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).