Metabolomics

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.

The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant.

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.

During a compatible interaction, the sebacinoid root‐associated fungi Piriformospora indica and Sebacina vermifera induce modification of root morphology and enhance shoot growth in Arabidopsis thaliana.The genomic traits common in these two fungi were investigated and compared with those of other root‐associated fungi and saprotrophs. The transcriptional responses of the two sebacinoid fungi and of Arabidopsis roots to colonization at three different symbiotic stages were analyzed by custom‐designed microarrays.We identified key genomic features characteristic of sebacinoid fungi, such as expansions for gene families involved in hydrolytic activities, carbohydrate‐binding and protein–protein interaction. Additionally, we show that colonization of Arabidopsis correlates with the induction of salicylic acid catabolism and accumulation of jasmonate and glucosinolates (GSLs). Genes involved in root developmental processes were specifically induced by S. vermifera at later stages during interaction.Using different Arabidopsis indole‐GSLs mutants and measurement of secondary metabolites, we demonstrate the importance of the indolic glucosinolate pathway in the growth restriction of P. indica and S. vermifera and we identify indole‐phytoalexins and specifically indole‐carboxylic acids derivatives as potential key players in the maintenance of a mutualistic interaction with root endophytes.