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Publications - Stress and Develop Biology

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Publications

Dietz, S.; Herz, K.; Döll, S.; Haider, S.; Jandt, U.; Bruelheide, H.; Scheel, D. Semi‐polar root exudates in natural grassland communities Ecol Evol 9, 5526-5541, (2019) DOI: 10.1002/ece3.5043

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.
Publications

Zembek, P.; Danilecka, A.; Hoser, R.; Eschen-Lippold, L.; Benicka, M.; Grech-Baran, M.; Rymaszewski, W.; Barymow-Filoniuk, I.; Morgiewicz, K.; Kwiatkowski, J.; Piechocki, M.; Poznanski, J.; Lee, J.; Hennig, J.; Krzymowska, M. Two Strategies of Pseudomonas syringae to Avoid Recognition of the HopQ1 Effector in Nicotiana Species Front Plant Sci 9, 978, (2018) DOI: 10.3389/fpls.2018.00978

Pseudomonas syringae employs a battery of type three secretion effectors to subvert plant immune responses. In turn, plants have developed receptors that recognize some of the bacterial effectors. Two strain-specific HopQ1 effector variants (for Hrp outer protein Q) from the pathovars phaseolicola 1448A (Pph) and tomato DC3000 (Pto) showed considerable differences in their ability to evoke disease symptoms in Nicotiana benthamiana. Surprisingly, the variants differ by only six amino acids located mostly in the N-terminal disordered region of HopQ1. We found that the presence of serine 87 and leucine 91 renders PtoHopQ1 susceptible to N-terminal processing by plant proteases. Substitutions at these two positions did not strongly affect PtoHopQ1 virulence properties in a susceptible host but they reduced bacterial growth and accelerated onset of cell death in a resistant host, suggesting that N-terminal mutations rendered PtoHopQ1 susceptible to processing in planta and, thus, represent a mechanism of recognition avoidance. Furthermore, we found that co-expression of HopR1, another effector encoded within the same gene cluster masks HopQ1 recognition in a strain-dependent manner. Together, these data suggest that HopQ1 is under high host-pathogen co-evolutionary selection pressure and P. syringae may have evolved differential effector processing or masking as two independent strategies to evade HopQ1 recognition, thus revealing another level of complexity in plant – microbe interactions.
Publications

Peters, K.; Gorzolka, K.; Bruelheide, H.; Neumann, S. Seasonal variation of secondary metabolites in nine different bryophytes Ecol Evol 8, 9105-9117, (2018) DOI: 10.1002/ece3.4361

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.
Publications

Chen, C.; Masi, R. D.; Lintermann, R.; Wirthmueller, L. Nuclear Import of Arabidopsis Poly(ADP-Ribose) Polymerase 2 Is Mediated by Importin-α and a Nuclear Localization Sequence Located Between the Predicted SAP Domains Front Plant Sci 9, 1581, (2018) DOI: 10.3389/fpls.2018.01581

Proteins of the Poly(ADP-Ribose) Polymerase (PARP) family modify target proteins by covalent attachment of ADP-ribose moieties onto amino acid side chains. In Arabidopsis, PARP proteins contribute to repair of DNA lesions and modulate plant responses to various abiotic and biotic stressors. Arabidopsis PARP1 and PARP2 are nuclear proteins and given that their molecular weights exceed the diffusion limit of nuclear pore complexes, an active import mechanism into the nucleus is likely. Here we use confocal microscopy of fluorescent protein-tagged Arabidopsis PARP2 and PARP2 deletion constructs in combination with site-directed mutagenesis to identify a nuclear localization sequence in PARP2 that is required for nuclear import. We report that in co-immunoprecipitation assays PARP2 interacts with several isoforms of the importin-α group of nuclear transport adapters and that PARP2 binding to IMPORTIN-α2 is mediated by the identified nuclear localization sequence. Our results demonstrate that PARP2 is a cargo protein of the canonical importin-α/β nuclear import pathway.
Publications

Herz, K.; Dietz, S.; Haider, S.; Jandt, U.; Scheel, D.; Bruelheide, H. Predicting individual plant performance in grasslands. Ecol Evol 7, 8958-8965, (2017) DOI: 10.1002/ece3.3393

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.
Publications

Strehmel, N.; Hoehenwarter, W.; Mönchgesang, S.; Majovsky, P.; Krüger, S.; Scheel, D.; Lee, J. Stress-reated 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.
Publications

Sheikh, A. H.; Eschen-Lippold, L.; Pecher, P.; Hoehenwarter, W.; Sinha, A. K.; Scheel, D.; Lee, J. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana Front Plant Sci 7, 61, (2016) DOI: 10.3389/fpls.2016.00061

Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense.
Publications

Lee, J.; Eschen-Lippold, L.; Lassowskat, I.; Böttcher, C.; Scheel, D. Cellular reprogramming through mitogen-activated protein kinases Front Plant Sci 6, 940, (2015) DOI: 10.3389/fpls.2015.00940

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.
Books and chapters

Hummel, J.; Strehmel, N.; Bölling, C.; Schmidt, S.; Walther D.; Kopka, J. Mass spectral search and analysis using the Golm metabolome. (Weckwerth, W.; Kahl, G.). 321-343, (2013) ISBN: 978-3-527-32777-5 DOI: 10.1002/9783527669882.ch18

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.
Publications

Wirthmueller, L.; Roth, C.; Banfield, M. J.; Wiermer, M. Hop-on hop-off: importin-α-guided tours to the nucleus in innate immune signaling Front Plant Sci 4, 149, (2013) DOI: 10.3389/fpls.2013.00149

Nuclear translocation of immune regulatory proteins and signal transducers is an essential process in animal and plant defense signaling against pathogenic microbes. Import of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors termed importins, typically dimers of a cargo-binding α-subunit and a β-subunit that mediates translocation through the nuclear pore complex. Here, we review recent reports of importin-α cargo specificity and mutant phenotypes in plant- and animal–microbe interactions. Using homology modeling of the NLS-binding cleft of nine predicted Arabidopsis α-importins and analyses of their gene expression patterns, we discuss functional redundancy and specialization within this transport receptor family. In addition, we consider how pathogen effector proteins that promote infection by manipulating host cell nuclear processes might compete with endogenous cargo proteins for nuclear uptake.
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