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Publications - Stress and Develop Biology

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Cotrim, C. A.; Weidner, A.; Strehmel, N.; Bisol, T. B.; Meyer, D.; Brandt, W.; Wessjohann, L. A.; Stubbs, M. T. A Distinct Aromatic Prenyltransferase Associated with the Futalosine Pathway ChemistrySelect 2, 9319-9325, (2017) DOI: 10.1002/slct.201702151

Menaquinone (MK) is an electron carrier molecule essential for respiration in most Gram positive bacteria. A crucial step in MK biosynthesis involves the prenylation of an aromatic molecule, catalyzed by integral membrane prenyltransferases of the UbiA (4‐hydroxybenzoate oligoprenyltransferase) superfamily. In the classical MK biosynthetic pathway, the prenyltransferase responsible is MenA (1,4‐dihydroxy‐2‐naphthoate octaprenyltransferase). Recently, an alternative pathway for formation of MK, the so‐called futalosine pathway, has been described in certain micro‐organisms. Until now, five soluble enzymes (MqnA‐MqnE) have been identified in the first steps. In this study, the genes annotated as ubiA from T. thermophilus and S. lividans were cloned, expressed and investigated for prenylation activity. The integral membrane proteins possess neither UbiA nor MenA activity and represent a distinct class of prenyltransferases associated with the futalosine pathway that we term MqnP. We identify a critical residue within a highly conserved Asp‐rich motif that serves to distinguish between members of the UbiA superfamily.

Rana, R.; Herz, K.; Bruelheide, H.; Dietz, S.; Haider, S.; Jandt, U.; Pena, R. Leaf Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) biochemical profile of grassland plant species related to land-use intensity Ecol Indic 84, 803-810, (2017) DOI: 10.1016/j.ecolind.2017.09.047

There is growing interest in the application of plant functional trait-based approaches for development of sustainable land-use strategies. In this context, one crucial task is to identify and measure plant traits, which respond to land-use intensity (response traits) and simultaneously have an impact on ecosystem functions (effect traits). We hypothesized that species-specific leaf chemical composition, which may function both as response and effect trait, can be derived from Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy tools in combination with multivariate statistical methods We investigated leaf ATR-FTIR spectra of two grasses, Poa pratensis L. and Dactylis glomerata L., and one forb, Achillea millefolium L. collected in grassland plots along a land-use intensity gradient in three regions of Germany. ATR-FTIR spectra appear to function as biochemical fingerprints unique to each species. The spectral response to land-use intensity was not consistent among species and less apparent in the two grasses than in the forb species. Whereas land-use intensification enhanced protein and cellulose content in A. millefolium, giving rise to changes in six spectral bands in the frequency range of 1088–1699 cm−1, only cellulose content increased in D. glomerata, affecting the bands of 1385–1394 cm−1. Poa pratensis spectra exhibited minimal changes under the influence of land-use, only in the spectral bands of 1373–1375 cm−1 associated with suberin-like aliphatic compounds. Our findings suggest that some species’ leaf chemical composition is responsive to land-use intensity, and thus, may have a predictive value for ecosystem services provided by those species within grassland vegetation (i.e., herbage yield quality).

Hempel, F.; Stenzel, I.; Heilmann, M.; Krishnamoorthy, P.; Menzel, W.; Golbik, R.; Helm, S.; Dobritzsch, D.; Baginsky, S.; Lee, J.; Hoehenwarter, W.; Heilmann, I. MAPKs influence pollen tube growth by controlling the formation of Phosphatidylinositol 4,5-Bisphosphate in an apical plasma membrane domain.  Plant Cell 29, 3030-3050, (2017) DOI: 10.1105/tpc.17.00543

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.

Strehmel, N.; Hoehenwarter, W.; Mönchgesang, S.; Majovsky, P.; Krüger, S.; Scheel, D.; Lee, J. Stress-reated mitogen-activated protein kinases stimulate the accumulation of small molecules and proteins in Arabidopsis thaliana root exudates. Front Plant Sci 8 , 1292, (2017) DOI: 10.3389/fpls.2017.01292

A delicate balance in cellular signaling is required for plants to respond to microorganisms or to changes in their environment. Mitogen-activated protein kinase (MAPK) cascades are one of the signaling modules that mediate transduction of extracellular microbial signals into appropriate cellular responses. Here, we employ a transgenic system that simulates activation of two pathogen/stress-responsive MAPKs to study release of metabolites and proteins into root exudates. The premise is based on our previous proteomics study that suggests upregulation of secretory processes in this transgenic system. An advantage of this experimental set-up is the direct focus on MAPK-regulated processes without the confounding complications of other signaling pathways activated by exposure to microbes or microbial molecules. Using non-targeted metabolomics and proteomics studies, we show that MAPK activation can indeed drive the appearance of dipeptides, defense-related metabolites and proteins in root apoplastic fluid. However, the relative levels of other compounds in the exudates were decreased. This points to a bidirectional control of metabolite and protein release into the apoplast. The putative roles for some of the identified apoplastic metabolites and proteins are discussed with respect to possible antimicrobial/defense or allelopathic properties. Overall, our findings demonstrate that sustained activation of MAPKs alters the composition of apoplastic root metabolites and proteins, presumably to influence the plant-microbe interactions in the rhizosphere. The reported metabolomics and proteomics data are available via Metabolights (Identifier: MTBLS441) and ProteomeXchange (Identifier: PXD006328), respectively.

Furlan, G.; Nakagami, H.; Eschen-Lippold, L.; Jiang, X.; Majovsky, P.; Kowarschik, K.; Hoehenwarter, W.; Lee, J.; Trujillo, M. Changes in PUB22 Ubiquitination Modes Triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 Dampen the Immune Response Plant Cell 29, 726-745, (2017) DOI: 10.1105/tpc.16.00654

Crosstalk between post-translational modifications such as ubiquitination and phosphorylation play key roles in controlling the duration and intensity of signalling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signalling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and thus autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo. 

Dobritzsch, M.; Lübken, T.; Eschen-Lippold, L.; Gorzolka, K.; Blum, E.; Matern, A.; Marillonnet, S.; Böttcher, C.; Dräger, B.; Rosahl, S. MATE Transporter-Dependent Export of Hydroxycinnamic Acid Amides. Plant Cell 28, 583-596, (2016) DOI: 10.1105/tpc.15.00706

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.

Eschen-Lippold, L.; Jiang, X.; Elmore, J. M.; Mackey, D.; Shan, L.; Coaker, G.; Scheel, D.; Lee, J. Bacterial AvrRpt2-like cysteine proteases block activation of the Arabidopsis mitogen-activated protein kinases, MPK4 and MPK11. Plant Physiol 171, 2223-2238, (2016) DOI: 10.1104/pp.16.00336

To establish infection, pathogens deliver effectors into host cells to target immune signalling components, including elements of mitogen-activated protein kinase (MPK) cascades. The virulence function of AvrRpt2, one of the first identified Pseudomonas syringae effectors, involves cleavage of the plant defence regulator, RIN4, and interference with plant auxin signalling. We show now that AvrRpt2 specifically suppresses flagellin-induced phosphorylation of Arabidopsis MPK4 and MPK11, but not MPK3 or MPK6. This inhibition requires the proteolytic activity of AvrRpt2, is associated with reduced expression of some plant defence genes, and correlates with enhanced pathogen infection in AvrRpt2-expressing transgenic plants. Diverse AvrRpt2-like homologs can be found in some phytopathogens, plant-associated and soil bacteria. Employing these putative bacterial AvrRpt2 homologs and inactive AvrRpt2 variants, we can uncouple the inhibition of MPK4/MPK11 activation from the cleavage of RIN4 and related members from the so-called NOI family, as well as from auxin signalling. Thus, this selective suppression of specific MAPKs is independent of the previously known AvrRpt2 targets and represents potentially a novel virulence function of AvrRpt2.

Brömme, T.; Schmitz, C.; Moszner, N.; Burtscher, P.; Strehmel, N.; Strehmel, B. Photochemical Oxidation of NIR Photosensitizers in the Presence of Radical Initiators and Their Prospective Use in Dental Applications ChemistrySelect 1, 524–532, (2016) DOI: 10.1002/slct.201600048

Photochemical oxidation of near infrared (NIR) photosensitizers in the presence of diaryl iodonium salts bearing either bis(trifluoromethylsulfonyl)imide or hexafluorophosphate was investigated by exposure with NIR LEDs emitting either at 790 nm, 830 nm, 850 nm or 870 nm. Four different cyanines with barbituryl group at the meso position exhibit similar absorption in the NIR. These photosensitizers initiate in combination with diaryliodonium salts radical photopolymerization of dental composites with the focus to cure large thicknesses. Furthermore, the mixture comprising the cyanine and the iodonium salt was used to generate brown color in dental composites on demand. This required to understand the mechanism of dye decomposition in more detail applying exposure kinetics and a coupling of Ultra Performance Liquid Chromatography (UPLC) with mass spectrometry (MS) to analyze the photoproducts formed. Data showed cleavage of the polymethine chain at typical positions in case of the oxidized species. These were formed as result of electron transfer between the excited state of the photosensitizer and the iodonium salt. UPLC-MS experiments additionally indicated a certain sensitivity of the system upon adding of acids and radicals generated by thermal treatment of azobisisobutyronitrile (AIBN). Thus, treatment of the photoinitiator composition led almost to the same products no matter the system was either exposed with NIR light or treated with acids or radicals generated by thermal decomposition of AIBN. These findings helped to understand the large curing depth of 14 mm upon NIR exposure at 850 nm and the brown color formed.

Ranf, S.; Scheel, D.; Lee, J. Challenges in the identification of microbe-associated molecular patterns in plant and animal innate immunity: a case study with bacterial lipopolysaccharide Mol Plant Pathol. 17, 1165–1169 , (2016) DOI: 10.1111/mpp.12452

Books and chapters

Faden, F.; Eschen-Lippold, L.; Dissmeyer, N. Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling (Lois, L. M. & Matthiesen, R., eds.). Methods Mol Biol 1450, 247-258, (2016) ISBN: 978-1-4939-3759-2 DOI: 10.1007/978-1-4939-3759-2_20

Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein–epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins—assumed to be constitutively expressed mostly independent of developmental and environmental states—can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein’s abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of protein expression by lane-to-lane, gel-to-gel, and blot-to-blot comparisons is facilitated especially with respect to experiments in the area of proteostasis dealing with highly variable protein levels and involving protein degradation mutants and treatments modulating protein abundance.
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