Untargeted metabolomics
The comprehensive exudate profiling of the different maize root types and zones exposed to single and combined abiotic stress was investigated using UHPLC-QTOF-MS untargeted metabolomic analysis. This approach allowed the putative annotation of 337 compounds, most of which belonged to phenylpropanoids and amino acids (Supplementary material 2). We solely found slight differences driven by root type or root zone (Supplemental material 3. However, the naïve HCA analysis showed a separation of exuded metabolic profiles according to the stress treatments (Figure 3). Therefore, we focused our analysis on understanding the differences among treatments. This latter HCA unsupervised model depicted two main clusters, separating control and drought-stressed plants from plants exposed to heat and combined stress, suggesting that temperature had a hierarchically stronger effect on exudate composition than drought. The supervised OPLS-DA further confirmed this separation as a function of the stress applied (Figure 4). The first latent vector allowed separating control plants and those exposed to drought from heat- and combined-stress plants. However, despite the closeness of exudates profile between control and drought-stressed plants, the second latent vector indicates that also the soil water content influenced exudate signatures (Figure 4). The VIP analysis highlighted the most discriminant compounds involved in OPLS-DA score plot separation (Supplementary material 4). The features having a VIP score > 1 were considered as markers. Among these compounds, phenylpropanoids appeared as the main class of compounds, possessing the highest VIP score (Supplementary material 4). Nevertheless, several organic acids and amino acids were also highlighted as VIP markers.
To further investigate the exudation pattern as a function of the stress, the volcano analysis was performed, to identify differential metabolites elicited under stress, compared to non-stress conditions. The differential metabolites for each treatment are provided in Supplementary material 5. According to the multivariate statistics (Figure 3 and Figure 4), plants exposed to heat and combined stresses presented the largest differences compared to control plants, pointing out 33 and 39 metabolites, respectively (supplementary material 5).
Venn analysis (Figure 5) shows that abiotic stress conditions shaped the exudation of compounds compared to the control conditions. Although several compounds are down-exuded in the presence of the stresses, the stress-treated plants presented a more pronounced up-exudation of compounds, especially under combined stress. Indeed, in front of 18 down-exuded metabolites, a total of 37 compounds were up-exuded, with 13 being observed in combined stress (Figure 5). In detail, all the stressed plants showed that among the shared compounds, hispidulin was strongly repressed, while 4-hydroxycoumarin, citramalic acid and malonic acid strongly increased (supplementary material 5).
Table 1 reports the specific and shared down- and up-exuded compounds in presence of single and combined stress. Drought specifically modulated the exudation of phenolic compounds. In particular, the water shortage repressed the exudation of the phenolic acid protocatechuic acid and dihydroisocoumarin mullein, but promoted the exudation of two conjugated flavonoids and the phytoalexin resveratrol (Table 1). The conjugated flavonoid kaempferol 3-O -(6”-acetyl-galactoside)7-O -rhamnoside was the only metabolite shared with the combined stress (Table 1). Similarly, heat stress largely promoted the exudation of phenolic compounds. In more detail, plants exposed to heat stress reduced the exudation of L-ascorbate, bisdemethoxycurcumin and apigenin 7-O -(6”-malonyl-apiosyl-glucoside) but increased those of 4-vinylphenol, luteolin 7-O -(2-apiosyl-6-malonyl)-glucoside, malate, 2,4-dihydroxybenzoic acid and benzoic acid (Table 1). Differently to the drought stress, the heat shared more metabolites with the combined stress: 4 and 8 down- and up-exuded, respectively (table 1).
Combined stress triggered a distinct exudation pattern, compared to the control or individual stresses. In this regard, 13 metabolites were up-exuded solely in the presence of the combined stress, most of which belonged to phenolic compounds. Among these, several flavonoids, the hydroxycinnamic acid trisinapoylgentiobiose, alk(en)ylresorcinol and nonadecenylresorcinol were identified. Moreover, palmitic acid, tryptophan, sinapine and the xanthophyll lutein could be highlighted under combined stress conditions (Table 1). A reduced exudation of alpha-aminoadipic acid, sorgolactone and 6-geranylnaringenin was also specifically observed in combined stress (Table 1).