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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

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

Mönchgesang, S.; Strehmel, N.; Schmidt, S.; Westphal, L.; Taruttis, F.; Müller, E.; Herklotz, S.; Neumann, S.; Scheel, D.; Natural variation of root 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.
Publikation

Kühnlenz, T.; Westphal, L.; Schmidt, H.; Scheel, D.; Clemens, S.; Expression of Caenorhabditis elegans PCS in the AtPCS1-deficient Arabidopsis thaliana cad1-3 mutant separates the metal tolerance and non-host resistance functions of phytochelatin synthases Plant Cell Environ. 38, 2239-2247, (2015) DOI: 10.1111/pce.12534

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.
Publikation

Geissler, K.; Eschen-Lippold, L.; Naumann, K.; Schneeberger, K.; Weigel, D.; Scheel, D.; Rosahl, S.; Westphal, L.; Mutations in the EDR1 Gene Alter the Response of Arabidopsis thaliana to Phytophthora infestans and the Bacterial PAMPs flg22 and elf18 Mol. Plant Microbe Interact. 28, 122-133, (2015) DOI: 10.1094/MPMI-09-14-0282-R

Mechanistically, nonhost resistance of Arabidopsis thaliana against the oomycete Phytophthora infestans is not well understood. Besides PEN2 and PEN3, which contribute to penetration resistance, no further components have been identified so far. In an ethylmethane sulphonate–mutant screen, we mutagenized pen2-1 and screened for mutants with an altered response to infection by P. infestans. One of the mutants obtained, enhanced response to Phytophthora infestans6 (erp6), was analyzed. Whole-genome sequencing of erp6 revealed a single nucleotide polymorphism in the coding region of the kinase domain of At1g08720, which encodes the putative MAPKKK ENHANCED DISEASE RESISTANCE1 (EDR1). We demonstrate that three independent lines with knock-out alleles of edr1 mount an enhanced response to P. infestans inoculation, mediated by increased salicylic acid signaling and callose deposition. Moreover, we show that the single amino acid substitution in erp6 causes the loss of in vitro autophosphorylation activity of EDR1. Furthermore, growth inhibition experiments suggest a so-far-unknown involvement of EDR1 in the response to the pathogen-associated molecular patterns flg22 and elf18. We conclude that EDR1 contributes to the defense response of A. thaliana against P. infestans. Our data position EDR1 as a negative regulator in postinvasive nonhost resistance.
Preprints

Thum, A.; Mönchgesang, S.; Westphal, L.; Lübken, T.; Rosahl, S.; Neumann, S.; Posch, S.; Supervised Penalized Canonical Correlation Analysis arXiv (2014)

The canonical correlation analysis (CCA) is commonly used to analyze data sets with paired data, e.g. measurements of gene expression and metabolomic intensities of the same experiments. This allows to find interesting relationships between the data sets, e.g. they can be assigned to biological processes. However, it can be difficult to interpret the processes and often the relationships observed are not related to the experimental design but to some unknown parameters.Here we present an extension of the penalized CCA, the supervised penalized approach (spCCA), where the experimental design is used as a third data set and the correlation of the biological data sets with the design data set is maximized to find interpretable and meaningful canonical variables. The spCCA was successfully tested on a data set of Arabidopsis thaliana with gene expression and metabolite intensity measurements and resulted in eight significant canonical variables and their interpretation. We provide an R-package under the GPL license.
Publikation

Heymann, T.; Westphal, L.; Wessjohann, L.; Glomb, M. A.; Growing and Processing Conditions Lead to Changes in the Carotenoid Profile of Spinach J. Agr. Food Chem. 62, 4960-4967, (2014) DOI: 10.1021/jf501136g

This study aimed to evaluate the influence of different light regimens during spinach cultivation on the isomeric composition of β-carotene. Irradiation with a halogen lamp, which has a wavelength spectrum close to that of daylight, was used to mimic field-grown conditions. The additional use of optical filters was established as a model system for greenhouse cultivation. Field-grown model systems led to a preferential increase of 9-cis-β-carotene, whereas 13-cis-β-carotene was just formed at the beginning of irradiation. Additionally 9,13-di-cis-β-carotene decreased significantly in the presence of energy-rich light. Isomerization of β-carotene was strongly suppressed during irradiation in greenhouse-grown model systems and led to significant differences. These results were verified in biological samples. Authentic field-grown spinach (Spinacia oleracea L.) showed among changes of other isomers a significantly higher level of 9-cis-isomers (7.52 ± 0.14%) and a significantly lower level of 9,13-di-cis-isomers (0.25 ± 0.03%) compared to authentic greenhouse-grown spinach (6.49 ± 0.11 and 0.76 ± 0.05%). Almost all analyzed commercial spinach samples (fresh and frozen) were identified as common field-grown cultivation. Further investigations resulted in a clear differentiation of frozen commercial samples from fresh spinach, caused by significantly higher levels of 13-cis- and 15-cis-β-carotene as a result of industrial blanching processes.
Publikation

Ranf, S.; Eschen-Lippold, L.; Fröhlich, K.; Westphal, L.; Scheel, D.; Lee, J.; Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1 BMC Plant Biol. 14, 374, (2014) DOI: 10.1186/s12870-014-0374-4

BackgroundPlant perception of conserved microbe-derived or damage-derived molecules (so-called microbe- or damage-associated molecular patterns, MAMPs or DAMPs, respectively) triggers cellular signaling cascades to initiate counteracting defence responses. Using MAMP-induced rise in cellular calcium levels as one of the earliest biochemical readouts, we initiated a genetic screen for components involved in early MAMP signaling in Arabidopsis thaliana.ResultsWe characterized here the “changed calcium elevation 5” (cce5) mutant, where five allelic cce5 mutants were isolated. They all show reduced calcium levels after elicitation with peptides representing bacteria-derived MAMPs (flg22 and elf18) and endogenous DAMP (AtPep1), but a normal response to chitin octamers. Mapping, sequencing of the mutated locus and complementation studies revealed CCE5 to encode the receptor-like cytoplasmic kinase (RLCK), avrPphB sensitive 1-like 1 (PBL1). Kinase activities of PBL1 derived from three of the cce5 alleles are abrogated in vivo. Validation with T-DNA mutants revealed that, besides PBL1, another RLCK, Botrytis-induced kinase 1 (BIK1), is also required for MAMP/DAMP-induced calcium elevations.ConclusionsHence, PBL1 and BIK1 (but not two related RLCKs, PBS1 and PBL2) are required for MAMP/DAMP-induced calcium signaling. It remains to be investigated if the many other RLCKs encoded in the Arabidopsis genome affect early calcium signal transduction – perhaps in dependence on the type of MAMP/DAMP ligands. A future challenge would be to identify the substrates of these various RLCKs, in order to elucidate their signaling role between the receptor complexes at the plasma membrane and downstream cellular signaling components.
Publikation

Kopischke, M.; Westphal, L.; Schneeberger, K.; Clark, R.; Ossowski, S.; Wewer, V.; Fuchs, R.; Landtag, J.; Hause, G.; Dörmann, P.; Lipka, V.; Weigel, D.; Schulze-Lefert, P.; Scheel, D.; Rosahl, S.; Impaired sterol ester synthesis alters the response of Arabidopsis thaliana to Phytophthora infestans Plant J. 73, 456-468, (2013) DOI: 10.1111/tpj.12046

Non‐host resistance of Arabidopsis thaliana against Phytophthora infestans, the causal agent of late blight disease of potato, depends on efficient extracellular pre‐ and post‐invasive resistance responses. Pre‐invasive resistance against P. infestans requires the myrosinase PEN2. To identify additional genes involved in non‐host resistance to P. infestans, a genetic screen was performed by re‐mutagenesis of pen2 plants. Fourteen independent mutants were isolated that displayed an enhanced response to Phytophthora (erp) phenotype. Upon inoculation with P. infestans, two mutants, pen2‐1 erp1‐3 and pen2‐1 erp1‐4, showed an enhanced rate of mesophyll cell death and produced excessive callose deposits in the mesophyll cell layer. ERP1 encodes a phospholipid:sterol acyltransferase (PSAT1) that catalyzes the formation of sterol esters. Consistent with this, the tested T‐DNA insertion lines of PSAT1 are phenocopies of erp1 plants. Sterol ester levels are highly reduced in all erp1/psat1 mutants, whereas sterol glycoside levels are increased twofold. Excessive callose deposition occurred independently of PMR4/GSL5 activity, a known pathogen‐inducible callose synthase. A similar formation of aberrant callose deposits was triggered by the inoculation of erp1psat1 plants with powdery mildew. These results suggest a role for sterol conjugates in cell non‐autonomous defense responses against invasive filamentous pathogens.
Publikation

Stegmann, M.; Anderson, R. G.; Westphal, L.; Rosahl, S.; McDowell, J. M.; Trujillo, M.; The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death Plant Signal Behav. 8, e27421, (2013) DOI: 10.4161/psb.27421

Components of the vesicle trafficking machinery are central to the immune response in plants. The role of vesicle trafficking during pre-invasive penetration resistance has been well documented. However, emerging evidence also implicates vesicle trafficking in early immune signaling. Here we report that Exo70B1, a subunit of the exocyst complex which mediates early tethering during exocytosis is involved in resistance. We show that exo70B1 mutants display pathogen-specific immuno-compromised phenotypes. We also show that exo70B1 mutants display lesion-mimic cell death, which in combination with the reduced responsiveness to pathogen-associated molecular patterns (PAMPs) results in complex immunity-related phenotypes.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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