Activation of camalexin biosynthesis in Arabidopsis thaliana in response to perception of bacterial lipopolysaccharides: a gene-to-metabolite study

Abstract

Lipopolysaccharides (LPS), as lipoglycan microbe-associated molecular pattern molecules, trigger activation of signal transduction pathways involved in defence that generate an enhanced defensive capacity in plants. The transcriptional regulation of the genes for tryptophan synthase B, TSB1, and the cytochrome P450 monooxygenases CYP79B2 and CYP71B15, involved in the camalexin biosynthetic pathway, were investigated in response to LPS treatment. GUS-reporter assays for CYP71B15 and CYP79B2 gene promoter activation were performed on transgenic plants and showed positive histochemical staining in response to LPS treatment, indicating activation of the promoters. Quantitative PCR revealed that transcripts of TSB1, CYP79B2 and CYP71B15 exhibited differential, transient up-regulation. TSB1 transcript levels were up-regulated between 6 and 9h after LPS-induction, while CYP71B15 and CYP79B2 both exhibited maxima at 12h. To obtain information on the gene-to-metabolite network, the effect of the transcriptome changes on the metabolome was correlated to camalexin production. Increases in camalexin concentration were quantified by ultra pressure liquid chromatography-mass spectrometry and both absorbance spectra and elemental composition confirmed its identity. The concentrations increased from 0.03 to 3.7μgg(-1) fresh weight over a 24-h time period, thus indicating that the up-regulation of the biosynthetic pathway in response to LPS was accompanied by a time-dependent increase in camalexin concentration. Metabolomic analysis through principal component analysis-derived scores plots revealed clusters of sample replicates for 0, 6, 12, 18 and 24h while loadings plots for LPS data identified camalexin as a biomarker that clearly demonstrated the variability between samples.