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Publikationen - Stress- und Entwicklungsbiologie

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Guerra, T.; Schilling, S.; Hake, K.; Gorzolka, K.; Sylvester, F.; Conrads, B.; Westermann, B.; Romeis, T. Calcium‐dependent protein kinase 5 links calcium‐signaling with N‐Hydroxy‐L‐pipecolic acid‐ and SARD1‐dependent immune memory in systemic acquired resistance New Phytol (2019) DOI: 10.1111/nph.16147

Systemic acquired resistance (SAR) prepares infected plants for faster and stronger defense activation upon subsequent attacks. SAR requires an information relay from primary infection to distal tissue and the initiation and maintenance of a self‐maintaining phytohormone salicylic acid (SA)‐defense loop.In spatial and temporal resolution, we show that calcium‐dependent protein kinase CPK5 contributes to immunity and SAR. In local basal resistance CPK5 functions upstream of SA‐synthesis, ‐perception, and ‐signaling. In systemic tissue, CPK5 signaling leads to accumulation of SAR inducing metabolite N‐Hydroxy‐L‐pipecolic acid (NHP) and SAR marker genes including Systemic Acquired Resistance Deficient 1 (SARD1)Plants of increased CPK5‐, but not CPK6‐ signaling, display an ‘enhanced SAR' phenotype toward a secondary bacterial infection. In sard1‐1 background, CPK5‐mediated basal resistance is still mounted, but NHP level is reduced and ‘enhanced SAR' is lost.The biochemical analysis determines CPK5 half maximal kinase activity for calcium K50 [Ca2+] to ~100 nM close to the cytoplasmic resting level. This low threshold uniquely qualifies CPK5 to decode subtle changes in calcium prerequisite to signal relay and onset and maintenance of priming at later time points in distal tissue. Our data explain why CPK5 functions as a hub in basal and systemic plant immunity.
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Teh, O.-K.; Lee, C.-W.; Ditengou, F. A.; Klecker, T.; Furlan, G.; Zietz, M.; Hause, G.; Eschen-Lippold, L.; Hoehenwarter, W.; Lee, J.; Ott, T.; Trujillo, M. Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function BioRxiv (2018) DOI: 10.1101/266171

The exocyst is a conserved hetero-octameric complex mediating early tethering during exocytosis. Its Exo70 subunit plays a critical role as a spatiotemporal regulator by mediating numerous protein and lipid interactions. However, a molecular understanding of the exocyst function remains challenging. We show that Exo70B2 locates to dynamic foci at the plasma membrane and transits through a BFA-sensitive compartment, reflecting its canonical function in secretion. However, treatment with the salicylic acid (SA) defence hormone analogue Benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We uncovered two ATG8-interacting motifs (AIMs) located in the C-terminal domain (C-domain) of Exo70B2 that mediate its recruitment into the vacuole. Moreover, we also show that Exo70B2 is phosphorylated near the AIMs and mimicking phosphorylation enhanced ATG8 interaction. Finally, Exo70B2 phosphonull lines were hypersensitive to BTH and more resistant to avirulent bacteria which induce SA production. Our results suggests a molecular mechanism in which phosphorylation of Exo70B2 by MPK3 functions in a feed-back system linking cellular signalling to the secretory pathway.
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Tabassum, N.; Eschen‐Lippold, L.; Athmer, B.; Baruah, M.; Brode, M.; Maldonado‐Bonilla, L. D.; Hoehenwarter, W.; Hause, G.; Scheel, D.; Lee, J. Phosphorylation‐dependent control of an RNA granule‐localized protein that fine‐tunes defence gene expression at a post‐transcriptional level Plant J (2019) DOI: 10.1111/tpj.14573

Mitogen‐activated protein kinase (MAPK) cascades are key signalling modules of plant defence responses to pathogen‐associated molecular patterns (PAMPs, e.g. bacterial flg22 peptide). The Tandem Zinc Finger Protein 9 (TZF9) is an RNA‐binding protein that is phosphorylated by two PAMP‐responsive MAPKs, MPK3 and MPK6. We mapped the major phosphosites in TZF9 and showed their importance for controlling in vitro RNA‐binding activity, in vivo flg22‐induced rapid disappearance of TZF9‐labelled processing body‐like structures and TZF9 protein turnover. Microarray analysis showed a strong discordance between transcriptome (total mRNA) and translatome (polysome‐associated mRNA) in the tzf9 mutant, with more mRNAs associated to ribosomes in the absence of TZF9. This suggests that TZF9 may sequester and inhibit translation of subsets of mRNAs. Fittingly, TZF9 physically interacts with poly(A)‐binding protein 2 (PAB2), a hallmark constituent of stress granules – a site for stress‐induced translational stalling/arrest. TZF9 even promotes stress granule assembly in the absence of stress. Hence, MAPKs may control defence gene expression post‐transcriptionally through release from translation arrest within TZF9‐PAB2‐containing RNA granules or perturbing PAB2 functions in translation control (e.g. in the mRNA closed‐loop model of translation).
Publikationen in Druck

Jiang, X.; Hoehenwarter, W.; Scheel, D.; Lee, J. Phosphorylation of the CAMTA3 transcription factor triggers its destabilization and nuclear export bioRxiv (2019) DOI: 10.1101/825323

The calmodulin-binding transcription activator 3 (CAMTA3) is a repressor of immunity-related genes but an activator of cold-induced genes in plants. Post-transcriptional or -translational mechanisms have been proposed to control CAMTA3’s role in the crosstalk between immune and chilling responses. Here, we show that treatment with the bacterial flg22 elicitor, but not cold stress, induces a phospho-mobility shift of CAMTA3 proteins. Correspondingly, CAMTA3 is directly phosphorylated by two flg22-responsive mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, which triggers CAMTA3 nuclear export and destabilization. SR1IP1, a substrate E3 ubiquitin ligase adaptor required for pathogen-induced CAMTA3 degradation, is shown here to be likely plasma-membrane-localized and therefore cannot physically interact with the nuclear CAMTA3. Despite the flg22-inducible re-localization of CAMTA3 to the cytoplasm, we failed to detect CAMTA3-SR1IP1 complexes. Hence, the role of SR1IP1 for CAMTA3 degradation needs to be re-evaluated. Surprisingly, flg22 elicitation can still induce nuclear export and phospho-mobility shift of a phospho-null CAMTA3 that cannot be phosphorylated by MAPKs, suggesting the participation of additional flg22-responsive kinase(s). A constitutively-active calcium-dependent protein kinase, CPK5, can stimulate a phospho-mobility shift in CAMTA3 similar to that induced by flg22. Although CPK5 can interact with CAMTA3, it did not directly phosphorylate CAMTA3, suggesting the requirement of a still unidentified downstream kinase or additional components. Overall, at least two flg22-responsive kinase pathways target CAMTA3 to induce degradation that presumably serves to remove CAMTA3 from target promoters and de-repress expression of defence genes.

Mönchgesang, S.; Strehmel, N.; Schmidt, S.; Westphal, L.; Taruttis, F.; Müller, E.; Herklotz, S.; Neumann, S.; Scheel, D. Natural variation of roots exudates in Arabidopsis thaliana - linking metabolomic and genomic data. Sci Rep 6, 29033 , (2016) DOI: 10.1038/srep29033

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.

Mönchgesang, S.; Strehmel, N.; Trutschel, D.; Westphal, L.; Neumann, S.; Scheel, D. Plant-to-plant variability in root metabolite profiles of 19 <i>Arabidopsis thaliana</i> accessions is substance-class-dependent Inter J Mol Sci 17, (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.

Ruttkies, C.; Strehmel, N.; Scheel, D.; Neumann, S. Annotation of metabolites from gas chromatography/atmospheric pressure chemical ionization tandem mass spectrometry data using an in silico generated compound database and MetFrag Rapid Commun Mass Spectrom 29, 1521-1529, (2015) DOI: 10.1002/rcm.7244

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.

Tautenhahn, R.; Böttcher, C.; Neumann, S. Annotation of LC/ESI-MS Mass Signals BIRD 2007 Proc. of BIRD 2007 - 1st International Conference on Bioinformatics Research and Development 2007 371-380, (2007) DOI: 10.1007/978-3-540-71233-6_29

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.

Peters, K.; Gorzolka, K.; Bruelheide, H.; Neumann, S. Computational workflow to study the seasonal variation of secondary metabolites in nine different bryophytes Sci Data 5, 180179, (2018) DOI: 10.1038/sdata.2018.179

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.

Peters, K.; Worrich, A.; Weinhold, A.; Alka, O.; Balcke, G.; Birkemeyer, C.; Bruelheide, H.; Calf, O. W.; Dietz, S.; Dührkop, K.; Gaquerel, E.; Heinig, U.; Kücklich, M.; Macel, M.; Müller, C.; Poeschl, Y.; Pohnert, G.; Ristok, C.; Rodríguez, V. M.; Ruttkies, C.; Schuman, M.; Schweiger, R.; Shahaf, N.; Steinbeck, C.; Tortosa, M.; Treutler, H.; Ueberschaar, N.; Velasco, P.; Weiß, B. M.; Widdig, A.; Neumann, S.; van Dam, N. M. Current Challenges in Plant Eco-Metabolomics Int J Mol Sci 19, 1385, (2018) DOI: 10.3390/ijms19051385

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
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