis-(\+)-12-oxophytodienoic acid, an intermediate in jasmonate biosynthesis and is encoded by a single copy gene in tomato. The full length AOC promoter isolated by genome walk contains 3600 bp. Transgenic tomato lines carrying a 1000 bp promoter fragment and the full length promoter, respectively, in front of the β-glucuronidase (GUS)-encoding uidA gene and several tobacco lines carrying the full length tomato AOC promoter before GUS were used to record organ- and tissue-specific promoter activities during development and in response to various stimuli. High promoter activities corresponding to immunocytochemically detected occurrence of the AOC protein were found in seeds and young seedlings and were confined to the root tip, hypocotyl and cotyledons of 3-d-old seedlings. In 10-d-old seedlings promoter activity appeared preferentially in the elongation zone. Fully developed tomato leaves were free of AOC promoter activity, but showed high activity upon wounding locally and systemically or upon treatment with JA, systemin or glucose. Tomato flowers showed high AOC promoter activities in ovules, sepals, anthers and pollen. Most of the promoter activity patterns found in tomato with the 1000 bp promoter fragment were also detected with the full length tomato AOC promoter in tobacco during development or in response to various stimuli. The data support a spatial and temporal regulation of JA biosynthesis during development and in response to environmental stimuli.} } @Article{IPB-1738, author = {Stehle, F. and Stubbs, M. T. and Strack, D. and Milkowski, C. and}, title = {{Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism}}, year = {2008}, pages = {775-787}, journal = {FEBS J.}, doi = {10.1111/j.1742-4658.2007.06244.x}, volume = {275}, abstract = {In plant secondary metabolism, β‐acetal ester‐dependent acyltransferases, such as the 1‐O ‐sinapoyl‐β‐glucose:l ‐malate sinapoyltransferase (SMT; EC 2.3.1.92), are homologous to serine carboxypeptidases. Mutant analyses and modeling of Arabidopsis SMT (AtSMT) have predicted amino acid residues involved in substrate recognition and catalysis, confirming the main functional elements conserved within the serine carboxypeptidase protein family. However, the functional shift from hydrolytic to acyltransferase activity and structure–function relationship of AtSMT remain obscure. To address these questions, a heterologous expression system for AtSMT has been developed that relies on Saccharomyces cerevisiae and an episomal leu2‐d vector. Codon usage adaptation of AtSMT cDNA raised the produced SMT activity by a factor of approximately three. N‐terminal fusion to the leader peptide from yeast proteinase A and transfer of this expression cassette to a high copy vector led to further increase in SMT expression by factors of 12 and 42, respectively. Finally, upscaling the biomass production by fermenter cultivation lead to another 90‐fold increase, resulting in an overall 3900‐fold activity compared to the AtSMT cDNA of plant origin. Detailed kinetic analyses of the recombinant protein indicated a random sequential bi‐bi mechanism for the SMT‐catalyzed transacylation, in contrast to a double displacement (ping‐pong) mechanism, characteristic of serine carboxypeptidases.} } @Article{IPB-1734, author = {Schliemann, W. and Ammer, C. and Strack, D. and}, title = {{Metabolite profiling of mycorrhizal roots of Medicago truncatula}}, year = {2008}, pages = {112-146}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2007.06.032}, volume = {69}, abstract = {Metabolite profiling of soluble primary and secondary metabolites, as well as cell wall-bound phenolic compounds from roots of barrel medic (Medicago truncatula) was carried out by GC–MS, HPLC and LC–MS. These analyses revealed a number of metabolic characteristics over 56 days of symbiotic interaction with the arbuscular mycorrhizal (AM) fungus Glomus intraradices, when compared to the controls, i.e. nonmycorrhizal roots supplied with low and high amounts of phosphate. During the most active stages of overall root mycorrhization, elevated levels of certain amino acids (Glu, Asp, Asn) were observed accompanied by increases in amounts of some fatty acids (palmitic and oleic acids), indicating a mycorrhiza-specific activation of plastidial metabolism. In addition, some accumulating fungus-specific fatty acids (palmitvaccenic and vaccenic acids) were assigned that may be used as markers of fungal root colonization. Stimulation of the biosynthesis of some constitutive isoflavonoids (daidzein, ononin and malonylononin) occurred, however, only at late stages of root mycorrhization. Increase of the levels of saponins correlated AM-independently with plant growth. Only in AM roots was the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives) observed. The structures of the unknown cyclohexenone derivatives were identified by spectroscopic methods as glucosides of blumenol C and 13-hydroxyblumenol C and their corresponding malonyl conjugates. During mycorrhization, the levels of typical cell wall-bound phenolics (e.g. 4-hydroxybenzaldehyde, vanillin, ferulic acid) did not change; however, high amounts of cell wall-bound tyrosol were exclusively detected in AM roots.Principal component analyses of nonpolar primary and secondary metabolites clearly separated AM roots from those of the controls, which was confirmed by an hierarchical cluster analysis. Circular networks of primary nonpolar metabolites showed stronger and more frequent correlations between metabolites in the mycorrhizal roots. The same trend, but to a lesser extent, was observed in nonmycorrhizal roots supplied with high amounts of phosphate. These results indicate a tighter control of primary metabolism in AM roots compared to control plants. Network correlation analyses revealed distinct clusters of amino acids and sugars/aliphatic acids with strong metabolic correlations among one another in all plants analyzed; however, mycorrhizal symbiosis reduced the cluster separation and enlarged the sugar cluster size. The amino acid clusters represent groups of metabolites with strong correlations among one another (cliques) that are differently composed in mycorrhizal and nonmycorrhizal roots. In conclusion, the present work shows for the first time that there are clear differences in development- and symbiosis-dependent primary and secondary metabolism of M. truncatula roots.} } @Article{IPB-1733, author = {Schliemann, W. and Ammer, C. and Strack, D. and}, title = {{Erratum to “Metabolite profiling of mycorrhizal roots of Medicago truncatula” [Phytochemistry 69 (2008) 112–146]}}, year = {2008}, pages = {1446-1447}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2008.02.004}, volume = {69}, } @Article{IPB-1732, author = {Schliemann, W. and Kolbe, B. and Schmidt, J. and Nimtz, M. and Wray, V. and}, title = {{Accumulation of apocarotenoids in mycorrhizal roots of leek (Allium porrum)}}, year = {2008}, pages = {1680-1688}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2008.02.015}, volume = {69}, abstract = {Colonization of the roots of leek (Allium porrum L.) by the arbuscular mycorrhizal fungus Glomus intraradices induced the formation of apocarotenoids, whose accumulation has been studied over a period of 25 weeks. Whereas the increase in the levels of the dominating cyclohexenone derivatives resembles the enhancement of root length colonization, the content of mycorradicin derivatives remains relatively low throughout. Structural analysis of the cyclohexenone derivatives by mass spectrometry and NMR spectroscopy showed that they are mono- and diglycosides of 13-hydroxyblumenol C and blumenol C acylated with 3-hydroxy-3-methyl-glutaric and/or malonic acid. Along with the isolation of three known compounds five others are shown to be hitherto unknown members of the fast-growing family of mycorrhiza-induced cyclohexenone conjugates.} } @Article{IPB-1730, author = {Schaarschmidt, S. and Hause, B. and}, title = {{Apoplastic invertases: Multi-faced players in the arbuscular mycorrhization}}, year = {2008}, pages = {317-319}, journal = {Plant Signal Behav.}, doi = {10.4161/psb.3.5.5307}, volume = {3}, abstract = {The mutualistic interaction of plants with arbuscular mycorrhizal (AM) fungi is characterized by an exchange of nutrients. The plant provides sugars in the form of hexoses to the heterotrophic fungus in return for phosphate as well as nitrogen, water, and micronutrients. Plant sucrose-cleaving enzymes are predicted to play a crucial role in hexose mobilization as these enzymes appear to be absent in the fungal partner. Here, recent findings concerning the function of plant apoplastic invertases in the AM symbiosis are discussed. Plants with modulated enzyme activity in roots and leaves provide additional insight on the complexity of the regulation of the AM interaction by apoplastic invertases as mycorrhization could be reduced or stimulated depending on the level of invertase activity and its tissue-specific expression.} } @Article{IPB-1729, author = {Rohde, B. and Hans, J. and Martens, S. and Baumert, A. and Hunziker, P. and Matern, U. and}, title = {{Anthranilate N-methyltransferase, a branch-point enzyme of acridone biosynthesis}}, year = {2008}, pages = {541-553}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2007.03360.x}, volume = {53}, abstract = {Acridone alkaloids formed by acridone synthase in Ruta graveolens L. are composed of N ‐methylanthraniloyl CoA and malonyl CoAs. A 1095 bp cDNA from elicited Ruta cells was expressed in Escherichia coli , and shown to encode S‐ adenosyl‐l ‐methionine‐dependent anthranilate N ‐methyltransferase. SDS–PAGE of the purified enzyme revealed a mass of 40 ± 2 kDa, corresponding to 40 059 Da for the translated polypeptide, whereas the catalytic activity was assigned to a homodimer. Alignments revealed closest relationships to catechol or caffeate O ‐methyltransferases at 56% and 55% identity (73% similarity), respectively, with little similarity (∼20%) to N ‐methyltransferases for purines, putrescine, glycine, or nicotinic acid substrates. Notably, a single Asn residue replacing Glu that is conserved in caffeate O ‐methyltransferases determines the catalytic efficiency. The recombinant enzyme showed narrow specificity for anthranilate, and did not methylate catechol, salicylate, caffeate, or 3‐ and 4‐aminobenzoate. Moreover, anthraniloyl CoA was not accepted. As Ruta graveolens acridone synthase also does not accept anthraniloyl CoA as a starter substrate, the anthranilate N ‐methylation prior to CoA activation is a key step in acridone alkaloid formation, channelling anthranilate from primary into secondary branch pathways, and holds promise for biotechnological applications. RT‐PCR amplifications and Western blotting revealed expression of the N ‐methyltransferase in all organs of Ruta plants, particularly in the flower and root, mainly associated with vascular tissues. This expression correlated with the pattern reported previously for expression of acridone synthase and acridone alkaloid accumulation.} } @Article{IPB-1717, author = {Meißner, D. and Albert, A. and Böttcher, C. and Strack, D. and Milkowski, C. and}, title = {{The role of UDP-glucose:hydroxycinnamate glucosyltransferases in phenylpropanoid metabolism and the response to UV-B radiation in Arabidopsis thaliana}}, year = {2008}, pages = {663-674}, journal = {Planta}, doi = {10.1007/s00425-008-0768-3}, volume = {228}, abstract = {Arabidopsis harbors four UDP-glycosyltransferases that convert hydroxycinnamates (HCAs) to 1-O-β-glucose esters, UGT84A1 (encoded by At4g15480), UGT84A2 (At3g21560), UGT84A3 (At4g15490), and UGT84A4 (At4g15500). To elucidate the role of the individual UGT84A enzymes in planta we analyzed gene expression, UGT activities and accumulation of phenylpropanoids in Arabidopsis wild type plants, ugt mutants and overexpressing lines. Individual ugt84A null alleles did not significantly reduce the gross metabolic flux to the accumulating compounds sinapoylcholine (sinapine) in seeds and sinapoylmalate in leaves. For the ugt84A2 mutant, LC/MS analysis revealed minor qualitative and quantitative changes of several HCA choline esters and of disinapoylspermidine in seeds. Overexpression of individual UGT84A genes caused increased enzyme activities but failed to produce significant changes in the pattern of accumulating HCA esters. For UGT84A3, our data tentatively suggest an impact on cell wall-associated 4-coumarate. Exposure of plants to enhanced UV-B radiation induced the UGT84A-encoding genes and led to a transient increase in sinapoylglucose and sinapoylmalate concentrations.} } @Article{IPB-1846, author = {Zum Felde, T. and Baumert, A. and Strack, D. and Becker, H. C. and Möllers, C. and}, title = {{Genetic variation for sinapate ester content in winter rapeseed (Brassica napus L.) and development of NIRS calibration equations}}, year = {2007}, pages = {291-296}, journal = {Plant Breed.}, doi = {10.1111/j.1439-0523.2007.01342.x}, volume = {126}, abstract = {Increasing the meal and protein quality of winter rapeseed (Brassica napus L.) for food and feed purposes is gaining importance in rapeseed breeding programmes. Rapeseed meal has a high content of phenolic acid esters, mainly sinapate esters, which have been shown to cause a dark colour and a bitter taste in rapeseed meal and derived protein products. The aim of the present study was to analyse the genetic variation for individual and total sinapate ester content, to develop Near Infrared Reflectance Spectroscopic (NIRS) calibrations, and to identify genotypes with a low sinapate ester content after testing in the field. The following sinapate esters were analysed by HPLC: sinapoylcholine (sinapine), sinapoylglucose, and a minor group of ‘other sinapate esters’ which includes free sinapate. A genotypically diverse set of seed samples of winter oilseed rape (old and new cultivars, breeding lines, resynthesized rapeseed) from different years and locations was collected, their NIRS spectra recorded and the samples were further analysed by HPLC. The complete NIRS calibration seed sample set (n \= 575) showed a large variation in total sinapate ester content, ranging from 3.2 to 12.7 mg sinapate equivalents per g seeds. The NIRS calibration equations showed high fractions of explained variances in cross validation () ranging from 0.75 (other sinapate esters) to 0.85 (sinapoylglucose). The standard errors of cross validation (SECV) ranged from 0.38 (other sinapate esters) to 0.70 mg/g seed (total sinapate esters). In validation and in independent validations the predicted results were not always acceptable, indicating that the NIRS calibrations need to be extended by analysing samples from new populations. Following replicated field experiments, a doubled haploid line obtained from the old Dutch cultivar Mansholts’ Hamburger Raps, and related DH lines from the cross DH Mansholts’ × Express were confirmed to have a 30–40% lower sinapate ester content compared to check cultivars.} } @Article{IPB-1782, author = {Fester, T. and Lohse, S. and Halfmann, K. and}, title = {{“Chromoplast” development in arbuscular mycorrhizal roots}}, year = {2007}, pages = {92-100}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2006.09.034}, volume = {68}, abstract = {The accumulation of apocarotenoids in arbuscular mycorrhizal (AM) roots suggests a dramatic reorganization of the plastids responsible for the biosynthesis of these compounds. This review describes the cytological and biochemical characterization of this phenomenon. The results presented suggest that plastids are key organelles for the establishment of the symbiotic interface of the AM symbiosis. In addition, a complex interplay of various plant cell components during the different functional phases of this interface is suggested. Arbuscule degradation appears to be of particular interest, as it correlates with the formation of the most extensive plastid structures and with apocarotenoid accumulation.} } @Article{IPB-1781, author = {Fester, T. and Hause, B. and}, title = {{Drought and symbiosis – why is abscisic acid necessary for arbuscular mycorrhiza?}}, year = {2007}, pages = {383-386}, journal = {New Phytol.}, doi = {10.1111/j.1469-8137.2007.02171.x}, volume = {175}, } @Article{IPB-1774, author = {Deepak, S. and Shailasree, S. and Kini, R. K. and Hause, B. and Shetty, S. H. and Mithöfer, A. and}, title = {{Role of hydroxyproline-rich glycoproteins in resistance of pearl millet against downy mildew pathogen Sclerospora graminicola}}, year = {2007}, pages = {323-333}, journal = {Planta}, doi = {10.1007/s00425-007-0484-4}, volume = {226}, abstract = {Hydroxyproline-rich glycoproteins (HRGPs) are important plant cell wall components involved in plant defense response to pathogen attack. In the present study, a resistant pearl millet (Pennisetum glaucum) cultivar, IP18292, was compared with a susceptible cultivar, 7042S, to investigate the contribution of HRGPs in the successful defense against the phytopathogenic oomycete S. graminicola. Northern hybridization using MeHRGP cDNA, a heterologous probe from cassava, indicated steady accumulation of HRGP transcripts, from 2 h.p.i. onwards with a maximum at 6 h.p.i., in the resistant cultivar. This is followed by HRGPs accumulation at about 8 h.p.i. as revealed by Western-blot analysis. Immunocytochemical localization by tissue printing and confocal immunofluorescence microscopy indicated cell walls of parenchymatic cells and the vascular tissue of coleoptile as sites of HRGP deposition. In vitro studies in the presence of horseradish peroxidase and H2O2 showed cross-linking of pearl millet HRGPs, which occurred parallel to isodityrosine accumulation. Inducible high isodityrosine content was also observed in vivo in the resistant cultivar. Here, H2O2 was found to accumulate as twin burst at 1 and 6 h.p.i., whereas in the susceptible cultivar only an early single peak was detectable. Moreover, the amount of hydroxyproline in HRGPs was about twice as high in the resistant as in the susceptible cultivar. These results suggest that cell wall strengthening in S. graminicola-infected resistant pearl millet is brought about by a combination of polypeptide cross-linking of isodityrosine as well as by the high content of hydroxyproline in HRGPs, and H2O2, in contrast to the susceptible plant.} } @Article{IPB-1772, author = {Cenzano, A. and Abdala, G. and Hause, B. and}, title = {{Cytochemical immuno-localization of allene oxide cyclase, a jasmonic acid biosynthetic enzyme, in developing potato stolons}}, year = {2007}, pages = {1449-1456}, journal = {J. Plant Physiol.}, doi = {10.1016/j.jplph.2006.10.007}, volume = {164}, abstract = {The involvement of jasmonates in the tuber development has been proved by the presence of many of these compounds in potato stolons, modification of their levels during the transition of the stolon into tuber, and induction of cell expansion upon exogenous jasmonates treatment. However, to date there is only little evidence of the presence of the jasmonic acid-biosynthetic enzymes in stolons or young tubers. As allene oxide cyclase represents the major control point for jasmonic acid biosynthesis, we studied the occurrence of allene oxide cyclase by immunological approaches in the early stages of tuber formation. In developing stolons, allene oxide cyclase as well as lipoxygenase were clearly detectable, but their levels did not change during development. Jasmonic acid treatment for 24 h, however, increased lipoxygenase and allene oxide cyclase protein levels in both developmental stages analyzed. In longitudinal sections of stolons of stages 1 and 2, allene oxide cyclase and lipoxygenase occurred in the apex and along the stolon axis. Allene oxide cyclase was clearly detectable in epidermal, cortical and pith parenchymatic cells, showing the highest levels in vascular tissues surrounding cells. Lipoxygenase was mainly located in the parenchymatic cortex cells. The occurrence of allene oxide cyclase in stolons together with the previous identification of jasmonates from developing stolons reveals that these organs are capable to synthesize and metabolize jasmonates.} } @Article{IPB-1770, author = {Burow, M. and Rice, M. and Hause, B. and Gershenzon, J. and Wittstock, U. and}, title = {{Cell- and tissue-specific localization and regulation of the epithiospecifier protein in Arabidopsis thaliana}}, year = {2007}, pages = {173-185}, journal = {Plant Mol. Biol.}, doi = {10.1007/s11103-007-9143-1}, volume = {64}, abstract = {The glucosinolate-myrosinase system found in plants of the order Brassicales is one of the best studied plant defense systems. Hydrolysis of the physiologically inert glucosinolates by hydrolytic enzymes called myrosinases, which only occurs upon tissue disruption, leads to the formation of biologically active compounds. The chemical nature of the hydrolysis products depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs promote the formation of epithionitriles and simple nitriles at the expense of the corresponding isothiocyanates which are formed through spontaneous rearrangement of the aglucone core structure. While isothiocyanates are toxic to a wide range of organisms, including insects, the ecological significance of nitrile formation and thus the role of ESP in plant-insect interactions is unclear. Here, we identified ESP-expressing cells in various organs and several developmental stages of different Arabidopsis thaliana ecotypes by immunolocalization. In the ecotype Landsberg erecta, ESP was found to be consistently present in the epidermal cells of all aerial parts except the anthers and in S-cells of the stem below the inflorescence. Analyses of ESP expression by quantitative real-time PCR, Western blotting, and ESP activity assays suggest that plants control the outcome of glucosinolate hydrolysis by regulation of ESP at both the transcriptional and the post-transcriptional levels. The localization of ESP in the epidermal cell layers of leaves, stems and reproductive organs supports the hypothesis that this protein has a specific function in defense against herbivores and pathogens.} } @Article{IPB-1814, author = {Pühler, A. and Strack, D. and}, title = {{Molecular basics of mycorrhizal symbioses}}, year = {2007}, pages = {6-7}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2006.09.001}, volume = {68}, } @Article{IPB-1813, author = {Phillips, M. A. and Walter, M. H. and Ralph, S. G. and Dąbrowska, P. and Luck, K. and Urós, E. M. and Boland, W. and Strack, D. and Rodríguez-Concepción, M. and Bohlmann, J. and Gershenzon, J. and}, title = {{Functional identification and differential expression of 1-deoxy-d-xylulose 5-phosphate synthase in induced terpenoid resin formation of Norway spruce (Picea abies)}}, year = {2007}, pages = {243-257}, journal = {Plant Mol. Biol.}, doi = {10.1007/s11103-007-9212-5}, volume = {65}, abstract = {Conifers produce terpenoid-based oleoresins as constitutive and inducible defenses against herbivores and pathogens. Much information is available about the genes and enzymes of the late steps of oleoresin terpenoid biosynthesis in conifers, but almost nothing is known about the early steps which proceed via the methylerythritol phosphate (MEP) pathway. Here we report the cDNA cloning and functional identification of three Norway spruce (Picea abies) genes encoding 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, and their differential expression in the stems of young saplings. Among them are representatives of both types of plant DXS genes. A single type I DXS gene is constitutively expressed in bark tissue and not affected by wounding or fungal application. In contrast, two distinct type II DXS genes, PaDXS2A and PaDXS2B, showed increased transcript abundance after these treatments as did two other genes of the MEP pathway tested, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 4-hydroxyl 3-methylbutenyl diphosphate reductase (HDR). We also measured gene expression in a Norway spruce cell suspension culture system that, like intact trees, accumulates monoterpenes after treatment with methyl jasmonate. These cell cultures were characterized by an up-regulation of monoterpene synthase gene transcripts and enzyme activity after elicitor treatment, as well as induced formation of octadecanoids, including jasmonic acid and 12-oxophytodienoic acid. Among the Type II DXS genes in cell cultures, PaDXS2A was induced by treatment with chitosan, methyl salicylate, and Ceratocystis polonica (a bark beetle-associated, blue-staining fungal pathogen of Norway spruce). However, PaDXS2B was induced by treatment with methyl jasmonate and chitosan, but was not affected by methyl salicylate or C. polonica. Our results suggest distinct functions of the three DXS genes in primary and defensive terpenoid metabolism in Norway spruce.} } @Article{IPB-1803, author = {Mittasch, J. and Strack, D. and Milkowski, C. and}, title = {{Secondary product glycosyltransferases in seeds of Brassica napus}}, year = {2007}, pages = {515-522}, journal = {Planta}, doi = {10.1007/s00425-006-0360-7}, volume = {225}, abstract = {This study describes a systematic screen for secondary product UDP-glycosyltransferases (UGTs; EC 2.4.1) involved in seed development of oilseed rape (Brassica napus) and was aimed at identifying genes related to UGT84A9 encoding UDP-glucose:sinapate glucosyltransferase (EC 2.4.1.120), a proven target for molecular breeding approaches to reduce the content of anti-nutritive sinapate esters. By RT-PCR with primers recognizing the conserved signature motif of UGTs, 13 distinct ESTs could be generated from seed RNA. Sequence analysis allowed to assign the isolated ESTs to groups B, D, E, and L of the UGT family. In an alternative approach, two open reading frames related to UGT84A9 were cloned from the B. napus genome and designated as UGT84A10 and UGT84A11, respectively. Functional expression of UGT84A10 revealed that the encoded enzyme catalyzes the formation of 1-O-acylglucosides (β-acetal esters) with several hydroxycinnamates whereas, in our hands, the recombinant UGT84A11 did not display this enzymatic activity. Semi-quantitative RT-PCR confirmed that the majority of potential UGTs specified by the isolated ESTs is differentially expressed. A pronounced transcriptional up-regulation during seed development was evident for UGT84A9 and one EST (BnGT3) clustering in group E of UGTs. UGT84A10 was highly induced in flowers and expressed to a moderate level in late seed maturation indicating a possible involvement in seed-specific sinapate ester biosynthesis.} } @Article{IPB-1794, author = {Hause, B. and Mrosk, C. and Isayenkov, S. and Strack, D. and}, title = {{Jasmonates in arbuscular mycorrhizal interactions}}, year = {2007}, pages = {101-110}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2006.09.025}, volume = {68}, abstract = {The mutualistic interaction between plants and arbuscular mycorrhizal (AM) fungi is believed to be regulated from the plant side among other signals by the action of phytohormones. Evidences for this are based mainly on application experiments and determination of phytohormone levels in AM roots by comparison to non-mycorrhizal roots. In case of jasmonates, additional proof is given by reverse genetic approaches, which led to first insights into their putative role in the establishment and functioning of the symbiosis. This review summarizes the current data about phytohormone action in AM roots and the role of jasmonates in particular.} } @Article{IPB-1791, author = {Guranowski, A. and Miersch, O. and Staswick, P. E. and Suza, W. and Wasternack, C. and}, title = {{Substrate specificity and products of side-reactions catalyzed by jasmonate:amino acid synthetase (JAR1)}}, year = {2007}, pages = {815-820}, journal = {FEBS Lett.}, doi = {10.1016/j.febslet.2007.01.049}, volume = {581}, abstract = {Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA‐amido conjugates, the most important of which appears to be JA‐Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono‐ and dinucleoside polyphosphates, which are side‐reaction products of many enzymes forming acyl ∼ adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile‐conjugates was observed for (±)‐JA and 9,10‐dihydro‐JA, while the rate of conjugation with 12‐hydroxy‐JA and OPC‐4 (3‐oxo‐2‐(2Z ‐pentenyl)cyclopentane‐1‐butyric acid) was only about 1–2% that for (±)‐JA. Of the two stereoisomers of JA, (−)‐JA and (\+)‐JA, rate of synthesis of the former was about 100‐fold faster than for (\+)‐JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg2\+, (−)‐JA and tripolyphosphate the ligase produces adenosine 5′‐tetraphosphate (p4A); (2) addition of isoleucine to that mixture halts the p4A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap3A) nor diadenosine tetraphosphate (Ap4A) and (4) Ap4A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA‐Ile.} } @Article{IPB-1789, author = {Gremillon, L. and Kiessling, J. and Hause, B. and Decker, E. L. and Reski, R. and Sarnighausen, E. and}, title = {{Filamentous temperature-sensitive Z (FtsZ) isoforms specifically interact in the chloroplasts and in the cytosol of Physcomitrella patens}}, year = {2007}, pages = {299-310}, journal = {New Phytol.}, doi = {10.1111/j.1469-8137.2007.02169.x}, volume = {176}, abstract = {Plant filamentous temperature‐sensitive Z (FtsZ) proteins have been reported to be involved in biological processes related to plastids. However, the precise functions of distinct isoforms are still elusive. Here, the intracellular localization of the FtsZ1‐1 isoform in a moss, Physcomitrella patens , was examined. Furthermore, the in vivo interaction behaviour of four distinct FtsZ isoforms was investigated.Localization studies of green fluorescent protein (GFP)‐tagged FtsZ1‐1 and fluorescence resonance energy transfer (FRET) analyses employing all dual combinations of four FtsZ isoforms were performed in transient protoplast transformation assays.FtsZ1‐1 is localized to network structures inside the chloroplasts and exerts influence on plastid division. Interactions between FtsZ isoforms occur in distinct ordered structures in the chloroplasts as well as in the cytosol.The results expand the view of the involvement of Physcomitrella FtsZ proteins in chloroplast and cell division. It is concluded that duplication and diversification of ftsZ genes during plant evolution were the main prerequisites for the successful remodelling and integration of the prokaryotic FtsZ‐dependent division mechanism into the cellular machineries of distinct complex processes in plants.} } @Article{IPB-1787, author = {Geissler, R. and Brandt, W. and Ziegler, J. and}, title = {{Molecular Modeling and Site-Directed Mutagenesis Reveal the Benzylisoquinoline Binding Site of the Short-Chain Dehydrogenase/Reductase Salutaridine Reductase}}, year = {2007}, pages = {1493-1503}, journal = {Plant Physiol.}, doi = {10.1104/pp.106.095166}, volume = {143}, abstract = {Recently, the NADPH-dependent short-chain dehydrogenase/reductase (SDR) salutaridine reductase (E.C. 1.1.1.248) implicated in morphine biosynthesis was cloned from Papaver somniferum. In this report, a homology model of the Papaver bracteatum homolog was created based on the x-ray structure of human carbonyl reductase 1. The model shows the typical α/β-folding pattern of SDRs, including the four additional helices αF′-1 to αF′-4 assumed to prevent the dimerization of the monomeric short-chain dehyrogenases/reductases. Site-directed mutagenesis of asparagine-152, serine-180, tyrosine-236, and lysine-240 resulted in enzyme variants with strongly reduced performance or inactive enzymes, showing the involvement of these residues in the proton transfer system for the reduction of salutaridine. The strong preference for NADPH over NADH could be abolished by replacement of arginine residues 44 and 48 by glutamic acid, confirming the interaction between the arginines and the 2′-phosphate group. Docking of salutaridine into the active site revealed nine amino acids presumably responsible for the high substrate specificity of salutaridine reductase. Some of these residues are arranged in the right position by an additional αE′ helix, which is not present in SDRs analyzed so far. Enzyme kinetic data from mutagenic replacement emphasize the critical role of these residues in salutaridine binding and provide the first data on the molecular interaction of benzylisoquinoline alkaloids with enzymes.} } @Article{IPB-1837, author = {Walter, M. H. and Floß, D. S. and Hans, J. and Fester, T. and Strack, D. and}, title = {{Apocarotenoid biosynthesis in arbuscular mycorrhizal roots: Contributions from methylerythritol phosphate pathway isogenes and tools for its manipulation}}, year = {2007}, pages = {130-138}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2006.09.032}, volume = {68}, abstract = {During colonization by arbuscular mycorrhizal (AM) fungi plant roots frequently accumulate two types of apocarotenoids (carotenoid cleavage products). Both compounds, C14 mycorradicin and C13 cyclohexenone derivatives, are predicted to originate from a common C40 carotenoid precursor. Mycorradicin is the chromophore of the “yellow pigment” responsible for the long-known yellow discoloration of colonized roots. The biosynthesis of apocarotenoids has been investigated with a focus on the two first steps of the methylerythritol phosphate (MEP) pathway catalyzed by 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR). In Medicago truncatula and other plants the DXS2 isogene appears to be specifically involved in the AM-mediated accumulation of apocarotenoids, whereas in the case of DXR a single gene contributes to both housekeeping and mycorrhizal (apo)carotenoid biosynthesis. Immunolocalization of DXR in mycorrhizal maize roots indicated an arbuscule-associated protein deposition, which occurs late in arbuscule development and accompanies arbuscule degeneration and breakdown. The DXS2 isogene is being developed as a tool to knock-down apocarotenoid biosynthesis in mycorrhizal roots by an RNAi strategy. Preliminary results from this approach provide starting points to suggest a new kind of function for apocarotenoids in mycorrhizal roots.} } @Article{IPB-1831, author = {Textor, S. and de Kraker, J.-W. and Hause, B. and Gershenzon, J. and Tokuhisa, J. G. and}, title = {{MAM3 Catalyzes the Formation of All Aliphatic Glucosinolate Chain Lengths in Arabidopsis}}, year = {2007}, pages = {60-71}, journal = {Plant Physiol.}, doi = {10.1104/pp.106.091579}, volume = {144}, abstract = {Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of ω-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in the Arabidopsis ecotype Columbia: MAM1 and MAM3 (formerly known as MAM-L). Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. Underlining its broad substrate specificity, MAM3 also accepts a range of non-Met-derived 2-oxoacids, e.g. converting pyruvate to citramalate and 2-oxoisovalerate to isopropylmalate, a step in leucine biosynthesis. To investigate its role in vivo, we identified plant lines with mutations in MAM3 that resulted in a complete lack or greatly reduced levels of long-chain glucosinolates. This phenotype could be complemented by reintroduction of a MAM3 expression construct. Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. The localization of MAM3 in the chloroplast suggests that this organelle is the site of Met chain elongation.} } @Article{IPB-1829, author = {ten Hoopen, P. and Hunger, A. and Muller, A. and Hause, B. and Kramell, R. and Wasternack, C. and Rosahl, S. and Conrad, U. and}, title = {{Immunomodulation of jasmonate to manipulate the wound response}}, year = {2007}, pages = {2525-2535}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/erm122}, volume = {58}, abstract = {Jasmonates are signals in plant stress responses and development. The exact mode of their action is still controversial. To modulate jasmonate levels intracellularly as well as compartment-specifically, transgenic Nicotiana tabacum plants expressing single-chain antibodies selected against the naturally occurring (3R,7R)-enantiomer of jasmonic acid (JA) were created in the cytosol and the endoplasmic reticulum. Consequently, the expression of anti-JA antibodies in planta caused JA-deficient phenotypes such as insensitivity of germinating transgenic seedlings towards methyl jasmonate and the loss of wound-induced gene expression. Results presented here suggest an essential role for cytosolic JA in the wound response of tobacco plants. The findings support the view that substrate availability takes part in regulating JA biosynthesis upon wounding. Moreover, high JA levels observed in immunomodulated plants in response to wounding suggest that tobacco plants are able to perceive a reduced level of physiologically active JA and attempt to compensate for this by increased JA accumulation.} } @Article{IPB-1826, author = {Stintzing, F. and Schliemann, W. and}, title = {{Pigments of Fly Agaric (Amanita muscaria)}}, year = {2007}, pages = {779-785}, journal = {Z. Naturforsch. C}, doi = {10.1515/znc-2007-11-1201}, volume = {62}, abstract = {The complex pigment pattern of fly agaric (Amanita muscaria) cap skins has been studied by LC-DAD and mass spectrometry. Among the betaxanthins the corresponding derivatives of serine, threonine, ethanolamine, alanine, Dopa, phenylalanine and tryptophan are reported for the first time to contribute to the pigment pattern of fly agarics. Betalamic acid, the chromophoric precursor of betaxanthins and betacyanins, muscaflavin and seco-dopas were also detected. Furthermore, the red-purple muscapurpurin and the red muscarubrin were tentatively assigned while further six betacyanin-like components could not be structurally allocated. Stability studies indicated a high susceptibility of pigment extracts to degradation which led to rapid colour loss thus rendering a complete characterization of betacyaninlike compounds impossible at present. Taking into account these difficulties the presented results may be a starting point for a comprehensive characterization of the pigment composition of fly agarics.} } @Article{IPB-1819, author = {Schaarschmidt, S. and González, M.-C. and Roitsch, T. and Strack, D. and Sonnewald, U. and Hause, B. and}, title = {{Regulation of Arbuscular Mycorrhization by Carbon. The Symbiotic Interaction Cannot Be Improved by Increased Carbon Availability Accomplished by Root-Specifically Enhanced Invertase Activity}}, year = {2007}, pages = {1827-1840}, journal = {Plant Physiol.}, doi = {10.1104/pp.107.096446}, volume = {143}, abstract = {The mutualistic interaction in arbuscular mycorrhiza (AM) is characterized by an exchange of mineral nutrients and carbon. The major benefit of AM, which is the supply of phosphate to the plant, and the stimulation of mycorrhization by low phosphate fertilization has been well studied. However, less is known about the regulatory function of carbon availability on AM formation. Here the effect of enhanced levels of hexoses in the root, the main form of carbohydrate used by the fungus, on AM formation was analyzed. Modulation of the root carbohydrate status was performed by expressing genes encoding a yeast (Saccharomyces cerevisiae)-derived invertase, which was directed to different subcellular locations. Using tobacco (Nicotiana tabacum) alc∷cwINV plants, the yeast invertase was induced in the whole root system or in root parts. Despite increased hexose levels in these roots, we did not detect any effect on the colonization with Glomus intraradices analyzed by assessment of fungal structures and the level of fungus-specific palmitvaccenic acid, indicative for the fungal carbon supply, or the plant phosphate content. Roots of Medicago truncatula, transformed to express genes encoding an apoplast-, cytosol-, or vacuolar-located yeast-derived invertase, had increased hexose-to-sucrose ratios compared to β-glucuronidase-transformed roots. However, transformations with the invertase genes did not affect mycorrhization. These data suggest the carbohydrate supply in AM cannot be improved by root-specifically increased hexose levels, implying that under normal conditions sufficient carbon is available in mycorrhizal roots. In contrast, tobacco rolC∷ppa plants with defective phloem loading and tobacco pyk10∷InvInh plants with decreased acid invertase activity in roots exhibited a diminished mycorrhization.} } @Article{IPB-1818, author = {Schaarschmidt, S. and Kopka, J. and Ludwig-Müller, J. and Hause, B. and}, title = {{Regulation of arbuscular mycorrhization by apoplastic invertases: enhanced invertase activity in the leaf apoplast affects the symbiotic interaction}}, year = {2007}, pages = {390-405}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2007.03150.x}, volume = {51}, abstract = {The effect of constitutive invertase overexpression on the arbuscular mycorrhiza (AM) is shown. The analysis of the enhanced potential for sucrose cleavage was performed with a heterozygous line of Nicotiana tabacum 35S::cwINV expressing a chimeric gene encoding apoplast‐located yeast‐derived invertase with the CaMV35S promoter. Despite the 35S promoter, roots of the transgenic plants showed no or only minor effects on invertase activity whereas the activity in leaves was increased at different levels. Plants with strongly elevated leaf invertase activity, which exhibited a strong accumulation of hexoses in source leaves, showed pronounced phenotypical effects like stunted growth and chlorosis, and an undersupply of the root with carbon. Moreover, transcripts of PR (pathogenesis related) genes accumulated in the leaves. In these plants, mycorrhization was reduced. Surprisingly, plants with slightly increased leaf invertase activity showed a stimulation of mycorrhization, particularly 3 weeks after inoculation. Compared with wild‐type, a higher degree of mycorrhization accompanied by a higher density of all fungal structures and a higher level of Glomus intraradices ‐specific rRNA was detected. Those transgenic plants showed no accumulation of hexoses in the source leaves, minor phenotypical effects and no increased PR gene transcript accumulation. The roots had even lower levels of phenolic compounds (chlorogenic acid and scopolin), amines (such as tyramine, dopamine, octopamine and nicotine) and some amino acids (including 5‐amino‐valeric acid and 4‐amino‐butyric acid), as well as an increased abscisic acid content compared with wild‐type. Minor metabolic changes were found in the leaves of these plants. The changes in metabolism and defense status of the plant and their putative role in the formation of an AM symbiosis are discussed.} } @INBOOK{IPB-118, author = {Wasternack, C. and Hause, B. and Stenzel, I. and Goetz, S. and Feussner, I. and Miersch, O. and}, title = {{Proceeding of the 17th Int. Symp. on Plant Lipids}}, year = {2007}, pages = {107-111}, chapter = {{Jasmonate signaling in tomato – The input of tissue-specific occurrence of allene oxide cyclase and JA metabolites}}, editor = {Benning C., Ollrogge, J.}, } @INBOOK{IPB-113, author = {Hinneburg, A. and Porzel, A. and Wolfram, K. and}, title = {{Bioinformatics Research and Development. BIRD 2007}}, year = {2007}, pages = {424-438}, chapter = {{An Evaluation of Text Retrieval Methods for Similarity Search of Multi-dimensional NMR-Spectra}}, journal = {Lecture Notes in Computer Science}, doi = {10.1007/978-3-540-71233-6_33}, volume = {4414}, abstract = {Searching and mining nuclear magnetic resonance (NMR)-spectra of naturally occurring substances is an important task to investigate new potentially useful chemical compounds. Multi-dimensional NMR-spectra are relational objects like documents, but consists of continuous multi-dimensional points called peaks instead of words. We develop several mappings from continuous NMR-spectra to discrete text-like data. With the help of those mappings any text retrieval method can be applied. We evaluate the performance of two retrieval methods, namely the standard vector space model and probabilistic latent semantic indexing (PLSI). PLSI learns hidden topics in the data, which is in case of 2D-NMR data interesting in its owns rights. Additionally, we develop and evaluate a simple direct similarity function, which can detect duplicates of NMR-spectra. Our experiments show that the vector space model as well as PLSI, which are both designed for text data created by humans, can effectively handle the mapped NMR-data originating from natural products. Additionally, PLSI is able to find meaningful ”topics” in the NMR-data.} } @Article{IPB-1927, author = {Ziegler, J. and Voigtländer, S. and Schmidt, J. and Kramell, R. and Miersch, O. and Ammer, C. and Gesell, A. and Kutchan, T. M. and}, title = {{Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis}}, year = {2006}, pages = {177-192}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2006.02860.x}, volume = {48}, abstract = {Plants of the order Ranunculales, especially members of the species Papaver , accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum ) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum , 69 show increased expression in morphinan alkaloid‐containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7‐epi ‐salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo‐keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.} } @Article{IPB-1882, author = {Kaiser, H. and Richter, U. and Keiner, R. and Brabant, A. and Hause, B. and Dräger, B. and}, title = {{Immunolocalisation of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tuber sprouts}}, year = {2006}, pages = {127-137}, journal = {Planta}, doi = {10.1007/s00425-006-0335-8}, volume = {225}, abstract = {Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.} } @Article{IPB-1880, author = {Isayenkova, J. and Wray, V. and Nimtz, M. and Strack, D. and Vogt, T. and}, title = {{Cloning and functional characterisation of two regioselective flavonoid glucosyltransferases from Beta vulgaris}}, year = {2006}, pages = {1598-1612}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2006.06.026}, volume = {67}, abstract = {Two full-length cDNAs encoding flavonoid-specific glucosyltransferases, UGT73A4 and UGT71F1, were isolated from a cDNA library of Beta vulgaris (Amaranthaceae) cell suspension cultures. They displayed high identity to position-specific betanidin and flavonoid glucosyltransferases from Dorotheanthus bellidiformis (Aizoaceae) and to enzymes with similar substrate specificities from various plant families. The open reading frame of the sequences encode proteins of 476 (UGT73A4) and 492 (UGT71F1) amino acids with calculated molecular masses of 54.07 kDa and 54.39 kDa, and isoelectric points of 5.8 and 5.6, respectively. Both enzymes were functionally expressed in Escherichia coli as His- and GST-tagged proteins, respectively. They exhibited a broad substrate specificity, but a distinct regioselectivity, glucosylating a variety of flavonols, flavones, flavanones, and coumarins. UGT73A4 showed a preference for the 4′- and 7-OH position in the flavonoids, whereas UGT71F1 preferentially glucosylated the 3- or the 7-OH position. Glucosylation of betanidin, the aglycone of the major betacyanin, betanin, in B. vulgaris was also observed to a low extent by both enzymes. Several O-glycosylated vitexin derivatives isolated from leaves of young B. vulgaris plants and rutin obtained from B. vulgaris tissue culture are discussed as potential endogenous products of UGT73A4 and UGT71F1. The results are analyzed with regard to evolution and specificity of plant natural product glucosyltransferases.} } @Article{IPB-1865, author = {Frenzel, A. and Tiller, N. and Hause, B. and Krajinski, F. and}, title = {{The conserved arbuscular mycorrhiza-specific transcription of the secretory lectin MtLec5 is mediated by a short upstream sequence containing specific protein binding sites}}, year = {2006}, pages = {792-800}, journal = {Planta}, doi = {10.1007/s00425-006-0262-8}, volume = {224}, abstract = {In Medicago truncatula a family of mycorrhiza-specific expressed lectins has been identified recently, but the function and regulation of these lectins during the arbuscular mycorrhiza symbiosis are still unknown. In order to characterize a first member of this protein family, MtLec5 was analyzed concerning its localization and regulation. Confocal laser scanning microscopy showed that MtLec5 is a secretory protein indicating a role as a vegetative storage protein, which is specifically expressed in mycorrhizal root systems. To study the molecular mechanisms leading to the mycorrhiza-specific transcription, deletion studies of pMtLec5 were done using reporter gene fusions. Potential cis-acting elements could be narrowed down to a 150 bp fragment that was located approximately at −300/−150 according to the transcription start, suggesting the binding of positive regulators to this area. Similar expression pattern of the reporter gene was found after transforming roots of the non-legume Nicotiana tabacum with the heterologous promoter–reporter fusions. This indicated that the observed mycorrhiza-specific transcriptional induction is not legume-specific. Electrophoretic mobility shift assays showed that several factors which were exclusively present in mycorrhizal roots bind within the 150 bp promoter area. This strengthens the hypothesis of positive regulators mediating the AM-specific gene expression.} } @Article{IPB-1860, author = {Delker, C. and Stenzel, I. and Hause, B. and Miersch, O. and Feussner, I. and Wasternack, C. and}, title = {{Jasmonate Biosynthesis in Arabidopsis thaliana - Enzymes, Products, Regulation}}, year = {2006}, pages = {297-306}, journal = {Plant Biol.}, doi = {10.1055/s-2006-923935}, volume = {8}, abstract = {Among the plant hormones jasmonic acid and related derivatives are known to mediate stress responses and several developmental processes. Biosynthesis, regulation, and metabolism of jasmonic acid in Arabidopsis thaliana are reviewed, including properties of mutants of jasmonate biosynthesis. The individual signalling properties of several jasmonates are described.} } @Article{IPB-1918, author = {Wasternack, C. and Stenzel, I. and Hause, B. and Hause, G. and Kutter, C. and Maucher, H. and Neumerkel, J. and Feussner, I. and Miersch, O. and}, title = {{The wound response in tomato – Role of jasmonic acid}}, year = {2006}, pages = {297-306}, journal = {J. Plant Physiol.}, doi = {10.1016/j.jplph.2005.10.014}, volume = {163}, abstract = {Plants respond to mechanical wounding or herbivore attack with a complex scenario of sequential, antagonistic or synergistic action of different signals leading to defense gene expression. Tomato plants were used as a model system since the peptide systemin and the lipid-derived jasmonic acid (JA) were recognized as essential signals in wound-induced gene expression. In this review recent data are discussed with emphasis on wound-signaling in tomato. The following aspects are covered: (i) systemin signaling, (ii) JA biosynthesis and action, (iii) orchestration of various signals such as JA, H2O2, NO, and salicylate, (iv) local and systemic response, and (v) amplification in wound signaling. The common occurrence of JA biosynthesis and systemin generation in the vascular bundles suggest JA as the systemic signal. Grafting experiments with JA-deficient, JA-insensitive and systemin-insensitive mutants strongly support this assumption.} } @Article{IPB-1908, author = {Strack, D. and Fester, T. and}, title = {{Isoprenoid metabolism and plastid reorganization in arbuscular mycorrhizal roots}}, year = {2006}, pages = {22-34}, journal = {New Phytol.}, doi = {10.1111/j.1469-8137.2006.01837.x}, volume = {172}, abstract = {Plant root‐colonizing arbuscular mycorrhizal (AM) fungi activate the methylerythritol phosphate pathway, carotenoid biosynthesis and oxidative carotenoid cleavage in roots, leading to C13 and C14 apocarotenoids, that is, cyclohexenone and mycorradicin derivatives. Mycorradicin causes the characteristic yellow coloration of many AM roots accumulating within a complex mixture of unknown components. The accumulating C13 cyclohexenones exhibit various ring substitutions and different glycosyl moieties. Transcript levels of the first two enzymes of the MEP pathway, 1‐deoxy‐d ‐xylulose 5‐phosphate synthase and 1‐deoxy‐d ‐xylulose 5‐phosphate reductoisomerase, and of the carotenoid pathway, phytoene desaturase and ζ‐carotene desaturase, along with a carotenoid‐cleaving dioxygenase, are markedly increased in AM roots. This correlates with proliferation and reorganization of root plastids. These results allow at this point only speculation about the significance of apocarotenoid accumulation: participation in the production of signaling molecules and control of fungal colonization or protection against soil‐borne pathogens; protection of root cells against oxidative damage of membranes by reactive oxygen species; and promotion of the symbiotic interactions between plant roots and AM fungi.} } @Article{IPB-1907, author = {Stehle, F. and Brandt, W. and Milkowski, C. and Strack, D. and}, title = {{Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism}}, year = {2006}, pages = {6366-6374}, journal = {FEBS Lett.}, doi = {10.1016/j.febslet.2006.10.046}, volume = {580}, abstract = {Structures of the serine carboxypeptidase‐like enzymes 1‐O ‐sinapoyl‐β‐glucose:l ‐malate sinapoyltransferase (SMT) and 1‐O ‐sinapoyl‐β‐glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the α/β‐hydrolase fold as scaffold for the catalytic triad Ser‐His‐Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1‐O ‐sinapoyl‐β‐glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1‐O ‐sinapoyl‐β‐glucose and l ‐malate. By conformational change, Arg322 transfers l ‐malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1‐O ‐sinapoyl‐β‐glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.} } @Article{IPB-1903, author = {Sharma, V. K. and Monostori, T. and Göbel, C. and Hänsch, R. and Bittner, F. and Wasternack, C. and Feussner, I. and Mendel, R. R. and Hause, B. and Schulze, J. and}, title = {{Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature}}, year = {2006}, pages = {264-276}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2005.11.009}, volume = {67}, abstract = {Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence.} } @Article{IPB-1900, author = {Schliemann, W. and Schneider, B. and Wray, V. and Schmidt, J. and Nimtz, M. and Porzel, A. and Böhm, H. and}, title = {{Flavonols and an indole alkaloid skeleton bearing identical acylated glycosidic groups from yellow petals of Papaver nudicaule}}, year = {2006}, pages = {191-201}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2005.11.002}, volume = {67}, abstract = {From yellow petals of Iceland poppy, besides the known flavonoid gossypitrin, seven kaempferol derivatives were isolated. In addition to kaempferol 3-O-β-sophoroside and kaempferol 3-O-β-sophoroside-7-O-β-glucoside, known from other plants, the mono- and dimalonyl conjugates of the latter were identified by MS and NMR spectroscopy. Structure analyses of a set of co-occurring pigments, the nudicaulins, revealed that they have the identical acylated glycoside moieties attached to a pentacyclic indole alkaloid skeleton for which the structure of 19-(4-hydroxyphenyl)-10H-1,10-ethenochromeno[2,3-b]indole-6,8,18-triol was deduced from MS and NMR as well as chemical and chiroptical methods.} } @Article{IPB-1899, author = {Schliemann, W. and Schmidt, J. and Nimtz, M. and Wray, V. and Fester, T. and Strack, D. and}, title = {{Erratum to “Accumulation of apocarotenoids in mycorrhizal roots of Ornithogalum umbellatum” [Phytochem. 67 (2006) 1196–1205]}}, year = {2006}, pages = {2090}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2006.07.018}, volume = {67}, } @Article{IPB-1898, author = {Schliemann, W. and Schmidt, J. and Nimtz, M. and Wray, V. and Fester, T. and Strack, D. and}, title = {{Accumulation of apocarotenoids in mycorrhizal roots of Ornithogalum umbellatum}}, year = {2006}, pages = {1196-1205}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2006.05.005}, volume = {67}, abstract = {Colonization of roots of Ornithogalum umbellatum by the arbuscular mycorrhizal fungus Glomus intraradices induced the accumulation of different types of apocarotenoids. In addition to the mycorrhiza-specific occurrence of cyclohexenone derivatives and the “yellow pigment” described earlier, free mycorradicin and numerous mycorradicin derivatives were detected in a complex apocarotenoid mixture for the first time. From the accumulation pattern of the mycorradicin derivatives their possible integration into the continuously accumulating “yellow pigment” is suggested. Structure analyses of the cyclohexenone derivatives by MS and NMR revealed that they are mono-, di- and branched triglycosides of blumenol C, 13-hydroxyblumenol C, and 13-nor-5-carboxy-blumenol C, some of which contain terminal rhamnose as sugar moiety.} } @Article{IPB-1897, author = {Schaarschmidt, S. and Roitsch, T. and Hause, B. and}, title = {{Arbuscular mycorrhiza induces gene expression of the apoplastic invertase LIN6 in tomato (Lycopersicon esculentum) roots}}, year = {2006}, pages = {4015-4023}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/erl172}, volume = {57}, abstract = {Extracellular invertases are suggested to play a crucial role in the arbuscular mycorrhiza (AM) symbiosis to fulfil the increased sink function of the mycorrhizal root and the supply of the obligate biotrophic AM fungus with hexoses. In tomato (Lycopersicon esculentum), LIN6 represents an apoplastic invertase which is described as a key enzyme in establishing and maintaining sink metabolism. In this study, transcript levels of LIN6 were analysed in tomato roots colonized with the AM fungus Glomus intraradices. Using real-time RT–PCR, a nearly 3-fold increase in LIN6 mRNA levels was detected at late stages of mycorrhization (11 weeks after inoculation). A 1.8-fold induction could already be achieved at earlier stages (5 weeks after inoculation) using higher inoculum concentrations, whereas wounding of non-mycorrhizal roots resulted in up to 12-fold enhanced LIN6 transcripts. As revealed by in situ hybridization, the expression of LIN6 upon mycorrhization was specifically restricted to colonized cells and to the central cylinder. Such a strongly localized pattern due to mycorrhizal cells and to the central core could also be shown for promoter activity using transgenic Nicotiana tabacum plants expressing the gene coding for β-glucuronidase under the control of the LIN6 promoter. The moderate induction of LIN6 expression in mycorrhizal tomato roots compared with stress-stimulated induction suggested a fine-tuning in the activation of sink metabolism in the mutualistic interaction, avoiding stress-induced defence reactions.} } @Article{IPB-1886, author = {Lohse, S. and Hause, B. and Hause, G. and Fester, T. and}, title = {{FtsZ Characterization and Immunolocalization in the Two Phases of Plastid Reorganization in Arbuscular Mycorrhizal Roots of Medicago truncatula}}, year = {2006}, pages = {1124-1134}, journal = {Plant Cell Physiol.}, doi = {10.1093/pcp/pcj083}, volume = {47}, abstract = {We have analyzed plastid proliferation in root cortical cells of Medicago truncatula colonized by arbuscular mycorrhizal (AM) fungi by concomitantly labeling fungal structures, root plastids, a protein involved in plastid division (FtsZ1) and a protein involved in the biosynthesis of AM-specific apocarotenoids. Antibodies directed against FtsZ1 have been generated after heterologous expression of the respective gene from M. truncatula and characterization of the gene product. Analysis of enzymatic activity and assembly experiments showed similar properties of this protein when compared with the bacterial proteins. Immunocytological experiments allowed two phases of fungal and plastid development to be clearly differentiated and plastid division to be monitored during these phases. In the early phase of arbuscule development, lens-shaped plastids, intermingled with the arbuscular branches, divide frequently. Arbuscule degradation, in contrast, is characterized by large, tubular plastids, decorated by a considerable number of FtsZ division rings.} } @INBOOK{IPB-128, author = {Wolfram, K. and Porzel, A. and Hinneburg, A. and}, title = {{Knowledge Discovery in Databases: PKDD 2006}}, year = {2006}, pages = {650-658}, chapter = {{Similarity Search for Multi-dimensional NMR-Spectra of Natural Products}}, journal = {Lecture Notes in Computer Science}, doi = {10.1007/11871637_67}, volume = {4213}, abstract = {Searching and mining nuclear magnetic resonance (NMR)-spectra of naturally occurring products is an important task to investigate new potentially useful chemical compounds. We develop a set-based similarity function, which, however, does not sufficiently capture more abstract aspects of similarity. NMR-spectra are like documents, but consists of continuous multi-dimensional points instead of words. Probabilistic semantic indexing (PLSI) is an retrieval method, which learns hidden topics. We develop several mappings from continuous NMR-spectra to discrete text-like data. The new mappings include redundancies into the discrete data, which proofs helpful for the PLSI-model used afterwards. Our experiments show that PLSI, which is designed for text data created by humans, can effectively handle the mapped NMR-data originating from natural products. Additionally, PLSI combined with the new mappings is able to find meaningful ”topics” in the NMR-data.} } @INBOOK{IPB-121, author = {Hause, B. and Frugier, F. and Crespi, M. and}, title = {{Medicago truncatula Handbook}}, year = {2006}, chapter = {{Immunolocalization}}, url = {https://www.noble.org/medicago-handbook/}, abstract = {This chapter presents an overview about the immunocytochemical techniques using in the model legume Medicago truncatula. After a short introduction into the basics of immunocytochemistry, detailed protocols that can be used to perform immunolabelling on light, confocal and electron microscopical level are listed. These protocols are successfully applied in the author’s laboratories to obtain information about the localization of various proteins in a range of tissues from Medicago truncatula. Depending on facilities to perform sectioning and the microscopical equipment, modifications might be required to suit individual demands. Therefore, please use these protocols as a starting point and adjust them after consulting experienced personnel at your local facilities.} } @Article{IPB-1969, author = {Lohse, S. and Schliemann, W. and Ammer, C. and Kopka, J. and Strack, D. and Fester, T. and}, title = {{Organization and Metabolism of Plastids and Mitochondria in Arbuscular Mycorrhizal Roots of Medicago truncatula}}, year = {2005}, pages = {329-340}, journal = {Plant Physiol.}, doi = {10.1104/pp.105.061457}, volume = {139}, abstract = {Colonization of root cortical cells by arbuscular mycorrhizal fungi leads to marked cytological changes of plastids and mitochondria. Plastids in particular are forming tubular extensions partially connecting individual organelles in a network-like way. These cytological changes correspond to an increased need for plastid and mitochondrial products during establishment and functioning of the symbiosis. The analysis of metabolite and transcript levels in mycorrhizal and nonmycorrhizal roots from Medicago truncatula revealed concomitant changes regarding a number of metabolic pathways. Our results indicate the activation of the mitochondrial tricarboxylic acid cycle and of plastid biosynthetic pathways producing fatty acids, amino acids, and apocarotenoids. These observations provide a general overview of structural and metabolic changes of plastids and mitochondria during colonization of root cortical cells by arbuscular mycorrhizal fungi.} } @Article{IPB-1968, author = {Liu, S. and Chen, K. and Schliemann, W. and Strack, D. and}, title = {{Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC-ESI[sol ]MS}}, year = {2005}, pages = {86-89}, journal = {Phytochem. Anal.}, doi = {10.1002/pca.816}, volume = {16}, abstract = {A simple method involving polyamide column chromatography in combination with HPLC‐PAD and HPLC‐ESI[sol ]MS for isolating and identifying two kinds of lignans, arctiin and arctigenin, in the leaves of burdock (Arctium lappa L.) has been established. After extraction of burdock leaves with 80% methanol, the aqueous phase of crude extracts was partitioned between water and chloroform and the aqueous phase was fractionated on a polyamide glass column. The fraction, eluting with 100% methanol, was concentrated and gave a white precipitate at 4°C from which two main compounds were purified by semi‐preparative HPLC. In comparison with the UV and ESI‐MS spectra and the HPLC retention time of authentic standards, the compounds were determined to be arctiin and arctigenin. The extraction[sol ]separation technique was validated using an internal standard method. Copyright © 2005 John Wiley \& Sons, Ltd.} } @Article{IPB-1960, author = {Kramell, R. and Schmidt, J. and Herrmann, G. and Schliemann, W. and}, title = {{N-(Jasmonoyl)tyrosine-Derived Compounds from Flowers of Broad Beans (Vicia faba)}}, year = {2005}, pages = {1345-1349}, journal = {J. Nat. Prod.}, doi = {10.1021/np0501482}, volume = {68}, abstract = {Two new amide-linked conjugates of jasmonic acid, N-[(3R,7R)-(−)-jasmonoyl]-(S)-dopa (3) and N-[(3R,7R)-(−)-jasmonoyl]-dopamine (5), were isolated in addition to the known compound N-[(3R,7R)-(−)-jasmonoyl]-(S)-tyrosine (2) from the methanolic extract of flowers of broad bean (Vicia faba). Their structures were proposed on the basis of spectroscopic data (LC-MS/MS) and chromatographic properties on reversed and chiral phases and confirmed by partial syntheses. Furthermore, tyrosine conjugates of two cucurbic acid isomers (7, 8) were detected and characterized by LC-MS. Crude enzyme preparations from flowers of V. faba hydroxylated both (±)-2 and N-[(3R,7R/3S,7S)-(−)-jasmonoyl]tyramine [(±)-4] to (±)-3 and (±)-5, respectively, suggesting a possible biosynthetic relationship. In addition, a commercial tyrosinase (mushroom) and a tyrosinase-containing extract from hairy roots of red beet exhibited the same catalytic properties, but with different substrate specificities. The conjugates (±)-2, (±)-3, (±)-4, and (±)-5 exhibited in a bioassay low activity to elicit alkaloid formation in comparison to free (±)-jasmonic acid [(±)-1].} } @Article{IPB-1959, author = {Isayenkov, S. and Mrosk, C. and Stenzel, I. and Strack, D. and Hause, B. and}, title = {{Suppression of Allene Oxide Cyclase in Hairy Roots of Medicago truncatula Reduces Jasmonate Levels and the Degree of Mycorrhization with Glomus intraradices}}, year = {2005}, pages = {1401-1410}, journal = {Plant Physiol.}, doi = {10.1104/pp.105.069054}, volume = {139}, abstract = {During the symbiotic interaction between Medicago truncatula and the arbuscular mycorrhizal (AM) fungus Glomus intraradices, an endogenous increase in jasmonic acid (JA) occurs. Two full-length cDNAs coding for the JA-biosynthetic enzyme allene oxide cyclase (AOC) from M. truncatula, designated as MtAOC1 and MtAOC2, were cloned and characterized. The AOC protein was localized in plastids and found to occur constitutively in all vascular tissues of M. truncatula. In leaves and roots, MtAOCs are expressed upon JA application. Enhanced expression was also observed during mycorrhization with G. intraradices. A partial suppression of MtAOC expression was achieved in roots following transformation with Agrobacterium rhizogenes harboring the MtAOC1 cDNA in the antisense direction under control of the cauliflower mosaic virus 35S promoter. In comparison to samples transformed with 35S∷uidA, roots with suppressed MtAOC1 expression exhibited lower JA levels and a remarkable delay in the process of colonization with G. intraradices. Both the mycorrhization rate, quantified by fungal rRNA, and the arbuscule formation, analyzed by the expression level of the AM-specific gene MtPT4, were affected. Staining of fungal material in roots with suppressed MtAOC1 revealed a decreased number of arbuscules, but these did not exhibit an altered structure. Our results indicate a crucial role for JA in the establishment of AM symbiosis.} } @Article{IPB-1958, author = {Hüsken, A. and Baumert, A. and Milkowski, C. and Becker, H. C. and Strack, D. and Möllers, C. and}, title = {{Resveratrol glucoside (Piceid) synthesis in seeds of transgenic oilseed rape (Brassica napus L.)}}, year = {2005}, pages = {1553-1562}, journal = {Theor. Appl. Genet.}, doi = {10.1007/s00122-005-0085-1}, volume = {111}, abstract = {Resveratrol is a phytoalexin produced in various plants like wine, peanut or pine in response to fungal infection or UV irradiation, but it is absent in members of the Brassicaceae. Moreover, resveratrol and its glucoside (piceid) are considered to have beneficial effects on human health, known to reduce heart disease, arteriosclerosis and cancer mortality. Therefore, the introduction of the gene encoding stilbene synthase for resveratrol production in rapeseed is a tempting approach to improve the quality of rapeseed products. The stilbene synthase gene isolated from grapevine (Vitis vinifera L.) was cloned under control of the seed-specific napin promotor and introduced into rapeseed (Brassica napus L.) by Agrobacterium-mediated co-transformation together with a ds-RNA-interference construct deduced from the sequence of the key enzyme for sinapate ester biosynthesis, UDP-glucose:sinapate glucosyltransferase (BnSGT1), assuming that the suppression of the sinapate ester biosynthesis may increase the resveratrol production in seeds through the increased availability of the precursor 4-coumarate. Resveratrol glucoside (piceid) was produced at levels up to 361 μg/g in the seeds of the primary transformants. This value exceeded by far piceid amounts reported from B. napus expressing VST1 in the wild type sinapine background. There was no significant difference in other important agronomic traits, like oil, protein, fatty acid and glucosinolate content in comparison to the control plants. In the third seed generation, up to 616 μg/g piceid was found in the seeds of a homozygous T3-plant with a single transgene copy integrated. The sinapate ester content in this homozygous T3-plant was reduced from 7.43 to 2.40 mg/g. These results demonstrate how the creation of a novel metabolic sink could divert the synthesis towards the production of piceid rather than sinapate ester, thereby increasing the value of oilseed products.} } @Article{IPB-1957, author = {Hüsken, A. and Baumert, A. and Strack, D. and Becker, H. C. and Möllers, C. and Milkowski, C. and}, title = {{Reduction of Sinapate Ester Content in Transgenic Oilseed Rape (Brassica napus) by dsRNAi-based Suppression of BnSGT1 Gene Expression}}, year = {2005}, pages = {127-138}, journal = {Mol. Breed.}, doi = {10.1007/s11032-005-6825-8}, volume = {16}, abstract = {Seeds of oilseed rape (Brassica napus) accumulate high amounts of antinutritive sinapate esters (SE) with sinapoylcholine (sinapine) as major component, accompanied by sinapoylglucose. These phenolic compounds compromise the use of the protein-rich valuable seed meal. Hence, a substantial reduction of the SE content is considered essential for establishing rape as a protein crop. The present work focuses on the suppression of sinapine synthesis in rape. Therefore, rape (spring cultivar Drakkar) was transformed with a dsRNAi construct designed to silence seed-specifically the BnSGT1 gene encoding UDP-glucose:sinapate glucosyltransferase (SGT1). This resulted in a substantial decrease of SE content in T2 seeds with a reduction reaching 61%. In T2 seeds a high and significant correlation between the contents of sinapoylglucose and all other sinapate esters has been observed. Among transgenic plants, no significant difference in other important agronomic traits, such as oil, protein, fatty acid and glucosinolate content in comparison to the control plants was observed. Maximal reduction of total SE content by 76% was observed in seeds of one homozygous T2 plant (T3 seeds) carrying the BnSGT1 suppression cassette as a single copy insert. In conclusion, this study is an initial proof of principle that suppression of sinapoylglucose formation leads to a strong reduction of SE in rape seeds and is thus a promising approach in establishing rape, currently an important oil crop, as a protein crop as well.} } @Article{IPB-1954, author = {Hause, B. and Fester, T. and}, title = {{Molecular and cell biology of arbuscular mycorrhizal symbiosis}}, year = {2005}, pages = {184-196}, journal = {Planta}, doi = {10.1007/s00425-004-1436-x}, volume = {221}, abstract = {The roots of most extant plants are able to become engaged in an interaction with a small group of fungi of the fungal order Glomales (Glomeromycota). This interaction—arbuscular mycorrhizal (AM) symbiosis—is the evolutionary precursor of most other mutualistic root-microbe associations. The molecular analysis of this interaction can elucidate basic principles regarding such associations. This review summarizes our present knowledge about cellular and molecular aspects of AM. Emphasis is placed on morphological changes in colonized cells, transfer of nutrients between both interacting partners, and plant defence responses. Similarities to and differences from other associations of plant and microorganisms are highlighted regarding defence reactions and signal perception.} } @Article{IPB-1953, author = {Hause, G. and Lischewski, S. and Wessjohann, L. A. and Hause, B. and}, title = {{Epothilone D affects cell cycle and microtubular pattern in plant cells}}, year = {2005}, pages = {2131-2137}, journal = {J. Exp. Bot.}, doi = {10.1093/jxb/eri211}, volume = {56}, abstract = {Epothilones, macrocyclic lactones from culture filtrates of the myxobacterium Sorangium cellulosum, are known as taxol-like microtubular drugs in human medicine. To date, nothing is known about the effect of epothilones on microtubules (MTs) in plant cells and/or on the plant cell cycle. As shown in this report, the treatment of tomato cell suspension cultures with epothilone D produced a continuous increase in the mitotic index. Dose–response curves revealed that epothilone D alters the mitotic index at concentrations as low as 1.5 μM. Mitotic arrest was already visible after only 2 h of treatment, and 55% of the cells were arrested after 24 h. As shown by immunocytological methods, abnormal spindles are formed during metaphase, which leads to a random distribution of chromosomes in the whole cell and prevents the formation of a metaphase plate. The process of chromosome decondensation does not seem to be affected, because micronuclei form at the same place with the distributed chromosomes. This suggests that epothilone D influences the stability of plant MTs mainly during metaphase of the mitotic cycle. In metaphase, the effects of epothilone D seem to be irreversible, because cells with an abnormal spindle could not be recovered after removal of the drug.} } @Article{IPB-1952, author = {Hartmann, T. and Kutchan, T. M. and Strack, D. and}, title = {{Evolution of metabolic diversity}}, year = {2005}, pages = {1198-1199}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2005.04.021}, volume = {66}, } @Article{IPB-1947, author = {Fester, T. and Wray, V. and Nimtz, M. and Strack, D. and}, title = {{Is stimulation of carotenoid biosynthesis in arbuscular mycorrhizal roots a general phenomenon?}}, year = {2005}, pages = {1781-1786}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2005.05.009}, volume = {66}, abstract = {The identification and quantification of cyclohexenone glycoside derivatives from the model legume Lotus japonicus revealed far higher levels than expected according to the stoichiometric relation to another, already determined carotenoid cleavage product, i.e., mycorradicin. Mycorradicin is responsible for the yellow coloration of many arbuscular mycorrhizal (AM) roots and is usually esterified in a complex way to other compounds. After liberation from such complexes it has been detected in AM roots of many, but not of all plants examined. The non-stoichiometric occurrence of this compound compared with other carotenoid cleavage products suggested that carotenoid biosynthesis might be activated upon mycorrhization even in plant species without detectable levels of mycorradicin. This assumption has been supported by inhibition of a key enzyme of carotenoid biosynthesis (phytoene desaturase) and quantification of the accumulating enzymic substrate (phytoene). Our observations suggest that the activation of carotenoid biosynthesis in AM roots is a general phenomenon and that quantification of mycorradicin is not always a good indicator for this activation.} } @Article{IPB-1946, author = {Fester, T. and Hause, G. and}, title = {{Accumulation of reactive oxygen species in arbuscular mycorrhizal roots}}, year = {2005}, pages = {373-379}, journal = {Mycorrhiza}, doi = {10.1007/s00572-005-0363-4}, volume = {15}, abstract = {We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H2O2 accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl3, intracellular accumulation of H2O2 was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H2O2 was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.} } @Article{IPB-2004, author = {Ziegler, J. and Diaz-Chávez, M. L. and Kramell, R. and Ammer, C. and Kutchan, T. M. and}, title = {{Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis}}, year = {2005}, pages = {458-471}, journal = {Planta}, doi = {10.1007/s00425-005-1550-4}, volume = {222}, abstract = {Benzylisoquinoline alkaloids constitute a group of about 2,500 structures and are mainly produced by plants of the order Ranunculales. But only the opium poppy, Papaver somniferum, and Papaver setigerum are able to produce morphine. In this study, we started to investigate by gene expression analysis the molecular basis for this exceptional biosynthetic ability. A sequencing project from P. somniferum seedlings was initiated using a method based on the amplified fragment length polymorphism technique that resulted in 849 UniGenes. These cDNAs were analysed on macroarrays for differential expression between morphine-containing P. somniferum plants and eight other Papaver species, which accumulate other benzylisoquinolines instead of morphine. Three cDNAs showing increased expression in P. somniferum compared to all the other Papaver species were identified. Whereas two showed no significant homology to any known protein, one putatively encoded an O-methyltransferase. Analysis of substrate specificity of the heterologously expressed protein and mass spectrometric identification of the enzymatic products identified this protein as S-adenosyl-L-methionine:(R,S)-3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (EC 2.1.1.116). Unlike other O-methyltransferases of different positional specificities implicated in benzylisoquinoline metabolism, the enzyme only accepted tetrahydroxylated tetrahydrobenzylisoquinolines as substrates; methylation was tolerated only at the 6-hydroxy position.} } @Article{IPB-1989, author = {Stumpe, M. and Carsjens, J.-G. and Stenzel, I. and Göbel, C. and Lang, I. and Pawlowski, K. and Hause, B. and Feussner, I. and}, title = {{Lipid metabolism in arbuscular mycorrhizal roots of Medicago truncatula}}, year = {2005}, pages = {781-791}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2005.01.020}, volume = {66}, abstract = {The peroxidation of polyunsaturated fatty acids, common to all eukaryotes, is mostly catalyzed by members of the lipoxygenase enzyme family of non-heme iron containing dioxygenases. Lipoxygenase products can be metabolized further in the oxylipin pathway by several groups of CYP74 enzymes. One prominent oxylipin is jasmonic acid (JA), a product of the 13-allene oxide synthase branch of the pathway and known as signaling substance that plays a role in vegetative and propagative plant development as well as in plant responses to wounding and pathogen attack. In barley roots, JA level increases upon colonization by arbuscular mycorrhizal fungi. Apart from this first result regarding JA, no information is available on the relevance of lipidperoxide metabolism in arbuscular mycorrhizal symbiosis. Thus we analyzed fatty acid and lipidperoxide patterns in roots of Medicago truncatula during mycorrhizal colonization. Levels of fungus-specific fatty acids as well as palmitic acid (16:0) and oleic acid (18:1 n − 9) were increased in mycorrhizal roots. Thus the degree of arbuscular mycorrhizal colonization of roots can be estimated via analysis of fungal specific esterified fatty acids. Otherwise, no significant changes were found in the profiles of esterified and free fatty acids. The 9- and 13-LOX products of linoleic and α-linolenic acid were present in all root samples, but did not show significant differences between mycorrhizal and non-mycorrhizal roots, except JA which showed elevated levels in mycorrhizal roots. In both types of roots levels of 13-LOX products were higher than those of 9-LOX products. In addition, three cDNAs encoding CYP74 enzymes, two 9/13-hydroperoxide lyases and a 13-allene oxide synthase, were isolated and characterized. The transcript accumulation of these three genes, however, was not increased in mycorrhizal roots of M. truncatula.} } @Article{IPB-1988, author = {Sharma, V. K. and Monostori, T. and Hause, B. and Maucher, H. and Göbel, C. and Hornung, E. and Hänsch, R. and Bittner, F. and Wasternack, C. and Feussner, I. and Mendel, R. R. and Schulze, J. and}, title = {{Genetic transformation of barley to modify expression of a 13-lipoxygenase}}, year = {2005}, pages = {33-34}, journal = {Acta Biol. Szeged.}, url = {http://abs.bibl.u-szeged.hu/index.php/abs/article/view/2409}, volume = {49}, abstract = {Immature scutella of barley were transformed with cDNA coding for a 13-lipoxygenase of barley (LOX-100) via particle bombardment. Regenerated plants were tested by PAT-assay, Western-analysis and PCR-screening. Immunocytochemical assay of T0 plants showed expression of the LOX cDNA both in the chloroplasts and in the cytosol, depending on the presence of the chloroplast signal peptide sequences in the cDNA. A few transgenic plants containing higher amounts of LOX-derived products have been found. These are the candidates for further analysis concerning pathogen resistance.} } @Article{IPB-1933, author = {Baumert, A. and Milkowski, C. and Schmidt, J. and Nimtz, M. and Wray, V. and Strack, D. and}, title = {{Formation of a complex pattern of sinapate esters in Brassica napus seeds, catalyzed by enzymes of a serine carboxypeptidase-like acyltransferase family?}}, year = {2005}, pages = {1334-1345}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2005.02.031}, volume = {66}, abstract = {Members of the Brassicaceae accumulate complex patterns of sinapate esters, as shown in this communication with seeds of oilseed rape (Brassica napus). Fifteen seed constituents were isolated and identified by a combination of high-field NMR spectroscopy and high resolution electrospray ionisation mass spectrometry. These include glucose, gentiobiose and kaempferol glycoside esters as well as sinapine (sinapoylcholine), sinapoylmalate and an unusual cyclic spermidine amide. One of the glucose esters (1,6-di-O-sinapoylglucose), two gentiobiose esters (1-O-caffeoylgentiobiose and 1,2,6′-tri-O-sinapoylgentiobiose) and two kaempferol conjugates [4′-(6-O-sinapoylglucoside)-3,7-di-O-glucoside and 3-O-sophoroside-7-O-(2-O-sinapoylglucoside)] seem to be new plant products. Serine carboxypeptidase-like (SCPL) acyltransferases catalyze the formation of sinapine and sinapoylmalate accepting 1-O-β-acetal esters (1-O-β-glucose esters) as acyl donors. To address the question whether the formation of other components of the complex pattern of the sinapate esters in B. napus seeds is catalyzed via 1-O-sinapoyl-β-glucose, we performed a seed-specific dsRNAi-based suppression of the sinapate glucosyltransferase gene (BnSGT1) expression. In seeds of BnSGT1-suppressing plants the amount of sinapoylglucose decreased below the HPLC detection limit resulting in turn in the disappearance or marked decrease of all the other sinapate esters, indicating that formation of the complex pattern of these esters in B. napus seeds is dependent on sinapoylglucose. This gives rise to the assumption that enzymes of an SCPL acyltransferase family catalyze the appropriate transfer reactions to synthesize the accumulating esters.} } @Article{IPB-2061, author = {Vogt, T. and}, title = {{Regiospecificity and kinetic properties of a plant natural product O-methyltransferase are determined by its N-terminal domain}}, year = {2004}, pages = {159-162}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(04)00163-2}, volume = {561}, abstract = {A recently discovered, S‐adenosyl‐L ‐methionine and bivalent cation‐dependent O‐methyltransferase from the ice plant, Mesembryanthemum crystallinum , is involved in the methylation of various flavonoid and phenylpropanoid conjugates. Differences in regiospecificity as well as altered kinetic properties of the recombinant as compared to the native plant O‐methyltransferase can be attributed to differences in the N‐terminal part of the protein. Upon cleavage of the first 11 amino acids, the recombinant protein displays essentially the same substrate specificity as observed earlier for the native plant enzyme. Product formation of the newly designed, truncated recombinant enzyme is consistent with light‐induced accumulation of methylated flavonoid conjugates in the ice plant. Therefore, substrate affinity and regiospecificity of an O‐methyltransferase in vivo and in vitro can be controlled by cleavage of an N‐terminal domain.} } @Article{IPB-2046, author = {Schaarschmidt, S. and Qu, N. and Strack, D. and Sonnewald, U. and Hause, B. and}, title = {{Local Induction of the alc Gene Switch in Transgenic Tobacco Plants by Acetaldehyde}}, year = {2004}, pages = {1566-1577}, journal = {Plant Cell Physiol.}, doi = {10.1093/pcp/pch177}, volume = {45}, abstract = {The alc promoter system, derived from the filamentous fungi Aspergillus nidulans, allows chemically regulated gene expression in plants and thereby the study of gene function as well as metabolic and developmental processes. In addition to ethanol, this system can be activated by acetaldehyde, described as the physiological inducer in A. nidulans. Here, we show that in contrast to ethanol, acetaldehyde allows tissue-specific activation of the alc promoter in transgenic tobacco plants. Soil drenching with aqueous acetaldehyde solutions at a concentration of 0.05% (v/v) resulted in the rapid and temporary induction of the alc gene expression system exclusively in roots. In addition, the split root system allows activation to be restricted to the treated part of the root. The temporary activation of the alc system by soil drenching with acetaldehyde could be prolonged over several weeks by subsequent applications at intervals of 7 d. This effect was demonstrated for the root-specific induction of a yeast-derived apoplast-located invertase under the control of the alcohol-inducible promoter system. In leaves, which exhibit a lower responsiveness to acetaldehyde than roots, the alc system was induced in the directly treated tissue only. Thus, acetaldehyde can be used as a local inducer of the alc gene expression system in tobacco plants.} } @Article{IPB-2039, author = {Milkowski, C. and Strack, D. and}, title = {{Serine carboxypeptidase-like acyltransferases}}, year = {2004}, pages = {517-524}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2003.12.018}, volume = {65}, abstract = {In plant secondary metabolism, an alternative pathway of ester formation is facilitated by acyltransferases accepting 1-O-β-acetal esters (1-O-β-glucose esters) as acyl donors instead of coenzyme A thioesters. Molecular data indicate homology of these transferases with hydrolases of the serine carboxypeptidase type defining them as serine carboxypeptidase-like (SCPL) acyltransferases. During evolution, they apparently have been recruited from serine carboxypeptidases and adapted to take over acyl transfer function. SCPL acyltransferases belong to the highly divergent class of α/β hydrolases. These enzymes make use of a catalytic triad formed by a nucleophile, an acid and histidine acting as a charge relay system for the nucleophilic attack on amide or ester bonds. In analogy to SCPL acyltransferases, bacterial thioesterase domains are known which favour transferase activity over hydrolysis. Structure elucidation reveals water exclusion and a distortion of the oxyanion hole responsible for the changed activity. In plants, SCPL proteins form a large family. By sequence comparison, a distinguished number of Arabidopsis SCPL proteins cluster with proven SCPL acyltransferases. This indicates the occurrence of a large number of SCPL proteins co-opted to catalyse acyltransfer reactions. SCPL acyltransferases are ideal systems to investigate principles of functional adaptation and molecular evolution of plant genes.Serine carboxypeptidase-like (SCPL) acyltransferases are involved in the formation of esters, accepting 1-O-β-glucose esters as acyl donors. SCPL proteins make use of a catalytic triad formed by a nucleophile, an acid and histidine acting as a charge relay system for the nucleophilic attack on amide or ester bonds. During evolution, these enzymes apparently have been recruited from serine carboxypeptidases and adapted to take over acyl transfer instead of hydrolysis functions. By sequence comparison, a distinguished number of Arabidopsis SCPL proteins cluster with proven SCPL acyltransferases. These enzymes might be ideal systems to investigate principles of functional adaptation and molecular evolution of plant genes.} } @Article{IPB-2038, author = {Milkowski, C. and Baumert, A. and Schmidt, D. and Nehlin, L. and Strack, D. and}, title = {{Molecular regulation of sinapate ester metabolism in Brassica napus: expression of genes, properties of the encoded proteins and correlation of enzyme activities with metabolite accumulation}}, year = {2004}, pages = {80-92}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2004.02036.x}, volume = {38}, abstract = {Members of the Brassicaceae family accumulate specific sinapate esters, i.e. sinapoylcholine (sinapine), which is considered as a major antinutritive compound in seeds of important crop plants like Brassica napus , and sinapoylmalate, which is implicated in UV‐B tolerance in leaves. We have studied the molecular regulation of the sinapate ester metabolism in B. napus , and we describe expression of genes, some properties of the encoded proteins and profiles of the metabolites and enzyme activities. The cloned cDNAs encoding the key enzymes of sinapine biosynthesis, UDP‐glucose (UDP‐Glc):B. napus sinapate glucosyltransferase (BnSGT1) and sinapoylglucose:B. napus choline sinapoyltransferase (BnSCT), were functionally expressed. BnSGT1 belongs to a subgroup of plant GTs catalysing the formation of 1‐O‐hydroxycinnamoyl‐β‐d ‐glucoses. BnSCT is another member of serine carboxypeptidase‐like (SCPL) family of acyltransferases. The B. napus genome contains at least two SGT and SCT genes, each derived from its progenitors B. oleracea and B. rapa . BnSGT1 and BnSCT activities are subjected to pronounced transcriptional regulation. BnSGT1 transcript level increases throughout early stages of seed development until the early cotyledonary stage, and stays constant in later stages. The highest level of BnSGT1 transcripts is reached in 2‐day‐old seedlings followed by a dramatic decrease. In contrast, expression of BnSCT is restricted to developing seeds. Regulation of gene expression at the transcript level seems to be responsible for changes of BnSGT1 and BnSCT activities during seed and seedling development of B. napus . Together with sinapine esterase (SCE) and sinapoylglucose:malate sinapoyltransferase (SMT), activities of BnSGT1 and BnSCT show a close correlation with the accumulation kinetics of the corresponding metabolites.} } @Article{IPB-2037, author = {Miersch, O. and Weichert, H. and Stenzel, I. and Hause, B. and Maucher, H. and Feussner, I. and Wasternack, C. and}, title = {{Constitutive overexpression of allene oxide cyclase in tomato (Lycopersicon esculentum cv. Lukullus) elevates levels of some jasmonates and octadecanoids in flower organs but not in leaves}}, year = {2004}, pages = {847-856}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2004.01.016}, volume = {65}, abstract = {The allene oxide cyclase (AOC), an enzyme in jasmonate biosynthesis, occurs in vascular bundles and ovules of tomato flowers which exhibit a tissue-specific oxylipin signature (Plant J. 24, 113-126, 2000). Constitutive overexpression of the AOC did not led to altered levels of jasmonates in leaves, but these levels increased upon wounding or other stresses suggesting regulation of jasmonate biosynthesis by substrate availability (Plant J. 33, 577-589, 2003). Here, we show dramatic changes in levels of jasmonic acid (JA), of 12-oxo-phytodienoic acid (OPDA), their methyl esters (JAME, OPDAME), and of dinor-OPDA in most flower organs upon constitutive overexpression of AOC. Beside a dominant occurrence of OPDAME and JA in most flower organs, the ratio among the various compounds was altered differentially in the organs of transgenic flowers, e.g. OPDAME increased up to 53-fold in stamen, and JA increased about 51-fold in buds and 7.5-fold in sepals. The increase in jasmonates and octadecanoids was accompanied by decreased levels of free lipid hydro(per)oxy compounds. Except for 16:2, the AOC overexpression led to a significant increase in free but not esterified polyunsaturated fatty acids in all flower organs. The data suggest different regulation of JA biosynthesis in leaves and flowers of tomato.Constitutive overexpression of the AOC increases in all flower organs levels of some jasmonates and octadecanoids, alters the ratios among the compounds, decreases levels of free lipid hydro(per)oxy compounds and increases levels of free but not of esterified polyunsaturated fatty acids.} } @Article{IPB-2035, author = {Maucher, H. and Stenzel, I. and Miersch, O. and Stein, N. and Prasad, M. and Zierold, U. and Schweizer, P. and Dorer, C. and Hause, B. and Wasternack, C. and}, title = {{The allene oxide cyclase of barley (Hordeum vulgare L.)—cloning and organ-specific expression}}, year = {2004}, pages = {801-811}, journal = {Phytochemistry}, doi = {10.1016/j.phytochem.2004.01.009}, volume = {65}, abstract = {The naturally occurring enantiomer of the various octadecanoids and jasmonates is established in a biosynthetic step catalyzed by the allene oxide cyclase (AOC). The AOC converts an allene oxide formed by an allene oxide synthase (AOS). Here, we show cloning and characterization of cDNAs encoding the AOC and a third AOS, respectively, in addition to the two AOSs previously published (Plant J. 21, 199–213, 2000). The ORF of the AOC-cDNA of 717 bp codes for a protein of 238 amino acid residues carrying a putative chloroplast target sequence. Overexpression without chloroplast target sequence revealed AOC activity. The AOC was found to be a single copy gene which mapped on chromosome 6H. AOC mRNA accumulation appeared in leaf segments upon treatment with various jasmonates, octadecanoids and ABA or during stress such as treatment with sorbitol or glucose solutions. Infection with powdery mildew activated AOC expression in susceptible and resistant lines of barley which correlated with PR1b expression. Among different tissues of barley seedlings, the scutellar node and leaf base accumulated AOC mRNA preferentially which correlated with accumulation of mRNAs for other biosynthetic enzymes (lipoxygenases, AOSs). AOC mRNA accumulation appeared also abundantly in parts of the root containing the tip and correlated with elevated levels of jasmonates. The data suggest a link of AOC expression and JA formation and support role of JA in stress responses and development of barley.Barley plants contain one allene oxide cyclase and three allene oxide synthases which are up-regulated during seedling development accompanied by elevated levels of jasmonate.} } @Article{IPB-2032, author = {Lukačin, R. and Matern, U. and Specker, S. and Vogt, T. and}, title = {{Cations modulate the substrate specificity of bifunctional class I O-methyltransferase from Ammi majus}}, year = {2004}, pages = {367-370}, journal = {FEBS Lett.}, doi = {10.1016/j.febslet.2004.10.032}, volume = {577}, abstract = {Caffeoyl‐coenzyme A O‐methyltransferase cDNA was cloned from dark‐grown Ammi majus L. (Apiaceae) cells treated with a crude fungal elicitor and the open reading frame was expressed in Escherichia coli . The translated polypeptide of 27.1‐kDa shared significant identity to other members of this highly conserved class of proteins and was 98.8% identical to the corresponding O‐methyltransferase from parsley. For biochemical characterization, the recombinant enzyme could be purified to apparent homogeneity by metal‐affinity chromatography, although the recombinant enzyme did not contain any affinity tag. Based on sequence analysis and substrate specificity, the enzyme classifies as a cation‐dependent O‐methyltransferase with pronounced preference for caffeoyl coenzyme A, when assayed in the presence of Mg2\+‐ions. Surprisingly, however, the substrate specificity changed dramatically, when Mg2\+ was replaced by Mn2\+ or Co2\+ in the assays. This effect could point to yet unknown functions and substrate specificities in situ and suggests promiscuous roles for the lignin specific cluster of plant O‐methyltransferases.} } @Article{IPB-2027, author = {Isayenkov, S. and Fester, T. and Hause, B. and}, title = {{Rapid determination of fungal colonization and arbuscule formation in roots of Medicago truncatula using real-time (RT) PCR}}, year = {2004}, pages = {1379-1383}, journal = {J. Plant Physiol.}, doi = {10.1016/j.jplph.2004.04.012}, volume = {161}, abstract = {The quantifications of root colonization and symbiotic activity in the arbuscular mycorrhizal (AM) association of Medicago truncatula and Glomus intraradices were performed by quantitative polymerase chain reaction (real-time PCR). A strong correlation between fungal colonization of the root system and the amounts of fungal rDNA and rRNA were shown. In contrast, the transcript levels of the AM-specific phosphate transporter 4 from M. truncatula (MtPT4) correlate with arbuscule formation rather than with fungal colonization. These results suggest (i) that real-time PCR assay is a rapid, useful, and accurate method for the determination of arbuscular mycorrhizal features, (ii) that the amount of fungal rDNA or rRNA is a good parameter to estimate fungal colonization, and (iii) that it is necessary to evaluate the amount of other transcripts—like the MtPT4 transcript—to obtain additional information about the symbiotic state of the colonized root system.} } @Article{IPB-2023, author = {Hans, J. and Brandt, W. and Vogt, T. and}, title = {{Site-directed mutagenesis and protein 3D-homology modelling suggest a catalytic mechanism for UDP-glucose-dependent betanidin 5-O-glucosyltransferase from Dorotheanthus bellidiformis}}, year = {2004}, pages = {319-333}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2004.02133.x}, volume = {39}, abstract = {In livingstone daisy (Dorotheanthus bellidiformis ), betanidin 5‐O‐glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site‐directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP‐glucose binding site, and His22, located close to the N‐terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP‐glucose‐dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an SN‐1 reaction, as one plausible alternative to the generally proposed SN‐2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.} } @Article{IPB-2011, author = {Camacho-Cristóbal, J. J. and Lunar, L. and Lafont, F. and Baumert, A. and González-Fontes, A. and}, title = {{Boron deficiency causes accumulation of chlorogenic acid and caffeoyl polyamine conjugates in tobacco leaves}}, year = {2004}, pages = {879-881}, journal = {J. Plant Physiol.}, doi = {10.1016/j.jplph.2003.12.003}, volume = {161}, abstract = {The effects of boron (B) deficiency on carbohydrate concentrations and the pattern of phenolic compounds were studied in leaves of tobacco plants (Nicotiana tabacum L.). Plants grown under B deficiency showed a notable increase in leaf carbohydrates and total phenolic compounds when compared to controls. The qualitative composition of phenolics was analyzed by HPLC-mass spectrometry. The level of caffeate conjugates (i.e., chlorogenic acid) increased in B-deficient plants. In addition, the accumulation of two caffeic acid amides (N-caffeoylputrescine and putative dicaffeoylspermidine) was observed.} } @Article{IPB-2010, author = {Bücking, H. and Förster, H. and Stenzel, I. and Miersch, O. and Hause, B. and}, title = {{Applied jasmonates accumulate extracellularly in tomato, but intracellularly in barley}}, year = {2004}, pages = {45-50}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(04)00178-4}, volume = {562}, abstract = {Jasmonic acid (JA) and its derivatives are well‐characterized signaling molecules in plant defense and development, but the site of their localization within plant tissue is entirely unknown. To address the question whether applied JA accumulates extracellularly or intracellularly, leaves of tomato and barley were fed with 14C‐labeled JA and the label was localized in cryofixed and lyophilized leaf tissues by microautoradiography. In tomato the radioactivity was detectable within the apoplast, but no label was found within the mesophyll cells. By contrast, in barley leaf tissues, radioactivity was detected within the mesophyll cells suggesting a cellular uptake of exogenously applied JA. JA, applied to leaves of both plants as in the labeling experiments, led in all leaf cells to the expression of JA‐inducible genes indicating that the perception is completed by JA signal transduction.} } @Article{IPB-2022, author = {Hans, J. and Hause, B. and Strack, D. and Walter, M. H. and}, title = {{Cloning, Characterization, and Immunolocalization of a Mycorrhiza-Inducible 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase in Arbuscule-Containing Cells of Maize}}, year = {2004}, pages = {614-624}, journal = {Plant Physiol.}, doi = {10.1104/pp.103.032342}, volume = {134}, abstract = {Colonization of plant roots by symbiotic arbuscular mycorrhizal fungi frequently leads to the accumulation of several apocarotenoids. The corresponding carotenoid precursors originate from the plastidial 2-C-methyl-d-erythritol 4-phosphate pathway. We have cloned and characterized 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), catalyzing the first committed step of the pathway, from maize (Zea mays). Functional identification was accomplished by heterologous expression of sequences coding for the mature protein in Escherichia coli. DXR is up-regulated in maize roots during mycorrhization as shown at transcript and protein levels, but is also abundant in leaves and young seedlings. Inspection of sequenced genomes and expressed sequence tag (EST) databases argue for a single-copy DXR gene. Immunolocalization studies in mycorrhizal roots using affinity-purified antibodies revealed a DXR localization in plastids around the main symbiotic structures, the arbuscules. DXR protein accumulation is tightly correlated with arbuscule development. The highest level of DXR protein is reached around maturity and initial senescence of these structures. We further demonstrate the formation of a DXR-containing plastidial network around arbuscules, which is highly interconnected in the mature, functional state of the arbuscules. Our findings imply a functional role of a still unknown nature for the apocarotenoids or their respective carotenoid precursors in the arbuscular life cycle.} } @INBOOK{IPB-135, author = {Fester, T. and}, title = {{Wurzelinduzierte Bodenvorgänge}}, year = {2004}, pages = {39-42}, chapter = {{Plastiden bei der arbuskulären Mykorrhizasymbiose}}, doi = {10.1007/978-3-322-80084-8_5}, abstract = {In arbuscular mycorrhizal symbioses, root cortical cell plastids catalyze central processes regarding the transported metabolites on the one hand and molecular building blocks for symbiotic structures on the other. These metabolic tasks correlate with a strong proliferation and structural changes of the organelles. Future work will address the molecular factors responsible for these metabolic and structural changes.} } @Article{IPB-2130, author = {Wang, W. and Hause, B. and Peumans, W. J. and Smagghe, G. and Mackie, A. and Fraser, R. and Van Damme, E. J. M. and}, title = {{The Tn Antigen-Specific Lectin from Ground Ivy Is an Insecticidal Protein with an Unusual Physiology}}, year = {2003}, pages = {1322-1334}, journal = {Plant Physiol.}, doi = {10.1104/pp.103.023853}, volume = {132}, abstract = {Leaves of ground ivy (Glechoma hederacea) contain a lectin (called Gleheda) that is structurally and evolutionary related to the classical legume lectins. Screening of a population of wild plants revealed that Gleheda accounts for more than one-third of the total leaf protein in some clones, whereas it cannot be detected in other clones growing in the same environment. Gleheda is predominantly expressed in the leaves where it accumulates during early leaf maturation. The lectin is not uniformly distributed over the leaves but exhibits a unique localization pattern characterized by an almost exclusive confinement to a single layer of palisade parenchyma cells. Insect feeding trials demonstrated that Gleheda is a potent insecticidal protein for larvae of the Colorado potato beetle (Leptinotarsa decemlineata). Because Gleheda is not cytotoxic, it is suggested that the insecticidal activity is linked to the carbohydrate-binding specificity of the lectin, which as could be demonstrated by agglutination assays with different types of polyagglutinable human erythrocytes is specifically directed against the Tn antigen structure (N-acetylgalactosamine O-linked to serine or threonine residues of proteins).} } @Article{IPB-2129, author = {Varet, A. and Hause, B. and Hause, G. and Scheel, D. and Lee, J. and}, title = {{The Arabidopsis NHL3 Gene Encodes a Plasma Membrane Protein and Its Overexpression Correlates with Increased Resistance to Pseudomonas syringae pv. tomato DC3000}}, year = {2003}, pages = {2023-2033}, journal = {Plant Physiol.}, doi = {10.1104/pp.103.020438}, volume = {132}, abstract = {The Arabidopsis genome contains a family of NDR1/HIN1-like (NHL) genes that show homology to the nonrace-specific disease resistance (NDR1) and the tobacco (Nicotiana tabacum) harpin-induced (HIN1) genes. NHL3 is a pathogen-responsive member of this NHL gene family that is potentially involved in defense. In independent transgenic NHL3-overexpressing plant lines, a clear correlation between increased resistance to virulent Pseudomonas syringae pv. tomato DC3000 and enhanced NHL3 transcript levels was seen. These transgenic plants did not show enhanced pathogenesis-related gene expression or reactive oxygen species accumulation. Biochemical and localization experiments were performed to assist elucidation of how NHL3 may confer enhanced disease resistance. Gene constructs expressing amino-terminal c-myc-tagged or carboxyl-terminal hemagglutinin epitope (HA)-tagged NHL3 demonstrated membrane localization in transiently transformed tobacco leaves. Stable Arabidopsis transformants containing the NHL3-HA construct corroborated the findings observed in tobacco. The detected immunoreactive proteins were 10 kD larger than the calculated size and could be partially accounted for by the glycosylation state. However, the expected size was not attained with deglycosylation, suggesting possibly additional posttranslational modification. Detergent treatment, but not chemicals used to strip membrane-associated proteins, could displace the immunoreactive signal from microsomal fractions, showing that NHL3 is tightly membrane associated. Furthermore, immunofluorescence and immunogold labeling, coupled with two-phase partitioning techniques, revealed plasma membrane localization of NHL3-HA. This subcellular localization of NHL3 positions it at an initial contact site to pathogens and may be important in facilitating interception of pathogen-derived signals.} } @Article{IPB-2126, author = {Strack, D. and Vogt, T. and Schliemann, W. and}, title = {{Recent advances in betalain research}}, year = {2003}, pages = {247-269}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(02)00564-2}, volume = {62}, abstract = {Betalains replace the anthocyanins in flowers and fruits of plants of most families of the Caryophyllales. Unexpectedly, they were also found in some higher fungi. Whereas the anthocyanin-analogous functions of betalains in flower and fruit colouration are obvious, their role in fungi remains obscure. The nature of newly identified betalains as well as final structure elucidation of earlier putatively described compounds published within the last decade is compiled in this report. Recent advances in research on betalain biosynthesis is also covered, including description of some ‘early’ reactions, i.e. betalain-specific dopa formation in plants and fungi and extradiolic dopa cleavage in fungi. Work on betalain-specific glucosyltransferases (GTs) has given new insights into the evolution of secondary plant enzymes. It is proposed that these GTs are phylogenetically related to flavonoid GTs. It was found that the decisive steps in betalain biosynthesis, i.e. condensation of the betalain chromophore betalamic acid with cyclo-dopa and amino acids or amines in the respective aldimine formation of the red-violet betacyanins and the yellow betaxanthins, are most likely to be non-enzymatic. Betalains have attracted workers in applied fields because of their use for food colouring and their antioxidant and radical scavenging properties for protection against certain oxidative stress-related disorders.This review describes structure elucidation of betalains published within the last decade. Recent advances in betalain biosynthesis are also covered, i.e. enzymatic steps of ‘early’ (dopa formation) and ‘late’ reactions (glucosylation and acylation) as well as non-enzymatic steps (cyclo-dopa and aldimine formation).} } @Article{IPB-2125, author = {Strack, D. and Fester, T. and Hause, B. and Schliemann, W. and Walter, M. H. and}, title = {{Arbuscular Mycorrhiza: Biological, Chemical, and Molecular Aspects}}, year = {2003}, pages = {1955-1979}, journal = {J. Chem. Ecol.}, doi = {10.1023/A:1025695032113}, volume = {29}, abstract = {Mycorrhizas are the most important mutualistic symbioses on earth. The most prevalent type are the arbuscular mycorrhizas (AMs) that develop between roots of most terrestrial plants and fungal species of the Zygomycota. The AM fungi are able to grow into the root cortex forming intercellular hyphae from which highly branched structures, arbuscules, originate within cortex cells. The arbuscules are responsible for nutrient exchange between the host and the symbiont, transporting carbohydrates from the plant to the fungus and mineral nutrients, especially phosphate, and water from the fungus to the plant. Plants adapt their phosphate uptake to the interaction with the AM fungus by synthesis of specific phosphate transporters. Colonization of root cells induces dramatic changes in the cytoplasmic organization: vacuole fragmentation, transformation of the plasma membrane to a periarbuscular membrane covering the arbuscule, increase of the cytoplasm volume and numbers of cell organelles, as well as movement of the nucleus into a central position. The plastids form a dense network covering the symbiotic interface. In some of these changes, microtubules are most likely involved. With regard to the molecular crosstalk between the two organisms, a number of phytohormones (cytokinins, abscisic acid, jasmonate) as well as various secondary metabolites have been examined: (i) Jasmonates occur at elevated level, which is accompanied by cell-specific expression of genes involved in jasmonate biosynthesis that might be linked to strong carbohydrate sink function of AM roots and induced defense reactions; (ii) apocarotenoids (derivatives of mycorradicin and glycosylated cyclohexenones) accumulate in most mycorrhizal roots examined so far. Their biosynthesis via the nonmevalonate methylerythritol phosphate (MEP) pathway has been studied resulting in new insights into AM-specific gene expression and biosynthesis of secondary isoprenoids.} } @Article{IPB-2124, author = {Stenzel, I. and Hause, B. and Miersch, O. and Kurz, T. and Maucher, H. and Weichert, H. and Ziegler, J. and Feussner, I. and Wasternack, C. and}, title = {{Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana}}, year = {2003}, pages = {895-911}, journal = {Plant Mol. Biol.}, doi = {10.1023/A:1023049319723}, volume = {51}, abstract = {In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.} } @Article{IPB-2089, author = {Hause, B. and Stenzel, I. and Miersch, O. and Wasternack, C. and}, title = {{Occurrence of the allene oxide cyclase in different organs and tissues of Arabidopsis thaliana}}, year = {2003}, pages = {971-980}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(03)00447-3}, volume = {64}, abstract = {Occurrence of an essential enzyme in jasmonate (JA) biosynthesis, the allene oxide cyclase, (AOC) was analyzed in different developmental stages and various organs of Arabidopsis thaliana plants by immuno blot analysis and immunocytological approaches. Levels of AOC and of the two preceding enzymes in JA biosynthesis increased during seedling development accompanied by increased levels of JA and 12-oxophytodienoic acid levels after 4 and 8 weeks. Most tissues including all vascular bundles and that of flower buds contain AOC protein. Flowers shortly before opening, however, contain AOC protein preferentially in ovules, stigma cells and vascular bundles, whereas in anthers and pollen AOC could not be detected. The putative roles of AOC and JA in development are discussed.The allene oxide cyclase (AOC) is an important enzyme in jasmonate biosynthesis. Levels and occurrence of AOC in different organs and tissues are altered during development of Arabidopsis thaliana.} } @Article{IPB-2088, author = {Hause, B. and Hause, G. and Kutter, C. and Miersch, O. and Wasternack, C. and}, title = {{Enzymes of Jasmonate Biosynthesis Occur in Tomato Sieve Elements}}, year = {2003}, pages = {643-648}, journal = {Plant Cell Physiol.}, doi = {10.1093/pcp/pcg072}, volume = {44}, abstract = {The allene oxide cyclase (AOC) is a plastid-located enzyme in the biosynthesis of the signaling compound jasmonic acid (JA). In tomato, AOC occurs specifically in ovules and vascular bundles [Hause et al. (2000)PlantJ. 24; 113]. Immunocytological analysis of longitudinal sections of petioles and flower stalks revealed the occurrence of AOC in companion cells (CC) and sieve elements (SE). Electron microscopic analysis led to the conclusion that the AOC-containing structures of SE are plastids. AOC was not detected in SE of 35S::AOCantisense plants. The enzymes preceding AOC in JA biosynthesis, the allene oxide synthase (AOS) and the lipoxygenase, were also detected in SE. In situ hybridization showed that the SE are free of AOC-mRNA suggesting AOC protein traffic from CC to SE via plasmodesmata. A control by in situ hybridization of AOS mRNA coding for a protein with a size above the exclusion limit of plasmodesmata indicated mRNA in CC and SE. The data suggest that SE carry the capacity to form 12-oxo-phytodienoic acid, the unique precursor of JA. Together with preferential generation of JA in vascular bundles [Stenzel et al. (2003)Plant J. 33: 577], the data support a role of JA in systemic wound signaling.} } @Article{IPB-2083, author = {Eckermann, C. and Schröder, G. and Eckermann, S. and Strack, D. and Schmidt, J. and Schneider, B. and Schröder, J. and}, title = {{Stilbenecarboxylate biosynthesis: a new function in the family of chalcone synthase-related proteins}}, year = {2003}, pages = {271-286}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(02)00554-X}, volume = {62}, abstract = {Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65–70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.Hydrangea macrophylla (Garden Hortensia) encodes a type III polyketide synthase synthesizing the stilbenecarboxylate backbone which is the basis for the biosynthesis of many secondary products in liverworts and in higher plants.} } @Article{IPB-2081, author = {Doll, J. and Hause, B. and Demchenko, K. and Pawlowski, K. and Krajinski, F. and}, title = {{A Member of the Germin-Like Protein Family is a Highly Conserved Mycorrhiza-Specific Induced Gene}}, year = {2003}, pages = {1208-1214}, journal = {Plant Cell Physiol.}, doi = {10.1093/pcp/pcg153}, volume = {44}, abstract = {A Medicago truncatula cDNA encoding a germin-like protein (GLP) was isolated from a suppression subtractive hybridization cDNA library enriched for arbuscular mycorrhiza (AM)-induced genes. The MtGLP1 amino acid sequence shows some striking differences to previously described plant GLP sequences and might therefore represent a new subgroup of this multigene family. The MtGlp1 mRNA was strongly induced in roots and root cultures colonized by the AM fungus Glomus intraradices. Whereas MtGlp1 is strongly induced in AM, no transcripts of the gene were detected in non-infected roots or in roots after infection with the oomycete root pathogen Aphanomyces euteiches or with Rhizobia. Increased phosphate levels during fertilization also could not stimulate MtGlp1 transcription. Hence, MtGlp1 induction seems to be an AM-specific phenomenon. In situ hybridization showed that MtGlp1 is localized in arbuscule containing cells. A putative orthologue of this AM-specific GLP gene could be localized in a second legume Lotus japonicus, indicating that the regulation of a member of the GLP family belongs to a conserved mechanism in AM regulation in different plant species.} } @Article{IPB-2077, author = {Cacace, S. and Schröder, G. and Wehinger, E. and Strack, D. and Schmidt, J. and Schröder, J. and}, title = {{A flavonol O-methyltransferase from Catharanthus roseus performing two sequential methylations}}, year = {2003}, pages = {127-137}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(02)00483-1}, volume = {62}, abstract = {Protein extracts from dark-grown cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) contained several O-methyltransferase (OMT) activities, including the 16-hydroxytabersonine O-methyltransferase (16HT-OMT) in indole alkaloid biosynthesis. This enzyme was enriched through several purification steps, including affinity chromatography on adenosine agarose. SDS-PAGE of the purified protein preparation revealed a protein band at the size expected for plant OMTs (38–43 kDa). Mass spectrometry indicated two dominant protein species of similar mass in this band, and sequences of tryptic peptides showed similarities to known OMTs. Homology-based RT-PCR identified cDNAs for four new OMTs. Two of these cDNAs (CrOMT2 and CrOMT4) encoded the proteins dominant in the preparation enriched for 16HT-OMT. The proteins were closely related (73% identity), but both shared only 48-53% identity with the closest relatives found in the public databases. The enzyme functions were investigated with purified recombinant proteins after cDNA expression in Escherichia coli. Unexpectedly, both proteins had no detectable 16HT-OMT activity, and CrOMT4 was inactive with all substrates investigated. CrOMT2 was identified as a flavonoid OMT that was expressed in dark-grown cell cultures and copurified with 16HT-OMT. It represented a new type of OMT that performs two sequential methylations at the 3′- and 5′-positions of the B-ring in myricetin (flavonol) and dihydromyricetin (dihydroflavonol). The resulting methylation pattern is characteristic for C. roseus flavonol glycosides and anthocyanins, and it is proposed that CrOMT2 is involved in their biosynthesis.Purification and molecular characterization of an unusual flavonoid O-dimethyltransferase that explains the 3′,5′-methylation in flavonols and anthocyanins of Madagascar periwinkle.} } @Article{IPB-2110, author = {Proels, R. K. and Hause, B. and Berger, S. and Roitsch, T. and}, title = {{Novel mode of hormone induction of tandem tomato invertase genes in floral tissues}}, year = {2003}, pages = {191-201}, journal = {Plant Mol. Biol.}, doi = {10.1023/A:1023973705403}, volume = {52}, abstract = {The genomic organization of two extracellular invertase genes from tomato (Lin5 and Lin7), which are linked in a direct tandem repeat, and their tissue-specific and hormone-inducible expression are shown. Transient expression analysis ofLin5 promoter sequences fused to the β-glucuronidase (GUS) reporter gene (uidA) demonstrates a specific expression of Lin5during tomato fruit development. A Lin5 promoter fragment was fused to the truncated nos promoter to analyse hormone induction via GUS reporter gene activity in transiently transformed tobacco leaves. A specific up-regulation of GUS activity conferred by this Lin5 promoter fragment in response to gibberellic acid (GA), auxin and abscisic acid (ABA) treatment was observed, indicating a critical role of the regulation of Lin5 by phytohormones in tomato flower and fruit development. In situ hybridization analysis of Lin7 shows a high tissue-specific expression in tapetum and pollen. These results support an important role for Lin5 and Lin7 extracellular invertases in the development of reproductive organs in tomato and contribute to unravel the underlying regulatory mechanisms.} } @Article{IPB-2109, author = {Peng, Z. F. and Strack, D. and Baumert, A. and Subramaniam, R. and Goh, N. K. and Tet Fatt, C. and Tan, S. N. and Chia, L. S. and}, title = {{Antioxidant flavonoids from leaves of Polygonum hydropiper L.}}, year = {2003}, pages = {219-228}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(02)00504-6}, volume = {62}, abstract = {Ten flavonoid compounds were isolated from the dried leaves of Polygonum hydropiper L. (Laksa leaves), and identified as 3-O-α-l-rhamnopyranosyloxy-3′,4′,5,7-tetrahydroxyflavone; 3-O-β-d-glucopyranosyloxy-4′,5,7-trihydroxyflavone; 6-hydroxyapigenin; 6″-O-(3,4,5-trihydroxybenzoyl) 3-O-β-d-glucopyranosyloxy-3′, 4′, 5, 7-tetrahydroxyflavone; scutillarein; 6-hydroxyluteolin; 3′,4′,5,6,7-pentahydroxyflavone; 6-hydroxyluteolin-7-O-β-d-glucopyranoside; quercetin 3-O-β-d-glucuronide; 2″-O-(3,4,5-trihydroxybenzoyl) quercitrin; quercetin. Evaluation of the antioxidative activity, conducted in vitro, by using electron spin resonance (ESR) and ultraviolet visible (UV–vis) spectrophotometric assays, showed that these isolated flavonoids possess strong antioxidative capabilities. Measurement of the Trolox equivalent antioxidant capacity (TEAC) values, against ABTS (2,2′-azinobis(3-ethyl-benzo-thiazoline-6-sulphonic acid) radicals and phenyl-tert-butyl nitrone (PBN) azo initiator (AI) also showed strong anti-oxidative activity. The most powerful of the antioxidants was 2″-O-(3,4,5-trihydroxybenzoyl) quercitrin (galloyl quercitrin). A combination of two flavonoid compounds was tested for synergistic anti-oxidative capacity, but no significant improvement was observed.Ten flavonoid compounds were isolated and identified from the dried leaves of Polygonum hydropiper L. Among these, 2″-O-(3,4,5-trihydroxybenzoyl) quercitrin (galloyl quercitrin) showed high-yield occurrence and the strongest antioxidant activity. A combination of two flavonoid compounds was tested for synergistic antioxidative capacity, but no significant improvement was observed.} } @Article{IPB-2107, author = {Opitz, S. and Schnitzler, J.-P. and Hause, B. and Schneider, B. and}, title = {{Histochemical analysis of phenylphenalenone-related compounds in Xiphidium caeruleum (Haemodoraceae)}}, year = {2003}, pages = {881-889}, journal = {Planta}, doi = {10.1007/s00425-002-0941-z}, volume = {216}, abstract = {Phenylphenalenones represent a typical group of secondary metabolites of the Haemodoraceae. Some of these phenolic compounds show organ-specific distribution within the plant. However, detailed information on cellular localisation is still lacking. To this end, confocal laser-scanning microscopy, microspectral photometry and high-performance liquid chromatography were used to study the tissue localisation of phenylphenalenone-type compounds in Xiphidium caeruleum Aubl. From the autofluorescence potential of these compounds, specific distribution of allophanylglucosides and non-glucosidic compounds of the phenylphenalenone-type in distinct cells of the roots (apical meristem, cortex, cap, epidermis) and the shoot system was revealed. Fluorescence enhancement using \"Naturstoff reagent A\" (NA) indicated the occurrence of NA-positive natural products in the vacuoles of leaf epidermal cells. The present results provide new insights into the possible functions of phenylphenalenone-related compounds in the context of their localisation. Additionally, the advantages and limitations of the techniques are discussed.} } @Article{IPB-2103, author = {Münzenberger, B. and Hammer, E. and Wray, V. and Schauer, F. and Schmidt, J. and Strack, D. and}, title = {{Detoxification of ferulic acid by ectomycorrhizal fungi}}, year = {2003}, pages = {117-121}, journal = {Mycorrhiza}, doi = {10.1007/s00572-003-0226-9}, volume = {13}, abstract = {The ectomycorrhizal fungi Laccaria amethystina and Lactarius deterrimus grown in liquid culture were used to study the fate of added ferulic acid. Laccaria amethystina degraded ferulic acid to the major metabolite vanillic acid. The intermediate vanillin was not detected. Lactarius deterrimus showed a completely different detoxification pattern. Two dimers and one trimer of ferulic acid could be identified as polymerization products of this fungus. A bioassay of the possible biological activities of ferulic acid and vanillic acid on these fungi revealed that vanillic acid was less toxic than ferulic acid for Laccaria amethystina but that both phenolic acids were toxic for Lactarius deterrimus. The results are discussed with respect to ectomycorrhizal fungal growth in the organic layer of forest soils and between living root cells of ectomycorrhizas.} } @Article{IPB-2102, author = {Monostori, T. and Schulze, J. and Sharma, V. K. and Maucher, H. and Wasternack, C. and Hause, B. and}, title = {{Novel plasmid vectors for homologous transformation of barley (Hordeum vulgare L.) with JIP23 cDNA in sense and antisense orientation}}, year = {2003}, pages = {17-24}, journal = {Cereal Res. Commun.}, doi = {10.1007/BF03543245}, volume = {31}, abstract = {The most abundant jasmonate-induced protein (JIP) in barley leaves is a 23 kDa protein (JIP23). Its function, however, is unknown. In order to analyze its function by homologous transformation, new plasmid vectors have been constructed. They carry the cDNA coding for JIP23 in sense or antisense orientation under the control of the Ubi-1-promoter as well as the pat resistance gene under the control of the 35S promoter. Barley mesophyll protoplasts were transiently transformed with the sense constructs. PAT activity and immunological detection of JIP23 could be achieved in transformed protoplasts but not in untransformed protoplasts indicating that the construct was active. Thus, these new vectors are suitable for stable transformation of barley. Carrying a multiple cloning site (MCS), these vectors can be used now in a wide range of transformation of barley.} } @Article{IPB-2097, author = {Liu, S. and Chen, K. and Schliemann, W. and Strack, D. and}, title = {{Isolation and identification of two flavone glycosides in burdock (Arctium lappa L.) leaves by polyamide column chromatography and high performance liquid chromatography in combination with lectrospray ionization-mass spectrometry}}, year = {2003}, pages = {1023}, journal = {Chin. J. Anal. Chem.}, volume = {31}, } @Article{IPB-2096, author = {Liu, S. and Chen, K. and Schliemann, W. and Schmidt, J. and Strack, D. and}, title = {{Isolation and Identification of Trace Lignans, Arctiin and Arctigenin, in Arctium lappa L. Leaves}}, year = {2003}, pages = {52-55}, journal = {Chin. J. Chromatogr.}, url = {http://open.oriprobe.com/articles/5545714/Isolation_and_Identification_of_Trace_Lignans__Arc.htm}, volume = {21}, } @Article{IPB-2094, author = {Krajinski, F. and Hause, B. and Gianinazzi-Pearson, V. and Franken, P. and}, title = {{Mtha1, a Plasma Membrane H\+-ATPase Gene from Medicago truncatula, Shows Arbuscule-Specific Induced Expression in Mycorrhizal Tissue}}, year = {2003}, pages = {754-761}, journal = {Plant Biol.}, doi = {10.1055/s-2002-37407}, volume = {4}, abstract = {Transport processes between plant and fungal cells are key elements in arbuscular mycorrhiza (AM), where H\+‐ATPases are considered to be involved in active uptake of nutrients from the symbiotic interface. Genes encoding H\+‐ATPases were identified in the genome of Medicago truncatula and three cDNA fragments of the H\+‐ATPase gene family (Mtha 1 ‐ 3) were obtained by RT‐PCR using RNA from M. truncatula mycorrhizal roots as template. While Mtha 2 and Mtha 3 appeared to be constitutively expressed in roots and unaffected by AM development, transcripts of Mtha 1 could only be detected in AM tissues and not in controls. Further analyses by RT‐PCR revealed that Mtha 1 transcripts are not detectable in shoots and phosphate availability did not affect RNA accumulation of the gene. Localization of transcripts by in situ hybridization on AM tissues showed that Mtha 1 RNA accumulates only in cells containing fungal arbuscules. This is the first report of arbuscule‐specific induced expression of a plant H\+‐ATPase gene in mycorrhizal tissues.} } @Article{IPB-2091, author = {Ibdah, M. and Zhang, X.-H. and Schmidt, J. and Vogt, T. and}, title = {{A Novel Mg2\+-dependent O-Methyltransferase in the Phenylpropanoid Metabolism of Mesembryanthemum crystallinum}}, year = {2003}, pages = {43961-43972}, journal = {J. Biol. Chem.}, doi = {10.1074/jbc.M304932200}, volume = {278}, abstract = {Upon irradiation with elevated light intensities, the ice plant (Mesembryanthemum crystallinum) accumulates a complex pattern of methylated and glycosylated flavonol conjugates in the upper epidermal layer. Identification of a flavonol methylating activity, partial purification of the enzyme, and sequencing of the corresponding peptide fragments revealed a novel S-adenosyl-l-methionine-dependent O-methyltransferase that was specific for flavonoids and caffeoyl-CoA. Cloning and functional expression of the corresponding cDNA verified that the new methyltransferase is a multifunctional 26.6-kDa Mg2\+-dependent enzyme, which shows a significant sequence similarity to the cluster of caffeoyl coenzyme A-methylating enzymes. Functional analysis of highly homologous members from chickweed (Stellaria longipes), Arabidopsis thaliana, and tobacco (Nicotiana tabacum) demonstrated that the enzymes from the ice plant, chickweed, and A. thaliana possess a broader substrate specificity toward o-hydroquinone-like structures than previously anticipated for Mg2\+-dependent O-methyltransferases, and are distinctly different from the tobacco enzyme. Besides caffeoyl-CoA and flavonols, a high specificity was also observed for caffeoylglucose, a compound never before reported to be methylated by any plant O-methyltransferase. Based on phylogenetic analysis of the amino acid sequence and differences in acceptor specificities among both animal and plant O-methyltransferases, we propose that the enzymes from the Centrospermae, along with the predicted gene product from A. thaliana, form a novel subclass within the caffeoyl coenzyme A-dependent O-methyltransferases, with potential divergent functions not restricted to lignin monomer biosynthesis.} } @Article{IPB-2122, author = {Stenzel, I. and Hause, B. and Maucher, H. and Pitzschke, A. and Miersch, O. and Ziegler, J. and Ryan, C. A. and Wasternack, C. and}, title = {{Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato - amplification in wound signalling}}, year = {2003}, pages = {577-589}, journal = {Plant J.}, doi = {10.1046/j.1365-313X.2003.01647.x}, volume = {33}, abstract = {The allene oxide cyclase (AOC)‐catalyzed step in jasmonate (JA) biosynthesis is important in the wound response of tomato. As shown by treatments with systemin and its inactive analog, and by analysis of 35S::prosysteminsense and 35S::prosysteminantisense plants, the AOC seems to be activated by systemin (and JA) leading to elevated formation of JA. Data are presented on the local wound response following activation of AOC and generation of JA, both in vascular bundles. The tissue‐specific occurrence of AOC protein and generation of JA is kept upon wounding or other stresses, but is compromised in 35S::AOCsense plants, whereas 35S::AOCantisense plants exhibited residual AOC expression, a less than 10% rise in JA, and no detectable expression of wound response genes. The (i) activation of systemin‐dependent AOC and JA biosynthesis occurring only upon substrate generation, (ii) the tissue‐specific occurrence of AOC in vascular bundles, where the prosystemin gene is expressed, and (iii) the tissue‐specific generation of JA suggest an amplification in the wound response of tomato leaves allowing local and rapid defense responses.} } @INBOOK{IPB-148, author = {Thorson, J. S. and Vogt, T. and}, title = {{Carbohydrate-Based Drug Discovery}}, year = {2003}, pages = {685-711}, chapter = {{Glycosylated Natural Products}}, doi = {10.1002/3527602437.ch25}, abstract = {IntroductionA Summary of Bioactive Glycosylated Secondary MetabolitesAgents that Interact with DNAEnediynesBleomycinsDiazobenzofluorenesAnthracyclinesPluramycinsAureolic AcidsAgents that Interact with RNAOrthosomycinsMacrolidesAminoglycosidesAmicetinsAgents that Interact with Cell Walls and Cell MembranesNon‐Ribosomal PeptidesPolyenesSaccharomicinsAgents that Interact with ProteinsIndolocarbazolesCoumarinsBenzoisochromanequinonesAvermectinsAngucyclinesCardiac GlycosidesLignansAnthraquinone GlycosidesGinsenosidesGlycoalkaloidsGlucosinolatesAgents that Interact with Other (or Undefined) TargetsPlant PhenolicsMono‐ and Triterpenoid GlycosidesPlant Polymeric Natural GlycosidesConclusionsReferences} } @INBOOK{IPB-147, author = {Stumpe, M. and Stenzel, I. and Weichert, H. and Hause, B. and Feussner, I. and}, title = {{Advanced Research on Plant Lipids}}, year = {2003}, pages = {287-290}, chapter = {{The Lipoxygenase Pathway in Mycorrhizal Roots of Medicago Truncatula}}, doi = {10.1007/978-94-017-0159-4_67}, abstract = {Mycorrhizas are by far the most frequent occurring beneficial symbiotic interactions between plants and fungi. Species in \>80% of extant plant families are capable of establishing an arbuscular mycorrhiza (AM). In relation to the development of the symbiosis the first molecular modifications are those associated with plant defense responses, which seem to be locally suppressed to levels compatible with symbiotic interaction (Gianinazzi-Pearson, 1996). AM symbiosis can, however, reduce root disease caused by several soil-borne pathogens. The mechanisms underlying this protective effect are still not well understood. In plants, products of the enzyme lipoxygenase (LOX) and the corresponding downstream enzymes, collectively named LOX pathway (Fig. 1B), are involved in wound healing, pest resistance, and signaling, or they have antimicrobial and antifungal activity (Feussner and Wasternack, 2002). The central reaction in this pathway is catalyzed by LOXs leading to formation of either 9- or 13-hydroperoxy octadeca(di/trien)oic acids (9/13-HPO(D/T); Brash, 1999). Thus LOXs may be divided into 9- and 13-LOXs (Fig. 1A). Seven different reaction branches within this pathway can use these hydroperoxy polyenoic fatty acids (PUFAs) leading to (i) keto PUFAs by a LOX; (ii) epoxy hydroxy-fatty acids by an epoxy alcohol synthase (EAS); (iii) octadecanoids and jasmonates via allene oxide synthase (AOS); (iv) leaf aldehydes and leaf alcohols via fatty acid hydroperoxide lyase (HPL); (v) hydroxy PUFAs (reductase); (vi) divinyl ether PUFAs via divinyl ether synthase (DES); and (vii) epoxy- or dihydrodiolPUFAs via peroxygenase (PDX; Feussner and Wasternack, 2002). AOS, HPL and DES belong to one subfamily of P450-containing enzymes, the CYP74 family (Feussner and Wasternack, 2002). Here, the involvement of this CYP74 enzyme family in mycorrhizal roots of M. truncatula during early stages of AM symbiosis formation was analyzed.} } @INBOOK{IPB-146, author = {Strack, D. and Milkowski, C. and}, title = {{Polyphenols 2002: Recent Advances in Polyphenols Research}}, year = {2003}, pages = {50-61}, chapter = {{Recruitment of serine carboxypeptidase-related proteins into phenylpropanoid metabolism}}, editor = {El Hadrami, I., Daayf, F., eds.}, volume = {1}, } @INBOOK{IPB-145, author = {Stenzel, I. and Hause, B. and Feussner, I. and Wasternack, C. and}, title = {{Advanced Research on Plant Lipids}}, year = {2003}, pages = {267-270}, chapter = {{Transcriptional Activation of Jasmonate Biosynthesis Enzymes is not Reflected at Protein Level}}, doi = {10.1007/978-94-017-0159-4_62}, abstract = {Jasmonic acid (JA) and its precursor 12-oxo phytodienoic acid (OPDA) are lipid-derived signals in plant stress responses and development (Wasternack and Hause, 2002). Within the wound-response pathway of tomato, a local response of expression of defense genes such as the proteinase inhibitor 2 gene (PIN2) is preceded by a rise in JA (Herde et al., 1996; Howe et al., 1996) and ethylene (O’Donnell et al., 1996). Mutants affected in JA biosynthesis such as defl (Howe et al., 1996) or spr-2 (Li et al., 2002) clearly indicated that JA biosynthesis is an ultimate part of wound signaling. It is less understood, however, how the rise in JA is regulated.} } @Article{IPB-2188, author = {Van Damme, E. J. M. and Hause, B. and Hu, J. and Barre, A. and Rougé, P. and Proost, P. and Peumans, W. J. and}, title = {{Two Distinct Jacalin-Related Lectins with a Different Specificity and Subcellular Location Are Major Vegetative Storage Proteins in the Bark of the Black Mulberry Tree}}, year = {2002}, pages = {757-769}, journal = {Plant Physiol.}, doi = {10.1104/pp.005892}, volume = {130}, abstract = {Using a combination of protein isolation/characterization and molecular cloning, we have demonstrated that the bark of the black mulberry tree (Morus nigra) accumulates large quantities of a galactose-specific (MornigaG) and a mannose (Man)-specific (MornigaM) jacalin-related lectin. MornigaG resembles jacalin with respect to its molecular structure, specificity, and co- and posttranslational processing indicating that it follows the secretory pathway and eventually accumulates in the vacuolar compartment. In contrast, MornigaM represents a novel type of highly active Man-specific jacalin-related lectin that is synthesized without signal peptide or other vacuolar targeting sequences, and accordingly, accumulates in the cytoplasm. The isolation and cloning, and immunocytochemical localization of MornigaG and MornigaM not only demonstrates that jacalin-related lectins act as vegetative storage proteins in bark, but also allows a detailed comparison of a vacuolar galactose-specific and a cytoplasmic Man-specific jacalin-related lectin from a single species. Moreover, the identification of MornigaM provides the first evidence, to our knowledge, that bark cells accumulate large quantities of a cytoplasmic storage protein. In addition, due to its high activity, abundance, and ease of preparation, MornigaM is of great potential value for practical applications as a tool and bioactive protein in biological and biomedical research.} } @Article{IPB-2168, author = {Ibdah, M. and Krins, A. and Seidlitz, H. K. and Heller, W. and Strack, D. and Vogt, T. and}, title = {{Spectral dependence of flavonol and betacyanin accumulation in Mesembryanthemum crystallinum under enhanced ultraviolet radiation}}, year = {2002}, pages = {1145-1154}, journal = {Plant Cell Environ.}, doi = {10.1046/j.1365-3040.2002.00895.x}, volume = {25}, abstract = {Mesembryanthemum crystallinum L. (Aizoaceae) is a drought‐ and salt‐tolerant halophyte that is able to endure harsh environmental conditions. Upon irradiation with high light irradiance (1200–1500 µ mol m−2 s−1) it displays a rapid cell‐specific accumulation of plant secondary metabolites in the upper leaf epidermis; a phenomenon that is not detectable with salt or drought treatment. The accumulation of these compounds, the betacyanins and acylated flavonol glycosides, increases if the plants are exposed to polychromatic radiation with a progressively decreasing short‐wave cut‐off in the ultraviolet range. The response is localized in the epidermal bladder cells on the tips of young leaves and epidermal layers of fully expanded leaves. It is demonstrated that the accumulation of flavonols and betacyanins can be described by a weakly sigmoid dose function in combination with an exponential decrease of the response function of the plant with increasing wavelength.} } @Article{IPB-2164, author = {Hause, B. and Maier, W. and Miersch, O. and Kramell, R. and Strack, D. and}, title = {{Induction of Jasmonate Biosynthesis in Arbuscular Mycorrhizal Barley Roots}}, year = {2002}, pages = {1213-1220}, journal = {Plant Physiol.}, doi = {10.1104/pp.006007}, volume = {130}, abstract = {Colonization of barley (Hordeum vulgare cv Salome) roots by an arbuscular mycorrhizal fungus, Glomus intraradices Schenck \& Smith, leads to elevated levels of endogenous jasmonic acid (JA) and its amino acid conjugate JA-isoleucine, whereas the level of the JA precursor, oxophytodienoic acid, remains constant. The rise in jasmonates is accompanied by the expression of genes coding for an enzyme of JA biosynthesis (allene oxide synthase) and of a jasmonate-induced protein (JIP23). In situ hybridization and immunocytochemical analysis revealed that expression of these genes occurred cell specifically within arbuscule-containing root cortex cells. The concomitant gene expression indicates that jasmonates are generated and act within arbuscule-containing cells. By use of a near-synchronous mycorrhization, analysis of temporal expression patterns showed the occurrence of transcript accumulation 4 to 6 d after the appearance of the first arbuscules. This suggests that the endogenous rise in jasmonates might be related to the fully established symbiosis rather than to the recognition of interacting partners or to the onset of interaction. Because the plant supplies the fungus with carbohydrates, a model is proposed in which the induction of JA biosynthesis in colonized roots is linked to the stronger sink function of mycorrhizal roots compared with nonmycorrhizal roots.} } @Article{IPB-2163, author = {Hause, B. and Meyer, K. and Viitanen, P. and Chapple, C. and Strack, D. and}, title = {{Immunolocalization of 1-O-sinapoylglucose:malate sinapoyltransferase in Arabidopsis thaliana}}, year = {2002}, pages = {26-32}, journal = {Planta}, doi = {10.1007/s00425-001-0716-y}, volume = {215}, abstract = {The serine carboxypeptidase-like protein 1-O-sinapoylglucose:malate sinapoyltransferase (SMT) catalyzes the transfer of the sinapoyl moiety of 1-O-sinapoylglucose to malate in the formation of sinapoylmalate in some members of the Brassicaceae. Rabbit polyclonal monospecific antibodies were raised against the recombinant SMT produced in Escherichia coli from the corresponding Arabidopsis thaliana (L.) Heynh. cDNA. Immunoblot analysis of protein from different Arabidopsis tissues showed that the SMT is produced in all plant organs, except in the seeds and young seedlings. The enzyme was most abundant in older seedlings as well as in rosette leaves and the flowering stem of the plant. Minor amounts were found in the cauline leaves, flower buds and siliques. Traces were detected in the root and flowers. Arabidopsis and transgenic tobacco (Nicotiana tabacum L.) plants expressing the full-length Arabidopsis SMT containing an N-terminal signal peptide showed apparent molecular masses of the protein of 52–55 kDa. The difference of ca. 8 kDa compared to the recombinant protein produced in E. coli was shown to be due to post-translational N-glycosylation of SMT in plants. Immunofluorescent labeling of Arabidopsis leaf sections localized SMT to the central vacuoles of mesophyll and epidermal cells. Comparable leaf sections of an SMT deletion mutant showed no vacuolar immunofluorescent labeling. We conclude that Arabidopsis SMT is synthesized as a precursor protein that is targeted to the endoplasmic reticulum where the signal peptide is removed. The correct N-terminus of the recombinantly produced SMT protein lacking the signal peptide was confirmed by Edman degradation. The protein is probably glycosylated in the Golgi apparatus from where it is subsequently routed to the vacuole.} } @Article{IPB-2155, author = {Fester, T. and Schmidt, D. and Lohse, S. and Walter, M. H. and Giuliano, G. and Bramley, P. M. and Fraser, P. D. and Hause, B. and Strack, D. and}, title = {{Stimulation of carotenoid metabolism in arbuscular mycorrhizal roots}}, year = {2002}, pages = {148-154}, journal = {Planta}, doi = {10.1007/s00425-002-0917-z}, volume = {216}, abstract = {Development of arbuscular mycorrhizal roots is correlated with accumulation of various isoprenoids, i.e. acyclic C14 polyene \'mycorradicin\' and C13 cyclohexenone derivatives. We present data indicating a strong stimulation of carotenoid metabolism in such roots. Carotenoid profiling revealed mycorrhiza-specific accumulation of ζ-carotene in Zea mays and Medicago truncatula. Precursor accumulation after inhibition of phytoene desaturase (Pds) activity by norflurazon indicated an increased phytoene biosynthetic capacity in mycorrhizal roots of all species analyzed. Nicotiana tabacum plants transformed with a PDS promoter-GUS construct showed a cell-specific induction of PDS promoter activity in root cells containing arbuscules. Mycorradicin biosynthesis and, partially, mycorrhization were impaired in maize mutants deficient in carotenoid biosynthesis. These data indicate that (1) mycorradicin is probably synthesized via a C40 precursor carotenoid, (2) carotenoid biosynthesis is induced in mycorrhizal roots, (3) induction occurs, at least partially, at the transcriptional level, and (4) that this may play a functional role during mycorrhization.} } @Article{IPB-2154, author = {Fester, T. and Hause, B. and Schmidt, D. and Halfmann, K. and Schmidt, J. and Wray, V. and Hause, G. and Strack, D. and}, title = {{Occurrence and Localization of Apocarotenoids in Arbuscular Mycorrhizal Plant Roots}}, year = {2002}, pages = {256-265}, journal = {Plant Cell Physiol.}, doi = {10.1093/pcp/pcf029}, volume = {43}, abstract = {The core structure of the yellow pigment from arbuscular mycorrhizal (AM) maize roots contains the apocarotenoids mycorradicin (an acyclic C14 polyene) and blumenol C cellobioside (a C13 cyclohexenone diglucoside). The pigment seems to be a mixture of different esterification products of these apocarotenoids. It is insoluble in water and accumulates as hydrophobic droplets in the vacuoles of root cortical cells. Screening 58 species from 36 different plant families, we detected mycorradicin in mycorrhizal roots of all Liliopsida analyzed and of a considerable number of Rosopsida, but also species were found in which mycorradicin was undetectable in mycorrhizal roots. Kinetic experiments and microscopic analyses indicate that accumulation of the yellow pigment is correlated with the concomitant degradation of arbuscules and the extensive plastid network covering these haustorium-like fungal structures. The role of the apocarotenoids in mycorrhizal roots is still unknown. The potential C40 carotenoid precursors, however, are more likely to be of functional importance in the development and functioning of arbuscules.} } @Article{IPB-2153, author = {Fester, T. and Kiess, M. and Strack, D. and}, title = {{A mycorrhiza-responsive protein in wheat roots}}, year = {2002}, pages = {219-222}, journal = {Mycorrhiza}, doi = {10.1007/s00572-002-0173-x}, volume = {12}, abstract = {A small protein, designated Myk15, was found to be strongly induced in wheat (Triticum aestivum) roots colonized by the arbuscular mycorrhizal fungus Glomus intraradices. This protein, which is most abundant in root fractions characterized by strong mycorrhizal colonization, has been characterized using two-dimensional polyacrylamide gel electrophoresis and microsequencing. It has an apparent molecular mass of 15 kDa and an isoelectric point of 4.5. The N-terminal sequence has high similarity to a peptide sequence deduced from an expressed sequence tag (EST) clone derived from Medicago truncatula roots colonized by G. intraradices. This EST clone is predicted to code for a protein with a similar size and isoelectric point as Myk15. The N-terminus of the deduced M. truncatula protein contains a highly hydrophobic stretch of 24 amino acid residues preceding the region with high similarity to the Myk15 N-terminus. This hydrophobic stretch is predicted to form a transmembrane α-helix and may correspond to a cleavable targeting domain.} } @Article{IPB-2194, author = {Walter, M. H. and Hans, J. and Strack, D. and}, title = {{Two distantly related genes encoding 1-deoxy-D-xylulose 5-phosphate synthases: differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots}}, year = {2002}, pages = {243-254}, journal = {Plant J.}, doi = {10.1046/j.1365-313X.2002.01352.x}, volume = {31}, abstract = {Isopentenyl diphosphate, the universal precursor of isoprenoids, is synthesized by two separate routes, one in the cytosol and the other in plastids. The initial step of the plastidial pathway is catalysed by 1‐deoxy‐d ‐xylulose 5‐phosphate synthase (DXS), which was previously thought to be encoded by a single‐copy gene. We have identified two distinct classes of DXS‐like cDNAs from the model legume Medicago truncatula . The deduced mature MtDXS1 and MtDXS2 proteins, excluding the predicted plastid‐targeting peptides, are similar in size (72.7 and 71.2 kDa) yet share only 70% identity in their amino acid sequences, and both encode functional DXS proteins as shown by heterologous expression in Escherichia coli. Available DXS sequences from other plants can easily be assigned to either class 1 or class 2. Partial sequences of multiple DXS genes in a single genome may be found in the databases of several monocot and dicot plants. Blot analyses of RNA from M. truncatula , maize, tomato and tobacco demonstrate preferential expression of DXS1 genes in many developing plant tissues except roots. By contrast, DXS2 transcript levels are low in most tissues but are strongly stimulated in roots upon colonization by mycorrhizal fungi, correlated with accumulation of carotenoids and apocarotenoids. Monoterpene‐synthesizing gland cells of leaf trichomes appear to be another site of DXS2 gene activity. The potential importance of DXS1 in many housekeeping functions and a still hypothetical role of DXS2 in the biosynthesis of secondary isoprenoids is discussed.} } @Article{IPB-2192, author = {Vogt, T. and}, title = {{Substrate specificity and sequence analysis define a polyphyletic origin of betanidin 5- and 6-O-glucosyltransferase from Dorotheanthus bellidiformis}}, year = {2002}, pages = {492-495}, journal = {Planta}, doi = {10.1007/s00425-001-0685-1}, volume = {214}, abstract = {Betanidin 6-O-glucosyltransferase (6-GT) is involved in the glycosylation of betacyanins, which replace the chromogenic anthocyanins as flower colorants in the Caryophyllales. The 6-GT cDNA was cloned from a cDNA library of Dorotheanthus bellidiformis (Burm. f.) N.E. Br., and the amino acid and nucleotide sequences were shown to be distinctly different from the corresponding betanidin 5-O-glucosyltransferase (5-GT) from the same plant species. Although both enzymes share very similar substrates, the proteins show only 19% amino acid sequence identity. In contrast, the protein sequence of the 6-GT showed significant identity to GTs from other species and may identify a new cluster of putative anthocyanidin GTs. Therefore, 6-GT and 5-GT apparently have evolved independently from ancestral glucosyltransferases involved in flavonoid biosynthesis.} } @Article{IPB-2147, author = {Chen, Y. and Peumans, W. J. and Hause, B. and Bras, J. and Kumar, M. and Proost, P. and Barre, A. and Rougé, P. and Van Damme, E. J. M. and}, title = {{Jasmonate methyl ester induces the synthesis of a cytoplasmic/nuclear chitooligosaccharide‐binding lectin in tobacco leaves}}, year = {2002}, pages = {905-907}, journal = {FASEB J.}, doi = {10.1096/fj.01-0598fje}, volume = {16}, abstract = {In contrast to animal lectins, no evidence has indicated the occurrence of plant lectins, which recognize and bind “endogenous” receptors and accordingly are involved in recognition mechanisms within the organism itself. Here we show that the plant hormone jasmonic acid methyl ester (JAME) induces in leaves of Nicotiana tabacum (var. Samsun NN) the expression of a lectin that is absent from untreated plants. The lectin specifically binds to oligomers of N‐acetylglucosamine and is detected exclusively in the cytoplasm and the nucleus. Both the subcellular location and specificity indicate that the Nicotiana tabacum agglutinin (called Nictaba) may be involved in the regulation of gene expression in stressed plants through specific protein‐carbohydrate interactions with regulatory cytoplasmic/nuclear glycoproteins. Searches in the databases revealed that many flowering plants contain sequences encoding putative homologues of the tobacco lectin, which suggest that Nictaba is the prototype of a widespread or possibly ubiquitous family of lectins with a specific endogenous role.} } @Article{IPB-2137, author = {Bachmann, A. and Hause, B. and Maucher, H. and Garbe, E. and Vörös, K. and Weichert, H. and Wasternack, C. and Feussner, I. and}, title = {{Jasmonate-Induced Lipid Peroxidation in Barley Leaves Initiated by Distinct 13-LOX Forms of Chloroplasts}}, year = {2002}, pages = {1645-1657}, journal = {Biol. Chem.}, doi = {10.1515/BC.2002.185}, volume = {383}, abstract = {In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [Vörös et al., Eur. J. Biochem. 251 (1998), 36 44], two fulllength cDNAs (LOX2:Hv:2, LOX2:Hv:3) were isolated from barley leaves (Hordeum vulgare cv. Salome) and characterized. Both of them encode 13-lipoxygenases with putative target sequences for chloroplast import. Immunogold labeling revealed preferential, if not exclusive, localization of lipoxygenase proteins in the stroma. The ultrastructure of the chloroplast was dramatically altered following methyl jasmonate treatment, indicated by a loss of thylakoid membranes, decreased number of stacks and appearance of numerous osmiophilic globuli. The three 13-lipoxygenases are differentially expressed during treatment with jasmonate, salicylate, glucose or sorbitol. Metabolite profiling of free linolenic acid and free linoleic acid, the substrates of lipoxygenases, in water floated or jasmonatetreated leaves revealed preferential accumulation of linolenic acid. Remarkable amounts of free 9- as well as 13-hydroperoxy linolenic acid were found. In addition, metabolites of these hydroperoxides, such as the hydroxy derivatives and the respective aldehydes, appeared following methyl jasmonate treatment. These findings were substantiated by metabolite profiling of isolated chloroplasts, and subfractions including the envelope, the stroma and the thylakoids, indicating a preferential occurrence of lipoxygenasederived products in the stroma and in the envelope. These data revealed jasmonateinduced activation of the hydroperoxide lyase and reductase branch within the lipoxygenase pathway and suggest differential activity of the three 13-lipoxygenases under different stress conditions.} } @INBOOK{IPB-159, author = {Wasternack, C. and Hause, B. and}, year = {2002}, pages = {165-221}, chapter = {{Jasmonates and octadecanoids: Signals in plant stress responses and development}}, journal = {Prog. Nucleic Acid Res. Mol. Biol.}, doi = {10.1016/S0079-6603(02)72070-9}, volume = {72}, abstract = {Plants are sessile organisms. Consequently they have to adapt constantly to fluctuations in the environment. Some of these changes involve essential factors such as nutrients, light, and water. Plants have evolved independent systems to sense nutrients such as phosphate and nitrogen. However, many of the environmental factors may reach levels which represent stress for the plant. The fluctuations can range between moderate and unfavorable, and the factors can be of biotic or abiotic origin. Among the biotic factors influencing plant life are pathogens and herbivores. In case of bacteria and fungi, symbiotic interactions such as nitrogen-fixating nodules and mycorrhiza, respectively, may be established. In case of insects, a tritrophic interaction of herbivores, carnivores, and plants may occur mutualistically or parasitically. Among the numerous abiotic factors are low temperature, frost, heat, high light conditions, ultraviolet light, darkness, oxidation stress, hypoxia, wind, touch, nutrient imbalance, salt stress, osmotic adjustment, water deficit, and desiccation.In the last decade jasmonates were recognized as being signals in plant responses to most of these biotic and abiotic factors. Signaling via jasmonates was found to occur intracellularly, and systemically as well as interorganismically. Jasmonates are a group of ubiquitously occurring plant growth regulators originally found as the major constituents in the etheric oil of jasmine, and were first suggested to play a role in senescence due to a strong senescence-promoting effect. Subsequently, numerous developmental processes were described in which jasmonates exhibited hormone-like properties. Recent knowledge is reviewed here on jasmonates and their precursors, the octadecanoids. After discussing occurrence and biosynthesis, emphasis is placed upon the signal transduction pathways in plant stress responses in which jasmonates act a signal. Finally, examples are described on the role of jasmonates in developmental processes.} } @INBOOK{IPB-157, author = {Riemann, D. and Röntsch, J. and Hause, B. and Langner, J. and Kehlen, A. and}, title = {{Cellular Peptidases in Immune Functions and Diseases 2}}, year = {2002}, pages = {57-66}, chapter = {{Cell-Cell Contact Between Lymphocytes and Fibroblast-Like Synovioctyes Induces Lymphocytic Expression of Aminopeptidase n/cd13 and Results in Lymphocytic Activation}}, journal = {Adv. Exp. Med. Biol.}, doi = {10.1007/0-306-46826-3_6}, volume = {477}, } @Article{IPB-2241, author = {Van Damme, E. J. M. and Hu, J. and Barre, A. and Hause, B. and Baggerman, G. and Rougé, P. and Peumans, W. J. and}, title = {{Purification, characterization, immunolocalization and structural analysis of the abundant cytoplasmic β-amylase from Calystegia sepium (hedge bindweed) rhizomes}}, year = {2001}, pages = {6263-6273}, journal = {Eur. J. Biochem.}, doi = {10.1046/j.0014-2956.2001.02584.x}, volume = {268}, abstract = {An abundant catalytically active β‐amylase (EC 3.2.1.2) was isolated from resting rhizomes of hedge bindweed (Calystegia sepium ). Biochemical analysis of the purified protein, molecular modeling, and cloning of the corresponding gene indicated that this enzyme resembles previously characterized plant β‐amylases with regard to its amino‐acid sequence, molecular structure and catalytic activities. Immunolocalization demonstrated that the β‐amylase is exclusively located in the cytoplasm. It is suggested that the hedge bindweed rhizome β‐amylase is a cytoplasmic vegetative storage protein.} } @Article{IPB-2219, author = {Hao, Q. and Van Damme, E. J. M. and Hause, B. and Barre, A. and Chen, Y. and Rougé, P. and Peumans, W. J. and}, title = {{Iris Bulbs Express Type 1 and Type 2 Ribosome-Inactivating Proteins with Unusual Properties}}, year = {2001}, pages = {866-876}, journal = {Plant Physiol.}, doi = {10.1104/pp.125.2.866}, volume = {125}, abstract = {Two closely related lectins from bulbs of the Dutch iris (Iris hollandica var. Professor Blaauw) have been isolated and cloned. Both lectins, called Iris agglutinin b and Iris agglutinin r, possess N-glycosidase activity and share a high sequence similarity with previously described type 2 ribosome-inactivating proteins (RIP). However, these lectins show only 57% to 59% sequence identity to a previously characterized type 1 RIP from iris, called IRIP. The identification of the iris lectins as type 2 RIP provides unequivocal evidence for the simultaneous occurrence of type 1 and type 2 RIP in iris bulbs and allowed a detailed comparison of type 1 and type 2 RIP from a single plant, which provides further insight into the molecular evolution of RIP. Binding studies and docking experiments revealed that the lectins exhibit binding activity not only toward Gal/N-acetylgalactosamine, but also toward mannose, demonstrating for the first time that RIP-binding sites can accommodate mannose.} } @Article{IPB-2212, author = {Fester, T. and Strack, D. and Hause, B. and}, title = {{Reorganization of tobacco root plastids during arbuscule development}}, year = {2001}, pages = {864-868}, journal = {Planta}, doi = {10.1007/s004250100561}, volume = {213}, abstract = {In the present paper we analyzed plastid populations labeled by the green fluorescent protein in non-mycorrhizal and mycorrhizal roots of tobacco (Nicotiana tabacum L.). We show by confocal laser scanning microscopy (i) a dramatic increase in these plastids in mycorrhizal roots and (ii) the formation of dense plastid networks covering the symbiotic interface of the arbuscular mycorrhiza, the arbuscule. These cytological observations point to an important role of root cortical cell plastids in the functioning of arbuscular mycorrhizal symbiosis.} } @Article{IPB-2206, author = {Cai, Y. and Sun, M. and Schliemann, W. and Corke, H. and}, title = {{Chemical Stability and Colorant Properties of Betaxanthin Pigments from Celosia argentea}}, year = {2001}, pages = {4429-4435}, journal = {J. Agr. Food Chem.}, doi = {10.1021/jf0104735}, volume = {49}, abstract = {The chemical stability and colorant properties of three betaxanthins recently identified from Celosia argentea varieties were evaluated. Lyophilized betaxanthin powders from yellow inflorescences of Celosia exhibited bright yellow color and high color purity with strong hygroscopicity. The aqueous solutions containing these betaxanthins were bright yellow in the pH range 2.2−7.0, and they were most stable at pH 5.5. The betaxanthins in a model system (buffer) were susceptible to heat, and found to be as unstable as red betacyanins (betanin and amaranthine) at high temperatures (\>40 °C), but more stable at 40 °C with the exclusion of light and air. The three betaxanthins had slightly higher pigment retention than amaranthine/isoamaranthine in crude extracts at 22 °C, as verified by HPLC analysis. Lyophilized betaxanthins had much better storage stability (mean 95.0% pigment retention) than corresponding aqueous solutions (14.8%) at 22 °C after 20 weeks. Refrigeration (4 °C) significantly increased pigment retention of aqueous betaxanthins to 75.5%.} } @Article{IPB-2200, author = {Baumert, A. and Mock, H.-P. and Schmidt, J. and Herbers, K. and Sonnewald, U. and Strack, D. and}, title = {{Patterns of phenylpropanoids in non-inoculated and potato virus Y-inoculated leaves of transgenic tobacco plants expressing yeast-derived invertase}}, year = {2001}, pages = {535-541}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(00)00422-2}, volume = {56}, abstract = {The patterns of secondary metabolites in leaves of yeast invertase-transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were analyzed. Plants expressing cytosolic yeast-derived invertase (cytInv) or apoplastic (cell wall associated) yeast invertase (cwInv) showed a characteristic phytochemical phenotype compared to untransformed controls (wild-type plants). The level of phenylpropanoids decreased in the cytInv plants but increased in the cwInv plants, which showed an induced de novo synthesis of a caffeic acid amide, i.e. N-caffeoylputrescine. In addition, the level of the coumarin glucoside scopolin was markedly enhanced. Increased accumulation of scopolin in the cwInv plants is possibly correlated with the induction of defense reactions and the appearance of necrotic lesions similar to the hypersensitive response caused by avirulent pathogens. This is consistent with results from potato virus Y-infected plants. Whereas there was no additional increase in the coumarins in leaves following infection in cwInv plants, wild-type plants showed a slight increase and cytInc a marked increase.} } @Article{IPB-2199, author = {Back, K. and Jang, S. M. and Lee, B.-C. and Schmidt, A. and Strack, D. and Kim, K.-M. and}, title = {{Cloning and Characterization of a Hydroxycinnamoyl-CoA:Tyramine N-(Hydroxycinnamoyl)Transferase Induced in Response to UV-C and Wounding from Capsicum annuum}}, year = {2001}, pages = {475-481}, journal = {Plant Cell Physiol.}, doi = {10.1093/pcp/pce060}, volume = {42}, abstract = {Hydroxycinnamoyl-CoA : tyramine N-(hydroxycinnamoyl) transferase (THT) is a pivotal enzyme in the synthesis of N-(hydroxycinnamoyl)-amines, which are associated with cell wall fortification in plants. The cDNA encoding THT was cloned from the leaves of UV-C treated Capsicum annuum (hot pepper) using a differential screening strategy. The predicted protein encoded by the THT cDNA is 250 amino acids in length and has a relative molecular mass of 28,221. The protein sequence derived from the cDNA shares 76% and 67% identity with the potato and tobacco THT protein sequences, respectively. The recombinant pepper THT enzyme was purified using a bacterial overexpression system. The purified enzyme has a broad substrate specificity including acyl donors such as cinnamoyl-, sinapoyl-, feruloyl-, caffeoyl-, and 4-coumaroyl-CoA and acceptors such as tyramine and octopamine. In UV-C treated plants, the THT mRNA was strongly induced in leaves, and the elevated level of expression was stable for up to 36 h. THT mRNA also increased in leaves that were detached from the plant but not treated with UV-C. THT expression was measured in different plant tissues, and was constitutive at a similar level in leaf, root, stem, flower and fruit. Induction of THT mRNA was correlated with an increase in THT protein.} } @Article{IPB-2237, author = {Strack, D. and Fester, T. and Hause, B. and Walter, M. H. and}, title = {{Die arbuskuläre Mykorrhiza: Eine unterirdische Lebensgemeinschaft}}, year = {2001}, pages = {286-295}, journal = {Biologie in unserer Zeit}, doi = {10.1002/1521-415X(200109)31:5<286::AID-BIUZ286>3.0.CO;2-G}, volume = {31}, abstract = {Pflanzen und bestimmte Pilze haben im Laufe ihrer Entwicklungsgeschichte „gelernt”︁, in einer engen Assoziation im Boden, der Mykorrhiza, eine äußerst erfolgreiche Symbiose miteinander einzugehen. Arbuskuläre Mykorrhizapilze helfen Pflanzen sich auf nährstoffarmen Böden ausreichend mit Wasser, Nährsalzen und Spurenelementen zu versorgen und fördern entscheidend Diversität und Produktivität von Pflanzengesellschaften. Darüber hinaus zeigen mykorrhizierte Pflanzen eine erhöhte Widerstandsfähigkeit gegen Pathogenbefall. Im Gegenzug „bezahlt”︁ die Pflanze den Pilz für diesen Gewinn mit Kohlenhydraten in Form einfacher Zucker (Glucose, Fructose). Durch manche Erfolge in der Erforschung der Mykorrhiza auf Metaboliten‐ und Genebene beginnen wir allmählich zu erahnen, wie komplex die molekularen Interaktionen dieser Symbiose sind. Es ist zu erwarten, dass das steigende Interesse an der Mykorrhizaforschung zu neuen Einsichten in die Strategien von Pflanzen und Pilzen in der Entwicklung mutualistisch‐symbiontischer Assoziationen führen wird.} } @Article{IPB-2236, author = {Strack, D. and Schliemann, W. and}, title = {{Bifunktionelle Polyphenoloxidasen: neuartige Funktionen in der Biosynthese pflanzlicher Farbstoffe}}, year = {2001}, pages = {3907-3911}, journal = {Angew. Chem.}, doi = {10.1002/1521-3757(20011015)113:20<3907::AID-ANGE3907>3.0.CO;2-J}, volume = {113}, abstract = {Bisher war die Funktion der Polyphenoloxidasen (PPO) unklar. Inzwischen konnte aber gezeigt werden, dass eine Tyrosinase an der Betacyan‐Biosynthese des Portulakröschens (siehe Bild) und der Roten Rübe sowie eine Chalkon‐spezifische PPO an der Auronbildung in gelben Löwenmaulblüten beteiligt ist.} } @Article{IPB-2235, author = {Strack, D. and Schliemann, W. and}, title = {{Bifunctional Polyphenol Oxidases: Novel Functions in Plant Pigment Biosynthesis}}, year = {2001}, pages = {3791-3794}, journal = {Angew. Chem. Int. Ed.}, doi = {10.1002/1521-3773(20011015)40:20<3791::AID-ANIE3791>3.0.CO;2-T}, volume = {40}, abstract = {Enzymes in search of a function, for polyphenol oxidases (PPOs), described as such, this situation has changed recently. A tyrosinase is involved in betacyanin biosynthesis in common portulaca (see picture) and red beet, and a chalcone‐specific PPO is responsible for the formation of aurones in yellow snapdragon flowers.} } @Article{IPB-2232, author = {Schliemann, W. and Cai, Y. and Degenkolb, T. and Schmidt, J. and Corke, H. and}, title = {{Betalains of Celosia argentea}}, year = {2001}, pages = {159-165}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(01)00141-8}, volume = {58}, abstract = {The betalains of yellow, orange and red inflorescences of common cockscomb (Celosia argentea var. cristata) were compared and proved to be qualitatively identical to those of feathered amaranth (Celosia argentea var. plumosa). In addition to the known compounds amaranthin and betalamic acid, the structures of three yellow pigments were elucidated to be immonium conjugates of betalamic acid with dopamine, 3-methoxytyramine and (S)-tryptophan by various spectroscopic techniques and comparison to synthesized reference compounds; the latter two are new to plants. Among the betacyanins occurring in yellow inflorescences in trace amounts, the presence of 2-descarboxy-betanidin, a dopamine-derived betacyanin, has been ascertained. The detection of high dopamine concentration may be of toxicological relevance in use of yellow inflorescences as a vegetable and in traditional Chinese medicine, common uses for the red inflorescences of common cockscomb.The betaxanthins of two Celosia argentea varieties were identified as betalamic acid conjugates of dopamine (1), 3-methoxytyramine (2) and (S)-tryptophan.} } @Article{IPB-2228, author = {Milkowski, C. and Krampe, S. and Weirich, J. and Hasse, V. and Boles, E. and Breunig, K. D. and}, title = {{Feedback Regulation of Glucose Transporter Gene Transcription in Kluyveromyces lactis by Glucose Uptake}}, year = {2001}, pages = {5223-5229}, journal = {J. Bacteriol.}, doi = {10.1128/JB.183.18.5223-5229.2001}, volume = {183}, abstract = {In the respirofermentative yeast Kluyveromyces lactis, only a single genetic locus encodes glucose transporters that can support fermentative growth. This locus is polymorphic in wild-type isolates carrying either KHT1and KHT2, two tandemly arranged HXT-like genes, or RAG1, a low-affinity transporter gene that arose by recombination between KHT1 andKHT2. Here we show that KHT1 is a glucose-induced gene encoding a low-affinity transporter very similar to Rag1p. Kht2p has a lower Km (3.7 mM) and a more complex regulation. Transcription is high in the absence of glucose, further induced by low glucose concentrations, and repressed at higher glucose concentrations. The response ofKHT1 and KHT2 gene regulation to high but not to low concentrations of glucose depends on glucose transport. The function of either Kht1p or Kht2p is sufficient to mediate the characteristic response to high glucose, which is impaired in akht1 kht2 deletion mutant. Thus, the KHTgenes are subject to mutual feedback regulation. Moreover, glucose repression of the endogenous β-galactosidase (LAC4) promoter and glucose induction of pyruvate decarboxylase were abolished in the kht1 kht2 mutant. These phenotypes could be partially restored by HXT gene family members fromSaccharomyces cerevisiae. The results indicate that the specific responses to high but not to low glucose concentrations require a high rate of glucose uptake.} } @Article{IPB-2226, author = {Lehmann, K. and Hause, B. and Altmann, D. and Köck, M. and}, title = {{Tomato Ribonuclease LX with the Functional Endoplasmic Reticulum Retention Motif HDEF Is Expressed during Programmed Cell Death Processes, Including Xylem Differentiation, Germination, and Senescence}}, year = {2001}, pages = {436-449}, journal = {Plant Physiol.}, doi = {10.1104/pp.010362}, volume = {127}, abstract = {We have studied the subcellular localization of the acid S-like ribonuclease (RNase) LX in tomato (Lycopersicon esculentum Mill.) cells using a combination of biochemical and immunological methods. It was found that the enzyme, unexpectedly excluded from highly purified vacuoles, accumulates in the endoplasmic reticulum. The evidence that RNase LX is a resident of the endoplasmic reticulum (ER) is supported by an independent approach showing that the C-terminal peptide HDEF of RNase LX acts as an alternative ER retention signal in plants. For functional testing, the cellular distribution of chimeric protein constructs based on a marker protein, Brazil nut (Bertholletia excelsa) 2S albumin, was analyzed immunochemically in transgenic tobacco (Nicotiana tabacum) plants. Here, we report that the peptide motif is necessary and sufficient to accumulate 2S albumin constructs of both vacuolar and extracellular final destinations in the ER. We have shown immunochemically that RNase LX is specifically expressed during endosperm mobilization and leaf and flower senescence. Using immunofluorescence, RNase LX protein was detected in immature tracheary elements, suggesting a function in xylem differentiation. These results support a physiological function of RNase LX in selective cell death processes that are also thought to involve programmed cell death. It is assumed that RNase LX accumulates in an ER-derived compartment and is released by membrane disruption into the cytoplasma of those cells that are intended to undergo autolysis. These processes are accompanied by degradation of cellular components supporting a metabolic recycling function of the intracellular RNase LX.} } @Article{IPB-2223, author = {Kobayashi, N. and Schmidt, J. and Wray, V. and Schliemann, W. and}, title = {{Formation and occurrence of dopamine-derived betacyanins}}, year = {2001}, pages = {429-436}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(00)00383-6}, volume = {56}, abstract = {In light of the fact that the main betaxanthin (miraxanthin V) and the major betacyanin (2-descarboxy-betanidin) in hairy root cultures of yellow beet (Beta vulgaris L.) are both dopamine-derived, the occurrence of similar structures for the minor betacyanins was also suggested. By HPLC comparison with the betacyanins obtained by dopamine administration to beet seedlings, enzymatic hydrolysis, LCMS and 1H NMR analyses, the minor betacyanins from hairy roots were identified as 2-descarboxy-betanin and its 6′-O-malonyl derivative. A short-term dopamine administration experiment with fodder beet seedlings revealed that the condensation step between 2-descarboxy-cyclo-Dopa and betalamic acid is the decisive reaction, followed by glucosylation and acylation. From these data a pathway for the biosynthesis of dopamine-derived betalains is proposed. Furthermore, the occurrence of these compounds in various cell and hairy root cultures as well as beet plants (Fodder and Garden Beet Group) is shown.} } @Article{IPB-2222, author = {Jones, P. and Vogt, T. and}, title = {{Glycosyltransferases in secondary plant metabolism: tranquilizers and stimulant controllers}}, year = {2001}, pages = {164-174}, journal = {Planta}, doi = {10.1007/s004250000492}, volume = {213}, abstract = {Plants are exposed to a wide range of toxic and bioactive low-molecular-weight molecules from both exogenous and endogenous sources. Glycosylation is one of the primary sedative mechanisms that plants utilise in order to maintain metabolic homeostasis. Recently, a range of glycosyltransferases has been characterized in detail with regard to substrate specificity. The next step in increasing our understanding of the biology of glycosylation will require information regarding the exact role of individual glycosyltransferases in planta, as well as an insight into their potential involvement in metabolon-complexes. Hopefully, this will answer how a large number of glycosyltransferases with broad, rather than narrow, substrate specificity can be constrained in order to avoid interfering with other pathways of primary and secondary metabolism. These and other topics are discussed.} } @INBOOK{IPB-162, author = {Strack, D. and}, title = {{Flavonoids and Other Polyphenols}}, year = {2001}, pages = {70-81}, chapter = {{Enzymes involved in hydroxycinnamate metabolism}}, journal = {Methods Enzymol.}, doi = {10.1016/S0076-6879(01)35232-1}, volume = {335}, } @Article{IPB-2288, author = {Wasternack, C. and Hause, B. and}, title = {{Stressabwehr und Entwicklung: Jasmonate — chemische Signale in Pflanzen}}, year = {2000}, pages = {312-320}, journal = {Biologie in unserer Zeit}, doi = {10.1002/1521-415X(200011)30:6<312::AID-BIUZ312>3.0.CO;2-8}, volume = {30}, abstract = {Chemische Signale wurden bereits im 19.Jahrhundert als Regulatoren von Wachstum und Entwicklung der Pflanzen postuliert.In den letzten 70 Jahren wurde die Wirkungsweise der klassischen Pflanzenhormone wie der Auxine, Gibberelline, Cytokinine, Ethylen und Abscisinsäure aufgeklärt. Doch erst im letzten Jahrzehnt entdeckte man die Bedeutung der Brassinosteroide, der Peptidhormone und der Jasmonate.} } @Article{IPB-2287, author = {Walter, M. H. and Fester, T. and Strack, D. and}, title = {{Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the \'yellow pigment\' and other apocarotenoids}}, year = {2000}, pages = {571-578}, journal = {Plant J.}, doi = {10.1046/j.1365-313x.2000.00708.x}, volume = {21}, abstract = {Plants and certain bacteria use a non‐mevalonate alternative route for the biosynthesis of many isoprenoids, including carotenoids. This route has been discovered only recently and has been designated the deoxyxylulose phosphate pathway or methylerythritol phosphate (MEP) pathway. We report here that colonisation of roots from wheat, maize, rice and barley by the arbuscular mycorrhizal fungal symbiont Glomus intraradices involves strong induction of transcript levels of two of the pivotal enzymes of the MEP pathway, 1‐deoxy‐D‐xylulose 5‐phosphate synthase (DXS) and 1‐deoxy‐D‐xylulose 5‐phosphate reductoisomerase (DXR). This induction is temporarily and spatially correlated with specific and concomitant accumulation of two classes of apocarotenoids, namely glycosylated C13 cyclohexenone derivatives and mycorradicin (C14) conjugates, the latter being a major component of the long‐known ‘yellow pigment’. A total of six cyclohexenone derivatives were characterised from mycorrhizal wheat and maize roots. Furthermore, the acyclic structure of mycorradicin described previously only from maize has been identified from mycorrhizal wheat roots after alkaline treatment of an ‘apocarotenoid complex’ of yellow root constituents. We propose a hypothetical scheme for biogenesis of both types of apocarotenoids from a common oxocarotenoid (xanthophyll) precursor. This is the first report demonstrating (i) that the plastidic MEP pathway is active in plant roots and (ii) that it can be induced by a fungus.} } @Article{IPB-2286, author = {Vogt, T. and Jones, P. and}, title = {{Glycosyltransferases in plant natural product synthesis: characterization of a supergene family}}, year = {2000}, pages = {380-386}, journal = {Trends Plant Sci.}, doi = {10.1016/S1360-1385(00)01720-9}, volume = {5}, abstract = {Glycosyltransferases of plant secondary metabolism transfer nucleotide-diphosphate-activated sugars to low molecular weight substrates. Until recently, glycosyltransferases were thought to have only limited influence on the basic physiology of the plant. This view has changed. Glycosyltransferases might in fact have an important role in plant defense and stress tolerance. Recent results obtained with several recombinant enzymes indicate that many glycosyltransferases are regioselective or regiospecific rather than highly substrate specific. This might indicate how plants evolve novel secondary products, placing enzymes with broad substrate specificities downstream of the conserved, early, pivotal enzymes of plant secondary metabolism.} } @Article{IPB-2285, author = {Vierheilig, H. and Maier, W. and Wyss, U. and Samson, J. and Strack, D. and Piché, Y. and}, title = {{Cyclohexenone derivative- and phosphate-levels in split-root systems and their role in the systemic suppression of mycorrhization in precolonized barley plants}}, year = {2000}, pages = {593-599}, journal = {J. Plant Physiol.}, doi = {10.1016/S0176-1617(00)80001-2}, volume = {157}, abstract = {In a split-root system root colonization by the arbuscular mycorrhizal fungus Glomus mosseae on one side is reduced when roots on the other side are already colonized by G. mosseae. Root colonization by arbuscular mycorrhizal fungi enhances the P-status of plants, thus the observed suppressional effect on further root colonization in precolonized barley plants could be P-level regulated. Split-root systems allow to separate plant mediated P-effects on root colonization by arbuscular mycorrhizal fungi from direct P-effects on arbuscular mycorrhizal fungi. By adding a KH2PO4-solution to one side of the split-root system of non-mycorrhizal control plants, higher P-levels were obtained as in split-root systems of G. mosseae precolonized plants. Subsequent inoculation with G. mosseae of the P-supplied and the precolonized plants resulted in an inhibition of root colonization in the precolonized plants, but not in the P-supplied plants, discarding the enhanced P-level as the responsible factor for the observed suppression. Cyclohexenone derivatives are secondary plant compounds only found in roots of mycorrhizal plants. Analysis of cyclohexenone derivatives in mycorrhizal and non-mycorrhizal roots in split-root systems revealed that cyclohexenone derivatives can be detected in mycorrhizal roots, but not in non-mycorrhizal roots of mycorrhizal plants. The presented results show clearly that cyclohexenone derivatives are not systemically accumulated and that the P-levels are not the responsible factors for the observed systemic suppression of mycorrhization in roots of precolonized barley plants.} } @Article{IPB-2284, author = {Vierheilig, H. and Gagnon, H. and Strack, D. and Maier, W. and}, title = {{Accumulation of cyclohexenone derivatives in barley, wheat and maize roots in response to inoculation with different arbuscular mycorrhizal fungi}}, year = {2000}, pages = {291-293}, journal = {Mycorrhiza}, doi = {10.1007/PL00009994}, volume = {9}, abstract = {Glomus intraradices, Glomus mosseae, and Gigaspora rosea leads to the accumulation of cyclohexenone derivatives. Mycorrhizal roots of all plants accumulate in response to all three fungi blumenin [9-O-(2′-O-glucuronosyl)-β-glucopyranoside of 6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one], 13-carboxyblumenol C 9-O-gentiobioside, nicoblumin [9-O-(6′-O-β-glucopyranosyl)-β-glucopyranoside of 13-hydroxy-6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one] and another, as yet unidentified, cyclohexenone derivative. The accumulation of all four compounds in three tested mycorrhizal plants colonized by the three arbuscular mycorrhizal fungi indicates no fungus-specific induction of these compounds.} } @Article{IPB-2279, author = {Roitsch, T. and Ehneß, R. and Goetz, M. and Hause, B. and Hofmann, M. and Sinha, A. K. and}, title = {{Regulation and function of extracellular invertase from higher plants in relation to assimilate partitioning, stress responses and sugar signalling}}, year = {2000}, pages = {815-825}, journal = {Funct. Plant Biol.}, doi = {10.1071/PP00001}, volume = {27}, abstract = {Carbohydrates are synthesised in photosynthetically active source tissues and exported, in most species in the form of sucrose, to photosynthetically less active or inactive sink tissues. Sucrose hydrolysis at the site of utilisation contributes to phloem unloading. This phenomenon links sink metabolism with phloem transport to, and partitioning between, sinks. Invertases catalyse the irreversible hydrolysis of sucrose and thus are expected to contribute to carbohydrate partitioning. Different invertase isoenzymes may be distinguished based on their intracellular location, their isoelectric points and pH optima. Extracellular, cell-wall-bound invertase is uniquely positioned to supply carbohydrates to sink tissues via an apoplasmic pathway, and links the transport sugar sucrose to hexose transporters. A number of studies demonstrate an essential function of this invertase isoenzyme for phloem unloading, carbohydrate partitioning and growth of sink tissues. Extracellular invertases were shown to be specifically expressed under conditions that require a high carbohydrate supply to sink tissues. Further, their expression is upregulated by a number of stimuli that affect source–sink relations. Substrate and reaction products of invertases are not only nutri-ents, but also signal molecules. Like hormones and in combination with hormones and other stimuli, they can regu-late many aspects of plant development from gene expression to long-distance nutrient allocation. Based on studies in Chenopodium rubrum, tomato (Lycopersicon esculentum) and tobacco (Nicotiana tabacum), the regulation of extracellular invertase and its function in assimilate partitioning, defence reactions and sugar signal transduction pathways are discussed.} } @Article{IPB-2276, author = {Peumans, W. J. and Hause, B. and Van Damme, E. J. M. and}, title = {{The galactose-binding and mannose-binding jacalin-related lectins are located in different sub-cellular compartments}}, year = {2000}, pages = {186-192}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(00)01801-9}, volume = {477}, abstract = {A galactose-specific and a mannose-specific lectin of the family of the jacalin-related lectins have been localized by immunofluorescence microscopy. The present localization studies provide for the first time unambiguous evidence for the cytoplasmic location of the mannose-specific jacalin-related lectin from rhizomes of Calystegia sepium, which definitely differs from the vacuolar location of the galactose-specific jacalin from Artocarpus integrifolia. These observations support the hypothesis that the galactose-specific jacalin-related lectins evolved from their mannose-specific homologues through the acquisition of vacuolar targeting sequences.} } @Article{IPB-2274, author = {Milkowski, C. and Baumert, A. and Strack, D. and}, title = {{Identification of four Arabidopsis genes encoding hydroxycinnamate glucosyltransferases}}, year = {2000}, pages = {183-184}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(00)02270-5}, volume = {486}, } @Article{IPB-2273, author = {Milkowski, C. and Baumert, A. and Strack, D. and}, title = {{Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase}}, year = {2000}, pages = {883-886}, journal = {Planta}, doi = {10.1007/s004250000411}, volume = {211}, abstract = {A cDNA encoding a UDP-glucose:sinapate glucosyltransferase (SGT) that catalyzes the formation of 1-O-sinapoylglucose, was isolated from cDNA libraries constructed from immature seeds and young seedlings of rape (Brassica napus L.). The open reading frame encoded a protein of 497 amino acids with a calculated molecular mass of 55,970 Da and an isoelectric point of 6.36. The enzyme, functionally expressed in Escherichia coli, exhibited broad substrate specificity, glucosylating sinapate, cinnamate, ferulate, 4-coumarate and caffeate. Indole-3-acetate, 4-hydroxybenzoate and salicylate were not conjugated. The amino acid sequence of the SGT exhibited a distinct sequence identity to putative indole-3-acetate glucosyltransferases from Arabidopsis thaliana and a limonoid glucosyltransferase from Citrus unshiu, indicating that SGT belongs to a distinct subgroup of glucosyltransferases that catalyze the formation of 1-O-acylglucosides (β-acetal esters).} } @Article{IPB-2272, author = {Mikkat, S. and Milkowski, C. and Hagemann, M. and}, title = {{The gene sll 0273 of the cyanobacterium Synechocystis sp. strain PCC6803 encodes a protein essential for growth at low Na\+/K\+ ratios}}, year = {2000}, pages = {549-559}, journal = {Plant Cell Environ.}, doi = {10.1046/j.1365-3040.2000.00565.x}, volume = {23}, abstract = {A mutant of Synechocystis sp. strain PCC6803 was obtained by random cartridge mutagenesis, which could not grow at low sodium concentrations. Genetic analyses revealed that partial deletion of the sll 0273 gene, encoding a putative Na\+ /H\+ exchanger, was responsible for this defect. Physiological characterization indicated that the sll 0273 mutant exhibited an increased sensitivity towards K\+ , even at low concentrations, which was compensated for by enhanced concentrations of Na\+ . This enhanced Na\+ demand could also be met by Li\+ . Furthermore, addition of monensin, an ionophore mediating electroneutral Na\+ /H\+ exchange, supported growth of the mutant at unfavourable Na\+ /K\+ ratios. Measurement of internal Na\+ and K\+ contents of wild‐type and mutant cells revealed a decreased Na\+ /K\+ ratio in mutant cells pre‐incubated at a low external Na\+ /K\+ ratio, while it remained at the level of the wild type after pre‐incubation at a high external Na\+ /K\+ ratio. We conclude that the Sll0273 protein is required for Na\+ influx, especially at low external Na\+ concentrations or low Na\+ /K\+ ratios. This system may be part of a sodium cycle and may permit re‐entry of Na\+ into the cells, if nutrient/Na\+ symporters are not functional or operating.} } @Article{IPB-2270, author = {Maucher, H. and Hause, B. and Feussner, I. and Ziegler, J. and Wasternack, C. and}, title = {{Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development}}, year = {2000}, pages = {199-213}, journal = {Plant J.}, doi = {10.1046/j.1365-313x.2000.00669.x}, volume = {21}, abstract = {Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase (LOX) pathway which leads to formation of jasmonic acid (JA). Two full‐length cDNAs of AOS designated as AOS1 and AOS2, respectively, were isolated from barley (H. vulgare cv. Salome) leaves, which represent the first AOS clones from a monocotyledonous species. For AOS1, the open reading frame encompasses 1461 bp encoding a polypeptide of 487 amino acids with calculated molecular mass of 53.4 kDa and an isoelectric point of 9.3, whereas the corresponding data of AOS2 are 1443 bp, 480 amino acids, 52.7 kDa and 7.9. Southern blot analysis revealed at least two genes. Despite the lack of a putative chloroplast signal peptide in both sequences, the protein co‐purified with chloroplasts and was localized within chloroplasts by immunocytochemical analysis. The barley AOSs, expressed in bacteria as active enzymes, catalyze the dehydration of LOX‐derived 9‐ as well as 13‐hydroperoxides of polyenoic fatty acids to the unstable allene oxides. In leaves, AOS mRNA accumulated upon treatment with jasmonates, octadecanoids and metabolizable carbohydrates, but not upon floating on abscisic acid, NaCl, Na‐salicylate or infection with powdery mildew. In developing seedlings, AOS mRNA strongly accumulated in the scutellar nodule, but less in the leaf base. Both tissues exhibited elevated JA levels. In situ hybridizations revealed the preferential occurrence of AOS mRNA in parenchymatic cells surrounding the vascular bundles of the scutellar nodule and in the young convoluted leaves as well as within the first internode. The properties of both barley AOSs, their up‐regulation of their mRNAs and their tissue specific expression suggest a role during seedling development and jasmonate biosynthesis.} } @Article{IPB-2269, author = {Maier, W. and Schmidt, J. and Nimtz, M. and Wray, V. and Strack, D. and}, title = {{Secondary products in mycorrhizal roots of tobacco and tomato}}, year = {2000}, pages = {473-479}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(00)00047-9}, volume = {54}, abstract = {Colonization of the roots of various tobacco species and cultivars (Nicotiana glauca Grah., N. longiflora Cav., N. rustica L., N. tabacum L., N. tabacum L. cv. Samsun NN, N. sanderae hort. Sander ex Wats.) as well as tomato plants (Lycopersicon esculentum L. cv. Moneymaker) by the arbuscular mycorrhizal fungus Glomus intraradices Schenck and Smith resulted in the accumulation of several glycosylated C13 cyclohexenone derivatives. Eight derivatives were isolated from the mycorrhizal roots by preparative high performance liquid chromatography (HPLC) and spectroscopically identified (MS and NMR) as mono-, di- and triglucosides of 6-(9-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one and monoglucosides of 6-(9-hydroxybutyl)-1,5-dimethyl-4-cyclohexen-3-one-1-carboxylic acid and 6-(9-hydroxybutyl)-1,1-dimethyl-4-cyclohexen-3-one-5-carboxylic acid. In contrast to the induced cyclohexenone derivatives, accumulation of the coumarins scopoletin and its glucoside (scopolin) in roots of N. glauca Grah. and N. tabacum L. cv. Samsun NN, was markedly suppressed.} } @Article{IPB-2267, author = {Lehfeldt, C. and Shirley, A. M. and Meyer, K. and Ruegger, M. O. and Cusumano, J. C. and Viitanen, P. V. and Strack, D. and Chapple, C. and}, title = {{Cloning of the SNG1 Gene of Arabidopsis Reveals a Role for a Serine Carboxypeptidase-like Protein as an Acyltransferase in Secondary Metabolism}}, year = {2000}, pages = {1295-1306}, journal = {Plant Cell}, doi = {10.1105/tpc.12.8.1295}, volume = {12}, abstract = {Serine carboxypeptidases contain a conserved catalytic triad of serine, histidine, and aspartic acid active-site residues. These enzymes cleave the peptide bond between the penultimate and C-terminal amino acid residues of their protein or peptide substrates. The Arabidopsis Genome Initiative has revealed that the Arabidopsis genome encodes numerous proteins with homology to serine carboxypeptidases. Although many of these proteins may be involved in protein turnover or processing, the role of virtually all of these serine carboxypeptidase-like (SCPL) proteins in plant metabolism is unknown. We previously identified an Arabidopsis mutant, sng1 (sinapoylglucose accumulator 1), that is defective in synthesis of sinapoylmalate, one of the major phenylpropanoid secondary metabolites accumulated by Arabidopsis and some other members of the Brassicaceae. We have cloned the gene that is defective in sng1 and have found that it encodes a SCPL protein. Expression of SNG1 in Escherichia coli demonstrates that it encodes sinapoylglucose:malate sinapoyltransferase, an enzyme that catalyzes a transesterification instead of functioning like a hydrolase, as do the other carboxypeptidases. This finding suggests that SCPL proteins have acquired novel functions in plant metabolism and provides an insight into the evolution of secondary metabolic pathways in plants.} } @Article{IPB-2266, author = {Lee, Y. K. and Hippe-Sanwald, S. and Jung, H. W. and Hong, J. K. and Hause, B. and Hwang, B. K. and}, title = {{In situ localization of chitinase mRNA and protein in compatible and incompatible interactions of pepper stems with Phytophthora capsici}}, year = {2000}, pages = {111-121}, journal = {Physiol. Mol. Plant Pathol.}, doi = {10.1006/pmpp.2000.0290}, volume = {57}, abstract = {The induction of chitinase (CAChi2) mRNA started as early as 6 h after inoculation and gradually increased in the incompatible interaction of pepper stems with Phytophthora capsici. In the compatible interaction, the induction of the chitinase transcripts was detected later than that in the incompatible interaction. The CAChi2 mRNA was usually localized in the vascular tissues and their expression was constricted in the phloem-related cells. These results showed that the spatial pattern of CAChi2 mRNA expression was similar in both compatible and incompatible interactions but the temporal patterns were different from each other. In addition, the early induction ofCAChi2 mRNA was quite distinct in the incompatible interaction. Immunogold labelling data showed specific labelling of chitinase on the cell wall of the oomycete in both compatible and incompatible interactions at 24 h after inoculation. In particular, numerous gold particles were deposited on the cell wall of P. capsici with a predominant accumulation over areas showing signs of degradation in the incompatible interaction. Chitinase labelling was also detected in the intercellular space and the host cytoplasm. However, healthy pepper stem tissue was nearly free of labelling.} } @Article{IPB-2264, author = {Kobayashi, N. and Schmidt, J. and Nimtz, M. and Wray, V. and Schliemann, W. and}, title = {{Betalains from Christmas cactus}}, year = {2000}, pages = {419-426}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(00)00129-1}, volume = {54}, abstract = {The presence of 14 betalain pigments have been detected by their characteristic spectral properties in flower petals of Christmas cactus (Schlumbergera x buckleyi). Along with the known vulgaxanthin I, betalamic acid, betanin and phyllocactin (6′-O-malonylbetanin), the structure of a new phyllocactin-derived betacyanin was elucidated by various spectroscopic techniques and carbohydrate analyses as betanidin 5-O-(2′-O-β-D-apiofuranosyl-6′-O-malonyl)-β-D-glucopyranoside. Among the more complex betacyanins occurring in trace amounts, the presence of a new diacylated betacyanin {betanidin 5-O-[(5″-O-E-feruloyl)-2′-O-β-D-apiofuranosyl-6′-O-malonyl)]-β-D-glucopyranoside} has been ascertained. Furthermore, the accumulation of betalains during flower development and their pattern in different organs of the flower has been examined.} } @Article{IPB-2263, author = {Irmler, S. and Schröder, G. and St-Pierre, B. and Crouch, N. P. and Hotze, M. and Schmidt, J. and Strack, D. and Matern, U. and Schröder, J. and}, title = {{Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase}}, year = {2000}, pages = {797-804}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.2000.00922.x}, volume = {24}, abstract = {The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis‐specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7‐deoxyloganin to loganin (deoxyloganin 7‐hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C . roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C . roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.} } @Article{IPB-2257, author = {Hause, B. and Stenzel, I. and Miersch, O. and Maucher, H. and Kramell, R. and Ziegler, J. and Wasternack, C. and}, title = {{Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles}}, year = {2000}, pages = {113-126}, journal = {Plant J.}, doi = {10.1046/j.1365-313x.2000.00861.x}, volume = {24}, abstract = {A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis (\+)12‐oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato . In stems, young leaves and young flowers, AOC mRNA accumulates to a low level , contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g−1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue‐specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue‐specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink–source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin‐mediated wound response of tomato.} } @Article{IPB-2256, author = {Hause, B. and Weichert, H. and Höhne, M. and Kindl, H. and Feussner, I. and}, title = {{Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies}}, year = {2000}, pages = {708-714}, journal = {Planta}, doi = {10.1007/s004250050671}, volume = {210}, abstract = {A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid.} } @Article{IPB-2250, author = {Breunig, K. D. and Bolotin–Fukuhara, M. and Bianchi, M. M. and Bourgarel, D. and Falcone, C. and Ferrero, I. and Frontali, L. and Goffrini, P. and Krijger, J. J. and Mazzoni, C. and Milkowski, C. and Steensma, H. Y. and Wésolowski–Louvel, M. and Zeeman, A. M. and}, title = {{Regulation of primary carbon metabolism in Kluyveromyces lactis}}, year = {2000}, pages = {771-780}, journal = {Enzyme Microb. Technol.}, doi = {10.1016/S0141-0229(00)00170-8}, volume = {26}, abstract = {In the recent past, through advances in development of genetic tools, the budding yeast Kluyveromyces lactis has become a model system for studies on molecular physiology of so-called “Nonconventional Yeasts.” The regulation of primary carbon metabolism in K. lactis differs markedly from Saccharomyces cerevisiae and reflects the dominance of respiration over fermentation typical for the majority of yeasts. The absence of aerobic ethanol formation in this class of yeasts represents a major advantage for the “cell factory” concept and large-scale production of heterologous proteins in K. lactis cells is being applied successfully. First insight into the molecular basis for the different regulatory strategies is beginning to emerge from comparative studies on S. cerevisiae and K. lactis. The absence of glucose repression of respiration, a high capacity of respiratory enzymes and a tight regulation of glucose uptake in K. lactis are key factors determining physiological differences to S. cerevisiae. A striking discrepancy exists between the conservation of regulatory factors and the lack of evidence for their functional significance in K. lactis. On the other hand, structurally conserved factors were identified in K. lactis in a new regulatory context. It seems that different physiological responses result from modified interactions of similar molecular modules.} } @Article{IPB-2246, author = {Binarová, P. and Cenklová, V. and Hause, B. and Kubátová, E. and Lysák, M. and Doležel, J. and Bögre, L. and Dráber, P. and}, title = {{Nuclear γ-Tubulin during Acentriolar Plant Mitosis}}, year = {2000}, pages = {433-442}, journal = {Plant Cell}, doi = {10.1105/tpc.12.3.433}, volume = {12}, abstract = {Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that γ-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of γ-tubulin in nuclei increased during the G2 phase, when cells are synchronized or sorted for particular phases of the cell cycle. γ-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear γ-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of γ-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.} } @Article{IPB-2292, author = {Ziegler, J. and Stenzel, I. and Hause, B. and Maucher, H. and Hamberg, M. and Grimm, R. and Ganal, M. and Wasternack, C. and}, title = {{Molecular Cloning of Allene Oxide Cyclase}}, year = {2000}, pages = {19132-19138}, journal = {J. Biol. Chem.}, doi = {10.1074/jbc.M002133200}, volume = {275}, abstract = {Allene oxide cyclase (EC 5.3.99.6) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5′-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (\>99%) formation of the 9S,13Senantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.} } @INBOOK{IPB-168, author = {Vogt, T. and}, title = {{Evolution of Metabolic Pathways}}, year = {2000}, pages = {317-347}, chapter = {{Glycosyltransferases Involved in Plant Secondary Metabolism}}, journal = {Recent Advances in Phytochemistry}, doi = {10.1016/S0079-9920(00)80011-8}, volume = {34}, } @Article{IPB-2340, author = {Winter, J. and Schneider, B. and Meyenburg, S. and Strack, D. and Adam, G. and}, title = {{Monitoring brassinosteroid biosynthetic enzymes by fluorescent tagging and HPLC analysis of their substrates and products}}, year = {1999}, pages = {237-242}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(98)00760-2}, volume = {51}, abstract = {Both the vicinal side chain hydroxyl groups and the 6-oxo function of brassinosteroids were modified by fluorescence tagging. Dansylaminophenylboronic acid was used as a derivatizing agent to form fluorescent esters of brassinosteroids containing a side-chain cis-diol structure. 6-Oxo type brassinosteroids were derivatized by means of dansylhydrazine. The modified brassinosteroids, as far as possible derivatized both at the diol and the oxo group, were separated by HPLC and the optimal emission wavelength was determined. By this approach almost all brassinosteroids, including biosynthetic precursors, were susceptible to highly sensitive analysis in the fmol range. This method has been verified as an analytical tool to determine brassinosteroids in cell culture extracts and to monitor brassinosteroid biosynthetic enzymes. 24-Epibrassinolide has been detected in tomato cell suspension cultures. Several steps of brassinosteroid biosynthesis, including the Baeyer–Villiger oxidation of 24-epicastasterone to give 24-epibrassinolide, were monitored in vitro with protein preparations of the same cell culture line.} } @Article{IPB-2338, author = {Weiss, M. and Schmidt, J. and Neumann, D. and Wray, V. and Christ, R. and Strack, D. and}, title = {{Phenylpropanoids in mycorrhizas of the Pinaceae}}, year = {1999}, pages = {491-502}, journal = {Planta}, doi = {10.1007/s004250050586}, volume = {208}, abstract = {Tissue-specific accumulation of phenylpropanoids was studied in mycorrhizas of the conifers, silver fir (Abies alba Mill.), Norway spruce [Picea abies (L.) Karst.], white pine (Pinus strobus L.), Scots pine (Pinus silvestris L.), and Douglas fir [Pseudotsuga menziesii (Mirbel) Franco], using high-performance liquid chromatography and histochemical methods. The compounds identified were soluble flavanols (catechin and epicatechin), proanthocyanidins (mainly dimeric catechins and/or epicatechins), stilbene glucosides (astringin and isorhapontin), one dihydroflavonol glucoside (taxifolin 3′-O-glucopyranoside), and a hydroxycinnamate derivative (unknown ferulate conjugate). In addition, a cell wall-bound hydroxycinnamate (ferulate) and a hydroxybenzaldehyde (vanillin) were analysed. Colonisation of the root by the fungal symbiont correlated with the distribution pattern of the above phenylpropanoids in mycorrhizas suggesting that these compounds play an essential role in restricting fungal growth. The levels of flavanols and cell wall-bound ferulate within the cortex were high in the apical part and decreased to the proximal side of the mycorrhizas. In both Douglas fir and silver fir, which allowed separation of inner and outer parts of the cortical tissues, a characteristic transversal distribution of these compounds was found: high levels in the inner non-colonised part of the cortex and low levels in the outer part where the Hartig net is formed. Restriction of fungal growth to the outer cortex may also be achieved by characteristic cell wall thickening of the inner cortex which exhibited flavanolic wall infusions in Douglas fir mycorrhizas. Long and short roots of conifers from natural stands showed similar distribution patterns of phenylpropanoids and cell wall thickening compared to the respective mycorrhizas. These results are discussed with respect to co-evolutionary adaptation of both symbiotic partners regarding root structure (anatomy) and root chemistry.} } @Article{IPB-2337, author = {Vogt, T. and Ibdah, M. and Schmidt, J. and Wray, V. and Nimtz, M. and Strack, D. and}, title = {{Light-induced betacyanin and flavonol accumulation in bladder cells of Mesembryanthemum crystallinum}}, year = {1999}, pages = {583-592}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(99)00151-X}, volume = {52}, abstract = {Treatment of the halophyte Mesembryanthemum crystallinum L. (ice plant) (Aizoaceae) with high intensities of white light resulted in a rapid cell-specific accumulation of betacyanins and flavonoids with 6-methoxyisorhamnetin 3-O-{[(2‴-E-feruloyl)-3‴-O-(β-d-glucopyranosyl)](2″-O-β-d-xylopyranosyl)}-β-d-glucopyranoside (mesembryanthin) as the predominant component, within bladder cells of the leaf epidermis. Induced accumulation of these metabolites was first detected 18 h after the initiation of light treatment in bladder cells located at the tip of young leaves followed by the bladder cells located on the epidermis of fully expanded leaves. UV-A light apparently is sufficient to induce accumulation of betacyanins and flavonoids. Application of 2-aminoindan 2-phosphonic acid, a specific inhibitor of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), not only inhibited the accumulation of flavonoids but also reduced betacyanin formation. Based on these observations we suggest these bladder cells as a model system to study regulation of betacyanin and flavonoid biosyntheses.} } @Article{IPB-2336, author = {Vogt, T. and Grimm, R. and Strack, D. and}, title = {{Cloning and expression of a cDNA encoding betanidin 5-O-glucosyltransferase, a betanidin- and flavonoid-specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae}}, year = {1999}, pages = {509-519}, journal = {Plant J.}, doi = {10.1046/j.1365-313X.1999.00540.x}, volume = {19}, abstract = {Based on protein sequence data and RT–PCR, a full length cDNA encoding betanidin 5‐O‐glucosyltransferase (5‐GT) was obtained from a cDNA library of Dorotheanthus bellidiformis (Burm.f.) N.E.Br. (Aizoaceae). 5‐GT catalyses the transfer of glucose from UDP‐glucose to the 5‐hydroxyl group of the chromogenic betanidin. Betanidin and its conjugates, referred to as betacyanins, are characteristic fruit and flower pigments in most members of the Caryophyllales, which fail to synthesise anthocyanins. The 5‐GT cDNA displayed homology to previously published glucosyltransferase sequences and exhibited high identity to sequences of several inducible glucosyltransferases of tobacco and tomato (Solanaceae). The open reading frame encodes a polypeptide of 489 amino acids with a calculated molecular mass of 55.24 kDa. The corresponding cDNA was expressed in Escherichia coli . The recombinant protein displayed identical substrate specificity compared to the native enzyme purified from D. bellidiformis cell suspension cultures. In addition to the natural substrate betanidin, ortho‐dihydroxylated flavonols and flavones were glycosylated preferentially at the B‐ring 4′‐hydroxyl group. 5‐GT is the first enzyme of betalain biosynthesis in plants, of which the corresponding cDNA has been cloned and expressed. The results are discussed in relation to molecular evolution of plant glucosyl‐ transferases.} } @Article{IPB-2332, author = {Steiner, U. and Schliemann, W. and Böhm, H. and Strack, D. and}, title = {{Tyrosinase involved in betalain biosynthesis of higher plants}}, year = {1999}, pages = {114-124}, journal = {Planta}, doi = {10.1007/s004250050541}, volume = {208}, abstract = {A tyrosine-hydroxylating enzyme was partially purified from betacyanin-producing callus cultures of Portulaca grandiflora Hook. by using hydroxyapatite chromatography and gel filtration. It was characterized as a tyrosinase (EC 1.14.18.1 and EC 1.10.3.1) by inhibition experiments with copper-chelating agents and detection of concomitant o-diphenol oxidase activity. The tyrosinase catalysed both the formation of L-(3,4-dihydroxyphenyl)-alanine (Dopa) and cyclo-Dopa which are the pivotal precursors in betalain biosynthesis. The hydroxylating activity with a pH optimum of 5.7 was specific for L-tyrosine and exhibited reaction velocities with L-tyrosine and D-tyrosine in a ratio of 1:0.2. Other monophenolic substrates tested were not accepted. The enzyme appeared to be a monomer with an apparent molecular mass of ca. 53 kDa as estimated by gel filtration and SDS-PAGE. Some other betalain-producing plants and cell cultures were screened for tyrosinase activity; however, activities could only be detected in red callus cultures and plants of P. grandiflora as well as in plants, hairy roots and cell cultures of Beta vulgaris L. subsp. vulgaris (Garden Beet Group), showing a clear correlation between enzyme activity and betacyanin content in young B. vulgaris plants. We propose that this tyrosinase is specifically involved in the betalain biosynthesis of higher plants.} } @Article{IPB-2330, author = {Schulz, B. and Römmert, A.-K. and Dammann, U. and Aust, H.-J. and Strack, D. and}, title = {{The endophyte-host interaction: a balanced antagonism?}}, year = {1999}, pages = {1275-1283}, journal = {Mycol. Res.}, doi = {10.1017/S0953756299008540}, volume = {103}, abstract = {Since secondary metabolites are involved in fungal-host interactions, those of endophytes and their hosts were studied to try to understand why endophytic infections remain symptomless. A screening of fungal isolates for biologically active secondary metabolites (antibacterial, antifungal, herbicidal) showed that the proportion of endophytic isolates that produced herbicidally active substances was three times that of the soil isolates and twice that of the phytopathogenic fungi. As markers for the plant defence reaction, the concentrations of certain phenolic metabolites were chosen. Those that differed in concentration were higher in the roots of plants infected with an endophyte than in those infected with a pathogen. The results presented here were regarded together with previous studies on other aspects of the plant defence response using dual cultures of plant host calli and endophytes, and of cell suspension cultures following endophytic as compared to pathogenic elicitation. The following hypothesis was developed: both the pathogen-host and the endophyte-host interactions involve constant mutual antagonisms at least in part based on the secondary metabolites the partners produce. Whereas the pathogen-host interaction is imbalanced and results in disease, that of the endophyte and its host is a balanced antagonism.} } @Article{IPB-2329, author = {Schröder, G. and Unterbusch, E. and Kaltenbach, M. and Schmidt, J. and Strack, D. and De Luca, V. and Schröder, J. and}, title = {{Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-hydroxylase}}, year = {1999}, pages = {97-102}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(99)01138-2}, volume = {458}, abstract = {Vinblastine and vincristine are two medically important bisindole alkaloids from Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. It starts with tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme. We now show that T16H is induced in the suspension culture by light and we report the cloning of the cDNA. The enzyme was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus, and the reaction product was identified by mass spectrometry. The protein (CYP71D12) shares 47–52% identity with other members of the CYP71D subfamily with unknown function. The induction by light was strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose). We discuss the possibility that the entire pathway to bisindoles can be expressed in suspension cultures.} } @Article{IPB-2328, author = {Schmidt, A. and Grimm, R. and Schmidt, J. and Scheel, D. and Strack, D. and Rosahl, S. and}, title = {{Cloning and Expression of a Potato cDNA Encoding Hydroxycinnamoyl-CoA:Tyramine N-(Hydroxycinnamoyl)transferase}}, year = {1999}, pages = {4273-4280}, journal = {J. Biol. Chem.}, doi = {10.1074/jbc.274.7.4273}, volume = {274}, abstract = {Hydroxycinnamoyl-CoA:tyramineN-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) catalyzes the transfer of hydroxycinnamic acids from the respective CoA esters to tyramine and other amines in the formation ofN-(hydroxycinnamoyl)amines. Expression of THT is induced byPhytophthora infestans, the causative agent of late blight disease in potato. The amino acid sequences of nine endopeptidase LysC-liberated peptides from purified potato THT were determined. Using degenerate primers, a THT-specific fragment was obtained by reverse transcription-polymerase chain reaction, and THT cDNA clones were isolated from a library constructed from RNA of elicitor-treated potato cells. The open reading frame encoding a protein of 248 amino acids was expressed in Escherichia coli. Recombinant THT exhibited a broad substrate specificity, similar to that of native potato THT, accepting cinnamoyl-, 4-coumaroyl-, caffeoyl-, feruloyl- and sinapoyl-CoA as acyl donors and tyramine, octopamine, and noradrenalin as acceptors tested. Elicitor-induced THT transcript accumulation in cultured potato cells peaked 5 h after initiation of treatment, whereas enzyme activity was highest from 5 to 30 h after elicitation. In soil-grown potato plants, THT mRNA was most abundant in roots. Genomic Southern analyses indicate that, in potato, THT is encoded by a multigene family.} } @Article{IPB-2327, author = {Schliemann, W. and Kobayashi, N. and Strack, D. and}, title = {{The Decisive Step in Betaxanthin Biosynthesis Is a Spontaneous Reaction}}, year = {1999}, pages = {1217-1232}, journal = {Plant Physiol.}, doi = {10.1104/pp.119.4.1217}, volume = {119}, abstract = {Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris“Golden Beet”) showed that all amino acids (S- andR-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris“Altamo”) plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly includingcyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.} } @Article{IPB-2316, author = {Maier, W. and Schmidt, J. and Wray, V. and Walter, M. H. and Strack, D. and}, title = {{The arbuscular mycorrhizal fungus, Glomus intraradices , induces the accumulation of cyclohexenone derivatives in tobacco roots}}, year = {1999}, pages = {620-623}, journal = {Planta}, doi = {10.1007/s004250050526}, volume = {207}, abstract = {Tobacco (Nicotiana tabacum L.) plants were grown with and without the arbuscular mycorrhizal fungus, Glomus intraradices Schenk \& Smith. High-performance liquid chromatographic analyses of methanolic extracts from mycorrhizal and non-mycorrhizal tobacco roots revealed marked fungus-induced changes in the patterns of UV-detectable products. The UV spectra of these products, obtained from an HPLC photodiode array detector, indicated the presence of several blumenol derivatives. The most predominant compound among these derivatives was spectroscopically identified as 13-hydroxyblumenol C 9-O-gentiobioside (“nicoblumin”), i.e. the 9-O-(6′-O-β-glucopyranosyl)-β-glucopyranoside of 13-hydroxy-6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one, a new natural product. This is the first report on the identification of blumenol derivatives in mycorrhizal roots of a non-gramineous plant.} } @Article{IPB-2308, author = {Hause, B. and Hertel, S. C. and Klaus, D. and Wasternack, C. and}, title = {{Cultivar-Specific Expression of the Jasmonate-Induced Protein of 23 kDa (JIP-23) Occurs in Hordeum vulgare L. by Jasmonates but not During Seed Germination}}, year = {1999}, pages = {83-89}, journal = {Plant Biol.}, doi = {10.1111/j.1438-8677.1999.tb00712.x}, volume = {1}, abstract = {Treatment of barley leaf segments with jasmonic acid methyl ester (JM) leads to the accumulation of a set of newly formed abundant proteins. Among them, the most abun dant protein exhibits a molecular mass of 23 kDa (JIP‐23). Here, data are presented on the occurrence and expression of the lIP‐23 genes in different cultivars of Hordeum vulgare . Southern blot analysis of 80 cultivars revealed the occurrence of 2 to 4 genes coding for JIP‐23 in all cultivars. By means of Northern blot and immunoblot analysis it is shown that some cultivars lack the ex pression of jip‐23 upon treatment of primary leaves with JM as well as upon stress performed by incubation with 1 M sorbitol solution. During germination, however, all tested cultivars ex hibited developmental expression of jip‐23 . The results are dis cussed in terms of possible functions of JIP‐23 in barley.} } @Article{IPB-2307, author = {Hause, B. and Vörös, K. and Kogel, K.-H. and Besser, K. and Wasternack, C. and}, title = {{A Jasmonate-responsive Lipoxygenase of Barley Leaves is Induced by Plant Activators but not by Pathogens}}, year = {1999}, pages = {459-462}, journal = {J. Plant Physiol.}, doi = {10.1016/S0176-1617(99)80283-1}, volume = {154}, abstract = {Using the recently isolated eDNA clone LOX2 : Hv : 1 which codes for the most abundant jasmonateinducible lipoxygenase (LOX) in barley leaves (Vörös et al., 1998), we analysed the capability of different activators of systemic activated resistance (SAR) to induce the expression of that LOX. Upon treatment of barley leaves with salicylate, 2,6-dichloroisonicotinic acid and benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester, all these compounds were able to induce the expression of the LOX2 : Hv : 1 gene, whereas upon infection with the powdery mildew fungus (Blumeria graminis f. sp. hordei) mRNA accumulation was not detectable in compatible or in incompatible interactions. The induction of the LOX2 : Hv : 1 protein by SAR activators and the expression of different sets of genes induced by jasmonate and salicylate, respectively, are discussed in relation to defense responses against pathogenic fungi.} } @Article{IPB-2306, author = {Hagemeier, J. and Batz, O. and Schmidt, J. and Wray, V. and Hahlbrock, K. and Strack, D. and}, title = {{Accumulation of phthalides in elicitor-treated cell suspension cultures of Petroselinum crispum}}, year = {1999}, pages = {629-635}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(99)00072-2}, volume = {51}, abstract = {The present study describes the effect of a Phytophthora sojae 25-amino acid oligopeptide (Pep25) elicitor on the secondary metabolism of parsley cell cultures (Petroselinum crispum L.). HPLC analysis of the accumulated compounds in the elicitor-treated cultures revealed the expected accumulation of furanocoumarins (e.g. marmesin and bergapten) as well as various non-coumarin compounds which have not been described previously to occur in this cell culture. These compounds were isolated by preparative HPLC and identified by spectroscopic methods (MS, NMR) as 5-hydroxy- and 7-hydroxy-3-butylidenephthalides including two novel conjugates of the 7-hydroxy derivative, i.e. 7-O-glucoside and 7-O-(6′-malonylglucoside).} } @Article{IPB-2303, author = {Fester, T. and Maier, W. and Strack, D. and}, title = {{Accumulation of secondary compounds in barley and wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and co-inoculation with rhizosphere bacteria}}, year = {1999}, pages = {241-246}, journal = {Mycorrhiza}, doi = {10.1007/s005720050240}, volume = {8}, abstract = {Colonization of Hordeum vulgare L. cv. Salome (barley)and Triticum aestivum L. cv. Caprimus (wheat) roots by the arbuscular mycorrhizal fungus Glomus intraradices Schenck \& Smith leads to de novo synthesis of isoprenoid cyclohexenone derivatives with blumenin [9-O-(2′-O-β-glucuronosyl)-β-glucopyranoside of 6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one] as the major constituent and to transient accumulation of hydroxycinnamate amides (4-coumaroylagmatine and -putrescine). Accumulation of these compounds in mycorrhizal wheat roots started 2 weeks after sowing together with the onset of arbuscule formation and proceeded with mycorrhizal progression. Highest levels were reached in 3- to 4-week-old secondary roots (root branches of first and higher order) characterized by the formation of vesicles. In the final developmental stages, the fungus produced massive amounts of spores, enclosing the stele of older root parts (older than 5 weeks) characterized by cortical death. In these root parts, the secondary compounds were detected in trace amounts only, indicating that they were located in the cortical tissues. Some rhizosphere bacteria tested, i.e. Agrobacterium rhizogenes, Pseudomonas fluorescens, and Rhizobium leguminosarum, markedly stimulated both fungal root colonization and blumenin accumulation, thus, acting as mycorrhiza-helper bacteria (MHB). Application of blumenin itself strongly inhibited fungal colonization and arbuscule formation at early stages of mycorrhiza development. This was associated with a markedly reduced accumulation of the hydroxycinnamate amides 4-coumaroylputrescine and -agmatine. The results suggest that both the isoprenoid and the phenylpropanoid metabolism are closely linked to the developmental stage and the extent of fungal colonization. Their possible involvement in the regulation of mycorrhiza development is discussed.} } @Article{IPB-2322, author = {Ortel, B. and Atzorn, R. and Hause, B. and Feussner, I. and Miersch, O. and Wasternack, C. and}, title = {{Jasmonate-induced gene expression of barley (Hordeum vulgare) leaves - the link between jasmonate and abscisic acid}}, year = {1999}, pages = {113-122}, journal = {Plant Growth Regul.}, doi = {10.1023/A:1006212017458}, volume = {29}, abstract = {In barley leaves a group of genes is expressed in response to treatment with jasmonates and abscisic acid (ABA) [21]. One of these genes coding for a jasmonate-induced protein of 23 kDa (JIP-23) was analyzed to find out the link between ABA and jasmonates by recording its expression upon modulating independently, the endogenous level of both of them. By use of inhibitors of JA synthesis and ABA degradation, and the ABA-deficient mutant Az34, as well as of cultivar-specific differences, it was shown that endogenous jasmonate increases are necessary and sufficient for expression of this gene. The endogenous rise of ABA did not induce synthesis of JIP-23, whereas exogenous ABA did not act via jasmonates. Different signalling pathways are suggested and discussed.} } @Article{IPB-2376, author = {Wasternack, C. and Miersch, O. and Kramell, R. and Hause, B. and Ward, J. and Beale, M. and Boland, W. and Parthier, B. and Feussner, I. and}, title = {{Jasmonic acid: biosynthesis, signal transduction, gene expression}}, year = {1998}, pages = {139-146}, journal = {Fett/Lipid}, doi = {10.1002/(SICI)1521-4133(19985)100:4/5<139::AID-LIPI139>3.0.CO;2-5}, volume = {100}, abstract = {Jasmonic acid (JA) is an ubiquitously occurring plant growth regulator which functions as a signal of developmentally or environmentally regulated expression of various genes thereby contributing to the defense status of plants [1–5]. The formation of jasmonates in a lipid‐based signalling pathway via octadecanoids seems to be a common principle for many plant species to express wound‐ and stressinduced genes [4, 5].There are various octadecanoid‐derived signals [3]. Among them, jasmonic acid and its amino acid conjugates are most active in barley, supporting arguments that β‐oxidation is an essential step in lipid‐based JA mediated responses. Furthermore, among derivatives of 12‐oxophytodienoic acid (PDA) carrying varying length of the carboxylic acid side‐chain, only those with a straight number of carbon atoms are able to induce JA responsive genes in barley leaves after treatment with these compounds. Barley leaves stressed by treatment with sorbitol solutions exhibit mainly an endogenous rise of JA and JA amino acid conjugates suggesting that both of them are stress signals. Data on organ‐ and tissue‐specific JA‐responsive gene expression will be presented and discussed in terms of “JA as a master switch” among various lipid‐derived signals.} } @Article{IPB-2375, author = {Wasternack, C. and Ortel, B. and Miersch, O. and Kramell, R. and Beale, M. and Greulich, F. and Feussner, I. and Hause, B. and Krumm, T. and Boland, W. and Parthier, B. and}, title = {{Diversity in octadecanoid-induced gene expression of tomato}}, year = {1998}, pages = {345-352}, journal = {J. Plant Physiol.}, doi = {10.1016/S0176-1617(98)80149-1}, volume = {152}, abstract = {In tomato plants wounding leads to up-regulation of various plant defense genes via jasmonates (Ryan, 1992; Bergey et al., 1996). Using this model system of jasmonic acid (JA) signalling, we analyzed activity of octadecanoids to express JA-responsive genes. Leaf treatments were performed with naturally occurring octadecanoids and their molecular mimics such as coronatine or indanone conjugates. JA responses were recorded in terms of up- or down-regulation of various genes by analyzing transcript accumulation, and at least partially in vitro translation products and polypeptide pattern of leaf extracts. The data suggest: (i) 12-Oxo-phytodienoic acid and other intermediates of the octadecanoid pathway has to be ß-oxidized to give a JA response, (ii) Octadecanoids which can not be ß-oxidized are inactive, (iii) JA, its methyl ester (JM), and its amino acid conjugates are most active signals in tomato leaves leading to up regulation of mainly wound-inducible genes and down-regulation of mainly \<house-keeping\> genes, (iv) Some compounds carrying a JA/JM- or JA amino acid conjugate-like structure induce/repress only a subset of genes suggesting diversity of JA signalling.} } @Article{IPB-2371, author = {Schröder, J. and Raiber, S. and Berger, T. and Schmidt, A. and Schmidt, J. and Soares-Sello, A. M. and Bardshiri, E. and Strack, D. and Simpson, T. J. and Veit, M. and Schröder, G. and}, title = {{Plant Polyketide Synthases: A Chalcone Synthase-Type Enzyme Which Performs a Condensation Reaction with Methylmalonyl-CoA in the Biosynthesis of C-Methylated Chalcones}}, year = {1998}, pages = {8417-8425}, journal = {Biochemistry}, doi = {10.1021/bi980204g}, volume = {37}, abstract = {Heterologous screening of a cDNA library from Pinus strobus seedlings identified clones for two chalcone synthase (CHS) related proteins (PStrCHS1 and PStrCHS2, 87.6% identity). Heterologous expression in Escherichia coli showed that PStrCHS1 performed the typical CHS reaction, that it used starter CoA-esters from the phenylpropanoid pathway, and that it performed three condensation reactions with malonyl-CoA, followed by the ring closure to the chalcone. PstrCHS2 was completely inactive with these starters and also with linear CoA-esters. Activity was detected only with a diketide derivative (N-acetylcysteamine thioester of 3-oxo-5-phenylpent-4-enoic acid) that corresponded to the CHS reaction intermediate postulated after the first condensation reaction. PstrCHS2 performed only one condensation, with 6-styryl-4-hydroxy-2-pyrone derivatives as release products. The enzyme preferred methylmalonyl-CoA against malonyl-CoA, if only methylmalonyl-CoA was available. These properties and a comparison with the CHS from Pinussylvestris suggested for PstrCHS2 a special function in the biosynthesis of secondary products. In contrast to P. sylvestris, P. strobus contains C-methylated chalcone derivatives, and the methyl group is at the position predicted from a chain extension with methylmalonyl-CoA in the second condensation of the biosynthetic reaction sequence. We propose that PstrCHS2 specifically contributes the condensing reaction with methylmalonyl-CoA to yield a methylated triketide intermediate. We discuss a model that the biosynthesis of C-methylated chalcones represents the simplest example of a modular polyketide synthase.} } @Article{IPB-2370, author = {Schmidt, A. and Scheel, D. and Strack, D. and}, title = {{Elicitor-stimulated biosynthesis of hydroxycinnamoyltyramines in cell suspension cultures of Solanum tuberosum}}, year = {1998}, pages = {51-55}, journal = {Planta}, doi = {10.1007/s004250050295}, volume = {205}, abstract = {Treatment of suspension-cultured potato cells (Solanum tuberosum L. cv. Desirée) with an elicitor from Phytophthora infestans induced increased incorporation of 4-hydroxybenzaldehyde, 4-hydroxybenzoate, and N-4-coumaroyl- and N-feruloyltyramine into the cell␣wall and secretion of N-4-coumaroyl- and N-feruloyltyramine into the culture medium. Induced metabolite accumulation was preceded by rapid and transient increases in activities of phenylalanine ammonia-lyase (EC 4.3.1.5) and tyrosine decarboxylase (TyrDC; EC 4.1.1.25), exhibiting maximal activities 5–10 h after initiation of elicitor treatment. Activities of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (EC 2.3.1.110), catalyzing the formation of N-4-coumaroyl- and N-feruloyltyramine, increased later and remained at high levels. The phenolic defense compounds appear to be involved in cell wall reinforcement and may further directly affect fungal growth in the apoplastic space.} } @Article{IPB-2367, author = {Schliemann, W. and Strack, D. and}, title = {{Intramolecular stabilization of acylated betacyanins}}, year = {1998}, pages = {585-588}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(98)00047-8}, volume = {49}, abstract = {Racemization and stability of the betacyanins, betanin (betanidin 5-O-glucoside) and amaranthin(betanidin 5-O-glucuronosylglucoside), under acidic conditions were compared with those of the corresponding feruloyl derivatives, lampranthin II and celosianin II. Both acylbetacyanins showed a reduced racemizationvelocity and celosianin II in addition an enhanced stability, possibly caused by intramolecular associationbetween the betanidin and the feruloyl moieties.} } @Article{IPB-2366, author = {Schliemann, W. and Steiner, U. and Strack, D. and}, title = {{Betanidin formation from dihydroxyphenylalanine in a model assay system}}, year = {1998}, pages = {1593-1598}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(98)00276-3}, volume = {49}, abstract = {Formation of betanidin, the aglycone of the red–violet betacyanins, has been demonstrated by a two-step model assay system. In the first step, dihydroxyphenylalanine (Dopa) was incubated with a Dopa dioxygenase preparation from Amanita muscaria, resulting in the formation of 4,5-seco-Dopa that spontaneously recyclized to betalamic acid. In the second step, a tyrosinase preparation from Portulaca grandiflora was added to the Dopa dioxygenase assay, resulting in Dopa oxidation followed by a spontaneous formation of cyclo-Dopa that, in turn, reacted spontaneously with betalamic acid to form betanidin. Thus, two enzymatic reactions, Dopa extradiol ring cleavage by the fungal enzyme and Dopa oxidation by the plant enzyme, initiate three spontaneous steps: the formation of cyclo-Dopa and betalamic acid and finally the condensation of these compounds to betanidin.} } @Article{IPB-2364, author = {Ratajczak, R. and Feussner, I. and Hause, B. and Böhm, A. and Parthier, B. and Wasternack, C. and}, title = {{Alteration of V-type H\+-ATPase during methyljasmonate-induced senescence in barley (Hordeum vulgare L. cv. Salome)}}, year = {1998}, pages = {199-206}, journal = {J. Plant Physiol.}, doi = {10.1016/S0176-1617(98)80133-8}, volume = {152}, abstract = {In barley leaves, the application of (−)-jasmonic acid or its methyl ester (JAME) induces a senescencelike phenotype. This is accompanied by the synthesis of abundant proteins, so-called jasmonate-induced proteins (JlPs). Here, we show that modifications of vacuolar H\+-ATPase (V-ATPase) subunits are jasmo-nate inducible. Using immunofluorescence analysis, we demonstrate that V-ATPase of barley leaves is exclusively located at the tonoplast also upon JAME treatment. Total ATP-hydrolysis activity of microsomal fractions increased by a factor of 10 during 72 h of JAME-treatment, while Bafilomycin Ai-sensitive ATP-hydrolysis activity, which is usually referred to V-ATPase activity, increased by a factor of about 2 in tono-plast-enriched membrane fractions. Moreover, due to JAME treatment there was a pronounced increase in ATP-hydrolysis activity at pH 6.2. This activity was not affected by inhibitors of P-, F-, or V-ATPases. However, biochemical analysis of partially purified V-ATPase suggests, that this activity might be due at least in part to the V-ATPase. JAME-treatment seems to change biochemical properties of the V-ATPase, i.e. a shift of the pH optimum of activity to a more acidic pH and a decrease in Bafilomycin A1 sensitivity. This is accompanied by the appearance of several additional forms of V-ATPase subunits which might represent either different isoforms or post-translationally modified proteins. We suggest that these changes in properties of the V-ATPase, which is involved in house-keeping and stress responses, may be due to JAME-induced senescence to overcome concomitant changes of the vacuolar membrane.} } @Article{IPB-2356, author = {Maier, W. and Schneider, B. and Strack, D. and}, title = {{Biosynthesis of sesquiterpenoid cyclohexenone derivatives in mycorrhizal barley roots proceeds via the glyceraldehyde 3-phosphate/pyruvate pathway}}, year = {1998}, pages = {521-524}, journal = {Tetrahedron Lett.}, doi = {10.1016/S0040-4039(97)10673-6}, volume = {39}, abstract = {Incorporation of [1-13C]- and [U-13C6]glucose indicates that the biosynthesis of sesquiterpenoid cyclohexenone derivatives in mycorrhizal barley roots proceeds via the glyceraldehyde 3-phosphate/pyruvate non-mevalonate pathway.Incorporation of label from [1-13C]glucose (•) and [U-13C6]glucose ( − ) into the aglycon part (blumenol C) of blumenin indicates that in barley roots the arbuscular mycorrhizal fungus Glomus intraradices induces the glyceraldehyde 3-phosphate/pyruvate non-mevalonate pathway leading to sesquiterpenoid cyclohexenone derivatives.} } @Article{IPB-2348, author = {Eckermann, S. and Schröder, G. and Schmidt, J. and Strack, D. and Edrada, R. A. and Helariutta, Y. and Elomaa, P. and Kotilainen, M. and Kilpeläinen, I. and Proksch, P. and Teeri, T. H. and Schröder, J. and}, title = {{New pathway to polyketides in plants}}, year = {1998}, pages = {387-390}, journal = {Nature}, doi = {10.1038/24652}, volume = {396}, abstract = {The repertoire of secondary metabolism (involving the production of compounds not essential for growth) in the plant kingdom is enormous, but the genetic and functional basis for this diversity is hard to analyse as many of the biosynthetic enzymes are unknown. We have now identified a key enzyme in the ornamental plant Gerbera hybrida (Asteraceae) that participates in the biosynthesis of compounds that contribute to insect and pathogen resistance. Plants transformed with an antisense construct of gchs2, a complementary DNA encoding a previously unknown function1,2, completely lack the pyrone derivatives gerberin and parasorboside. The recombinant plant protein catalyses the principal reaction in the biosynthesis of these derivatives: GCHS2 is a polyketide synthase that uses acetyl-CoA and two condensation reactions with malonyl-CoA to form the pyrone backbone of thenatural products. The enzyme also accepts benzoyl-CoA to synthesize the backbone of substances that have become of interest as inhibitors of the HIV-1 protease3,4,5. GCHS2 is related to chalcone synthase (CHS) and its properties define a new class of function in the protein superfamily. It appears that CHS-related enzymes are involved in the biosynthesis of a much larger range of plant products than was previously realized.} } @Article{IPB-2346, author = {Churin, J. and Hause, B. and Feussner, I. and Maucher, H. P. and Feussner, K. and Börner, T. and Wasternack, C. and}, title = {{Cloning and expression of a new cDNA from monocotyledonous plants coding for a diadenosine 5′,5′′′-P1,P4-tetraphosphate hydrolase from barley (Hordeum vulgare)}}, year = {1998}, pages = {481-485}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(98)00819-9}, volume = {431}, abstract = {From a cDNA library generated from mRNA of white leaf tissues of the ribosome‐deficient mutant ‘albostrians\' of barley (Hordeum vulgare cv. Haisa) a cDNA was isolated carrying 54.2% identity to a recently published cDNA which codes for the diadenosine‐5′,5′′′‐P1,P4‐tetraphosphate (Ap4A) hydrolase of Lupinus angustifolius (Maksel et al. (1998) Biochem. J. 329, 313–319), and 69% identity to four partial peptide sequences of Ap4A hydrolase of tomato. Overexpression in Escherichia coli revealed a protein of about 19 kDa, which exhibited Ap4A hydrolase activity and cross‐reactivity with an antibody raised against a purified tomato Ap4A hydrolase (Feussner et al. (1996) Z. Naturforsch. 51c, 477–486). Expression studies showed an mRNA accumulation in all organs of a barley seedling. Possible functions of Ap4A hydrolase in plants will be discussed.} } @Article{IPB-2342, author = {Binarová, P. and Hause, B. and Doležel, J. and Dráber, P. and}, title = {{Association of γ-tubulin with kinetochore/centromeric region of plant chromosomes}}, year = {1998}, pages = {751-757}, journal = {Plant J.}, doi = {10.1046/j.1365-313x.1998.00166.x}, volume = {14}, abstract = {Monoclonal antibodies raised against a phylogenetically conserved peptide from the C‐terminal domain of γ‐tubulin molecule were used for immunofluorescence detection of γ‐tubulin in acentriolar mitotic spindles of plant cells. The antibodies stained kinetochore fibres along their whole length, including the close vicinity of kinetochores. After microtubule disassembly by the antimicrotubular drugs amiprophos‐methyl, oryzalin and colchicine, γ‐tubulin was found on remnants of kinetochore fibres attached to chromosomes. In cells recovering from the amiprophos‐methyl treatment, γ‐tubulin was localized with the re‐growing kinetochore microtubule fibres nucleated or captured by kinetochore/centromeric regions. On isolated chromosomes, γ‐tubulin co‐localized with α‐tubulin in the kinetochore/centromeric region. The data presented suggest that in acentriolar higher plant cells γ‐tubulin might be directly or indirectly involved in modulation and/or stabilization of kinetochore–microtubule interactions.} } @Article{IPB-2393, author = {Görschen, E. and Dunaeva, M. and Hause, B. and Reeh, I. and Wasternack, C. and Parthier, B. and}, title = {{Expression of the ribosome-inactivating protein JIP60 from barley in transgenic tobacco leads to an abnormal phenotype and alterations on the level of translation}}, year = {1997}, pages = {470-478}, journal = {Planta}, doi = {10.1007/s004250050151}, volume = {202}, abstract = {In this paper we report the in-planta activity of the ribosome-inactivating protein JIP60, a 60-kDa jasmonate-induced protein from barley (Hordeum vulgare L.), in transgenic tobacco (Nicotiana tabacum L.) plants. All plants expressing the complete JIP60 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited conspicuous and similar phenotypic alterations, such as slower growth, shorter internodes, lanceolate leaves, reduced root development, and premature senescence of leaves. Microscopic inspection of developing leaves showed a loss of residual meristems and higher degree of vacuolation of mesophyll cells as compared to the wild type. When probed with an antiserum which was immunoreactive against both the N- and the C-terminal half of JIP60, a polypeptide with a molecular mass of about 30 kDa, most probably a processed JIP60 product, could be detected. Phenotypic alterations could be correlated with the differences in the detectable amount of the JIP60 mRNA and processed JIP60 protein. The protein biosynthesis of the transformants was characterized by an increased polysome/monosome ratio but a decreased in-vivo translation activity. These findings suggest that JIP60 perturbs the translation machinery in planta. An immunohistological analysis using the JIP60 antiserum indicated that the immunoreactive polypeptide(s) are located mainly in the nucleus of transgenic tobacco leaf cells and to a minor extent in the cytoplasm.} } @Article{IPB-2386, author = {Feussner, I. and Fritz, I. G. and Hause, B. and Ullrich, W. R. and Wasternack, C. and}, title = {{Induction of a new Lipoxygenase Form in Cucumber Leaves by Salicylic Acid or 2,6-Dichloroisonicotinic Acid}}, year = {1997}, pages = {101-108}, journal = {Bot. Acta}, doi = {10.1111/j.1438-8677.1997.tb00616.x}, volume = {110}, abstract = {Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6‐dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX‐95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX‐97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.} } @Article{IPB-2382, author = {Baumert, A. and Schumann, B. and Porzel, A. and Schmidt, J. and Strack, D. and}, title = {{Triterpenoids from Pisolithus tinctorius isolates and ectomycorrhizas}}, year = {1997}, pages = {499-504}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(97)00007-1}, volume = {45}, abstract = {Two new triterpenoids have been identified by spectroscopic methods from mycelia of Pisolithus tinctorius as 24-ethyllanosta-8,24(241)-diene-3β,22ξ-diol and (22S)-24,25-dimethyllanosta-8-en-22,241-epoxy-3β-ol-241-one (25-methylpisolactone) along with the two known triterpenoids 24-methyllanosta-8,24(241)-diene-3β,22ξ-diol and (22S)-24-methyllanosta-8-en-22,241-epoxy-3β-ol-241-one (pisolactone). Quantification of these compounds in fungal isolates (surface and suspension cultures) and Pinus sylvestris ectomycorrhizas showed that the amount of the new triterpenoids was markedly higher in the mycorrhizas as in the isolates.} } @Article{IPB-2424, author = {Ziegler, J. and Vogt, T. and Miersch, O. and Strack, D. and}, title = {{Concentration of Dilute Protein Solutions Prior to Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis}}, year = {1997}, pages = {257-260}, journal = {Anal. Biochem.}, doi = {10.1006/abio.1997.2248}, volume = {250}, } @Article{IPB-2422, author = {Winter, J. and Schneider, B. and Strack, D. and Adam, G. and}, title = {{Role of a cytochrome P450-dependent monooxygenase in the hydroxylation of 24-epi-brassinolide}}, year = {1997}, pages = {233-237}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(96)00827-8}, volume = {45}, abstract = {24-epi-Brassinolide, exogenously applied to cell suspension cultures of Lycopersicon esculentum is hydroxylated at C-25 and C-26, respectively, followed by glucosylation of the newly formed hydroxyl group. Treatment of the cell cultures with the specific cytochrome P450 inhibitors, clotrimazole and ketoconazole, resulted in a strong decrease of only the C-25 hydroxylation, whereas hydroxylation at C-26 was not affected. The common cytochrome P450 inducers, ethanol, MnCl2, phenobarbital, pregnenolone 16α-carbonitrile or clofibrate, did not induce hydroxylation activity at C-25 or at C-26. In addition, substrate analogues (22S,23S-homobrassinolide, 24-epi-castasterone, ecdysone, and 20-OH-ecdysone) were not accepted. Only application of 24-epi-brassinolide and brassinolide resulted in an increased activity of both the C-25- and C-26-hydroxylases. For further examination of the molecular level of this inducing effect, the influence of the protein biosynthesis inhibitor cycloheximide has been studied. Thus, increase of both hydroxylase activities is obviously based on gene expression by action of the substrates, 24-epi-brassinolide and brassinolide.} } @Article{IPB-2416, author = {Weiss, M. and Mikolajewski, S. and Peipp, H. and Schmitt, U. and Schmidt, J. and Wray, V. and Strack, D. and}, title = {{Tissue-Specific and Development-Dependent Accumulation of Phenylpropanoids in Larch Mycorrhizas}}, year = {1997}, pages = {15-27}, journal = {Plant Physiol.}, doi = {10.1104/pp.114.1.15}, volume = {114}, abstract = {The tissue-specific and development-dependent accumulation of secondary products in roots and mycorrhizas of larch (Larix decidua Mill.; Pinaceae) was studied using high-performance liquid chromatography and histochemical methods. The compounds identified were soluble catechin, epicatechin, quercetin 3-O-[alpha]-rhamnoside, cyanidin- and peonidin 3-O-[beta]-glucoside, 4-O-[beta]-hydroxybenzoyl-O-[beta]-glucose, 4-hydroxybenzoate 4-O-[beta]-glucoside, maltol 3-O-[beta]-glucoside, and the wall-bound 4-hydroxybenzaldehyde, vanillin, and ferulate. In addition, we partially identified a tetrahydroxystilbene monoglycoside, a quercetin glycoside, and eight oligomeric proanthocyanidins. Comparison between the compounds accumulating in the apical tissue of fine roots, long roots, and in vitro grown mycorrhizas (L. decidua-Suillus tridentinus) showed elevated levels of the major compounds catechin and epicatechin as well as the minor compound 4-hydroxybenzoate 4-O-[beta]-glucoside specifically in the root apex of young mycorrhizas. The amounts of wall-bound 4-hydroxybenzaldehyde and vanillin were increased in all of the mycorrhizal sections examined. During the early stages of mycorrhization the concentrations of these compounds increased rapidly, perhaps induced by the mycorrhizal fungus. In addition, studies of L. decidua-Boletinus cavipes mycorrhizas from a natural stand showed that the central part of the subapical cortex tissue and the endodermis both accumulate massive concentrations of catechin, epicatechin, and wall-bound ferulate compared with the outer part of the cortex, where the Hartig net is being formed.} } @Article{IPB-2412, author = {Vogt, T. and Zimmermann, E. and Grimm, R. and Meyer, M. and Strack, D. and}, title = {{Are the characteristics of betanidin glucosyltransferases from cell-suspension cultures of Dorotheanthus bellidiformis indicative of their phylogenetic relationship with flavonoid glucosyltransferases?}}, year = {1997}, pages = {349-361}, journal = {Planta}, doi = {10.1007/s004250050201}, volume = {203}, abstract = {Uridine 5′-diphosphoglucose:betanidin 5-O- and 6-O-glucosyltransferases (5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific formation of betanin (betanidin 5-O-β-glucoside) and gomphrenin I (betanidin 6-O-β-glucoside), respectively. Both enzymes were purified to near homogeneity from cell-suspension cultures of Dorotheanthus bellidiformis, the 5-GT by classical chromatographic techniques and the 6-GT by affinity dye-ligand chromatography using UDP-glucose as eluent. Data obtained with highly purified enzymes indicate that 5-GT and 6-GT catalyze the indiscriminate transfer of glucose from UDP-glucose to hydroxyl groups of betanidin, flavonols, anthocyanidins and flavones, but discriminate between individual hydroxyl groups of the respective acceptor compounds. The 5-GT catalyzes the transfer of glucose to the C-4′ hydroxyl group of quercetin as its best substrate, and the 6-GT to the C-3 hydroxyl group of cyanidin as its best substrate. Both enzymes also catalyze the formation of the respective 7-O-glucosides, but to a minor extent. Although the enzymes were not isolated to homogeneity, chromatographic, electrophoretic and kinetic properties proved that the respective enzyme activities were based on the presence of single enzymes, i.e. 5-GT and 6-GT. The N terminus of the 6-GT revealed high sequence identity to a proposed UDP-glucose:flavonol 3-O-glucosyltransferase (UF3GT) of Manihot esculenta. In addition to the 5-GT and 6-GT, we isolated a UF3GT from D. bellidiformis cell cultures that preferentially accepted myricetin and quercetin, but was inactive with betanidin. The same result was obtained with a UF3GT from Antirrhinum majus and a flavonol 4′-O-glucosyltransferase from Allium cepa. Based on these results, the main question to be addressed reads: Are the characteristics of the 5-GT and 6-GT indicative of their phylogenetic relationship with flavonoid glucosyltransferases?} } @Article{IPB-2408, author = {Peipp, H. and Maier, W. and Schmidt, J. and Wray, V. and Strack, D. and}, title = {{Arbuscular mycorrhizal fungus-induced changes in the accumulation of secondary compounds in barley roots}}, year = {1997}, pages = {581-587}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(96)00561-4}, volume = {44}, abstract = {Hordeum vulgare (barley) was grown in a defined nutritional medium with and without the arbuscular mycorrhizal fungus Glomus intraradices. HPLC of methanolic extracts from the roots of mycorrhized and non-mycorrhized plants revealed fungus-induced accumulation of some secondary metabolites. These compounds were isolated and identified by spectroscopic methods (NMR, MS) to be the hydroxycinnamic acid amides N-(E)-4-coumaroylputrescine, N-(E)-feruloylputrescine, N-(E)-4-coumaroylagmatine and N-(E)-feruloylagmatine, exhibiting a transient accumulation, and the cyclohexenone derivatives 4-(3-O-β-glucopyranosyl-butyl)-3-(hydroxymethyl)-5,5-dimethyl-2-cyclohexen-1-one and 4-{3-O-[(2′-O-β-glucuronosyl)-β-glucopyranosyl]-butyl}-3,5,5-trimethyl-2-cyclohexen-1-one (blumenin), exhibiting a continuous accumulation. A third cyclohexenone derivative, 4-{3-O-[(2′-O-β-glucuronosyl)-β-glucopyranosyl]-1-butenyl}-3,5,5-trimethyl-2-cyclohexen-1-one, was detectable only in minute amounts. It is suggested that accumulation of the amides in early developmental stages of barley mycorrhization reflects initiation of a defence response. However, the continuous accumulation of the cyclohexenone derivatives, especially blumenin, seems to correlate with the establishment of a functional barley mycorrhiza.} } @Article{IPB-2406, author = {Maier, W. and Hammer, K. and Dammann, U. and Schulz, B. and Strack, D. and}, title = {{Accumulation of sesquiterpenoid cyclohexenone derivatives induced by an arbuscular mycorrhizal fungus in members of the Poaceae}}, year = {1997}, pages = {36-42}, journal = {Planta}, doi = {10.1007/s004250050100}, volume = {202}, abstract = {Sixty one members of the Poaceae, including various cereals, were grown in defined nutrient media with and without the arbuscular mycorrhizal (AM) fungus, Glomus intraradices Schenk \& Smith. The roots of all species investigated were colonized by the AM fungus, however, to different degrees and independent of their systematic position. High-performance liquid chromatographic analyses of methanolic extracts from the roots of mycorrhizal and nonmycorrhizal species revealed dramatic changes in the patterns of UV-detectable products along with a widespread occurrence of AM-fungus-induced accumulation of sesquiterpenoid cyclohexenone derivatives. The latter occur most often in the tribes Poeae, Triticeae and Aveneae. Some additional control experiments on plant infection with pathogens (Gaeumannomyces graminis) and Drechslera sp.) or an endophyte (Fusarium sp.), as well as application of abiotic stress, proved that the metabolism of these terpenoids is part of a response pattern of many gramineous roots in their specific reaction to AM fungal colonization.} } @Article{IPB-2404, author = {Lee, J. and Vogt, T. and Schmidt, J. and Parthier, B. and Löbler, M. and}, title = {{Methyljasmonate-induced accumulation of coumaroyl conjugates in barley leaf segments}}, year = {1997}, pages = {589-592}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(96)00562-6}, volume = {44}, abstract = {The effect of methyljasmonate on the induction of phenolic components in barley leaf segments was investigated. RP-HPLC of methanol extracts showed that three compounds accumulate to high concentrations in response to methyljasmonate treatment. Two of them were identified as N-(E)-4-coumaroylputrescine and N-(E)-4-coumaroylagmatine by UV-spectroscopy and mass spectrometry.} } @Article{IPB-2403, author = {Lee, J. E. and Vogt, T. and Hause, B. and Löbler, M. and}, title = {{Methyl Jasmonate Induces an O-Methyltransferase in Barley}}, year = {1997}, pages = {851-862}, journal = {Plant Cell Physiol.}, doi = {10.1093/oxfordjournals.pcp.a029244}, volume = {38}, abstract = {We have previously described a truncated cDNA clone for a barley (Hordeum vulgare L. cv. Salome) jasmonate regulated gene, JRG5, which shows homology to caffeic acid O-methyltransferase (COMT). A cDNA encompassing the coding region was amplified by PCR and cloned for overexpression in E. coli. Western blot analyses indicate that the recombinant protein crossreacts with the antibodies directed against the tobacco class II OMT and only weakly with the antibodies for the tobacco class I OMT. An immunoreactive band in the protein extract of jasmo-nate-treated leaf segments suggests that JRG5 transcripts that accumulate after jasmonate treatment are also translated. Specific methylating activities on caffeic acid and catechol were obtained from the recombinant protein through renaturation of protein extracted from inclusion bodies or from bacteria grown and induced at low temperature. On Northern blots, the JRG5 transcripts were detected in the leaf sheath but not the leaf lamina, stem, root or inflorescence and accumulated in leaf segments after jasmonate application. Several hormone or stress treatments did not induce JRG5 mRNA accumulation. This includes sor-bitol stress which is known to lead to enhanced endogenous jasmonate levels and the implications for jasmonate signaling are discussed. Based on quantitative measurements and fluorescence microscopy, jasmonate-induced accumulation of ferulic acid and phenolic polymers in the cell wall were detected and the possibility of cell wall strengthening mediated through phenolic crosslinks is discussed} } @Article{IPB-2401, author = {Kramell, R. and Miersch, O. and Hause, B. and Ortel, B. and Parthier, B. and Wasternack, C. and}, title = {{Amino acid conjugates of jasmonic acid induce jasmonate-responsive gene expression in barley (Hordeum vulgare L.) leaves}}, year = {1997}, pages = {197-202}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(97)01005-3}, volume = {414}, abstract = {Leaves of barley (Hordeum vulgare L. cv. Salome ) treated with jasmonic acid (JA), its methyl ester (JM), or its amino acid conjugates exhibit up‐regulation of specific genes and down‐regulation of house‐keeping genes. This transcriptional regulation exhibits several specificities. (i) The (−)‐enantiomers are more active, and conjugates are mainly active if they carry an l ‐amino acid moiety. (ii) The various JA‐responsive genes respond differentially to enantiomeric and chiralic forms. (iii) Both JA and its amino acid conjugates exhibiting no or negligible interconversion induce/repress genes.} } @Article{IPB-2396, author = {Hause, B. and Feussner, K. and Wasternack, C. and}, title = {{Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures}}, year = {1997}, pages = {452-457}, journal = {Bot. Acta}, doi = {10.1111/j.1438-8677.1997.tb00662.x}, volume = {110}, abstract = {Diadenosine 5′,5′”‐P1,P4‐tetraphosphate (Ap4A) cleaving enzymes are assumed to regulate intracellular levels of Ap4A, a compound known to affect cell proliferation and stress responses. From plants an Ap4A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap4A hydrolase in 4‐day‐old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein‐5‐isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap4A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6‐diamidino‐2‐phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap4A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap4A in triggering DNA synthesis and cell proliferation.} } @Article{IPB-2395, author = {Hause, B. and Kogel, K.-H. and Parthier, B. and Wasternack, C. and}, title = {{In barley leaf cells, jasmonates do not act as a signal during compatible or incompatible interactions with the powdery mildew fungus (Erysiphe graminis f. sp. hordei)}}, year = {1997}, pages = {127-132}, journal = {J. Plant Physiol.}, doi = {10.1016/S0176-1617(97)80191-5}, volume = {150}, abstract = {We have studied a possible function of jasmonates as mediators in the host-pathogen interaction of barley (Hordeum vulgare L.) with the powdery mildew fungus Egh (Erysiphe graminis f. sp. hordei). Previous findings from whole-leaf extracts demonstrated that (i) extracts from infected barley leaves did not contain enhanced levels of jasmonates, (ii) transcripts of jasmonate-inducible genes were not expressed upon infection, and (iii) exogenous application of jasmonates did not induce resistance to Egh (Kogel et al., 1995). Nevertheless, the question arises whether or not jasmonates are involved in the interaction of barley with the powdery mildew fungus at the local site of infection. Using an immunocytological approach the analysis of leaf cross-sections from a susceptible barley cultivar and its near-isogenic mlo5-resistant line revealed no accumulation of JIP-23, the most abundant jasmonate inducible protein, neither in epidermal cells attacked by the pathogen nor in adjacent mesophyll cells. As a positive control, cross-sections from methyl jasmonate-treated leaf segments showed a strong signal for JIP-23 accumulation. Because the presence of the jasmonate-inducible protein is highly indicative for an already low threshold level of endogenous jasmonate (Lehmann et al., 1995), the lack of JIP-23 accumulation at the sites of attempted fungal infection clearly demonstrates the absence of enhanced levels of jasmonates. This excludes even a local rise of jasmonate confined to those single cells penetrated (Mlo genotype) or attacked (mlo5 genotype) by the fungus.} } @INBOOK{IPB-184, author = {Strack, D. and Schliemann, W. and}, title = {{Römpp-Lexikon Naturstoffe}}, year = {1997}, chapter = {{Farbstoffe}}, editor = {Fugmann, B., Lang-Fugmann, S., Steglich, W.}, } @Article{IPB-2445, author = {Steiner, U. and Schliemann, W. and Strack, D. and}, title = {{Assay for Tyrosine Hydroxylation Activity of Tyrosinase from Betalain-Forming Plants and Cell Cultures}}, year = {1996}, pages = {72-75}, journal = {Anal. Biochem.}, doi = {10.1006/abio.1996.0253}, volume = {238}, abstract = {In our studies on tyrosinase-catalyzed tyrosine hydroxylation, possibly involved in betalain biosynthesis, we have evaluated different assays for the detection and quantification of the enzymatic product Dopa with respect to sensitivity, simplicity, and suitability for automatization. A tyrosinase assay including reversed-phase high-performance liquid chromatography with isocratic elution and fluorescence detection has been developed (native fluorescence of Dopa; excitation at 281 nm, emission at 314 nm). This improved assay was sensitive (detection limit: 2 pmol Dopa) and showed a wide linear range of Dopa detection (10 pmol–20 nmol Dopa). The method proved to be suitable for high-performance liquid chromatography with an autosampler and has been applied for measuring tyrosinase activity of cell cultures and different tissues ofPortulaca grandiflora.} } @Article{IPB-2444, author = {Schliemann, W. and Joy, R. W. and Komamine, A. and Metzger, J. W. and Nimtz, M. and Wray, V. and Strack, D. and}, title = {{Betacyanins from plants and cell cultures of Phytolacca americana}}, year = {1996}, pages = {1039-1046}, journal = {Phytochemistry}, doi = {10.1016/0031-9422(96)00100-8}, volume = {42}, } @Article{IPB-2439, author = {Lorenzen, M. and Racicot, V. and Strack, D. and Chapple, C. and}, title = {{Sinapic Acid Ester Metabolism in Wild Type and a Sinapoylglucose-Accumulating Mutant of Arabidopsis}}, year = {1996}, pages = {1625-1630}, journal = {Plant Physiol.}, doi = {10.1104/pp.112.4.1625}, volume = {112}, abstract = {Sinapoylmalate is one of the major phenylpropanoid metabolites that is accumulated in the vegetative tissue of Arabidopsis thaliana. A thin-layer chromatography-based mutant screen identified two allelic mutant lines that accumulated sinapoylglucose in their leaves in place of sinapoylmalate. Both mutations were found to be recessive and segregated as single Mendelian genes. These mutants define a new locus called SNG1 for sinapoylglucose accumulator. Plants that are homozygous for the sng1 mutation accumulate normal levels of malate in their leaves but lack detectable levels of the final enzyme in sinapate ester biosynthesis, sinapoylglucose:malate sinapoyltransferase. A study of wild-type and sng1 seedlings found that sinapic acid ester biosynthesis in Arabidopsis is developmentally regulated and that the accumulation of sinapate esters is delayed in sng1 mutant seedlings.} } @Article{IPB-2438, author = {Leopold, J. and Hause, B. and Lehmann, J. and Graner, A. and Parthier, B. and Wasternack, C. and}, title = {{Isolation, characterization and expression of a cDNA coding for a jasmonate-inducible protein of 37 kDa in barley leaves}}, year = {1996}, pages = {675-684}, journal = {Plant Cell Environ.}, doi = {10.1111/j.1365-3040.1996.tb00402.x}, volume = {19}, abstract = {In barley leaves, there is a dramatic alteration of gene expression upon treatment with jasmonates leading to the accumulation of newly formed proteins, designated as jasmonate‐inducible proteins (JIPs). In the present study, a new jasmonate‐inducible cDNA, designated pHvJS37, has been isolated by differential screening of a γgt10 cDNA library constructed from mRNA of jasmonate‐treated barley leaf segments. The open reading frame (ORF) encodes a 39‐9 kDa polypeptide which cross‐reacts with antibodies raised against the in vivo JIP‐37. The hydropathic plot suggests that the protein is mainly hydrophilic, containing two hydrophilic domains near the C‐terminus. Database searches did not show any sequence homology of pHv.JS37 to known sequences. Southern analysis revealed at least two genes coding for JIP‐37 which map to the distal portion of the long arm of chromosome 3 and are closely related to genes coding for JIP‐23. The expression pattern of the JIP‐37 genes over time shows differential responses to jasmonate, abscisic acid (ABA), osmotic stress (such as sorbitol treatment) and desiccation stress. No expression was found under salt stress. From experiments using an inhibitor and intermediates of jasmonate synthesis such as α‐linolenic acid and 12‐oxophytodienoic acid, we hypothesize that there is a stress‐induced lipid‐based signalling pathway in which an endogenous rise of jasmonate switches on JIP‐37 gene expression. Using immunocytochemical techniques, JIP‐37 was found to be simultaneously located in the nucleus, the cytoplasm and the vacuoles.} } @Article{IPB-2437, author = {Keller, H. and Hohlfeld, H. and Wray, V. and Hahlbrock, K. and Scheel, D. and Strack, D. and}, title = {{Changes in the accumulation of soluble and cell wall-bound phenolics in elicitor-treated cell suspension cultures and fungus-infected leaves of Solanum tuberosum}}, year = {1996}, pages = {389-396}, journal = {Phytochemistry}, doi = {10.1016/0031-9422(95)00866-7}, volume = {42}, abstract = {Cell suspension cultures of potato (Solanum tuberosum cv. Datura) treated with an elicitor preparation from Phytophthora infestans and potato leaves infected with the same fungus were used to study changes in the accumulation patterns of soluble and cell wall-bound phenolics. The compounds were identified by chromatographic comparison with authentic substances and by spectroscopic methods (FAB mass spectrometry, 1H and 13C NMR). The soluble phenolics were 4-O-β-glucopyranosylhydroquinone (arbutin), 4-O-β-glucopyranosylbenzoate, 3-methoxy-4-O-β-glucopyranosylbenzoate (vanillate glucoside), N-(E)-caffeoylputrescine, 2-O-β-glucopyranosylbenzoate (salicylate glucoside), N-(E)-feruloylputrescine, and N-(E)-feruloylaspartate. The cell wall-bound phenolics were 4-hydroxybenzoate, 4-hydroxybenzaldehyde, 3-methoxy-4-hydroxybenzaldehyde (vanillin), 4-(E)-coumarate, (E)-ferulate, N-4-(E)-coumaroyltyramine, and N-(E)-feruloyltyramine. The most prominent phenolics showing elicitor- or fungus-induced increases in accumulation rates were the soluble putrescine amides and cell wall-bound 4-hydroxybenzaldehyde and tyramine amides. In addition, there was a secretion of large amounts of coumaroyltyramine into the cell culture medium.} } @Article{IPB-2436, author = {Hohlfeld, H. and Scheel, D. and Strack, D. and}, title = {{Purification of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase from cell-suspension cultures of Solanum tuberosum L. cv. Datura}}, year = {1996}, pages = {166-168}, journal = {Planta}, doi = {10.1007/BF00196893}, volume = {199}, abstract = {A pathogen-elicitor-inducible acyltransferase [tyramine hydroxycinnamoyltransferase (THT); EC 2.3.1], which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was purified to apparent homogeneity from cell-suspension cultures of potato (Solanum tuberosum L. cv. Datura), with a 1400-fold enrichment, a 5% recovery and a final specific activity of 208 mkat·(kg protein)−1. Affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent was the decisive step in the purification sequence. The purified protein showed a native molecular mass of ca. 49 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and in the absence of a reducing agent (2-mercaptoethanol) indicated that THT is a heterodimer in which the protein subunits (ca. 25 kDa) are non-covalently associated. The enzyme was stimulated fivefold by 10 mM Ca2\+. The apparent K m value for tyramine was dependent on the nature of the hydroxycinnamoyl-CoA present. Thus, the K m value for tyramine was about tenfold greater (174 μM) in the presence of 4-coumaroyl-CoA than in the presence of feruloyl-CoA (20 μM).} } @Article{IPB-2435, author = {Hohlfeld, M. and Veit, M. and Strack, D. and}, title = {{Hydroxycinnamoyltransferases Involved in the Accumulation of Caffeic Acid Esters in Gametophytes and Sporophytes of Equisetum arvense}}, year = {1996}, pages = {1153-1159}, journal = {Plant Physiol.}, doi = {10.1104/pp.111.4.1153}, volume = {111}, abstract = {Four hydroxycinnamoyltransferases from Equisetum arvense L. were studied that catalyze the formation of mono-O-caffeoyl-meso-tartrate, di-O-caffeoyl-meso-tartrate, 5-O-caffeoylshikimate (dactylifrate), and 5-O-caffeoylquinate (chlorogenate). The enzymes were classified as coenzyme A (CoA)-ester-dependent acyltransferases (EC 2.3.1), i.e. hydroxycinnamoyl-CoA:meso-tartrate hydroxycinnamoyltransferase (CTT), hydroxycinnamoyl-CoA:caf-feoyl-meso-tartrate hydroxycinnamoyltransferase (CCT), hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (CST), and hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase. The CTT, CCT, and CST were partially purified and separated from E. arvense gametophytes by hydrophobic interaction chromatography on Fractogel TSK Butyl-650 followed by molecular exclusion on fast protein liquid chromatography-Superdex-75 with 87-, 62-, and 130- fold enrichments and 12, 8, and 11% yields, respectively. The enzyme activities obtained with caffeoyl-CoA were 95 (CTT), 74 (CCT), and 200 [mu]kat (CST) kg-1 protein. The apparent native relative molecular weight values were found to be approximately 45,000 (CTT), 52,000 (CCT), and 50,000 (CST). Each enzyme showed highest activities at pH 7.5, the CCT and CST in Tris-HCl (1.2 and 1.0 M) and the CTT in imidazole-HCl (1.25 M). Enzyme activities were stimulated more than 3-fold by 100 mM ascorbate. The apparent energies of activation (kilojoules mol-1) were calculated to be 56 (CTT), 69 (CST), and 76 (CCT). The enzymes accepted cinnamoyl-CoA and various hydroxycinnamoyl-CoAs. The time course of the transferase activities along with that of a fourth one, hydroxycinnamoyl-CoA:quinate hydroxycinnamoyltransferase, and the pattern of product accumulation were determined during a 1-year growth period of the E. arvense sporophytes.} } @Article{IPB-2434, author = {Heuer, S. and Vogt, T. and Böhm, H. and Strack, D. and}, title = {{Partial purification and characterization of UDP-glucose: betanidin 5-O- and 6-O-glucosyltransferases from cell suspension cultures of Dorotheanthus bellidiformis (Burm. f.) N.E.Br.}}, year = {1996}, pages = {244-250}, journal = {Planta}, doi = {10.1007/BF00196565}, volume = {199}, abstract = {Uridine 5′-diphosphoglucose-dependent glucosyl-transferases (UDP-glucose:betanidin 5-O- and 6-O-glucosyltransferases; 5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific transfer of glucose to the 5- and 6-hydroxy group of betanidin in the formation of betanin and gomphrenin I, respectively. Both GT activities were partially purified from cell suspension cultures of Dorotheanthus bellidiformis (Burm. f.) N.E. Br. Isoelectric focusing of crude protein extracts indicated the presence of three 5-GT isoforms and a single 6-GT form. The 5-GT isoforms were partially separated from each other and completely from the 6-GT. Studies of the glucosyltransferase activities were focused on the major isoform of the 5-GTs and the 6-GT, which displayed the same pH optimum near 7.5 in K-phosphate buffer. A 3- and 2.5-fold enrichment and 11% and 10% recovery of the 5-GT and 6-GT, respectively, were routinely achieved; however, a 3300-fold enrichment of the major 5-GT isoform and a 6-fold enrichment of the 6-GT were also achieved. Both enzymes are monomers and displayed apparent native Mrs near 55 000. The maxima of the reaction temperature were at 50 °C for the 5-GT and at 37°C for the 6-GT with respective apparent energies of activation of 51 and 53 kJ · mol−1. Kinetic studies indicated that the apparent Michaelis constants (apparent K m) of the GTs for one substrate were dependent on the concentration of the second substrate. However, the relationship between the apparent K m values and the dissociation constants (K i) were different; m \> K i applies for the 5-GT and K m \< K i for the 6-GT activity. Consequently, this results in a predominant formation of betanin at low substrate concentrations, but a predominant formation of gomphrenin I at high substrate concentrations, assuming that both enzymes may compete freely for their substrates. This might explain why we could not observe a correlation between extractable 5-GT and 6-GT activities and the in-vivo accumulation of the respective products from cell-suspension cultures of D. bellidiformis.} } @Article{IPB-2432, author = {Hause, B. and Demus, U. and Teichmann, C. and Parthier, B. and Wasternack, C. and}, title = {{Developmental and Tissue-Specific Expression of JIP-23, a Jasmonate-Inducible Protein of Barley}}, year = {1996}, pages = {641-649}, journal = {Plant Cell Physiol.}, doi = {10.1093/oxfordjournals.pcp.a028993}, volume = {37}, abstract = {Developmental expression of a 23 kDa jasmonate-induced protein (JIP-23) of barley leaves (Hordeum vulgare cv. Salome) was studied by measuring the time-dependent accumulation of transcript and protein during germination. Tissue-specific expression of JIP-23 was analyzed immunocytochemically and by in situ hybridizations, respectively. During seed germination JIP-23 mRNA was found to accumulate transiently with a maximum at 32 h, whereas the protein was steadily detectable after the onset of expression. The occurrence of new isoforms of JIP-23 during germination in comparison to jasmonate-treated leaves suggests, that the JIP-23 gene family of barley is able to express different subsets of isoforms dependent on the developmental stage.JIP-23 and its transcript were found mainly in the scutellum, the scutellar nodule and in lower parts of the primary leaf of 6 days old seedlings. All these tissues exhibited high levels of endogenous jasmonates. In situ hybridization revealed specific accumulation of JIP-23 mRNA in companion cells of the phloem in the nodule plate of the scutellum. In accordance with that, JIP-23 was detected immunocytochemically in phloem cells of the root as well as of the scutellar nodule and in parenchymatic cells of the scutellum. The cell type-specific occurrence of JIP-23 was restricted to cells, which are known to be highly stressed osmotically by active solute transport. This observation suggests, that the expression of this protein might be a response to osmotic stress during development.} } @Article{IPB-2431, author = {Feussner, I. and Hause, B. and Nellen, A. and Wasternack, C. and Kindl, H. and}, title = {{Lipid-body lipoxygenase is expressed in cotyledons during germination prior to other lipoxygenase forms}}, year = {1996}, pages = {288-293}, journal = {Planta}, doi = {10.1007/BF00206255}, volume = {198}, abstract = {Lipid bodies are degraded during germination. Whereas some proteins, e.g. oleosins, are synthesized during the formation of lipid bodies of maturating seeds, a new set of proteins, including a specific form of lipoxygenase (LOX; EC 1.13.11.12), is detectable in lipid bodies during the stage of fat degradation in seed germination. In cotyledons of cucumber (Cucumis sativus L.) seedlings at day 4 of germination, the most conspicuous staining with anti-LOX antibodies was observed in the cytosol. At very early stages of germination, however, the LOX form present in large amounts and synthesized preferentially was the lipid-body LOX. This was demonstrated by immunocytochemical staining of cotyledons from 1-h and 24-h-old seedlings: the immunodecoration of sections of 24-h-old seedlings with anti-LOX antiserum showed label exclusively correlated with lipid bodies of around 3 μm in diameter. In accordance, the profile of LOX protein isolated from lipid bodies during various stages of germination showed a maximum at day 1. By measuring biosynthesis of the protein in vivo we demonstrated that the highest rates of synthesis of lipid-body LOX occurred at day 1 of germination. The early and selective appearance of a LOX form associated with lipid bodies at this stage of development is discussed.} } @Article{IPB-2461, author = {Maier, W. and Peipp, H. and Schmidt, J. and Wray, V. and Strack, D. and}, title = {{Levels of a Terpenoid Glycoside (Blumenin) and Cell Wall-Bound Phenolics in Some Cereal Mycorrhizas}}, year = {1995}, pages = {465-470}, journal = {Plant Physiol.}, doi = {10.1104/pp.109.2.465}, volume = {109}, abstract = {Four cereals, Hordeum vulgare (barley), Triticum aestivum (wheat), Secale cereale (rye), and Avena sativa (oat), were grown in a defined nutritional medium with and without the arbuscular mycorrhizal fungus Glomus intraradices. Levels of soluble and cell wall-bound secondary metabolites in the roots of mycorrhizal and nonmycorrhizal plants were determined by high-performance liquid chromatography during the first 6 to 8 weeks of plant development. Whereas there was no difference in the levels of the cell wall-bound hydroxycinnamic acids, 4-coumaric and ferulic acids, there was a fungus-induced change of the soluble secondary root metabolites. The most obvious effect observed in all four cereals was the induced accumulation of a terpenoid glycoside. This compound was isolated and identified by spectroscopic methods (nuclear magnetic resonance, mass spectrometry) to be a cyclohexenone derivative, i.e. blumenol C 9-O-(2[prime]-O-[beta]-glucuronosyl)-[beta]-glucoside. The level of this compound was found to be directly correlated with the degree of root colonization.} } @Article{IPB-2456, author = {Hohlfeld, H. and Schurmann, W. and Scheel, D. and Strack, D. and}, title = {{Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum}}, year = {1995}, pages = {545-552}, journal = {Plant Physiol.}, doi = {10.1104/pp.107.2.545}, volume = {107}, abstract = {A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2\+ or Mg2\+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1.} } @Article{IPB-2454, author = {Feussner, I. and Hause, B. and Vörös, K. and Parthier, B. and Wasternack, C. and}, title = {{Jasmonate-induced lipoxygenase forms are localized in chloroplasts of barley leaves (Hordeum vulgare cv. Salome)}}, year = {1995}, pages = {949-957}, journal = {Plant J.}, doi = {10.1046/j.1365-313X.1995.07060949.x}, volume = {7}, abstract = {Barley leaves respond to application of (−)‐jasmonic acid (JA), or its methylester (JM) with the synthesis of abundant proteins, so‐called jasmonate induced proteins (JIPs). Here Western blot analysis is used to show a remarkable increase upon JM treatment of a 100 kDa lipoxygenase (LOX), and the appearance of two new LOX forms of 98 and 92 kDa. The temporal increase of LOX‐100 protein upon JM treatment was clearly distinguishable from the additionally detectable LOX forms. JM‐induced LOX forms in barley leaves were compared with those of Arabidopsis and soybean leaves. Both dicot species showed a similar increase of one LOX upon JM induction, whereas, leaves from soybean responded with additional synthesis of a newly formed LOX of 94 kDa.Using immunofluorescence analysis and isolation of intact chloroplasts, it is demonstrated that JM‐induced LOX forms of barley leaves are exclusively located in the chloroplasts of all chloroplast‐containing cells. Analogous experiments carried out with Arabidopsis and soybean revealed a similar plastidic location of JM‐induced LOX forms in Arabidopsis but a different situation for soybean. In untreated soybean leaves the LOX protein was mainly restricted to vacuoles of paraveinal mesophyll cells. Additionally, LOX forms could be detected in cytoplasm and nuclei of bundle sheath cells. Upon JM treatment cytosolic LOX was detectable in spongy mesophyll cells, too. The intracellular location of JM‐induced LOX is discussed in terms of stress‐related phenomena mediated by JM.} } @Article{IPB-2475, author = {Schneider, G. and Schliemann, W. and}, title = {{Gibberellin conjugates: an overview}}, year = {1994}, pages = {247-260}, journal = {Plant Growth Regul.}, doi = {10.1007/BF00029898}, volume = {15}, abstract = {This article surveys the currently isolated and identified GA conjugates, their synthesis and evaluates modern methods for analysing GA glucose conjugates. The metabolism of applied GAs in higher plant systems leading, in most cases, to GA conjugates is also considered. The enzymology of the formation and hydrolysis of GA glucose conjugates is discussed in connection with their possible physiological function.} } @Article{IPB-2473, author = {Hause, B. and zur Nieden, U. and Lehmann, J. and Wasternack, C. and Parthier, B. and}, title = {{Intracellular Localization of Jasmonate-Induced Proteins in Barley Leaves}}, year = {1994}, pages = {333-341}, journal = {Bot. Acta}, doi = {10.1111/j.1438-8677.1994.tb00804.x}, volume = {107}, abstract = {The plant growth substance jasmonic acid and its methyl ester (JA‐Me) induce a set of proteins (jasmonate‐induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one‐dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno‐gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP‐23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP‐37 was detected in vacuoles and in the nucleoplasm; JIP‐66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP‐100 was found within the stromal fraction of chloroplasts; JIP‐70 is present in the peroxisome and the nucleus; JIP‐50 and JIP‐6 accumulate in vacuoles. The location of JIP‐66 and JIP‐6 confirms their possible physiological role deduced from molecular analysis of their cDNA.} } @Article{IPB-2472, author = {Gottstein, D. and Schliemann, W. and}, title = {{Purine glucosylating activity in cell suspension cultures ofSolanum tuberosum L.}}, year = {1994}, pages = {265-268}, journal = {Plant Cell Tiss. Organ Cult.}, doi = {10.1007/BF00037730}, volume = {36}, abstract = {Cell suspension cultures ofSolanum tuberosum L. cv. Adretta were established from leaf-derived calluses. In the search for purine glucosylating activity, the metabolism of 6-benzylaminopurine was studied. The main metabolite of BA was isolated and identified as 6-benzylaminopurine 7-β-d-glucopyranoside indicating the occurrence of purine glucosylating activity.} } @Article{IPB-2482, author = {Schliemann, W. and Schaller, B. and Jensen, E. and Schneider, G. and}, title = {{Native gibberellin-O-glucosides from mature seeds of Phaseolus coccineus}}, year = {1993}, pages = {35-38}, journal = {Phytochemistry}, doi = {10.1016/S0031-9422(00)90504-1}, volume = {35}, abstract = {Using an improved purification and derivatization procedure, the endogenous gibberellin-O-glucosides in mature runner beans (Phaseolus coccineus) were analysed by combined gas chromatography-mass spectrometry. In addition to the previously characterized GA1-3-O-glucoside and GA8-2-O-glucoside, from runner beans, the following GA-O-glucosides were identified as endogenously occurring compounds by comparison of their full scan mass spectra and Kovats retention indices with those of standards: GA1-13-O-glucoside, 3-epiGA1-3-O-glucoside, GA5-13-O-glucoside and GA29-2-O-glucoside. The first three are reported in higher plants for the first time. The physiological relevance of the detected GA-O-glucosides is discussed.} } @Article{IPB-2481, author = {Schliemann, W. and Schneider, G. and}, title = {{Gibberellins in Gramineae}}, year = {1993}, pages = {91-98}, journal = {Plant Growth Regul.}, doi = {10.1007/BF00144588}, volume = {12}, abstract = {Data on the occurrence of free and conjugated gibberellins in different tribes of Gramineae are compiled and discussed with regard to their biosynthetic pathways. From the gibberellins detected so far the functioning of both the early 13-hydroxylation and the non-3,13-hydroxylation pathway of GA biosynthesis in gramineous plants can be deduced and the discovery of further gibberellin conjugates may be expected.} }