Untargeted metabolomics profiling of Arabidopsis thalianaroots by UHPLC/QTOF-MS
The untargeted metabolomics approach was used to investigate the effect of heat and drought stresses and their combination on the metabolomic profile of A. thaliana roots. The metabolomics raw data are submitted to a public repository (https://www.ebi.ac.uk/metabolights/MTBLS6421). This approach allowed us to putatively annotate more than 1100 compounds, further used to infer the biological processes involved in plant stress responses. The list of the annotated metabolites, with composite mass spectra and abundance, is provided as supplementary material (Supplementary Table S1).
Unsupervised hierarchical cluster analysis and supervised OPLS discriminant analysis were successfully performed to identify patterns between the conditions under investigation. The clusters produced from the heatmap based on fold-change highlighted distinct metabolomic profiles in roots, depending on the stress applied. In fact, two main clusters were generated, one featuring H and H+D and the other including the control and D (Supplementary Figure S1). These findings were corroborated by the supervised OPLS-DA, where all stress conditions separated from the control and underlined a separation of D from the other conditions (control, H and H+D) (Figure 1). The score plot also showed that the combined stress (H+D) influenced metabolic profile closer to the H and far from the control and D. Afterwards, VIP analysis was carried out to find the compounds with the highest contribution to the OPLS-DA discrimination (VIP score > 1.2 , Supplementary Table S2). Overall, phenylpropanoids and nitrogen-containing compounds, hormones, and amino acids exhibited the highest discriminant potential.
Then, Volcano Plot analysis identified 405 differential compounds significantly differing from the control (p-value < 0.05; FC ≥ 2). Despite the large number of compounds modulated in response to the stresses, only 65 metabolites overlap for all the H, D and H+D (Supplementary Table S3). Moreover, the combined treatment (H+D) presented several compounds common with H stress, indicating a hierarchical prevalence of the latter. In contrast, D alone provoked a distinct response, as suggested by OPLS-DA. Figure 2 depicts the modulation of the biosynthetic pathways in response to the specific stresses resulting from differential compounds accumulation. Overall, all the individual and combined stress had a detrimental effect on root metabolism, particularly specialised metabolism. In agreement with multivariate analysis, roots exposed to H stress showed a comparatively stronger modulation of specialised metabolism. In general terms, several plant defense mechanisms were activated in a stress-specific manner. Despite the general decrease of specialised metabolites, glucosinolates increased in response to stress (H, D, H+D). In this sense, phytoalexins related to glucosinolates pathways (i.e. indole-3-carboxaldehyde and indole-3-carbinol) and glutathione-related compounds were also modulated under combined stress conditions (H+D).
Phenylpropanoids decreased in response to H and, to a lesser extent, to H+D while increasing in response to D, which promoted the accumulation of flavonoids and anthocyanins. Regarding terpenes, H decreased these compounds while H+D and D alone elicited this pathway, including their precursors. Although the detrimental effect of H on plant metabolism, plants exposed to H increased in phospholipids while sterols accumulated in the two other stress conditions considered (D, H+D).
The stresses also influenced the phytohormone profile, with distinctive modulations as a function of the condition considered (Figure 3). In more detail, cytokinins (CKs) were accumulated by the combined stress (H+D) and D stress. Moreover, in H, CKs and abscisic acid (ABA) synthesis were negatively modulated, while gibberellins (GAs) and jasmonates (JAs) were down accumulated under water deficit conditions (D). Ascorbate-related compounds were also modulated in plants exposed to the stress, with dehydroascorbate accumulated under H and D stress.