Multivariate analysis of metabolome in leaves
To further study the effect of H2S and rhizobia on water
deficiency, a metabolomic analysis was performed in the control, Q8 and
Q8+NaHS -treated soybean seedlings under NW and SW conditions. The
OPLS-DA results (Fig. 11A, C and E, Fig. S2A, B, Fig. 12A, B) showed
different responses in levels of metabolites by leaves of the three
treatments to water stress. The importance of metabolites in sample
discrimination associated with difference treatments were easily visible
in the volcano plot which combined univariate and multivariate analyses
(−log (P ) obtained in the two-way ANOVA was plotted against the
loading (pcorr) along axis (Fig. 11B, D, F). As can be
seen from the volcano plot, in the control, SW condition resulted in
more obvious metabolic response than NW condition (Fig. 11B), while the
differential metabolites were less changed under the Q8 treatment (Fig.
11D). In addition, Q8+NaHS treatment seemed to have more up-regulation
metabolites under SW conditions than under NW condition (Fig. 11F). The
metabolites in the leaves were changed depending on the Q8 and Q8+NaHS
treatments under water deficiency (Table 1). Our results showed that
fatty acyl metabolites including methyl furfuracrylate and 2-Octenedioic
acid synthesis were appreciably regulated by control treatment under SW
condition. In addition, under SW condition, fatty acyl metabolites and
isoflavone metabolites (Biochanin A) were sharply influenced by Q8
treatment. Furthermore, Q8+NaHS treatment regulated more metabolites
than control and Q8 treatments under water deficiency. We focused on
separate analysis of lipid metabolism changes between different
components (Table S2), and based on P ≤0.05, Q8+NaHS treatment
significantly increased up-regulation metabolites compared with control.
Furthermore, the results showed that the addition of rhizobia
appreciably affected the changes of metabolites under NW condition (Fig.
S2C, D, Table S4). In SW condition, control/Q8 treatment led to a more
distinct metabolic response than treatments with Q8/Q8+NaHS (Table 2).
We could visually observe the change of metabolites under different
treatments by the volcano plot (Fig. 12C, D). Under SW condition, there
were 37 different metabolites based on P ≤0.001 in the comparison
between control and Q8 treatments (Table 2). Among them, we separately
analyzed the composition of lipids among different treatments (Table
S5). The number of up-regulated lipid metabolites were remarkably
increased between control and Q8 treatments under SW condition. Under SW
condition, the up-regulated lipid metabolites were similar to the NW
condition between Q8 and Q8+NaHS treatments. It is worth noting that
metabolomics that was indicated H2S and rhizobia
synergistically regulated lipid metabolites under SW condition,
including PE, PG, agavoside A, and dephospho-CoA in leaves, showing an
effective H2S and rhizobia synergistically regulation
and improved tolerance to water deficiency.