Untargeted metabolomics aims to gather information on as many metabolites as possible in biological systems by taking into account all information present in the data sets. Here we describe a detailed protocol for large-scale untargeted metabolomics of plant tissues, based on reversed phase liquid chromatography coupled to high-resolution mass spectrometry (LC-QTOF MS) of aqueous methanol extracts. Dedicated software, MetAlign, is used for automated baseline correction and alignment of all extracted mass peaks across all samples, producing detailed information on the relative abundance of thousands of mass signals representing hundreds of metabolites. Subsequent statistics and bioinformatics tools can be used to provide a detailed view on the differences and similarities between (groups of) samples or to link metabolomics data to other systems biology information, genetic markers and/or specific quality parameters. The complete procedure from metabolite extraction to assembly of a data matrix with aligned mass signal intensities takes about 6 days for 50 samples.

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Systemic resistance induced in plants by nonpathogenic rhizobacteria is typically effective against multiple pathogens. Here, we show that root-colonizing Pseudomonas fluorescens strain SS101 (Pf.SS101) enhanced resistance in Arabidopsis (Arabidopsis thaliana) against several bacterial pathogens, including Pseudomonas syringae pv tomato (Pst) and the insect pest Spodoptera exigua. Transcriptomic analysis and bioassays with specific Arabidopsis mutants revealed that, unlike many other rhizobacteria, the Pf.SS101-induced resistance response to Pst is dependent on salicylic acid signaling and not on jasmonic acid and ethylene signaling. Genome-wide transcriptomic and untargeted metabolomic analyses showed that in roots and leaves of Arabidopsis plants treated with Pf.SS101, approximately 1,910 genes and 50 metabolites were differentially regulated relative to untreated plants. Integration of both sets of "omics" data pointed to a prominent role of camalexin and glucosinolates in the Pf.SS101-induced resistance response. Subsequent bioassays with seven Arabidopsis mutants (myb51, cyp79B2cyp79B3, cyp81F2, pen2, cyp71A12, cyp71A13, and myb28myb29) disrupted in the biosynthesis pathways for these plant secondary metabolites showed that camalexin and glucosinolates are indeed required for the induction of Pst resistance by Pf. SS101. Also for the insect S. exigua, the indolic glucosinolates appeared to play a role in the Pf.SS101-induced resistance response. This study provides, to our knowledge for the first time, insight into the substantial biochemical and temporal transcriptional changes in Arabidopsis associated with the salicylic acid-dependent resistance response induced by specific rhizobacteria.