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Publications - Stress and Develop Biology

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Books and chapters

Doell, S.; Arens, N.; Mock, H. Liquid Chromatography and Liquid Chromatography–Mass Spectrometry of Plants: Techniques and Applications (Meyers, R. A., ed.). (2019) ISBN: 9780470027318 DOI: 10.1002/9780470027318.a9912.pub2

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.
Publications

Schön, M.; Töller, A.; Diezel, C.; Roth, C.; Westphal, L.; Wiermer, M.; Somssich, I. E. Analyses of wrky18 wrky40 Plants Reveal Critical Roles of SA/EDS1 Signaling and Indole-Glucosinolate Biosynthesis for Golovinomyces orontii Resistance and a Loss-of Resistance Towards Pseudomonas syringae pv. tomato AvrRPS4 Mol Plant Microbe Interact 26, 758-767, (2013) DOI: 10.1094/MPMI-11-12-0265-R

Simultaneous mutation of two WRKY-type transcription factors, WRKY18 and WRKY40, renders otherwise susceptible wild-type Arabidopsis plants resistant towards the biotrophic powdery mildew fungus Golovinomyces orontii. Resistance in wrky18 wrky40 double mutant plants is accompanied by massive transcriptional reprogramming, imbalance in salicylic acid (SA) and jasmonic acid (JA) signaling, altered ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) expression, and accumulation of the phytoalexin camalexin. Genetic analyses identified SA biosynthesis and EDS1 signaling as well as biosynthesis of the indole-glucosinolate 4MI3G as essential components required for loss-of-WRKY18 WRKY40–mediated resistance towards G. orontii. The analysis of wrky18 wrky40 pad3 mutant plants impaired in camalexin biosynthesis revealed an uncoupling of pre- from postinvasive resistance against G. orontii. Comprehensive infection studies demonstrated the specificity of wrky18 wrky40–mediated G. orontii resistance. Interestingly, WRKY18 and WRKY40 act as positive regulators in effector-triggered immunity, as the wrky18 wrky40 double mutant was found to be strongly susceptible towards the bacterial pathogen Pseudomonas syringae DC3000 expressing the effector AvrRPS4 but not against other tested Pseudomonas strains. We hypothesize that G. orontii depends on the function of WRKY18 and WRKY40 to successfully infect Arabidopsis wild-type plants while, in the interaction with P. syringae AvrRPS4, they are required to mediate effector-triggered immunity.
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

Bethke, G.; Pecher, P.; Eschen-Lippold, L.; Tsuda, K.; Katagiri, F.; Glazebrook, J.; Scheel, D.; Lee, J. Activation of the Arabidopsis thaliana mitogen-activated protein kinase MPK11 by the flagellin-derived elicitor peptide, flg22 Mol Plant Microbe Interact 25, 471-480, (2012) DOI: 10.1094/MPMI-11-11-0281

Mitogen-activated protein kinases (MAPKs) mediate cellular signal transduction during stress responses, as well as diverse growth and developmental processes in eukaryotes. Pathogen infection or treatments with conserved pathogen-associated molecular patterns (PAMPs) such as the bacterial flagellin-derived flg22 peptide are known to activate three Arabidopsis thaliana MAPKs, MPK3, MPK4 and MPK6. Several stresses, including flg22 treatment, are known to increase MPK11 expression but activation of MPK11 has not been shown. Here, we show that MPK11 activity can indeed be increased through flg22 elicitation. A small-scale microarray for profiling defense-related genes revealed that cinnamyl alcohol dehyrogenase 5 (CAD5) requires MPK11 for full flg22-induced expression. An mpk11 mutant showed increased flg22-mediated growth inhibition but no altered susceptibility to Pseudomonas syringae, Botrytis cinerea or Alternaria brassicicola. In mpk3, mpk6, or mpk4 backgrounds, MPK11 is required for embryo/seed development or general viability. Although this developmental deficiency in double mutants and the lack of or only subtle mpk11 phenotypes suggest functional MAPK redundancies, comparison to the paralogous MPK4 reveals distinct functions. Taken together, future investigations of MAPK roles in stress signalling should include MPK11 as a fourth PAMP-activated MAPK.
Publications

Kirsten, S.; Navarro-Quezada, A.; Penselin, D.; Wenzel, C.; Matern, A.; Leitner, A.; Baum, T.; Seiffert, U.; Knogge, W. Necrosis-Inducing Proteins of Rhynchosporium commune, Effectors in Quantitative Disease Resistance Mol Plant Microbe Interact 25, 1314-1325, (2012) DOI: 10.1094/MPMI-03-12-0065-R

The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an “active” NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner.
Publications

Rasche, F.; Svatoš, A.; Maddula, R. K.; Böttcher, C.; Böcker, S. Computing Fragmentation Trees from Tandem Mass Spectrometry Data Anal Chem 83, 1243-1251, (2011) DOI: 10.1021/ac101825k

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.
Publications

Eschen-Lippold, L.; Altmann, S.; Rosahl, S. DL-β-Aminobutyric Acid–Induced Resistance of Potato Against Phytophthora infestans Requires Salicylic Acid but Not Oxylipins Mol Plant Microbe Interact 23, 585-592, (2010) DOI: 10.1094/MPMI-23-5-0585

Inducing systemic resistance responses in crop plants is a promising alternative way of disease management. To understand the underlying signaling events leading to induced resistance, functional analyses of plants defective in defined signaling pathway steps are required. We used potato, one of the economically most-important crop plants worldwide, to examine systemic resistance against the devastating late blight pathogen Phytophthora infestans, induced by treatment with dl-β-aminobutyric acid (BABA). Transgenic plants impaired in either the 9-lipoxygenase pathway, which produces defense-related compounds, or the 13-lipoxygenase pathway, which generates jasmonic acid–derived signals, expressed wild-type levels of BABA-induced resistance. Plants incapable of accumulating salicylic acid (SA), on the other hand, failed to mount this type of induced resistance. Consistently, treatment of these plants with the SA analog 2,6-dichloroisonicotinic acid restored BABA-induced resistance. Together, these results demonstrate the indispensability of a functional SA pathway for systemic resistance in potato induced by BABA.
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