Development of a two‐step screening ESI‐TOF‐MS method for rapid determination of significant stress‐induced metabolome modifications in plant leaf extracts: The wound response in Arabidopsis thaliana as a case study

Abstract

To study the stress-induced effects caused by wounding under a new perspective, a metabolomic strategy based on HPLC-MS has been devised for the model plant Arabidopsis thaliana. To detect induced metabolites and precisely localise these compounds among the numerous constitutive metabolites, HPLC-MS analyses were performed in a two-step strategy. In a first step, rapid direct TOF-MS measurements of the crude leaf extract were performed with a ballistic gradient on a short LC-column. The HPLC-MS data were investigated by multivariate analysis as total mass spectra (TMS). Principal components analysis (PCA) and hierarchical cluster analysis (HCA) on principal coordinates were combined for data treatment. PCA and HCA demonstrated a clear clustering of plant specimens selecting the highest discriminating ions given by the complete data analysis, leading to the specific detection of discrete-induced ions (m/z values). Furthermore, pool constitution with plants of homogeneous behaviour was achieved for confirmatory analysis. In this second step, long high-resolution LC profilings on an UPLC-TOF-MS system were used on pooled samples. This allowed to precisely localise the putative biological marker induced by wounding and by specific extraction of accurate m/z values detected in the screening procedure with the TMS spectra.

Transient increase in salicylic acid and its glucose conjugates after wounding in Arabidopsis leaves

“…An immediate induction of jasmonic acid with maximum accumulation 60 to 90 minutes after wounding, followed by a decline to the basal level, was seen, which is the typical induction pattern of the compound reported in Arabidopsis (Glauser et al 2008;Grata et al 2007). Thus, the specimens treated with silicon carbide particles appear to be suitable for wounding research.…”

“…It is generally observed that jasmonates are immediately induced after mechanical wounding, reach a peak around 60 minutes afterward, and then decline to the basal level. More recently, metabolic changes in jasmonates after wounding of Arabidopsis leaves were detected by metabolome analysis (Glauser et al 2008;Grata et al 2007). Following the initial response to wounding, accumulation of lignin and suberin at the wound site occurs to seal the wounded surface to impede further invasions by pathogens and fluid loss (Almagro et al 2009).…”

Spatial and Temporal Dynamics of Jasmonate Synthesis and Accumulation in Arabidopsis in Response to Wounding

“…Metabolomics-A non-targeted metabolomic approach using UPLC-TOF-MS was devised to search for wound-inducible metabolites in A. thaliana (24). The profiles of wounded and unwounded WT leaves (Col-0 accession) as well as those of the JA synthesis mutant opr3 (20) in the Wassilewskija background were compared for the detection of jasmonates.…”

Rice Metabolomics

“…There have been many reports on the application of PCA to metabolomic data [7,12,19,54,65,71,75,93,94,95]. In addition, several statistical analytical methods have been used for analysis of metabolomic datasets; for example, hierarchical cluster analysis (HCA) [39,78], partial least squares discriminant analysis (PLS-DA) [52,65], and batch-learning self-organizing map (BL-SOM) [43,44,54]. Depending on the objective of each study, the most appropriate statistical analytical method should be exploited to analyze the metabolomic data.…”