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.