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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.
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

Avrova, A.; Knogge, W. Rhynchosporium commune: a persistent threat to barley cultivation Mol Plant Pathol 13, 986-997, (2012) DOI: 10.1111/j.1364-3703.2012.00811.x

Rhynchosporium commune is a haploid fungus causing scald or leaf blotch on barley, other Hordeum spp. and Bromus diandrus.TaxonomyRhynchosporium commune is an anamorphic Ascomycete closely related to the teleomorph Helotiales genera Oculimacula and Pyrenopeziza.Disease symptomsRhynchosporium commune causes scald‐like lesions on leaves, leaf sheaths and ears. Early symptoms are generally pale grey oval lesions. With time, the lesions acquire a dark brown margin with the centre of the lesion remaining pale green or pale brown. Lesions often merge to form large areas around which leaf yellowing is common. Infection frequently occurs in the leaf axil, which can lead to chlorosis and eventual death of the leaf.Life cycleRhynchosporium commune is seed borne, but the importance of this phase of the disease is not fully understood. Debris from previous crops and volunteers, infected from the stubble from previous crops, are considered to be the most important sources of the disease. Autumn‐sown crops can become infected very soon after sowing. Secondary spread of disease occurs mainly through splash dispersal of conidia from infected leaves. Rainfall at the stem extension growth stage is the major environmental factor in epidemic development.Detection and quantificationRhynchosporium commune produces unique beak‐shaped, one‐septate spores both on leaves and in culture. The development of a specific polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) has allowed the identification of asymptomatic infection in seeds and during the growing season.Disease controlThe main measure for the control of R. commune is the use of fungicides with different modes of action, in combination with the use of resistant cultivars. However, this is constantly under review because of the ability of the pathogen to adapt to host plant resistance and to develop fungicide resistance.
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

Haapalainen, M.; Engelhardt, S.; Küfner, I.; Li, C.-M.; Nürnberger, T.; Lee, J.; Romantschuk, M.; Taira, S. Functional mapping of harpin HrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization, lipid interactions and plant defence induction Mol Plant Pathol 12, 151-166, (2011) DOI: 10.1111/j.1364-3703.2010.00655.x

Harpin HrpZ is one of the most abundant proteins secreted through the pathogenesis-associated type III secretion system of the plant pathogen Pseudomonas syringae. HrpZ shows membrane-binding and pore-forming activities in vitro, suggesting that it could be targeted to the host cell plasma membrane. We studied the native molecular forms of HrpZ and found that it forms dimers and higher order oligomers. Lipid binding by HrpZ was tested with 15 different membrane lipids, with HrpZ interacting only with phosphatidic acid. Pore formation by HrpZ in artificial lipid vesicles was found to be dependent on the presence of phosphatidic acid. In addition, HrpZ was able to form pores in vesicles prepared from Arabidopsis thaliana plasma membrane, providing evidence for the suggested target of HrpZ in the host. To map the functions associated with HrpZ, we constructed a comprehensive series of deletions in the hrpZ gene derived from P. syringae pv. phaseolicola, and studied the mutant proteins. We found that oligomerization is mainly mediated by a region near the C-terminus of the protein, and that the same region is also essential for membrane pore formation. Phosphatidic acid binding seems to be mediated by two regions separate in the primary structure. Tobacco, a nonhost plant, recognizes, as a defence elicitor, a 24-amino-acid HrpZ fragment which resides in the region indispensable for the oligomerization and pore formation functions of HrpZ.
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