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Publikation

Hettwer, K.; Böttcher, C.; Frolov, A.; Mittasch, J.; Albert, A.; von Roepenack-Lahaye, E.; Strack, D.; Milkowski, C.; Dynamic metabolic changes in seeds and seedlings of Brassica napus (oilseed rape) suppressing UGT84A9 reveal plasticity and molecular regulation of the phenylpropanoid pathway Phytochemistry 124, 46-57, (2016) DOI: 10.1016/j.phytochem.2016.01.014

In Brassica napus, suppression of the key biosynthetic enzyme UDP-glucose:sinapic acid glucosyltransferase (UGT84A9) inhibits the biosynthesis of sinapine (sinapoylcholine), the major phenolic component of seeds. Based on the accumulation kinetics of a total of 158 compounds (110 secondary and 48 primary metabolites), we investigated how suppression of the major sink pathway of sinapic acid impacts the metabolome of developing seeds and seedlings. In UGT84A9-suppressing (UGT84A9i) lines massive alterations became evident in late stages of seed development affecting the accumulation levels of 58 secondary and 7 primary metabolites. UGT84A9i seeds were characterized by decreased amounts of various hydroxycinnamic acid (HCA) esters, and increased formation of sinapic and syringic acid glycosides. This indicates glycosylation and β-oxidation as metabolic detoxification strategies to bypass intracellular accumulation of sinapic acid. In addition, a net loss of sinapic acid upon UGT84A9 suppression may point to a feedback regulation of HCA biosynthesis. Surprisingly, suppression of UGT84A9 under control of the seed-specific NAPINC promoter was maintained in cotyledons during the first two weeks of seedling development and associated with a reduced and delayed transformation of sinapine into sinapoylmalate. The lack of sinapoylmalate did not interfere with plant fitness under UV-B stress. Increased UV-B radiation triggered the accumulation of quercetin conjugates whereas the sinapoylmalate level was not affected.
Publikation

Clauß, K.; von Roepenack-Lahaye, E.; Böttcher, C.; Roth, M. R.; Welti, R.; Erban, A.; Kopka, J.; Scheel, D.; Milkowski, C.; Strack, D.; Overexpression of Sinapine Esterase BnSCE3 in Oilseed Rape Seeds Triggers Global Changes in Seed Metabolism Plant Physiol. 155, 1127-1145, (2011) DOI: 10.1104/pp.110.169821

Sinapine (O-sinapoylcholine) is the predominant phenolic compound in a complex group of sinapate esters in seeds of oilseed rape (Brassica napus). Sinapine has antinutritive activity and prevents the use of seed protein for food and feed. A strategy was developed to lower its content in seeds by expressing an enzyme that hydrolyzes sinapine in developing rape seeds. During early stages of seedling development, a sinapine esterase (BnSCE3) hydrolyzes sinapine, releasing choline and sinapate. A portion of choline enters the phospholipid metabolism, and sinapate is routed via 1-O-sinapoyl-β-glucose into sinapoylmalate. Transgenic oilseed rape lines were generated expressing BnSCE3 under the control of a seed-specific promoter. Two distinct single-copy transgene insertion lines were isolated and propagated to generate homozygous lines, which were subjected to comprehensive phenotyping. Sinapine levels of transgenic seeds were less than 5% of wild-type levels, whereas choline levels were increased. Weight, size, and water content of transgenic seeds were significantly higher than those of wild-type seeds. Seed quality parameters, such as fiber and glucosinolate levels, and agronomically important traits, such as oil and protein contents, differed only slightly, except that amounts of hemicellulose and cellulose were about 30% higher in transgenic compared with wild-type seeds. Electron microscopic examination revealed that a fraction of the transgenic seeds had morphological alterations, characterized by large cavities near the embryonic tissue. Transgenic seedlings were larger than wild-type seedlings, and young seedlings exhibited longer hypocotyls. Examination of metabolic profiles of transgenic seeds indicated that besides suppression of sinapine accumulation, there were other dramatic differences in primary and secondary metabolism. Mapping of these changes onto metabolic pathways revealed global effects of the transgenic BnSCE3 expression on seed metabolism.
Bücher und Buchkapitel

Böttcher, C.; von Roepenack-Lahaye, E.; Scheel, D.; Resources for Metabolomics (Schmidt, R. & Bancroft, I., eds.). Plant Genetics and Genomics: Crops and Models 9, 469-503, (2011) ISBN: 978-1-4419-7118-0 DOI: 10.1007/978-1-4419-7118-0_17

Metabolomics is developing toward an integral component of functional genomics approaches. The large structural diversity of plant metabolites requires different analytical techniques for broad metabolite analysis. In addition, new bioinformatics tools and databases are necessary for data analysis and storage. This chapter describes the resources available for comprehensive analysis of plant secondary metabolites focusing on Arabidopsis thaliana and Brassica species. In particular, a platform for non-targeted profiling of semi-polar plant metabolites based on liquid chromatography coupled to mass spectrometry is described.
Publikation

Bartsch, M.; Bednarek, P.; Vivancos, P. D.; Schneider, B.; von Roepenack-Lahaye, E.; Foyer, C. H.; Kombrink, E.; Scheel, D.; Parker, J. E.; Accumulation of Isochorismate-derived 2,3-Dihydroxybenzoic 3-O-β-D-Xyloside in Arabidopsis Resistance to Pathogens and Ageing of Leaves J. Biol. Chem. 285, 25654-25665, (2010) DOI: 10.1074/jbc.M109.092569

An intricate network of hormone signals regulates plant development and responses to biotic and abiotic stress. Salicylic acid (SA), derived from the shikimate/isochorismate pathway, is a key hormone in resistance to biotrophic pathogens. Several SA derivatives and associated modifying enzymes have been identified and implicated in the storage and channeling of benzoic acid intermediates or as bioactive molecules. However, the range and modes of action of SA-related metabolites remain elusive. In Arabidopsis, Enhanced Disease Susceptibility 1 (EDS1) promotes SA-dependent and SA-independent responses in resistance against pathogens. Here, we used metabolite profiling of Arabidopsis wild type and eds1 mutant leaf extracts to identify molecules, other than SA, whose accumulation requires EDS1 signaling. Nuclear magnetic resonance and mass spectrometry of isolated and purified compounds revealed 2,3-dihydroxybenzoic acid (2,3-DHBA) as an isochorismate-derived secondary metabolite whose accumulation depends on EDS1 in resistance responses and during ageing of plants. 2,3-DHBA exists predominantly as a xylose-conjugated form (2-hydroxy-3-β-O-d-xylopyranosyloxy benzoic acid) that is structurally distinct from known SA-glucose conjugates. Analysis of DHBA accumulation profiles in various Arabidopsis mutants suggests an enzymatic route to 2,3-DHBA synthesis that is under the control of EDS1. We propose that components of the EDS1 pathway direct the generation or stabilization of 2,3-DHBA, which as a potentially bioactive molecule is sequestered as a xylose conjugate.
Publikation

Böttcher, C.; von Roepenack-Lahaye, E.; Schmidt, J.; Clemens, S.; Scheel, D.; Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray- tandem mass spectrometry J. Mass Spectrom. 44, 466-476, (2009) DOI: 10.1002/jms.1522

Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI‐QTOF‐MS and direct infusion ESI‐FTICR‐MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision‐induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl‐ and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed‐phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI‐MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class. Copyright © 2008 John Wiley & Sons, Ltd.
Publikation

Böttcher, C.; Centeno, D.; Freitag, J.; Höfgen, R.; Köhl, K.; Kopka, J.; Kroymann, J.; Matros, A.; Mock, H.-P.; Neumann, S.; Pfalz, M.; von Roepenack-Lahaye, E.; Schauer, N.; Trenkamp, S.; Zubriggen, M.; Fernie, A. R.; Teaching (and learning from) metabolomics: The 2006 PlantMetaNet ETNA Metabolomics Research School Physiol. Plant. (2008) DOI: 10.1111/j.1399-3054.2007.00990.x

Under the auspices of the European Training and Networking Activity programme of the European Union, a ‘Metabolic Profiling and Data Analysis’ Plant Genomics and Bioinformatics Summer School was hosted in Potsdam, Germany between 20 and 29 September 2006. Sixteen early career researchers were invited from the European Union partner nations and the so‐called developing nations (Appendix). Lectures from invited leading European researchers provided an overview of the state of the art of these fields and seeded discussion regarding major challenges for their future advancement. Hands‐on experience was provided by an example experiment – that of defining the metabolic response of Arabidopsis to treatment of a commercial herbicide of defined mode of action. This experiment was performed throughout the duration of the course in order to teach the concepts underlying extraction and machine handling as well as to provide a rich data set with which the required computation and statistical skills could be illustrated. Here we review the state of the field by describing both key lectures given at and practical aspects taught at the summer school. In addition, we disclose results that were obtained using the four distinct technical platforms at the different participating institutes. While the effects of the chosen herbicide are well documented, this study looks at a broader number of metabolites than in previous investigations. This allowed, on the one hand, not only to characterise further effects of the herbicide than previously observed but also to detect molecules other than the herbicide that were obviously present in the commercial formulation. These data and the workshop in general are all discussed in the context of the teaching of metabolomics.
Publikation

Böttcher, C.; von Roepenack-Lahaye, E.; Schmidt, J.; Schmotz, C.; Neumann, S.; Scheel, D.; Clemens, S.; Metabolome Analysis of Biosynthetic Mutants Reveals a Diversity of Metabolic Changes and Allows Identification of a Large Number of New Compounds in Arabidopsis Plant Physiol. 147, 2107-2120, (2008) DOI: 10.1104/pp.108.117754

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.
Publikation

Roth, U.; von Roepenack-Lahaye, E.; Clemens, S.; Proteome changes in Arabidopsis thaliana roots upon exposure to Cd2+ J. Exp. Bot. 57, 4003-4013, (2006) DOI: 10.1093/jxb/erl170

Cadmium is a major environmental pollutant that enters human food via accumulation in crop plants. Responses of plants to cadmium exposure—which directly influence accumulation rates—are not well understood. In general, little is known about stress-elicited changes in plants at the proteome level. Alterations in the root proteome of hydroponically grown Arabidopsis thaliana plants treated with 10 μM Cd2+ for 24 h are reported here. These conditions trigger the synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, shown here as PC2 accumulation. Two-dimensional gel electrophoresis using different pH gradients in the first dimension detected on average ∼1100 spots per gel type. Forty-one spots indicated significant changes in protein abundance upon Cd2+ treatment. Seventeen proteins found in 25 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Selected results were independently confirmed by western analysis and selective enrichment of a protein family (glutathione S-transferases) through affinity chromatography. Most of the identified proteins belong to four different classes: metabolic enzymes such as ATP sulphurylase, glycine hydroxymethyltransferase, and trehalose-6-phosphate phosphatase; glutathione S-transferases; latex allergen-like proteins; and unknown proteins. These results represent a basis for reverse genetics studies to better understand plant responses to toxic metal exposure and to the generation of internal sinks for reduced sulphur.
Publikation

Harada, E.; von Roepenack-Lahaye, E.; Clemens, S.; A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to γ-glutamylcysteine and lacks phytochelatin synthase activity Phytochemistry 65, 3179-3185, (2004) DOI: 10.1016/j.phytochem.2004.09.017

Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to γ-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC–MSMS analysis was unequivocally identified as γ-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to γ-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.
Publikation

von Roepenack-Lahaye, E.; Degenkolb, T.; Zerjeski, M.; Franz, M.; Roth, U.; Wessjohann, L.; Schmidt, J.; Scheel, D.; Clemens, S.; Profiling of Arabidopsis Secondary Metabolites by Capillary Liquid Chromatography Coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry Plant Physiol. 134, 548-559, (2004) DOI: 10.1104/pp.103.032714

Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2,000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling.
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