TY - JOUR ID - 440 TI - Tissue-specific signatures of metabolites and proteins in asparagus roots and exudates JO - Hortic Res PY - 2021 SP - 86 AU - Döll, S. AU - Djalali Farahani-Kofoet, R. AU - Zrenner, R. AU - Henze, A. AU - Witzel, K. AU - VL - 8 UR - https://doi.org/10.1038/s41438-021-00510-5 DO - 10.1038/s41438-021-00510-5 AB - Comprehensive untargeted and targeted analysis of root exudate composition has advanced our understanding of rhizosphere processes. However, little is known about exudate spatial distribution and regulation. We studied the specific metabolite signatures of asparagus root exudates, root outer (epidermis and exodermis), and root inner tissues (cortex and vasculature). The greatest differences were found between exudates and root tissues. In total, 263 non-redundant metabolites were identified as significantly differentially abundant between the three root fractions, with the majority being enriched in the root exudate and/or outer tissue and annotated as ‘lipids and lipid-like molecules’ or ‘phenylpropanoids and polyketides’. Spatial distribution was verified for three selected compounds using MALDI-TOF mass spectrometry imaging. Tissue-specific proteome analysis related root tissue-specific metabolite distributions and rhizodeposition with underlying biosynthetic pathways and transport mechanisms. The proteomes of root outer and inner tissues were spatially very distinct, in agreement with the fundamental differences between their functions and structures. According to KEGG pathway analysis, the outer tissue proteome was characterized by a high abundance of proteins related to ‘lipid metabolism’, ‘biosynthesis of other secondary metabolites’and ‘transport and catabolism’, re flecting its main functions of providing a hydrophobic barrier, secreting secondary metabolites, and mediating water and nutrient uptake. Proteins more abundant in the inner tissue related to ‘transcription’, ‘translation’ and ‘folding, sorting and degradation’, in accord with the high activity of cortical and vasculature cell layers in growth-and development-related processes. In summary, asparagus root fractions accumulate specific metabolites. This expands our knowledge of tissue-specific plant cell function. A2 - C1 - ER - TY - DATA ID - 2508 TI - PSYCHE - a valuable experiment in plant NMR-metabolomics JO - RADAR PY - 2020 SP - AU - Stark, P. AU - Zab, C. AU - Porzel, A. AU - Franke, K. AU - Rizzo, P. AU - Wessjohann, L. A. AU - VL - UR - https://www.radar-service.eu/radar/en/dataset/nUwWpwHaWREjQglm DO - 10.22000/338 AB - Dataset: NMR raw dataInstrument: Agilent VNMRS 600 NMR spectrometer A2 - C1 - Bioorganic Chemistry ER - TY - JOUR ID - 616 TI - Drivers of the composition of active rhizosphere bacterial communities in temperate grasslands JO - ISME J. PY - 2020 SP - 463-475 AU - Vieira, S. AU - Sikorski, J. AU - Dietz, S. AU - Herz, K. AU - Schrumpf, M. AU - Bruelheide, H. AU - Scheel, D. AU - Friedrich, M. W. AU - Overmann, J. AU - VL - 14 UR - DO - 10.1038/s41396-019-0543-4 AB - The active bacterial rhizobiomes and root exudate profiles of phytometers of six plant species growing in central European temperate grassland communities were investigated in three regions located up to 700 km apart, across diverse edaphic conditions and along a strong land use gradient. The recruitment process from bulk soil communities was identified as the major direct driver of the composition of active rhizosphere bacterial communities. Unexpectedly, the effect of soil properties, particularly soil texture, water content, and soil type, strongly dominated over plant properties and the composition of polar root exudates of the primary metabolism. While plant species-specific selection of bacteria was minor, the RNA-based composition of active rhizosphere bacteria substantially differed between rhizosphere and bulk soil. Although other variables could additionally be responsible for the consistent enrichment of particular bacteria in the rhizosphere, distinct bacterial OTUs were linked to the presence of specific polar root exudates independent of individual plant species. Our study also identified numerous previously unknown taxa that are correlated with rhizosphere dynamics and hence represent suitable targets for future manipulations of the plant rhizobiome. A2 - C1 - ER - TY - JOUR ID - 597 TI - UHPLC-MS metabolome based classification of umbelliferous fruit taxa: a prospect for phyto-equivalency of its different accessions and in response to roasting JO - RSC Adv. PY - 2020 SP - 76-85 AU - Serag, A. AU - Baky, M. H. AU - Döll, S. AU - Farag, M. A. AU - VL - 10 UR - DOI https://doi.org/10.1039/C9RA07841J DO - 10.1039/C9RA07841J AB - Herbs of the Umbelliferae family are popular spices valued worldwide for their many nutritional and health benefits. Herein, five chief umbelliferous fruits viz., cumin, fennel, anise, coriander and caraway were assessed for its secondary metabolites diversity along with compositional changes incurring upon roasting as analyzed via ultra-high performance liquid chromatography coupled to photodiode array and electrospray ionization mass detectors UHPLC-qToF/MS. A total of 186 metabolites were annotated, according to metabolomics society guidelines, belonging mainly to flavonoids, fatty acids and phenolic acids. Multivariate models viz., PCA, HCA and OPLS-DA were further employed to assess fruits\' heterogeneity in an untargeted manner and determine mechanistic changes in bioactive makeup post roasting viz., glycosidic cleavage, lipid degradation and Maillard reaction. Finally, the fruits\' antioxidant activity showed decline upon roasting and in correlation with its total phenolic content. This study presents the first complete map of umbelliferous fruit metabolome, compositional differences and its roasting effect. A2 - C1 - ER - TY - JOUR ID - 545 TI - Root exudate composition of grass and forb species in natural grasslands JO - Sci. Rep. PY - 2020 SP - 10691 AU - Dietz, S. AU - Herz, K. AU - Gorzolka, K. AU - Jandt, U. AU - Bruelheide, H. AU - Scheel, D. AU - VL - 10 UR - DO - 10.1038/s41598-019-54309-5 AB - Plants exude a diverse cocktail of metabolites into the soil as response to exogenous and endogenous factors. So far, root exudates have mainly been studied under artificial conditions due to methodological difficulties. In this study, each five perennial grass and forb species were investigated for polar and semi-polar metabolites in exudates under field conditions. Metabolite collection and untargeted profiling approaches combined with a novel classification method allowed the designation of 182 metabolites. The composition of exuded polar metabolites depended mainly on the local environment, especially soil conditions, whereas the pattern of semi-polar metabolites was primarily affected by the species identity. The profiles of both polar and semi-polar metabolites differed between growth forms, with grass species being generally more similar to each other and more responsive to the abiotic environment than forb species. This study demonstrated the feasibility of investigating exudates under field conditions and to identify the driving factors of exudate composition. A2 - C1 - Biochemistry of Plant Interactions ER - TY - CHAP ID - 18 TI - Proteomic Approaches to Identify Proteins Responsive to Cold Stress T2 - Plant Cold Acclimation PB - Methods Mol. Biol. PY - 2020 SP - 161-170 AU - Jozefowicz, A. M. AU - Döll, S. AU - Mock, H.-P. AU - VL - 2156 UR - SN - 978-1-0716-0660-5 DO - 10.1007/978-1-0716-0660-5_12 AB - Changing environmental conditions greatly affect the accumulation of many proteins; therefore, the analysis of alterations in the proteome is essential to understand the plant response to abiotic stress. Proteomics provides a platform for the identification and quantification of plant proteins responsive to cold stress and taking part in cold acclimation. Here, we describe the preparation of proteins for LC-MS measurement to monitor the changes of protein patterns during cold treatment in Arabidopsis thaliana. In our protocol, proteins are precipitated using TCA/acetone, quantified with 2D Quant Kit and digested with trypsin using a filter-based method and analyzed using an LC-MS approach. The acquired results can be further applied for label-free protein quantification. A2 - Hincha, D. K. & Zuther, E., eds. C1 - ER - TY - JOUR ID - 695 TI - A substrate of the ABC transporter PEN3 stimulates bacterial flagellin (flg22)-induced callose deposition in Arabidopsis thaliana JO - J. Biol. Chem. PY - 2019 SP - 6857-6870 AU - Matern, A. AU - Böttcher, C. AU - Eschen-Lippold, L. AU - Westermann, B. AU - Smolka, U. AU - Döll, S. AU - Trempel, F. AU - Aryal, B. AU - Scheel, D. AU - Geisler, M. AU - Rosahl, S. AU - VL - 294 UR - DO - 10.1074/jbc.RA119.007676 AB - Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-yl-methyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro. Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca2+ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogen-associated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens. A2 - C1 - Biochemistry of Plant Interactions; Bioorganic Chemistry ER - TY - JOUR ID - 675 TI - Correction: Linking root exudates to functional plant traits JO - PLOS ONE PY - 2019 SP - e0213965 AU - Herz, K. AU - Dietz, S. AU - Gorzolka, K. AU - Haider, S. AU - Jandt, U. AU - Scheel, D. AU - Bruelheide, H. AU - VL - 14 UR - DO - 10.1371/journal.pone.0213965 AB - A2 - C1 - ER - TY - JOUR ID - 705 TI - Early Pep-13-induced immune responses are SERK3A/B-dependent in potato JO - Sci. Rep. PY - 2019 SP - 18380 AU - Nietzschmann, L. AU - Gorzolka, K. AU - Smolka, U. AU - Matern, A. AU - Eschen-Lippold, L. AU - Scheel, D. AU - Rosahl, S. AU - VL - 9 UR - DO - 10.1038/s41598-019-54944-y AB - Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 645 TI - Semi‐polar root exudates in natural grassland communities JO - Ecol. Evol. PY - 2019 SP - 5526-5541 AU - Dietz, S. AU - Herz, K. AU - Döll, S. AU - Haider, S. AU - Jandt, U. AU - Bruelheide, H. AU - Scheel, D. AU - VL - 9 UR - DO - 10.1002/ece3.5043 AB - In the rhizosphere, plants are exposed to a multitude of different biotic and abiotic factors, to which they respond by exuding a wide range of secondary root metabolites. So far, it has been unknown to which degree root exudate composition is species‐specific and is affected by land use, the local impact and local neighborhood under field conditions. In this study, root exudates of 10 common grassland species were analyzed, each five of forbs and grasses, in the German Biodiversity Exploratories using a combined phytometer and untargeted liquid chromatography‐mass spectrometry (LC‐MS) approach. Redundancy analysis and hierarchical clustering revealed a large set of semi‐polar metabolites common to all species in addition to species‐specific metabolites. Chemical richness and exudate composition revealed that forbs, such as Plantago lanceolata and Galium species, exuded more species‐specific metabolites than grasses. Grasses instead were primarily affected by environmental conditions. In both forbs and grasses, plant functional traits had only a minor impact on plant root exudation patterns. Overall, our results demonstrate the feasibility of obtaining and untargeted profiling of semi‐polar metabolites under field condition and allow a deeper view in the exudation of plants in a natural grassland community. A2 - C1 - ER - TY - JOUR ID - 725 TI - Supporting non-target identification by adding hydrogen deuterium exchange MS/MS capabilities to MetFrag JO - Anal. Bioanal. Chem. PY - 2019 SP - 4683-4700 AU - Ruttkies, C. AU - Schymanski, E. L. AU - Strehmel, N. AU - Hollender, J. AU - Neumann, S. AU - Williams, A. J. AU - Krauss, M. AU - VL - 411 UR - DO - 10.1007/s00216-019-01885-0 AB - Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is increasingly popular for the non-targeted exploration of complex samples, where tandem mass spectrometry (MS/MS) is used to characterize the structure of unknown compounds. However, mass spectra do not always contain sufficient information to unequivocally identify the correct structure. This study investigated how much additional information can be gained using hydrogen deuterium exchange (HDX) experiments. The exchange of “easily exchangeable” hydrogen atoms (connected to heteroatoms), with predominantly [M+D]+ ions in positive mode and [M-D]− in negative mode was observed. To enable high-throughput processing, new scoring terms were incorporated into the in silico fragmenter MetFrag. These were initially developed on small datasets and then tested on 762 compounds of environmental interest. Pairs of spectra (normal and deuterated) were found for 593 of these substances (506 positive mode, 155 negative mode spectra). The new scoring terms resulted in 29 additional correct identifications (78 vs 49) for positive mode and an increase in top 10 rankings from 80 to 106 in negative mode. Compounds with dual functionality (polar head group, long apolar tail) exhibited dramatic retention time (RT) shifts of up to several minutes, compared with an average 0.04 min RT shift. For a smaller dataset of 80 metabolites, top 10 rankings improved from 13 to 24 (positive mode, 57 spectra) and from 14 to 31 (negative mode, 63 spectra) when including HDX information. The results of standard measurements were confirmed using targets and tentatively identified surfactant species in an environmental sample collected from the river Danube near Novi Sad (Serbia). The changes to MetFrag have been integrated into the command line version available at http://c-ruttkies.github.io/MetFrag and all resulting spectra and compounds are available in online resources and in the Electronic Supplementary Material (ESM). A2 - C1 - ER - TY - CHAP ID - 23 TI - Liquid Chromatography and Liquid Chromatography–Mass Spectrometry of Plants: Techniques and Applications T2 - Encyclopedia of Analytical Chemistry PB - PY - 2019 SP - AU - Doell, S. AU - Arens, N. AU - Mock, H. AU - VL - UR - SN - 9780470027318 DO - 10.1002/9780470027318.a9912.pub2 AB - Mass spectrometry coupled with LC (liquid chromatography) separation has developed into a technique routinely applied for targeted as well as for nontargeted analysis of complex biological samples, not only in plant biochemistry. Earlier on, LC‐MS (liquid chromatography–mass spectrometry) was mostly part of the efforts for identification of one or few unknown metabolites of interest as part of a phytochemical study. As a major strategy, unknown compounds had to be purified in sufficient quantities. The purified fractions were then subjected to LC‐MS/MS as part of the structural elucidation, mostly complemented by NMR (nuclear magnetic resonance) analysis. With the advance of mass spectrometry instrumentation, LC‐MS is now widely applied for analysis of crude plant extracts and large numbers (100s to 1000s) of samples. It has become an essential part of metabolomic studies (see Metabolomics), aiming at the comprehensive coverage of the metabolite profiles of cells, tissues, or organs. Owing to the huge chemical diversity of small molecules, conditions for the extraction will restrict the subfraction of the metabolome, which can be actually analyzed. The conditions for LC have to be adjusted to allow good separation of the particular metabolites from the respective extract. Major consideration will be the selection of an appropriate column and suitable eluents, the establishment of gradient profiles, temperature conditions, and so on. A2 - Meyers, R. A., ed. C1 - ER - TY - JOUR ID - 821 TI - Seasonal variation of secondary metabolites in nine different bryophytes JO - Ecol. Evol. PY - 2018 SP - 9105-9117 AU - Peters, K. AU - Gorzolka, K. AU - Bruelheide, H. AU - Neumann, S. AU - VL - 8 UR - DO - 10.1002/ece3.4361 AB - Bryophytes occur in almost all land ecosystems and contribute to global biogeochemical cycles, ecosystem functioning, and influence vegetation dynamics. As growth and biochemistry of bryophytes are strongly dependent on the season, we analyzed metabolic variation across seasons with regard to ecological characteristics and phylogeny. Using bioinformatics methods, we present an integrative and reproducible approach to connect ecology with biochemistry. Nine different bryophyte species were collected in three composite samples in four seasons. Untargeted liquid chromatography coupled with mass spectrometry (LC/MS) was performed to obtain metabolite profiles. Redundancy analysis, Pearson's correlation, Shannon diversity, and hierarchical clustering were used to determine relationships among species, seasons, ecological characteristics, and hierarchical clustering. Metabolite profiles of Marchantia polymorpha and Fissidens taxifolius which are species with ruderal life strategy (R‐selected) showed low seasonal variability, while the profiles of the pleurocarpous mosses and Grimmia pulvinata which have characteristics of a competitive strategy (C‐selected) were more variable. Polytrichum strictum and Plagiomnium undulatum had intermediary life strategies. Our study revealed strong species‐specific differences in metabolite profiles between the seasons. Life strategies, growth forms, and indicator values for light and soil were among the most important ecological predictors. We demonstrate that untargeted Eco‐Metabolomics provide useful biochemical insight that improves our understanding of fundamental ecological strategies. A2 - C1 - ER - TY - JOUR ID - 820 TI - Current Challenges in Plant Eco-Metabolomics JO - Int. J. Mol. Sci. PY - 2018 SP - 1385 AU - Peters, K. AU - Worrich, A. AU - Weinhold, A. AU - Alka, O. AU - Balcke, G. AU - Birkemeyer, C. AU - Bruelheide, H. AU - Calf, O. W. AU - Dietz, S. AU - Dührkop, K. AU - Gaquerel, E. AU - Heinig, U. AU - Kücklich, M. AU - Macel, M. AU - Müller, C. AU - Poeschl, Y. AU - Pohnert, G. AU - Ristok, C. AU - Rodríguez, V. M. AU - Ruttkies, C. AU - Schuman, M. AU - Schweiger, R. AU - Shahaf, N. AU - Steinbeck, C. AU - Tortosa, M. AU - Treutler, H. AU - Ueberschaar, N. AU - Velasco, P. AU - Weiß, B. M. AU - Widdig, A. AU - Neumann, S. AU - van Dam, N. M. AU - VL - 19 UR - DO - 10.3390/ijms19051385 AB - The relatively new research discipline of Eco-Metabolomics is the application of metabolomics techniques to ecology with the aim to characterise biochemical interactions of organisms across different spatial and temporal scales. Metabolomics is an untargeted biochemical approach to measure many thousands of metabolites in different species, including plants and animals. Changes in metabolite concentrations can provide mechanistic evidence for biochemical processes that are relevant at ecological scales. These include physiological, phenotypic and morphological responses of plants and communities to environmental changes and also interactions with other organisms. Traditionally, research in biochemistry and ecology comes from two different directions and is performed at distinct spatiotemporal scales. Biochemical studies most often focus on intrinsic processes in individuals at physiological and cellular scales. Generally, they take a bottom-up approach scaling up cellular processes from spatiotemporally fine to coarser scales. Ecological studies usually focus on extrinsic processes acting upon organisms at population and community scales and typically study top-down and bottom-up processes in combination. Eco-Metabolomics is a transdisciplinary research discipline that links biochemistry and ecology and connects the distinct spatiotemporal scales. In this review, we focus on approaches to study chemical and biochemical interactions of plants at various ecological levels, mainly plant–organismal interactions, and discuss related examples from other domains. We present recent developments and highlight advancements in Eco-Metabolomics over the last decade from various angles. We further address the five key challenges: (1) complex experimental designs and large variation of metabolite profiles; (2) feature extraction; (3) metabolite identification; (4) statistical analyses; and (5) bioinformatics software tools and workflows. The presented solutions to these challenges will advance connecting the distinct spatiotemporal scales and bridging biochemistry and ecology A2 - C1 - Cell and Metabolic Biology ER - TY - JOUR ID - 819 TI - Computational workflow to study the seasonal variation of secondary metabolites in nine different bryophytes JO - Sci. Data PY - 2018 SP - 180179 AU - Peters, K. AU - Gorzolka, K. AU - Bruelheide, H. AU - Neumann, S. AU - VL - 5 UR - DO - 10.1038/sdata.2018.179 AB - In Eco-Metabolomics interactions are studied of non-model organisms in their natural environment and relations are made between biochemistry and ecological function. Current challenges when processing such metabolomics data involve complex experiment designs which are often carried out in large field campaigns involving multiple study factors, peak detection parameter settings, the high variation of metabolite profiles and the analysis of non-model species with scarcely characterised metabolomes. Here, we present a dataset generated from 108 samples of nine bryophyte species obtained in four seasons using an untargeted liquid chromatography coupled with mass spectrometry acquisition method (LC/MS). Using this dataset we address the current challenges when processing Eco-Metabolomics data. Here, we also present a reproducible and reusable computational workflow implemented in Galaxy focusing on standard formats, data import, technical validation, feature detection, diversity analysis and multivariate statistics. We expect that the representative dataset and the reusable processing pipeline will facilitate future studies in the research field of Eco-Metabolomics. A2 - C1 - ER - TY - JOUR ID - 790 TI - Linking root exudates to functional plant traits JO - PLOS ONE PY - 2018 SP - e0204128 AU - Herz, K. AU - Dietz, S. AU - Gorzolka, K. AU - Haider, S. AU - Jandt, U. AU - Scheel, D. AU - Bruelheide, H. AU - VL - 13 UR - DO - 10.1371/journal.pone.0204128 AB - Primary and secondary metabolites exuded by plant roots have mainly been studied under laboratory conditions, while knowledge of root exudate patterns of plants growing in natural communities is very limited. Focusing on ten common European grassland plant species, we asked to which degree exuded metabolite compositions are specific to species or growth forms (forbs and grasses), depend on environments and local neighbourhoods, and reflect traditional plant functional traits. Root exudates were collected under field conditions and analysed using a non-targeted gas chromatography coupled mass spectrometry (GC-MS) approach. In total, we annotated 153 compounds of which 36 were identified by structure and name as metabolites mainly derived from the primary metabolism. Here we show by using variance partitioning, that the composition of exuded polar metabolites was mostly explained by plot identity, followed by plant species identity while plant species composition of the local neighbourhood played no role. Total and root dry biomass explained the largest proportion of variance in exudate composition, with additional variance explained by traditional plant traits. Although the exudate composition was quite similar between the two growth forms, we found some metabolites that occurred only in one of the two growth forms. Our study demonstrated the feasibility of measuring polar exudates under non-sterile field conditions by mass spectrometry, which opens new avenues of research for functional plant ecology. A2 - C1 - ER - TY - JOUR ID - 961 TI - Arabidopsis Transporter ABCG37/PDR9 contributes primarily highly oxygenated Coumarins to Root Exudation JO - Sci. Rep. PY - 2017 SP - 3704 AU - Ziegler, J. AU - Schmidt, S. AU - Strehmel, N. AU - Scheel, D. AU - Abel, S. AU - VL - 7 UR - DO - 10.1038/s41598-017-03250-6 AB - The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and mutants deficient in the expression of ABCG36 (PDR8/PEN3), ABCG37 (PDR9) or both transporters. Comparison of the metabolite profiles indicated distinct roles for each ABC transporter in root exudation. Thymidine exudation could be attributed to ABCG36 function, whereas coumarin exudation was strongly reduced only in ABCG37 deficient plants. However, coumarin exudation was compromised in abcg37 mutants only with respect to certain metabolites of this substance class. The specificity of ABCG37 for individual coumarins was further verified by a targeted LC-MS based coumarin profiling method. The response to iron deficiency, which is known to strongly induce coumarin exudation, was also investigated. In either treatment, the distribution of individual coumarins between roots and exudates in the investigated genotypes suggested the involvement of ABCG37 in the exudation specifically of highly oxygenated rather than monohydroxylated coumarins. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 960 TI - Arabidopsis thaliana root and root exudate metabolism is altered by the growth-promoting bacterium Kosakonia radicincitans DSM 16656T JO - Plant Soil PY - 2017 SP - 557-573 AU - Witzel, K. AU - Strehmel, N. AU - Baldermann, S. AU - Neugart, S. AU - Becker, Y. AU - Becker, M. AU - Berger, B. AU - Scheel, D. AU - Grosch, R. AU - Schreiner, M. AU - Ruppel, S. AU - VL - 419 UR - DO - 10.1007/s11104-017-3371-1 AB - AimsPlant growth-promoting bacteria (PGPB) affect host physiological processes in various ways. This study aims at elucidating the dependence of bacterial-induced growth promotion on the plant genotype and characterizing plant metabolic adaptations to PGPB.MethodsEighteen Arabidopsis thaliana accessions were inoculated with the PGPB strain Kosakonia radicincitans DSM 16656T. Colonisation pattern was assessed by enhanced green fluorescent protein (eGFP)-tagged K. radicincitans in three A. thaliana accessions differing in their growth response. Metabolic impact of bacterial colonisation was determined for the best responding accession by profiling distinct classes of plant secondary metabolites and root exudates.ResultsInoculation of 18 A. thaliana accessions resulted in a wide range of growth responses, from repression to enhancement. Testing the bacterial colonisation of three accessions did not reveal a differential pattern. Profiling of plant secondary metabolites showed a differential accumulation of glucosinolates, phenylpropanoids and carotenoids in roots. Analysis of root exudates demonstrated that primary and secondary metabolites were predominantly differentially depleted by bacterial inoculation.ConclusionsThe plant genotype controls the bacterial growth promoting traits. Levels of lutein and β-carotene were elevated in inoculated roots. Supplementing a bacterial suspension with β-carotene increased bacterial growth, while this was not the case when lutein was applied, indicating that β-carotene could be a positive regulator of plant growth promotion. A2 - C1 - ER - TY - JOUR ID - 891 TI - Predicting individual plant performance in grasslands JO - Ecol. Evol. PY - 2017 SP - 8958-8965 AU - Herz, K. AU - Dietz, S. AU - Haider, S. AU - Jandt, U. AU - Scheel, D. AU - Bruelheide, H. AU - VL - 7 UR - DO - 10.1002/ece3.3393 AB - Plant functional traits are widely used to predict community productivity. However, they are rarely used to predict individual plant performance in grasslands. To assess the relative importance of traits compared to environment, we planted seedlings of 20 common grassland species as phytometers into existing grassland communities varying in land‐use intensity. After 1 year, we dug out the plants and assessed root, leaf, and aboveground biomass, to measure plant performance. Furthermore, we determined the functional traits of the phytometers and of all plants growing in their local neighborhood. Neighborhood impacts were analyzed by calculating community‐weighted means (CWM) and functional diversity (FD) of every measured trait. We used model selection to identify the most important predictors of individual plant performance, which included phytometer traits, environmental conditions (climate, soil conditions, and land‐use intensity), as well as CWM and FD of the local neighborhood. Using variance partitioning, we found that most variation in individual plant performance was explained by the traits of the individual phytometer plant, ranging between 19.30% and 44.73% for leaf and aboveground dry mass, respectively. Similarly, in a linear mixed effects model across all species, performance was best predicted by phytometer traits. Among all environmental variables, only including land‐use intensity improved model quality. The models were also improved by functional characteristics of the local neighborhood, such as CWM of leaf dry matter content, root calcium concentration, and root mass per volume as well as FD of leaf potassium and root magnesium concentration and shoot dry matter content. However, their relative effect sizes were much lower than those of the phytometer traits. Our study clearly showed that under realistic field conditions, the performance of an individual plant can be predicted satisfyingly by its functional traits, presumably because traits also capture most of environmental and neighborhood conditions. A2 - C1 - ER - TY - JOUR ID - 890 TI - Drivers of intraspecific trait variation of grass and forb species in German meadows and pastures JO - J. Veg. Sci. PY - 2017 SP - 705-716 AU - Herz, K. AU - Dietz, S. AU - Haider, S. AU - Jandt, U. AU - Scheel, D. AU - Bruelheide, H. AU - VL - 28 UR - DO - 10.1111/jvs.12534 AB - QuestionsTo what extent is trait variation in grasses and forbs driven by land‐use intensity, climate, soil conditions and plant diversity of the local neighbourhood? Do grass and forb species differ in the degree of intraspecific trait variation?LocationManaged grasslands in three regions of Germany.MethodsUsing a phytometer approach, we raised 20 common European grassland species (ten forbs and ten grasses) and planted them into 54 plots of different land‐use types (pasture, meadow, mown pasture). After 1 yr in the field, we measured above‐ and below‐ground plant functional traits. Linear mixed effects models (LMEM) were used to identify the most powerful predictors for every trait. Variation partitioning was applied to assess the amount of inter‐ and intraspecific trait variation in grasses and forbs explained by environmental conditions (land‐use intensity, climate and soil conditions) and plant species diversity of the local neighbourhood.ResultsFor 12 out of the 14 traits studied, either land‐use intensity or local neighbourhood diversity were predictors in the best LMEM. Land‐use intensity had considerably stronger effects than neighbourhood diversity. Root dry matter content and root phosphorus concentration of forbs were more affected by land‐use intensity than those of grasses. For almost all traits, intraspecific trait variation of grasses was much higher than that of forbs, while traits of forbs varied more among species. Overall, inter‐ and intraspecific variation was of the same magnitude.ConclusionThe similar magnitude of intra‐ and interspecific trait variation suggests that both sources should be considered in grassland studies at a scale similar to that of our study. The high amount of intraspecific trait variation that was explained by environmental factors and local neighbourhood diversity clearly demonstrates the high potential of species to adjust to local conditions, which would be ignored when only considering species mean trait values. A2 - C1 - ER - TY - JOUR ID - 871 TI - A Distinct Aromatic Prenyltransferase Associated with the Futalosine Pathway JO - ChemistrySelect PY - 2017 SP - 9319-9325 AU - Cotrim, C. A. AU - Weidner, A. AU - Strehmel, N. AU - Bisol, T. B. AU - Meyer, D. AU - Brandt, W. AU - Wessjohann, L. A. AU - Stubbs, M. T. AU - VL - 2 UR - DO - 10.1002/slct.201702151 AB - Menaquinone (MK) is an electron carrier molecule essential for respiration in most Gram positive bacteria. A crucial step in MK biosynthesis involves the prenylation of an aromatic molecule, catalyzed by integral membrane prenyltransferases of the UbiA (4‐hydroxybenzoate oligoprenyltransferase) superfamily. In the classical MK biosynthetic pathway, the prenyltransferase responsible is MenA (1,4‐dihydroxy‐2‐naphthoate octaprenyltransferase). Recently, an alternative pathway for formation of MK, the so‐called futalosine pathway, has been described in certain micro‐organisms. Until now, five soluble enzymes (MqnA‐MqnE) have been identified in the first steps. In this study, the genes annotated as ubiA from T. thermophilus and S. lividans were cloned, expressed and investigated for prenylation activity. The integral membrane proteins possess neither UbiA nor MenA activity and represent a distinct class of prenyltransferases associated with the futalosine pathway that we term MqnP. We identify a critical residue within a highly conserved Asp‐rich motif that serves to distinguish between members of the UbiA superfamily. A2 - C1 - Bioorganic Chemistry ER - TY - JOUR ID - 928 TI - Leaf Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) biochemical profile of grassland plant species related to land-use intensity JO - Ecol. Indic. PY - 2017 SP - 803-810 AU - Rana, R. AU - Herz, K. AU - Bruelheide, H. AU - Dietz, S. AU - Haider, S. AU - Jandt, U. AU - Pena, R. AU - VL - 84 UR - DO - 10.1016/j.ecolind.2017.09.047 AB - There is growing interest in the application of plant functional trait-based approaches for development of sustainable land-use strategies. In this context, one crucial task is to identify and measure plant traits, which respond to land-use intensity (response traits) and simultaneously have an impact on ecosystem functions (effect traits). We hypothesized that species-specific leaf chemical composition, which may function both as response and effect trait, can be derived from Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy tools in combination with multivariate statistical methods We investigated leaf ATR-FTIR spectra of two grasses, Poa pratensis L. and Dactylis glomerata L., and one forb, Achillea millefolium L. collected in grassland plots along a land-use intensity gradient in three regions of Germany. ATR-FTIR spectra appear to function as biochemical fingerprints unique to each species. The spectral response to land-use intensity was not consistent among species and less apparent in the two grasses than in the forb species. Whereas land-use intensification enhanced protein and cellulose content in A. millefolium, giving rise to changes in six spectral bands in the frequency range of 1088–1699 cm−1, only cellulose content increased in D. glomerata, affecting the bands of 1385–1394 cm−1. Poa pratensis spectra exhibited minimal changes under the influence of land-use, only in the spectral bands of 1373–1375 cm−1 associated with suberin-like aliphatic compounds. Our findings suggest that some species’ leaf chemical composition is responsive to land-use intensity, and thus, may have a predictive value for ecosystem services provided by those species within grassland vegetation (i.e., herbage yield quality). A2 - C1 - ER - TY - JOUR ID - 917 TI - PYK10 myrosinase reveals a functional coordination between endoplasmic reticulum bodies and glucosinolates in Arabidopsis thaliana JO - Plant J. PY - 2017 SP - 204-220 AU - Nakano, R. T. AU - Piślewska-Bednarek, M. AU - Yamada, K. AU - Edger, P. P. AU - Miyahara, M. AU - Kondo, M. AU - Böttcher, C. AU - Mori, M. AU - Nishimura, M. AU - Schulze-Lefert, P. AU - Hara-Nishimura, I. AU - Bednarek, P. AU - VL - 89 UR - DO - 10.1111/tpj.13377 AB - The endoplasmic reticulum body (ER body) is an organelle derived from the ER that occurs in only three families of the order Brassicales and is suggested to be involved in plant defense. ER bodies in Arabidopsis thaliana contain large amounts of β‐glucosidases, but the physiological functions of ER bodies and these enzymes remain largely unclear. Here we show that PYK10, the most abundant β‐glucosidase in A. thaliana root ER bodies, hydrolyzes indole glucosinolates (IGs) in addition to the previously reported in vitro substrate scopolin. We found a striking co‐expression between ER body‐related genes (including PYK10), glucosinolate biosynthetic genes and the genes for so‐called specifier proteins affecting the terminal products of myrosinase‐mediated glucosinolate metabolism, indicating that these systems have been integrated into a common transcriptional network. Consistent with this, comparative metabolite profiling utilizing a number of A. thaliana relatives within Brassicaceae identified a clear phylogenetic co‐occurrence between ER bodies and IGs, but not between ER bodies and scopolin. Collectively, our findings suggest a functional link between ER bodies and glucosinolate metabolism in planta. In addition, in silico three‐dimensional modeling, combined with phylogenomic analysis, suggests that PYK10 represents a clade of 16 myrosinases that arose independently from the other well‐documented class of six thioglucoside glucohydrolases. These findings provide deeper insights into how glucosinolates are metabolized in cruciferous plants and reveal variation of the myrosinase–glucosinolate system within individual plants. A2 - C1 - ER - TY - JOUR ID - 981 TI - MATE Transporter-Dependent Export of Hydroxycinnamic Acid Amides JO - Plant Cell PY - 2016 SP - 583-596 AU - Dobritzsch, M. AU - Lübken, T. AU - Eschen-Lippold, L. AU - Gorzolka, K. AU - Blum, E. AU - Matern, A. AU - Marillonnet, S. AU - Böttcher, C. AU - Dräger, B. AU - Rosahl, S. AU - VL - 28 UR - DO - 10.1105/tpc.15.00706 AB - The ability of Arabidopsis thaliana to successfully prevent colonization by Phytophthora infestans, the causal agent of late blight disease of potato (Solanum tuberosum), depends on multilayered defense responses. To address the role of surface-localized secondary metabolites for entry control, droplets of a P. infestans zoospore suspension, incubated on Arabidopsis leaves, were subjected to untargeted metabolite profiling. The hydroxycinnamic acid amide coumaroylagmatine was among the metabolites secreted into the inoculum. In vitro assays revealed an inhibitory activity of coumaroylagmatine on P. infestans spore germination. Mutant analyses suggested a requirement of the p-coumaroyl-CoA:agmatine N4-p-coumaroyl transferase ACT for the biosynthesis and of the MATE transporter DTX18 for the extracellular accumulation of coumaroylagmatine. The host plant potato is not able to efficiently secrete coumaroylagmatine. This inability is overcome in transgenic potato plants expressing the two Arabidopsis genes ACT and DTX18. These plants secrete agmatine and putrescine conjugates to high levels, indicating that DTX18 is a hydroxycinnamic acid amide transporter with a distinct specificity. The export of hydroxycinnamic acid amides correlates with a decreased ability of P. infestans spores to germinate, suggesting a contribution of secreted antimicrobial compounds to pathogen defense at the leaf surface. A2 - C1 - Cell and Metabolic Biology; Biochemistry of Plant Interactions ER - TY - JOUR ID - 976 TI - Photochemical Oxidation of NIR Photosensitizers in the Presence of Radical Initiators and Their Prospective Use in Dental Applications JO - ChemistrySelect PY - 2016 SP - 524-532 AU - Brömme, T. AU - Schmitz, C. AU - Moszner, N. AU - Burtscher, P. AU - Strehmel, N. AU - Strehmel, B. AU - VL - 1 UR - DO - 10.1002/slct.201600048 AB - Photochemical oxidation of near infrared (NIR) photosensitizers in the presence of diaryl iodonium salts bearing either bis(trifluoromethylsulfonyl)imide or hexafluorophosphate was investigated by exposure with NIR LEDs emitting either at 790 nm, 830 nm, 850 nm or 870 nm. Four different cyanines with barbituryl group at the meso position exhibit similar absorption in the NIR. These photosensitizers initiate in combination with diaryliodonium salts radical photopolymerization of dental composites with the focus to cure large thicknesses. Furthermore, the mixture comprising the cyanine and the iodonium salt was used to generate brown color in dental composites on demand. This required to understand the mechanism of dye decomposition in more detail applying exposure kinetics and a coupling of Ultra Performance Liquid Chromatography (UPLC) with mass spectrometry (MS) to analyze the photoproducts formed. Data showed cleavage of the polymethine chain at typical positions in case of the oxidized species. These were formed as result of electron transfer between the excited state of the photosensitizer and the iodonium salt. UPLC‐MS experiments additionally indicated a certain sensitivity of the system upon adding of acids and radicals generated by thermal treatment of azobisisobutyronitrile (AIBN). Thus, treatment of the photoinitiator composition led almost to the same products no matter the system was either exposed with NIR light or treated with acids or radicals generated by thermal decomposition of AIBN. These findings helped to understand the large curing depth of 14 mm upon NIR exposure at 850 nm and the brown color formed. A2 - C1 - ER - TY - JOUR ID - 1023 TI - Plant-to-Plant Variability in Root Metabolite Profiles of 19 Arabidopsis thaliana Accessions Is Substance-Class-Dependent JO - Int. J. Mol. Sci. PY - 2016 SP - 1565 AU - Mönchgesang, S. AU - Strehmel, N. AU - Trutschel, D. AU - Westphal, L. AU - Neumann, S. AU - Scheel, D. AU - VL - 17 UR - DO - 10.3390/ijms17091565 AB - Natural variation of secondary metabolism between different accessions of Arabidopsis thaliana (A. thaliana) has been studied extensively. In this study, we extended the natural variation approach by including biological variability (plant-to-plant variability) and analysed root metabolic patterns as well as their variability between plants and naturally occurring accessions. To screen 19 accessions of A. thaliana, comprehensive non-targeted metabolite profiling of single plant root extracts was performed using ultra performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS). Linear mixed models were applied to dissect the total observed variance. All metabolic profiles pointed towards a larger plant-to-plant variability than natural variation between accessions and variance of experimental batches. Ratios of plant-to-plant to total variability were high and distinct for certain secondary metabolites. None of the investigated accessions displayed a specifically high or low biological variability for these substance classes. This study provides recommendations for future natural variation analyses of glucosinolates, flavonoids, and phenylpropanoids and also reference data for additional substance classes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1022 TI - Natural variation of root exudates in Arabidopsis thaliana-linking metabolomic and genomic data JO - Sci. Rep. PY - 2016 SP - 29033 AU - Mönchgesang, S. AU - Strehmel, N. AU - Schmidt, S. AU - Westphal, L. AU - Taruttis, F. AU - Müller, E. AU - Herklotz, S. AU - Neumann, S. AU - Scheel, D. AU - VL - 6 UR - DO - 10.1038/srep29033 AB - Many metabolomics studies focus on aboveground parts of the plant, while metabolism within roots and the chemical composition of the rhizosphere, as influenced by exudation, are not deeply investigated. In this study, we analysed exudate metabolic patterns of Arabidopsis thaliana and their variation in genetically diverse accessions. For this project, we used the 19 parental accessions of the Arabidopsis MAGIC collection. Plants were grown in a hydroponic system, their exudates were harvested before bolting and subjected to UPLC/ESI-QTOF-MS analysis. Metabolite profiles were analysed together with the genome sequence information. Our study uncovered distinct metabolite profiles for root exudates of the 19 accessions. Hierarchical clustering revealed similarities in the exudate metabolite profiles, which were partly reflected by the genetic distances. An association of metabolite absence with nonsense mutations was detected for the biosynthetic pathways of an indolic glucosinolate hydrolysis product, a hydroxycinnamic acid amine and a flavonoid triglycoside. Consequently, a direct link between metabolic phenotype and genotype was detected without using segregating populations. Moreover, genomics can help to identify biosynthetic enzymes in metabolomics experiments. Our study elucidates the chemical composition of the rhizosphere and its natural variation in A. thaliana, which is important for the attraction and shaping of microbial communities. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1003 TI - Dynamic metabolic changes in seeds and seedlings of Brassica napus (oilseed rape) suppressing UGT84A9 reveal plasticity and molecular regulation of the phenylpropanoid pathway JO - Phytochemistry PY - 2016 SP - 46-57 AU - Hettwer, K. AU - Böttcher, C. AU - Frolov, A. AU - Mittasch, J. AU - Albert, A. AU - von Roepenack-Lahaye, E. AU - Strack, D. AU - Milkowski, C. AU - VL - 124 UR - DO - 10.1016/j.phytochem.2016.01.014 AB - 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. A2 - C1 - Cell and Metabolic Biology ER - TY - JOUR ID - 1070 TI - Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation JO - J. Exp. Bot. PY - 2016 SP - 1421-1432 AU - Ziegler, J. AU - Schmidt, S. AU - Chutia, R. AU - Müller, J. AU - Böttcher, C. AU - Strehmel, N. AU - Scheel, D. AU - Abel, S. AU - VL - 67 UR - DO - 10.1093/jxb/erv539 AB - Plants have evolved two major strategies to cope with phosphate (Pi) limitation. The systemic response, mainly comprising increased Pi uptake and metabolic adjustments for more efficient Pi use, and the local response, enabling plants to explore Pi-rich soil patches by reorganization of the root system architecture. Unlike previous reports, this study focused on root exudation controlled by the local response to Pi deficiency. To approach this, a hydroponic system separating the local and systemic responses was developed. Arabidopsis thaliana genotypes exhibiting distinct sensitivities to Pi deficiency could be clearly distinguished by their root exudate composition as determined by non-targeted reversed-phase ultraperformance liquid chromatography electrospray ionization quadrupole-time-of-flight mass spectrometry metabolite profiling. Compared with wild-type plants or insensitive low phosphate root 1 and 2 (lpr1 lpr2) double mutant plants, the hypersensitive phosphate deficiency response 2 (pdr2) mutant exhibited a reduced number of differential features in root exudates after Pi starvation, suggesting the involvement of PDR2-encoded P5-type ATPase in root exudation. Identification and analysis of coumarins revealed common and antagonistic regulatory pathways between Pi and Fe deficiency-induced coumarin secretion. The accumulation of oligolignols in root exudates after Pi deficiency was inversely correlated with Pi starvation-induced lignification at the root tips. The strongest oligolignol accumulation in root exudates was observed for the insensitive lpr1 lpr2 double mutant, which was accompanied by the absence of Pi deficiency-induced lignin deposition, suggesting a role of LPR ferroxidases in lignin polymerization during Pi starvation. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1056 TI - Discovering Regulated Metabolite Families in Untargeted Metabolomics Studies JO - Anal. Chem. PY - 2016 SP - 8082-8090 AU - Treutler, H. AU - Tsugawa, H. AU - Porzel, A. AU - Gorzolka, K. AU - Tissier, A. AU - Neumann, S. AU - Balcke, G. U. AU - VL - 88 UR - DO - 10.1021/acs.analchem.6b01569 AB - The identification of metabolites by mass spectrometry constitutes a major bottleneck which considerably limits the throughput of metabolomics studies in biomedical or plant research. Here, we present a novel approach to analyze metabolomics data from untargeted, data-independent LC-MS/MS measurements. By integrated analysis of MS1 abundances and MS/MS spectra, the identification of regulated metabolite families is achieved. This approach offers a global view on metabolic regulation in comparative metabolomics. We implemented our approach in the web application “MetFamily”, which is freely available at http://msbi.ipb-halle.de/MetFamily/. MetFamily provides a dynamic link between the patterns based on MS1-signal intensity and the corresponding structural similarity at the MS/MS level. Structurally related metabolites are annotated as metabolite families based on a hierarchical cluster analysis of measured MS/MS spectra. Joint examination with principal component analysis of MS1 patterns, where this annotation is preserved in the loadings, facilitates the interpretation of comparative metabolomics data at the level of metabolite families. As a proof of concept, we identified two trichome-specific metabolite families from wild-type tomato Solanum habrochaites LA1777 in a fully unsupervised manner and validated our findings based on earlier publications and with NMR. A2 - C1 - Bioorganic Chemistry; Cell and Metabolic Biology ER - TY - JOUR ID - 1052 TI - Piriformospora indica Stimulates Root Metabolism of Arabidopsis thaliana JO - Int. J. Mol. Sci. PY - 2016 SP - 1091 AU - Strehmel, N. AU - Mönchgesang, S. AU - Herklotz, S. AU - Krüger, S. AU - Ziegler, J. AU - Scheel, D. AU - VL - 17 UR - DO - 10.3390/ijms17071091 AB - Piriformospora indica is a root-colonizing fungus, which interacts with a variety of plants including Arabidopsis thaliana. This interaction has been considered as mutualistic leading to growth promotion of the host. So far, only indolic glucosinolates and phytohormones have been identified as key players. In a comprehensive non-targeted metabolite profiling study, we analyzed Arabidopsis thaliana’s roots, root exudates, and leaves of inoculated and non-inoculated plants by ultra performance liquid chromatography/electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC/(ESI)-QTOFMS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS), and identified further biomarkers. Among them, the concentration of nucleosides, dipeptides, oligolignols, and glucosinolate degradation products was affected in the exudates. In the root profiles, nearly all metabolite levels increased upon co-cultivation, like carbohydrates, organic acids, amino acids, glucosinolates, oligolignols, and flavonoids. In the leaf profiles, we detected by far less significant changes. We only observed an increased concentration of organic acids, carbohydrates, ascorbate, glucosinolates and hydroxycinnamic acids, and a decreased concentration of nitrogen-rich amino acids in inoculated plants. These findings contribute to the understanding of symbiotic interactions between plant roots and fungi of the order of Sebacinales and are a valid source for follow-up mechanistic studies, because these symbioses are particular and clearly different from interactions of roots with mycorrhizal fungi or dark septate endophytes A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1085 TI - Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae JO - PLOS ONE PY - 2015 SP - e0138242 AU - Buhtz, A. AU - Witzel, K. AU - Strehmel, N. AU - Ziegler, J. AU - Abel, S. AU - Grosch, R. AU - VL - 10 UR - DO - 10.1371/journal.pone.0138242 AB - The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1150 TI - Annotation of metabolites from gas chromatography/atmospheric pressure chemical ionization tandem mass spectrometry data using an in silico generated compound database and MetFrag JO - Rapid Commun. Mass Spectrom. PY - 2015 SP - 1521-1529 AU - Ruttkies, C. AU - Strehmel, N. AU - Scheel, D. AU - Neumann, S. AU - VL - 29 UR - DO - 10.1002/rcm.7244 AB - RationaleGas chromatography (GC) coupled to atmospheric pressure chemical ionization quadrupole time‐of‐flight mass spectrometry (APCI‐QTOFMS) is an emerging technology in metabolomics. Reference spectra for GC/APCI‐MS/MS barely exist; therefore, in silico fragmentation approaches and structure databases are prerequisites for annotation. To expand the limited coverage of derivatised structures in structure databases, in silico derivatisation procedures are required.MethodsA cheminformatics workflow has been developed for in silico derivatisation of compounds found in KEGG and PubChem, and validated on the Golm Metabolome Database (GMD). To demonstrate this workflow, these in silico generated databases were applied together with MetFrag to APCI‐MS/MS spectra acquired from GC/APCI‐MS/MS profiles of Arabidopsis thaliana and Solanum tuberosum. The Metabolite‐Likeness of the original candidate structure was included as additional scoring term aiming at candidate structures of natural origin.ResultsThe validation of our in silico derivatisation workflow on the GMD showed a true positive rate of 94%. MetFrag was applied to two datasets. In silico derivatisation of the KEGG and PubChem database served as a candidate source. For both datasets the Metabolite‐Likeness score improved the identification performance. The derivatised data sources have been included into the MetFrag web application for the annotation of GC/APCI‐MS/MS spectra.ConclusionsWe demonstrated that MetFrag can support the identification of components from GC/APCI‐MS/MS profiles, especially in the (common) case where reference spectra are not available. This workflow can be easily adapted to other types of derivatisation and is freely accessible together with the generated structure databases. A2 - C1 - ER - TY - JOUR ID - 1121 TI - Cellular reprogramming through mitogen-activated protein kinases JO - Front. Plant Sci. PY - 2015 SP - 940 AU - Lee, J. AU - Eschen-Lippold, L. AU - Lassowskat, I. AU - Böttcher, C. AU - Scheel, D. AU - VL - 6 UR - DO - 10.3389/fpls.2015.00940 AB - Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1120 TI - Mutualistic root endophytism is not associated with the reduction of saprotrophic traits and requires a noncompromised plant innate immunity JO - New Phytol. PY - 2015 SP - 841-857 AU - Lahrmann, U. AU - Strehmel, N. AU - Langen, G. AU - Frerigmann, H. AU - Leson, L. AU - Ding, Y. AU - Scheel, D. AU - Herklotz, S. AU - Hilbert, M. AU - Zuccaro, A. AU - VL - 207 UR - DO - 10.1111/nph.13411 AB - During a compatible interaction, the sebacinoid root‐associated fungi Piriformospora indica and Sebacina vermifera induce modification of root morphology and enhance shoot growth in Arabidopsis thaliana.The genomic traits common in these two fungi were investigated and compared with those of other root‐associated fungi and saprotrophs. The transcriptional responses of the two sebacinoid fungi and of Arabidopsis roots to colonization at three different symbiotic stages were analyzed by custom‐designed microarrays.We identified key genomic features characteristic of sebacinoid fungi, such as expansions for gene families involved in hydrolytic activities, carbohydrate‐binding and protein–protein interaction. Additionally, we show that colonization of Arabidopsis correlates with the induction of salicylic acid catabolism and accumulation of jasmonate and glucosinolates (GSLs). Genes involved in root developmental processes were specifically induced by S. vermifera at later stages during interaction.Using different Arabidopsis indole‐GSLs mutants and measurement of secondary metabolites, we demonstrate the importance of the indolic glucosinolate pathway in the growth restriction of P. indica and S. vermifera and we identify indole‐phytoalexins and specifically indole‐carboxylic acids derivatives as potential key players in the maintenance of a mutualistic interaction with root endophytes. A2 - C1 - ER - TY - JOUR ID - 1270 TI - Profiling of secondary metabolites in root exudates of Arabidopsis thaliana JO - Phytochemistry PY - 2014 SP - 35-46 AU - Strehmel, N. AU - Böttcher, C. AU - Schmidt, S. AU - Scheel, D. AU - VL - 108 UR - DO - 10.1016/j.phytochem.2014.10.003 AB - To explore the chemical composition of root exudates of the model plant Arabidopsis thaliana a workflow for nontargeted metabolite profiling of the semipolar fraction of root exudates was developed. It comprises hydroponic plant cultivation and sampling of root exudates under sterile conditions, sample preparation by solid-phase extraction and analysis by reversed-phase UPLC/ESI-QTOFMS. Following the established workflow, root exudates of six-week-old plants were profiled and a set of reproducibly occurring molecular features was compiled. To structurally elucidate the corresponding metabolites, accurate mass tandem mass spectrometry and on-line hydrogen/deuterium exchange were applied. Currently, a total of 103 compounds were detected and annotated by elemental composition of which more than 90 were structurally characterized or classified. Among them, 42 compounds were rigorously identified using an authenticated standard. The compounds identified so far include nucleosides, deoxynucleosides, aromatic amino acids, anabolites and catabolites of glucosinolates, dipeptides, indolics, salicylic and jasmonic acid catabolites, coumarins, mono-, di- and trilignols, hydroxycinnamic acid derivatives and oxylipins and exemplify the high chemical diversity of plant root exudates. A2 - C1 - ER - TY - JOUR ID - 1269 TI - Annotating unknown components from GC/EI-MS-based metabolite profiling experiments using GC/APCI(+)-QTOFMS JO - Metabolomics PY - 2014 SP - 324-336 AU - Strehmel, N. AU - Kopka, J. AU - Scheel, D. AU - Böttcher, C. AU - VL - 10 UR - DO - 10.1007/s11306-013-0569-y AB - GC/EI-MS-based metabolite profiling of derivatized polar fractions of crude plant extracts typically reveals several hundred components. Thereof, only up to one half can be identified using mass spectral and retention index libraries, the rest remains unknown. In the present work, the utility of GC/APCI(+)-QTOFMS for the annotation of unknown components was explored. Hence, EI and APCI(+) mass spectra of ~100 known components were extracted from GC/EI-QMS and GC/APCI(+)-QTOFMS profiles obtained from a methoximated and trimethylsilylated root extract of Arabidopsis thaliana. Based on this reference set, adduct and fragment ion formation under APCI(+) conditions was examined and the calculation of elemental compositions evaluated. During these studies, most of the components formed dominating protonated molecular ions. Despite the high mass accuracy (|Δm| ≤ 3 mDa) and isotopic pattern accuracy (mSigma ≤ 30) the determination of a component’s unique native elemental composition requires additional information, namely the number of trimethylsilyl and methoxime moieties as well as the analysis of corresponding collision-induced dissociation (CID) mass spectra. After all, the reference set was used to develop a strategy for the pairwise assignment of EI and APCI(+) mass spectra. Proceeding from these findings, the annotation of unidentified components detected by GC/EI-QMS using GC/APCI(+)-QTOFMS and corresponding deuterated derivatization reagents was attempted. For a total of 25 unknown components, pairs of EI and APCI(+) mass spectra were compiled and elemental compositions determined. Integrative interpretation of EI and CID mass spectra resulted in 14 structural hypotheses, of which seven were confirmed using authenticated standards. A2 - C1 - ER - TY - JOUR ID - 1259 TI - Metabolome Analysis of Arabidopsis thaliana Roots Identifies a Key Metabolic Pathway for Iron Acquisition JO - PLOS ONE PY - 2014 SP - e102444 AU - Schmidt, H. AU - Günther, C. AU - Weber, M. AU - Spörlein, C. AU - Loscher, S. AU - Böttcher, C. AU - Schobert, R. AU - Clemens, S. AU - VL - 9 UR - DO - 10.1371/journal.pone.0102444 AB - Fe deficiency compromises both human health and plant productivity. Thus, it is important to understand plant Fe acquisition strategies for the development of crop plants which are more Fe-efficient under Fe-limited conditions, such as alkaline soils, and have higher Fe density in their edible tissues. Root secretion of phenolic compounds has long been hypothesized to be a component of the reduction strategy of Fe acquisition in non-graminaceous plants. We therefore subjected roots of Arabidopsis thaliana plants grown under Fe-replete and Fe-deplete conditions to comprehensive metabolome analysis by gas chromatography-mass spectrometry and ultra-pressure liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. Scopoletin and other coumarins were found among the metabolites showing the strongest response to two different Fe-limited conditions, the cultivation in Fe-free medium and in medium with an alkaline pH. A coumarin biosynthesis mutant defective in ortho-hydroxylation of cinnamic acids was unable to grow on alkaline soil in the absence of Fe fertilization. Co-cultivation with wild-type plants partially rescued the Fe deficiency phenotype indicating a contribution of extracellular coumarins to Fe solubilization. Indeed, coumarins were detected in root exudates of wild-type plants. Direct infusion mass spectrometry as well as UV/vis spectroscopy indicated that coumarins are acting both as reductants of Fe(III) and as ligands of Fe(II). A2 - C1 - ER - TY - JOUR ID - 1258 TI - Feruloyl-CoA 6'-Hydroxylase1-Dependent Coumarins Mediate Iron Acquisition from Alkaline Substrates in Arabidopsis JO - Plant Physiol. PY - 2014 SP - 160-172 AU - Schmid, N. B. AU - Giehl, R. F. H. AU - Döll, S. AU - Mock, H.-P. AU - Strehmel, N. AU - Scheel, D. AU - Kong, X. AU - Hider, R. C. AU - von Wirén, N. AU - VL - 164 UR - DO - 10.1104/pp.113.228544 AB - Although iron (Fe) is one of the most abundant elements in the earth’s crust, its low solubility in soils restricts Fe uptake by plants. Most plant species acquire Fe by acidifying the rhizosphere and reducing ferric to ferrous Fe prior to membrane transport. However, it is unclear how these plants access Fe in the rhizosphere and cope with high soil pH. In a mutant screening, we identified 2-oxoglutarate-dependent dioxygenase Feruloyl-CoA 6′-Hydroxylase1 (F6′H1) to be essential for tolerance of Arabidopsis (Arabidopsis thaliana) to high pH-induced Fe deficiency. Under Fe deficiency, F6′H1 is required for the biosynthesis of fluorescent coumarins that are released into the rhizosphere, some of which possess Fe(III)-mobilizing capacity and prevent f6′h1 mutant plants from Fe deficiency-induced chlorosis. Scopoletin was the most prominent coumarin found in Fe-deficient root exudates but failed to mobilize Fe(III), while esculetin, i.e. 6,7-dihydroxycoumarin, occurred in lower amounts but was effective in Fe(III) mobilization. Our results indicate that Fe-deficient Arabidopsis plants release Fe(III)-chelating coumarins as part of the strategy I-type Fe acquisition machinery. A2 - C1 - ER - TY - JOUR ID - 1189 TI - The Biosynthetic Pathway of Indole-3-Carbaldehyde and Indole-3-Carboxylic Acid Derivatives in Arabidopsis JO - Plant Physiol. PY - 2014 SP - 841-853 AU - Böttcher, C. AU - Chapman, A. AU - Fellermeier, F. AU - Choudhary, M. AU - Scheel, D. AU - Glawischnig, E. AU - VL - 165 UR - DO - 10.1104/pp.114.235630 AB - Indolic secondary metabolites play an important role in pathogen defense in cruciferous plants. In Arabidopsis (Arabidopsis thaliana), in addition to the characteristic phytoalexin camalexin, derivatives of indole-3-carbaldehyde (ICHO) and indole-3-carboxylic acid (ICOOH) are synthesized from tryptophan via the intermediates indole-3-acetaldoxime and indole-3-acetonitrile. Based on feeding experiments combined with nontargeted metabolite profiling, their composition in nontreated and silver nitrate (AgNO3)-treated leaf tissue was comprehensively analyzed. As major derivatives, glucose conjugates of 5-hydroxyindole-3-carbaldehyde, ICOOH, and 6-hydroxyindole-3-carboxylic acid were identified. Quantification of ICHO and ICOOH derivative pools after glucosidase treatment revealed that, in response to AgNO3 treatment, their total accumulation level was similar to that of camalexin. ARABIDOPSIS ALDEHYDE OXIDASE1 (AAO1), initially discussed to be involved in the biosynthesis of indole-3-acetic acid, and Cytochrome P450 (CYP) 71B6 were found to be transcriptionally coexpressed with camalexin biosynthetic genes. CYP71B6 was expressed in Saccharomyces cerevisiae and shown to efficiently convert indole-3-acetonitrile into ICHO and ICOOH, thereby releasing cyanide. To evaluate the role of both enzymes in the biosynthesis of ICHO and ICOOH derivatives, knockout and overexpression lines for CYP71B6 and AAO1 were established and analyzed for indolic metabolites. The observed metabolic phenotypes suggest that AAO1 functions in the oxidation of ICHO to ICOOH in both nontreated and AgNO3-treated leaves, whereas CYP71B6 is relevant for ICOOH derivative biosynthesis specifically after induction. In summary, a model for the biosynthesis of ICHO and ICOOH derivatives is presented. A2 - C1 - ER - TY - JOUR ID - 1231 TI - Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana JO - Front. Plant Sci. PY - 2014 SP - 554 AU - Lassowskat, I. AU - Böttcher, C. AU - Eschen-Lippold, L. AU - Scheel, D. AU - Lee, J. AU - VL - 5 UR - DO - 10.3389/fpls.2014.00554 AB - Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3, and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g., WRKY transcription factors and proteins encoded by the genes from the “PEN” pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control. (Proteomics data are available with the identifier PXD001252 via ProteomeXchange, http://proteomecentral.proteomexchange.org). A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1230 TI - The ABC Transporter ABCG1 Is Required for Suberin Formation in Potato Tuber Periderm JO - Plant Cell PY - 2014 SP - 3403-3415 AU - Landgraf, R. AU - Smolka, U. AU - Altmann, S. AU - Eschen-Lippold, L. AU - Senning, M. AU - Sonnewald, S. AU - Weigel, B. AU - Frolova, N. AU - Strehmel, N. AU - Hause, G. AU - Scheel, D. AU - Böttcher, C. AU - Rosahl, S. AU - VL - 26 UR - DO - 10.1105/tpc.114.124776 AB - The lipid biopolymer suberin plays a major role as a barrier both at plant-environment interfaces and in internal tissues, restricting water and nutrient transport. In potato (Solanum tuberosum), tuber integrity is dependent on suberized periderm. Using microarray analyses, we identified ABCG1, encoding an ABC transporter, as a gene responsive to the pathogen-associated molecular pattern Pep-13. Further analyses revealed that ABCG1 is expressed in roots and tuber periderm, as well as in wounded leaves. Transgenic ABCG1-RNAi potato plants with downregulated expression of ABCG1 display major alterations in both root and tuber morphology, whereas the aerial part of the ABCG1-RNAi plants appear normal. The tuber periderm and root exodermis show reduced suberin staining and disorganized cell layers. Metabolite analyses revealed reduction of esterified suberin components and hyperaccumulation of putative suberin precursors in the tuber periderm of RNA interference plants, suggesting that ABCG1 is required for the export of suberin components. A2 - C1 - Biochemistry of Plant Interactions; Bioorganic Chemistry ER - TY - JOUR ID - 1355 TI - Cell phenotypic changes of mouse connective tissue fibroblasts (L-929) to poly(ethylene glycol)-based gels JO - Biomater. Sci. PY - 2013 SP - 850-859 AU - Strehmel, C. AU - Zhang, Z. AU - Strehmel, N. AU - Lensen, M. C. AU - VL - 1 UR - DO - 10.1039/C3BM60055F AB - Cellular responses to various gels fabricated by photoinitiated crosslinking using acrylated linear and multi-arm poly(ethylene glycol) (PEG)-based and poly(propylene glycol)-b-poly(ethylene glycol) precursors were investigated. While no proteinadsorption and cell adhesion were observed on the hydrophilic PEG-based gels, proteinadsorption and cell adhesion did occur on the more hydrophobic gel generated from the block copolymer precursor. Murine fibroblast viability on the poly(ethylene glycol)-based gels was studied in the course of 72 h and the results indicated no cytotoxicity. In a systematic study, extra- and intracellular metabolites of the murine fibroblasts cultured on these PEG-based gels were examined by GC-MS. Distinct intra- and extracellular changes in primary metabolism, namely amino acid metabolism, glycolysis and fatty acid metabolism, were observed. Cells cultured on the polymeric gels induced more intense intracellular changes in the metabolite profile by means of higher metabolite intensities with time in comparison to cells cultured on the reference substrate (tissue culture polystyrene). In contrast, extracellular changes of metabolite intensities were comparable. A2 - C1 - ER - TY - JOUR ID - 1332 TI - Reprogramming the Phenylpropanoid Metabolism in Seeds of Oilseed Rape by Suppressing the Orthologs of REDUCED EPIDERMAL FLUORESCENCE1 JO - J. Plant Physiol. PY - 2013 SP - 1656-1669 AU - Mittasch, J. AU - Böttcher, C. AU - Frolov, A. AU - Strack, D. AU - Milkowski, C. AU - VL - 161 UR - DO - 10.1104/pp.113.215491 AB - As a result of the phenylpropanoid pathway, many Brassicaceae produce considerable amounts of soluble hydroxycinnamate conjugates, mainly sinapate esters. From oilseed rape (Brassica napus), we cloned two orthologs of the Arabidopsis (Arabidopsis thaliana) gene REDUCED EPIDERMAL FLUORESCENCE1 (REF1) encoding a coniferaldehyde/sinapaldehyde dehydrogenase. The enzyme is involved in the formation of ferulate and sinapate from the corresponding aldehydes, thereby linking lignin and hydroxycinnamate biosynthesis as a potential branch-point enzyme. We used RNA interference to silence REF1 genes in seeds of oilseed rape. Nontargeted metabolite profiling showed that BnREF1-suppressing seeds produced a novel chemotype characterized by reduced levels of sinapate esters, the appearance of conjugated monolignols, dilignols, and trilignols, altered accumulation patterns of kaempferol glycosides, and changes in minor conjugates of caffeate, ferulate, and 5-hydroxyferulate. BnREF1 suppression affected the level of minor sinapate conjugates more severely than that of the major component sinapine. Mapping of the changed metabolites onto the phenylpropanoid metabolic network revealed partial redirection of metabolic sequences as a major impact of BnREF1 suppression. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1307 TI - An UPLC-MS/MS Method for the Simultaneous Identification and Quantitation of Cell Wall Phenolics in Brassica napus Seeds JO - J. Agr. Food Chem. PY - 2013 SP - 1219-1227 AU - Frolov, A. AU - Henning, A. AU - Böttcher, C. AU - Tissier, A. AU - Strack, D. AU - VL - 61 UR - DO - 10.1021/jf3042648 AB - The seed residues left after pressing of rapeseed oil are rich in proteins and could be used for human nutrition and animal feeding. These press cakes contain, however, antinutritives, with fiber being the most abundant one. The analysis of fiber phenolic component (localized to seed coat cell walls) is, therefore, important in breeding and food quality control. However, correct structure and content assignments of cell wall-bound phenolics are challenging due to their low stability during sample preparation. Here, a novel LC-MS/MS-based method for the simultaneous identification and quantitation of 66 cell wall-bound phenolics and their derivatives is described. The method was internally standardized, corrected for degradation effects during sample preparation, and cross-validated with a well-established UV-based procedure. This approach was successfully applied to the analysis of cell wall phenolic patterns in different B. napus cultivars and proved to be suitable for marker compound search as well as assay development. A2 - C1 - Cell and Metabolic Biology ER - TY - JOUR ID - 1335 TI - Nearline acquisition and processing of liquid chromatography-tandem mass spectrometry data JO - Metabolomics PY - 2013 SP - 84-91 AU - Neumann, S. AU - Thum, A. AU - Böttcher, C. AU - VL - 9 UR - DO - 10.1007/s11306-012-0401-0 AB - Liquid chromatography–mass spectrometry (LC–MS) is a commonly used analytical platform for non-targeted metabolite profiling experiments. Although data acquisition, processing and statistical analyses are almost routine in such experiments, further annotation and subsequent identification of chemical compounds are not. For identification, tandem mass spectra provide valuable information towards the structure of chemical compounds. These are typically acquired online, in data-dependent mode, or offline, using handcrafted acquisition methods and manually extracted from raw data. Here, we present several methods to fast-track and improve both the acquisition and processing of LC–MS/MS data. Our nearly online (nearline) data-dependent tandem MS strategy creates a minimal set of LC–MS/MS acquisition methods for relevant features revealed by a preceding non-targeted profiling experiment. Using different filtering criteria, such as intensity or ion type, the acquisition of irrelevant spectra is minimized. Afterwards, LC–MS/MS raw data are processed with feature detection and grouping algorithms. The extracted tandem mass spectra can be used for both library search and de-novo identification methods. The algorithms are implemented in the R package MetShot and support the export to Bruker, Agilent or Waters QTOF instruments and the vendor-independent TraML standard. We evaluate the performance of our workflow on a Bruker micrOTOF-Q by comparison of automatically acquired and extracted tandem mass spectra obtained from a mixture of natural product standards against manually extracted reference spectra. Using Arabidopsis thaliana wild-type and biosynthetic gene knockout plants, we characterize the metabolic products of a biosynthetic pathway and demonstrate the integration of our approach into a typical non-targeted metabolite profiling workflow. A2 - C1 - ER - TY - CHAP ID - 80 TI - Mass Spectral Search and Analysis Using the Golm Metabolome Database T2 - The Handbook of Plant Metabolomics PB - PY - 2013 SP - 321-343 AU - Hummel, J. AU - Strehmel, N. AU - Bölling, C. AU - Schmidt, S. AU - Walther, D. AU - Kopka, J. AU - VL - UR - SN - 9783527669882 DO - 10.1002/9783527669882.ch18 AB - The novel “omics” technologies of the postgenomic era generate large multiplexed phenotyping datasets, which can only inadequately be published in the traditional journal and supplemental formats. For this reason, public databases have been developed that utilize the efficient communication of knowledge through the World Wide Web. This trend also applies to the metabolomics field, which is, after genomics, transcriptomics, and proteomics, the fourth major systems‐level phenotyping platform. Each different analytical technology used in metabolomics studies requires specific reference data for metabolite identification and optimal data formats for reporting the complex metabolite profiling data features. Therefore, we envision that every technology platform or even each high‐throughput metabolomic laboratory will establish dedicated databases, which will communicate between each other and will be integrated by meta‐databases and web services. The Golm Metabolome Database (GMD) (http://gmd.mpimp‐golm.mpg.de/) is a metabolomic database, maintained by the Max Planck Institute of Molecular Plant Physiology, that was initiated around a nucleus of reference data from gas chromatography–mass spectrometry metabolite profiling data and is now developing toward a general mass spectrometry‐based repository of reference metabolite profiles for essential plant tissues and typical variations of growth conditions. This chapter describes the mass spectral searches and analyses currently supported by the GMD. We specifically address the searches for the different chemical entities within GMD, namely the metabolites, reference substances, and the chemically derivatized analytes. We report the diverse options for mass spectral analyses and highlight the decision tree‐supported prediction of chemical substructures, a feature of GMD that currently appears to be a unique among the many tools for the analysis of gas chromatography–electron ionization mass spectra. A2 - Weckwerth, W. & Kahl, G., eds. C1 - ER - TY - JOUR ID - 1429 TI - The O-Carbamoyltransferase TobZ Catalyzes an Ancient Enzymatic Reaction JO - Angew. Chem. Int. Ed. PY - 2012 SP - 4046-4052 AU - Parthier, C. AU - Görlich, S. AU - Jaenecke, F. AU - Breithaupt, C. AU - Bräuer, U. AU - Fandrich, U. AU - Clausnitzer, D. AU - Wehmeier, U. F. AU - Böttcher, C. AU - Scheel, D. AU - Stubbs, M. T. AU - VL - 51 UR - DO - 10.1002/anie.201108896 AB - An ancient reaction vessel: TobZ carbamoylates the antibiotic tobramycin to form nebramycin 5′. The YrdC‐like domain (blue) catalyzes the formation of the novel intermediate carbamoyladenylate, which is channeled through a common “reaction chamber” to the Kae1‐like domain (brown), site of carbamoyl transfer. A2 - C1 - ER - TY - JOUR ID - 1428 TI - Die O-Carbamoyltransferase TobZ katalysiert eine enzymatische Reaktion frühen Ursprungs JO - Angew. Chem. PY - 2012 SP - 4122-4128 AU - Parthier, C. AU - Görlich, S. AU - Jaenecke, F. AU - Breithaupt, C. AU - Bräuer, U. AU - Fandrich, U. AU - Clausnitzer, D. AU - Wehmeier, U. F. AU - Böttcher, C. AU - Scheel, D. AU - Stubbs, M. T. AU - VL - 124 UR - DO - 10.1002/ange.201108896 AB - Ein uraltes Reaktionsgefäß: TobZ carbamoyliert das Antibiotikum Tobramycin unter Bildung von Nebramycin‐5′. Dabei katalysiert die YrdC‐ähnliche Domäne (blau) die Bildung eines intermediären Carbamoyladenylats, das innerhalb einer tunnelartigen Reaktionskammer zur Kae1‐ähnlichen Domäne (braun), dem Ort des Carbamoyltransfers, transferiert wird. A2 - C1 - ER - TY - JOUR ID - 1419 TI - CAMERA: An Integrated Strategy for Compound Spectra Extraction and Annotation of Liquid Chromatography/Mass Spectrometry Data Sets JO - Anal. Chem. PY - 2012 SP - 283-289 AU - Kuhl, C. AU - Tautenhahn, R. AU - Böttcher, C. AU - Larson, T. R. AU - Neumann, S. AU - VL - 84 UR - DO - 10.1021/ac202450g AB - Liquid chromatography coupled to mass spectrometry is routinely used for metabolomics experiments. In contrast to the fairly routine and automated data acquisition steps, subsequent compound annotation and identification require extensive manual analysis and thus form a major bottleneck in data interpretation. Here we present CAMERA, a Bioconductor package integrating algorithms to extract compound spectra, annotate isotope and adduct peaks, and propose the accurate compound mass even in highly complex data. To evaluate the algorithms, we compared the annotation of CAMERA against a manually defined annotation for a mixture of known compounds spiked into a complex matrix at different concentrations. CAMERA successfully extracted accurate masses for 89.7% and 90.3% of the annotatable compounds in positive and negative ion modes, respectively. Furthermore, we present a novel annotation approach that combines spectral information of data acquired in opposite ion modes to further improve the annotation rate. We demonstrate the utility of CAMERA in two different, easily adoptable plant metabolomics experiments, where the application of CAMERA drastically reduced the amount of manual analysis. A2 - C1 - ER - TY - JOUR ID - 1403 TI - Glucosinolates are produced in trichomes of Arabidopsis thaliana JO - Front. Plant Sci. PY - 2012 SP - 242 AU - Frerigmann, H. AU - Böttcher, C. AU - Baatout, D. AU - Gigolashvili, T. AU - VL - 3 UR - DO - 10.3389/fpls.2012.00242 AB - Glucosinolates (GS) are important plant secondary metabolites in plant resistance to herbivores, bacteria, and fungi, which have been shown to be accumulating in different organs and tissue types at varying concentrations. There are more than 200 GS species found in order Brassicales and presence of these compounds is well documented on organ-specific but not on cell-specific level. We used UPLC/ESI-QTOF-MS to measure the presence of GS and qRT-PCR to analyse the expression of GS biosynthetic and regulatory genes in isolated Arabidopsis thaliana trichomes. Trichomes of Arabidopsis are shown to synthesize chemoprotective aliphatic glucosinolates (AGS) and indolic glucosinolates (IGS), which are known for their biological activities against fungi, bacterial pathogens, or herbivores. UPLC/ESI-QTOF-MS analysis of various IGS mutants reveal increased or decreased levels of IGS in trichomes of gain- and loss-of-function mutants correspondingly. Using pMYB51/HIG1-uidA and pMYB28/PMG1/HAG1-uidA reporter plants we demonstrate that production of these important compounds is activated in trichomes of leaves or inflorescences in response to wounding. Since trichomes represent the first interface in plant-environment interactions, the possible role of GS containing trichomes in plant defense or signaling is discussed. A2 - C1 - ER - TY - JOUR ID - 1516 TI - Use of recombinantly produced 15N3-labelled nicotianamine for fast and sensitive stable isotope dilution ultra-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry JO - Anal. Bioanal. Chem. PY - 2011 SP - 1355-1361 AU - Schmidt, H. AU - Böttcher, C. AU - Trampczynska, A. AU - Clemens, S. AU - VL - 399 UR - DO - 10.1007/s00216-010-4436-7 AB - Nicotianamine (NA) is an important metal chelator, implicated in the intra- and intercellular trafficking of several transition metal ions in plants. To decipher its roles in physiological processes such as micronutrient acquisition, distribution or storage, fast and sensitive analytical techniques for quantification of this non-proteinogenic amino acid will be required. The use of a recombinant Schizosaccharomyces pombe strain expressing a nicotianamine synthase (NAS) gene allowed for the production of [15N3]-NA, which was enriched from cell extracts through cation exchange and used for stable isotope dilution analysis of NA. Such an approach should be widely applicable to important bioanalytes that are difficult to synthesize. The analytical procedure comprises mild aqueous extraction and rapid Fmoc derivatization, followed by fast separation using ultra-performance liquid chromatography (UPLC) and sensitive detection by positive ion electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) with a chromatographic cycle time of only 8 min. Derivatization was optimized with respect to incubation time and species suitable for quantification. The limit of detection was 0.14 to 0.23 pmol in biological matrices with the response being linear up to 42 pmol. Recovery rates were between 83% and 104% in various biological matrices including fission yeast cells, fungal mycelium, plant leaves and roots. A2 - C1 - ER - TY - JOUR ID - 1515 TI - Verticillin A is likely not produced by Verticillium sp. JO - J. Antibiot. PY - 2011 SP - 523-524 AU - Schenke, D. AU - Böttcher, C. AU - Lee, J. AU - Scheel, D. AU - VL - 64 UR - DO - 10.1038/ja.2011.36 AB - A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1514 TI - Crosstalk between abiotic ultraviolet-B stress and biotic (flg22) stress signalling in Arabidopsis prevents flavonol accumulation in favor of pathogen defence compound production JO - Plant Cell Environ. PY - 2011 SP - 1849-1864 AU - Schenke, D. AU - Böttcher, C. AU - Scheel, D. AU - VL - 34 UR - DO - 10.1111/j.1365-3040.2011.02381.x AB - Plants respond to both abiotic and biotic stresses with alterations in the expression of genes required to produce protective metabolites. Sometimes plants can be challenged with different stresses simultaneously and as they cannot evade from this situation, priorities have to be set to deal with the most urgent threat. The abiotic stress ultraviolet‐B (UV‐B) light induces the production of UV‐protective flavonols in Arabidopsis Col‐0 cell suspension cultures and this accumulation is attenuated by concurrent application of the bacterial elicitor flg22 (simulating biotic stress). This inhibition correlates with strong suppression of the flavonol biosynthesis genes. In parallel, flg22 induces the production of defence‐related compounds, such as the phytoalexins, camalexin and scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread. This correlated positively with flg22‐mediated expression of enzymes for lignin, scopoletin and camalexin production. As flavonols, lignin and scopoletin are all derived from phenylalanine, it appears that the plant focuses the metabolism on production of scopoletin and lignin at the expense of flavonol production. Furthermore, it appears that this crosstalk involves antagonistic regulation of two opposing MYB transcription factors, the positive regulator of the flavonol pathway MYB12 (UV‐B‐induced and flg22‐suppressed) and the negative regulator MYB4 (UV‐B‐ and flg22‐induced). A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1510 TI - Computing Fragmentation Trees from Tandem Mass Spectrometry Data JO - Anal. Chem. PY - 2011 SP - 1243-1251 AU - Rasche, F. AU - Svatoš, A. AU - Maddula, R. K. AU - Böttcher, C. AU - Böcker, S. AU - VL - 83 UR - DO - 10.1021/ac101825k AB - The structural elucidation of organic compounds in complex biofluids and tissues remains a significant analytical challenge. For mass spectrometry, the manual interpretation of collision-induced dissociation (CID) mass spectra is cumbersome and requires expert knowledge, as the fragmentation mechanisms of ions formed from small molecules are not completely understood. The automated identification of compounds is generally limited to searching in spectral libraries. Here, we present a method for interpreting the CID spectra of the organic compound’s protonated ions by computing fragmentation trees that establish not only the molecular formula of the compound and all fragment ions but also the dependencies between fragment ions. This is an important step toward the automated identification of unknowns from the CID spectra of compounds that are not in any database. A2 - C1 - ER - TY - JOUR ID - 1489 TI - Cytosolic γ-Glutamyl Peptidases Process Glutathione Conjugates in the Biosynthesis of Glucosinolates and Camalexin in Arabidopsis JO - Plant Cell PY - 2011 SP - 2456-2469 AU - Geu-Flores, F. AU - Møldrup, M. E. AU - Böttcher, C. AU - Olsen, C. E. AU - Scheel, D. AU - Halkier, B. A. AU - VL - 23 UR - DO - 10.1105/tpc.111.083998 AB - The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the γ-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant γ-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways. A2 - C1 - ER - TY - JOUR ID - 1478 TI - Overexpression of Sinapine Esterase BnSCE3 in Oilseed Rape Seeds Triggers Global Changes in Seed Metabolism JO - Plant Physiol. PY - 2011 SP - 1127-1145 AU - Clauß, K. AU - von Roepenack-Lahaye, E. AU - Böttcher, C. AU - Roth, M. R. AU - Welti, R. AU - Erban, A. AU - Kopka, J. AU - Scheel, D. AU - Milkowski, C. AU - Strack, D. AU - VL - 155 UR - DO - 10.1104/pp.110.169821 AB - 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. A2 - C1 - ER - TY - CHAP ID - 92 TI - Resources for Metabolomics T2 - Genetics and Genomics of the Brassicaceae PB - Plant Genetics and Genomics: Crops and Models PY - 2011 SP - 469-503 AU - Böttcher, C. AU - von Roepenack-Lahaye, E. AU - Scheel, D. AU - VL - 9 UR - SN - 978-1-4419-7118-0 DO - 10.1007/978-1-4419-7118-0_17 AB - 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. A2 - Schmidt, R. & Bancroft, I., eds. C1 - ER - TY - JOUR ID - 1533 TI - Accumulation of Isochorismate-derived 2,3-Dihydroxybenzoic 3-O-β-D-Xyloside in Arabidopsis Resistance to Pathogens and Ageing of Leaves JO - J. Biol. Chem. PY - 2010 SP - 25654-25665 AU - Bartsch, M. AU - Bednarek, P. AU - Vivancos, P. D. AU - Schneider, B. AU - von Roepenack-Lahaye, E. AU - Foyer, C. H. AU - Kombrink, E. AU - Scheel, D. AU - Parker, J. E. AU - VL - 285 UR - DO - 10.1074/jbc.M109.092569 AB - 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. A2 - C1 - ER - TY - JOUR ID - 1561 TI - Calcium promotes cadmium elimination as vaterite grains by tobacco trichomes JO - Geochim. Cosmochim. Acta PY - 2010 SP - 5817-5834 AU - Isaure, M.-P. AU - Sarret, G. AU - Harada, E. AU - Choi, Y.-E. AU - Marcus, M. A. AU - Fakra, S. C. AU - Geoffroy, N. AU - Pairis, S. AU - Susini, J. AU - Clemens, S. AU - Manceau, A. AU - VL - 74 UR - DO - 10.1016/j.gca.2010.07.011 AB - In tobacco plants, elimination of Zn and Cd via the production of Ca-containing grains at the top of leaf hairs, called trichomes, is a potent detoxification mechanism. This study examines how Cd is incorporated in these biominerals, and how calcium growth supplement modifies their nature. Scanning electron microscopy coupled with energy dispersive X-ray microanalysis (SEM-EDX), microfocused X-ray diffraction (μ-XRD), and microfocused X-ray absorption near edge structure (μ-XANES) spectroscopy were used to image the morphology of the grains, identify the crystallized mineral phases, and speciate Cd, respectively. The mineralogy of the grains and chemical form of Cd varied with the amount of Ca. When tobacco plants were grown in a nutrient solution containing 25 μM Cd and low Ca supplement (Ca/Cd = 11 mol ratio), most of the grains were oblong-shaped and low-Cd-substituted calcite. When exposed to the same amount of Cd and high Ca supplement (Ca/Cd = 131 mol ratio), grains were more abundant and diverse in compositions, and in total more Cd was eliminated. Most grains in the high Ca/Cd experiment were round-shaped and composed predominantly of Cd-substituted vaterite, a usually metastable calcium carbonate polymorph, and subordinate calcite. Calcium oxalate and a Ca amorphous phase were detected occasionally in the two treatments, but were devoid of Cd. The biomineralization of cadmium and implications of results for Cd exposure of smokers and phytoremediation are discussed. A2 - C1 - ER - TY - JOUR ID - 1557 TI - Expression Profiling of Tobacco Leaf Trichomes Identifies Genes for Biotic and Abiotic Stresses JO - Plant Cell Physiol. PY - 2010 SP - 1627-1637 AU - Harada, E. AU - Kim, J.-A. AU - Meyer, A. J. AU - Hell, R. AU - Clemens, S. AU - Choi, Y.-E. AU - VL - 51 UR - DO - 10.1093/pcp/pcq118 AB - Nicotiana tabacum (tobacco) plants have short and long glandular trichomes. There is evidence that tobacco trichomes play several roles in the defense against biotic and abiotic stresses. cDNA libraries were constructed from control and cadmium (Cd)-treated leaf trichomes. Almost 2,000 expressed sequence tag (EST) cDNA clones were sequenced to analyze gene expression in control and Cd-treated leaf trichomes. Genes for stress response as well as for primary metabolism scored highly, indicating that the trichome is a biologically active and stress-responsive tissue. Reverse transcription–PCR (RT–PCR) analysis demonstrated that antipathogenic T-phylloplanin-like proteins, glutathione peroxidase and several classes of pathogenesis-related (PR) proteins were expressed specifically or dominantly in trichomes. Cysteine-rich PR proteins, such as non-specific lipid transfer proteins (nsLTPs) and metallocarboxypeptidase inhibitors, are candidates for the sequestration of metals. The expression of osmotin and thaumatin-like proteins was induced by Cd treatment in both leaves and trichomes. Confocal laser scanning microscopy (CLSM) showed that glutathione levels in tip cells of both long and short trichomes were higher than those in other types of leaf cells, indicating the presence of an active sulfur-dependent protective system in trichomes. Our results revealed that the trichome-specific transcriptome approach is a powerful tool to investigate the defensive functions of trichomes against both abiotic and biotic stress. Trichomes are shown to be an enriched source of useful genes for molecular breeding towards stress-tolerant plants. A2 - C1 - ER - TY - JOUR ID - 1661 TI - Phytochelatin Synthesis Is Essential for the Detoxification of Excess Zinc and Contributes Significantly to the Accumulation of Zinc JO - Plant Physiol. PY - 2009 SP - 938-948 AU - Tennstedt, P. AU - Peisker, D. AU - Böttcher, C. AU - Trampczynska, A. AU - Clemens, S. AU - VL - 149 UR - DO - 10.1104/pp.108.127472 AB - The synthesis of phytochelatins (PCs) is essential for the detoxification of nonessential metals and metalloids such as cadmium and arsenic in plants and a variety of other organisms. To our knowledge, no direct evidence for a role of PCs in essential metal homeostasis has been reported to date. Prompted by observations in Schizosaccharomyces pombe and Saccharomyces cerevisiae indicating a contribution of PC synthase expression to Zn2+ sequestration, we investigated a known PC-deficient Arabidopsis (Arabidopsis thaliana) mutant, cad1-3, and a newly isolated second strong allele, cad1-6, with respect to zinc (Zn) homeostasis. We found that in a medium with low cation content PC-deficient mutants show pronounced Zn2+ hypersensitivity. This phenotype is of comparable strength to the well-documented Cd2+ hypersensitivity of cad1 mutants. PC deficiency also results in significant reduction in root Zn accumulation. To be able to sensitively measure PC accumulation, we established an assay using capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry of derivatized extracts. Plants grown under control conditions consistently showed PC2 accumulation. Analysis of plants treated with same-effect concentrations revealed that Zn2+-elicited PC2 accumulation in roots reached about 30% of the level of Cd2+-elicited PC2 accumulation. We conclude from these data that PC formation is essential for Zn2+ tolerance and provides driving force for the accumulation of Zn. This function might also help explain the mysterious occurrence of PC synthase genes throughout the plant kingdom and in a wide range of other organisms. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1625 TI - Phenylpropanoid polyamine conjugate biosynthesis in Arabidopsis thaliana flower buds JO - Phytochemistry PY - 2009 SP - 1392-1400 AU - Fellenberg, C. AU - Böttcher, C. AU - Vogt, T. AU - VL - 70 UR - DO - 10.1016/j.phytochem.2009.08.010 AB - 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. A2 - C1 - Cell and Metabolic Biology ER - TY - JOUR ID - 1610 TI - The Multifunctional Enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) Converts Cysteine-Indole-3-Acetonitrile to Camalexin in the Indole-3-Acetonitrile Metabolic Network of Arabidopsis thaliana JO - Plant Cell PY - 2009 SP - 1830-1845 AU - Böttcher, C. AU - Westphal, L. AU - Schmotz, C. AU - Prade, E. AU - Scheel, D. AU - Glawischnig, E. AU - VL - 21 UR - DO - 10.1105/tpc.109.066670 AB - Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, γ-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate–treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1609 TI - Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray- tandem mass spectrometry JO - J. Mass Spectrom. PY - 2009 SP - 466-476 AU - Böttcher, C. AU - von Roepenack-Lahaye, E. AU - Schmidt, J. AU - Clemens, S. AU - Scheel, D. AU - VL - 44 UR - DO - 10.1002/jms.1522 AB - 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. A2 - C1 - Bioorganic Chemistry ER - TY - JOUR ID - 1743 TI - Highly sensitive feature detection for high resolution LC/MS JO - BMC Bioinformatics PY - 2008 SP - 504 AU - Tautenhahn, R. AU - Böttcher, C. AU - Neumann, S. AU - VL - 9 UR - DO - 10.1186/1471-2105-9-504 AB - BackgroundLiquid chromatography coupled to mass spectrometry (LC/MS) is an important analytical technology for e.g. metabolomics experiments. Determining the boundaries, centres and intensities of the two-dimensional signals in the LC/MS raw data is called feature detection. For the subsequent analysis of complex samples such as plant extracts, which may contain hundreds of compounds, corresponding to thousands of features – a reliable feature detection is mandatory.ResultsWe developed a new feature detection algorithm centWave for high-resolution LC/MS data sets, which collects regions of interest (partial mass traces) in the raw-data, and applies continuous wavelet transformation and optionally Gauss-fitting in the chromatographic domain. We evaluated our feature detection algorithm on dilution series and mixtures of seed and leaf extracts, and estimated recall, precision and F-score of seed and leaf specific features in two experiments of different complexity.ConclusionThe new feature detection algorithm meets the requirements of current metabolomics experiments. centWave can detect close-by and partially overlapping features and has the highest overall recall and precision values compared to the other algorithms, matchedFilter (the original algorithm of XCMS) and the centroidPicker from MZmine. The centWave algorithm was integrated into the Bioconductor R-package XCMS and is available from http://www.bioconductor.org/ A2 - C1 - ER - TY - JOUR ID - 1696 TI - Tapetum-specific location of a cation-dependent O-methyltransferase in Arabidopsis thaliana JO - Plant J. PY - 2008 SP - 132-145 AU - Fellenberg, C. AU - Milkowski, C. AU - Hause, B. AU - Lange, P.-R. AU - Böttcher, C. AU - Schmidt, J. AU - Vogt, T. AU - VL - 56 UR - DO - 10.1111/j.1365-313X.2008.03576.x AB - 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 . A2 - C1 - Bioorganic Chemistry; Cell and Metabolic Biology; Molecular Signal Processing ER - TY - JOUR ID - 1683 TI - Teaching (and learning from) metabolomics: The 2006 PlantMetaNet ETNA Metabolomics Research School JO - Physiol. Plant. PY - 2008 SP - AU - Böttcher, C. AU - Centeno, D. AU - Freitag, J. AU - Höfgen, R. AU - Köhl, K. AU - Kopka, J. AU - Kroymann, J. AU - Matros, A. AU - Mock, H.-P. AU - Neumann, S. AU - Pfalz, M. AU - von Roepenack-Lahaye, E. AU - Schauer, N. AU - Trenkamp, S. AU - Zubriggen, M. AU - Fernie, A. R. AU - VL - UR - DO - 10.1111/j.1399-3054.2007.00990.x AB - 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. A2 - C1 - ER - TY - JOUR ID - 1682 TI - Metabolome Analysis of Biosynthetic Mutants Reveals a Diversity of Metabolic Changes and Allows Identification of a Large Number of New Compounds in Arabidopsis JO - Plant Physiol. PY - 2008 SP - 2107-2120 AU - Böttcher, C. AU - von Roepenack-Lahaye, E. AU - Schmidt, J. AU - Schmotz, C. AU - Neumann, S. AU - Scheel, D. AU - Clemens, S. AU - VL - 147 UR - DO - 10.1104/pp.108.117754 AB - 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. A2 - C1 - Bioorganic Chemistry ER - TY - JOUR ID - 1678 TI - Identification and characterization of novel senescence-associated genes from barley (Hordeum vulgare) primary leaves JO - Plant Biol. PY - 2008 SP - 121-135 AU - Ay, N. AU - Clauß, K. AU - Barth, O. AU - Humbeck, K. AU - VL - 10 UR - DO - 10.1111/j.1438-8677.2008.00092.x AB - Leaf senescence is the final developmental stage of a leaf. The progression of barley primary leaf senescence was followed by measuring the senescence‐specific decrease in chlorophyll content and photosystem II efficiency. In order to isolate novel factors involved in leaf senescence, a differential display approach with mRNA populations from young and senescing primary barley leaves was applied. In this approach, 90 senescence up‐regulated cDNAs were identified. Nine of these clones were, after sequence analyses, further characterized. The senescence‐associated expression was confirmed by Northern analyses or quantitative RealTime‐PCR. In addition, involvement of the phytohormones ethylene and abscisic acid in regulation of these nine novel senescence‐induced cDNA fragments was investigated. Two cDNA clones showed homologies to genes with a putative regulatory function. Two clones possessed high homologies to barley retroelements, and five clones may be involved in degradation or transport processes. One of these genes was further analysed. It encodes an ADP ribosylation factor 1‐like protein (HvARF1) and includes sequence motifs representing a myristoylation site and four typical and well conserved ARF‐like protein domains. The localization of the protein was investigated by confocal laser scanning microscopy of onion epidermal cells after particle bombardment with chimeric HvARF1‐GFP constructs. Possible physiological roles of these nine novel SAGs during barley leaf senescence are discussed. A2 - C1 - ER - TY - JOUR ID - 1718 TI - The role of UDP-glucose:hydroxycinnamate glucosyltransferases in phenylpropanoid metabolism and the response to UV-B radiation in Arabidopsis thaliana JO - Planta PY - 2008 SP - 663-674 AU - Meißner, D. AU - Albert, A. AU - Böttcher, C. AU - Strack, D. AU - Milkowski, C. AU - VL - 228 UR - DO - 10.1007/s00425-008-0768-3 AB - 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. A2 - C1 - Cell and Metabolic Biology ER - TY - JOUR ID - 1769 TI - Evaluation of Matrix Effects in Metabolite Profiling Based on Capillary Liquid Chromatography Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry JO - Anal. Chem. PY - 2007 SP - 1507-1513 AU - Böttcher, C. AU - Roepenack-Lahaye, E. v. AU - Willscher, E. AU - Scheel, D. AU - Clemens, S. AU - VL - 79 UR - DO - 10.1021/ac061037q AB - The coupling of liquid chromatography to electrospray ionization quadrupole time-of-flight mass spectrometry can be a powerful tool for metabolomics, i.e., the comprehensive detection of low molecular weight compounds in biological systems. There have, however, been doubts about the feasibility and reliability of this approach, because LC−MSespecially with electrospray ionizationcan be subject to matrix effects. We evaluated matrix effects for our metabolomics platform in three ways: (i) postextraction addition of a set of reference compounds to different complex biological matrixes to determine absolute and relative matrix effects, (ii) postcolumn infusion of two reference compounds, and (iii) mixing of two complex matrixes. Our data demonstrate that there are indeed significant absolute matrix effects when comparing highly divergent samples. However, relative matrix effects are negligibleunless extremely divergent matrixes are comparedand do not compromise the relative quantification that is aimed for in nontargeted metabolomics studies. In conclusion, employing LC-coupled ESI-QTOF-MS for metabolomics studies is feasible yet rigorous validation is necessary. A2 - C1 - ER - TY - CHAP ID - 117 TI - Annotation of LC/ESI-MS Mass Signals T2 - Bioinformatics Research and Development. BIRD 2007 PB - Lecture Notes in Computer Science PY - 2007 SP - 371-380 AU - Tautenhahn, R. AU - Böttcher, C. AU - Neumann, S. AU - VL - 4414 UR - SN - 978-3-540-71233-6 DO - 10.1007/978-3-540-71233-6_29 AB - Mass spectrometry is the work-horse technology of the emerging field of metabolomics. The identification of mass signals remains the largest bottleneck for a non-targeted approach: due to the analytical method, each metabolite in a complex mixture will give rise to a number of mass signals. In contrast to GC/MS measurements, for soft ionisation methods such as ESI-MS there are no extensive libraries of reference spectra or established deconvolution methods. We present a set of annotation methods which aim to group together mass signals measured from a single metabolite, based on rules for mass differences and peak shape comparison.Availability: The software and documentation is available as an R package on http://msbi.ipb-halle.de/ A2 - C1 - ER - TY - JOUR ID - 1906 TI - Hexafluoroacetone as Protecting and Activating Reagent: New Routes to Amino, Hydroxy, and Mercapto Acids and Their Application for Peptide and Glyco- and Depsipeptide Modification JO - Chem. Rev. PY - 2006 SP - 4728-4746 AU - Spengler, J. AU - Böttcher, C. AU - Albericio, F. AU - Burger, K. AU - VL - 106 UR - DO - 10.1021/cr0509962 AB - A2 - C1 - ER - TY - JOUR ID - 1896 TI - Proteome changes in Arabidopsis thaliana roots upon exposure to Cd2+ JO - J. Exp. Bot. PY - 2006 SP - 4003-4013 AU - Roth, U. AU - von Roepenack-Lahaye, E. AU - Clemens, S. AU - VL - 57 UR - DO - 10.1093/jxb/erl170 AB - 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. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 1888 TI - Analysis of fungal cyclopentenone derivatives from Hygrophorus spp. by liquid chromatography/electrospray-tandem mass spectrometry JO - J. Mass Spectrom. PY - 2006 SP - 361-371 AU - Lübken, T. AU - Arnold, N. AU - Wessjohann, L. AU - Böttcher, C. AU - Schmidt, J. AU - VL - 41 UR - DO - 10.1002/jms.996 AB - Fruitbodies of the genus Hygrophorus (Basidiomycetes) contain a series of anti‐biologically active compounds. These substances named hygrophorones possess a cyclopentenone skeleton. LC/ESI‐MS/MS presents a valuable tool for the identification of such compounds. The mass spectral behaviour of typical selected members of this group under positive and negative ion electrospray conditions is discussed. Using the ESI collision‐induced dissociation (CID) mass spectra of the [M + H]+ and [M − H]− ions, respectively, the compounds can be classified with respect to the substitution pattern at the cyclopentenone ring and the type of oxygenation at C‐6 (hydroxy/acetoxy or oxo function) of the side chain. The elemental composition of the fragment ions was determined by ESI‐QqTOF measurements. Thus, in case of the negative ion CID mass spectra an unusual loss of CO2 from the deprotonated molecular ions could be observed. A2 - C1 - Bioorganic Chemistry ER - TY - JOUR ID - 1915 TI - The transition metal chelator nicotianamine is synthesized by filamentous fungi JO - FEBS Lett. PY - 2006 SP - 3173-3178 AU - Trampczynska, A. AU - Böttcher, C. AU - Clemens, S. AU - VL - 580 UR - DO - 10.1016/j.febslet.2006.04.073 AB - Nicotianamine is an important metal ligand in plants. Surprisingly, recent genome sequencing revealed that ascomycetes encode proteins with similarity to plant nicotianamine synthases (NAS). By expression in a Zn2+‐hypersensitive fission yeast mutant we show for a protein from Neurospora crassa that it indeed possesses NAS activity. Using electrospray‐ionization‐quadrupole‐time‐of‐flight mass spectrometry we prove the formation of nicotianamine in N. crassa . Transcript level is strongly upregulated under Zn deficiency as shown by real‐time PCR. These findings demonstrate that nicotianamine is more widespread in nature than anticipated and provide further evidence for a function of nicotianamine as a cytosolic chelator of Zn2+ ions. A2 - C1 - Biochemistry of Plant Interactions ER - TY - CHAP ID - 120 TI - Capillary HPLC Coupled to Electrospray Ionization Quadrupole Time-of-flight Mass Spectrometry T2 - Biotechnology in Agriculture and Forestry PB - Biotechnology in Agriculture and Forestry PY - 2006 SP - 65-79 AU - Clemens, S. AU - Böttcher, C. AU - Franz, M. AU - Willscher, E. AU - v. Roepenack-Lahaye, E. AU - Scheel, D. AU - VL - 57 UR - SN - 978-3-540-29782-6 DO - 10.1007/3-540-29782-0_5 AB - A2 - C1 - ER - TY - JOUR ID - 2025 TI - A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to γ-glutamylcysteine and lacks phytochelatin synthase activity JO - Phytochemistry PY - 2004 SP - 3179-3185 AU - Harada, E. AU - von Roepenack-Lahaye, E. AU - Clemens, S. AU - VL - 65 UR - DO - 10.1016/j.phytochem.2004.09.017 AB - 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. A2 - C1 - Biochemistry of Plant Interactions ER - TY - JOUR ID - 2063 TI - Profiling of Arabidopsis Secondary Metabolites by Capillary Liquid Chromatography Coupled to Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry JO - Plant Physiol. PY - 2004 SP - 548-559 AU - von Roepenack-Lahaye, E. AU - Degenkolb, T. AU - Zerjeski, M. AU - Franz, M. AU - Roth, U. AU - Wessjohann, L. AU - Schmidt, J. AU - Scheel, D. AU - Clemens, S. AU - VL - 134 UR - DO - 10.1104/pp.103.032714 AB - 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. A2 - C1 - Bioorganic Chemistry; Biochemistry of Plant Interactions ER -