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Publikation

Ruttkies, C.; Schymanski, E. L.; Strehmel, N.; Hollender, J.; Neumann, S.; Williams, A. J.; Krauss, M.; Supporting non-target identification by adding hydrogen deuterium exchange MS/MS capabilities to MetFrag Anal. Bioanal. Chem. 411, 4683-4700, (2019) DOI: 10.1007/s00216-019-01885-0

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).
Publikation

Westphal, L.; Strehmel, N.; Eschen-Lippold, L.; Bauer, N.; Westermann, B.; Rosahl, S.; Scheel, D.; Lee, J.; pH effects on plant calcium fluxes: lessons from acidification-mediated calcium elevation induced by the γ-glutamyl-leucine dipeptide identified from Phytophthora infestans Sci. Rep. 9, 4733, (2019) DOI: 10.1038/s41598-019-41276-0

Cytosolic Ca2+ ([Ca2+]cyt) elevation is an early signaling response upon exposure to pathogen-derived molecules (so-called microbe-associated molecular patterns, MAMPs) and has been successfully used as a quantitative read-out in genetic screens to identify MAMP receptors or their associated components. Here, we isolated and identified by mass spectrometry the dipeptide γ-Glu-Leu as a component of a Phytophthora infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with synthetic γ-Glu-Leu revealed stimulatory effects on defense signaling, including a weak enhancement of the expression of some MAMP-inducible genes or affecting the refractory period to a second MAMP elicitation. However, γ-Glu-Leu is not a classical MAMP since pH adjustment abolished these activities and importantly, the observed effects of γ-Glu-Leu could be recapitulated by mimicking extracellular acidification. Thus, although γ-Glu-Leu can act as a direct agonist of calcium sensing receptors in animal systems, the Ca2+-mobilizing activity in plants reported here is due to acidification. Low pH also shapes the Ca2+ signature of well-studied MAMPs (e.g. flg22) or excitatory amino acids such as glutamate. Overall, this work serves as a cautionary reminder that in defense signaling studies where Ca2+ flux measurements are concerned, it is important to monitor and consider the effects of pH.
Publikation

Strehmel, N.; Hoehenwarter, W.; Mönchgesang, S.; Majovsky, P.; Krüger, S.; Scheel, D.; Lee, J.; Stress-Related Mitogen-Activated Protein Kinases Stimulate the Accumulation of Small Molecules and Proteins in Arabidopsis thaliana Root Exudates Front. Plant Sci. 8, 1292, (2017) DOI: 10.3389/fpls.2017.01292

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

Mönchgesang, S.; Strehmel, N.; Trutschel, D.; Westphal, L.; Neumann, S.; Scheel, D.; Plant-to-Plant Variability in Root Metabolite Profiles of 19 Arabidopsis thaliana Accessions Is Substance-Class-Dependent Int. J. Mol. Sci. 17, 1565, (2016) DOI: 10.3390/ijms17091565

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.
Publikation

Ziegler, J.; Schmidt, S.; Chutia, R.; Müller, J.; Böttcher, C.; Strehmel, N.; Scheel, D.; Abel, S.; 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 J. Exp. Bot. 67, 1421-1432, (2016) DOI: 10.1093/jxb/erv539

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.
Publikation

Strehmel, N.; Mönchgesang, S.; Herklotz, S.; Krüger, S.; Ziegler, J.; Scheel, D.; Piriformospora indica Stimulates Root Metabolism of Arabidopsis thaliana Int. J. Mol. Sci. 17, 1091, (2016) DOI: 10.3390/ijms17071091

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
Publikation

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

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

Buhtz, A.; Witzel, K.; Strehmel, N.; Ziegler, J.; Abel, S.; Grosch, R.; Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae PLOS ONE 10, e0138242, (2015) DOI: 10.1371/journal.pone.0138242

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.
Publikation

Strehmel, N.; Böttcher, C.; Schmidt, S.; Scheel, D.; Profiling of secondary metabolites in root exudates of Arabidopsis thaliana Phytochemistry 108, 35-46, (2014) DOI: 10.1016/j.phytochem.2014.10.003

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.
Publikation

Landgraf, R.; Smolka, U.; Altmann, S.; Eschen-Lippold, L.; Senning, M.; Sonnewald, S.; Weigel, B.; Frolova, N.; Strehmel, N.; Hause, G.; Scheel, D.; Böttcher, C.; Rosahl, S.; The ABC Transporter ABCG1 Is Required for Suberin Formation in Potato Tuber Periderm Plant Cell 26, 3403-3415, (2014) DOI: 10.1105/tpc.114.124776

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.
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