@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-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-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-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-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-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-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-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-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-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-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-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.} } @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-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-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.} } @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.} } @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-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-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.} } @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-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-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-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-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-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-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-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-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-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-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-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}, } @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-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-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-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-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-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-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-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-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-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-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-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-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-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.} } @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.} } @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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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.} } @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-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}, } @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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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-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.} }