Modulation of metabolomic profile
Here, ultra-high-performance liquid chromatography
quadrupole-time-of-flight high-resolution mass spectrometry (UHPLC-QTOF)
and annotation in publicly available databases and large metabolite
groups were conducted to obtain wide metabolome coverage. We applied
UHPLC-QTOF-based untargeted metabolomic profiling of crude extracts to
assess relative differences in the vegetative stage (43 DAT) and
reproductive stage (131 DAT) leaf metabolite profiles between inoculated
and uninoculated plants. A principal component analysis (PCA) explained
79% of the overall variance. The PCA score plot (Figure 1) showed two
main clusters accounting for the vegetative and reproductive stages,
respectively. Within each cluster, good separation was achieved between
the metabolomic profiles of leaves from inoculated plants and those of
leaves harvested from the uninoculated control. This finding was
anticipated as the metabolism dramatically shifts as the plant
transitions from the vegetative to reproductive phases. This outcome was
relevant as PCA provides unsupervised descriptions of
relatedness/unrelatedness. We ran a t -test ANOVA (P <
0.01) to identify differentially accumulated metabolites at each plant
growth stage. This analysis disclosed > 466 annotated
metabolites (Supplementary Tables 2 and 3) that had significantly
changed between the vegetative and reproductive stages. Of these, 327
were common to both the first and second sampling points (Figure 2). In
contrast, 68 and 71 metabolites differentially accumulated during the
vegetative and reproductive stages, respectively (Figure 2). The
interactions between microbial inoculants and plants are complex.
Nevertheless, metabolomics effectively included the metabolic responses
and mechanisms involved in the plant-microbe interactions. As 70% of
the metabolites were common to the vegetative and reproductive
phenological stages (Figure 2), biostimulant-mediated shifts in the leaf
metabolome were recorded for both of them. On the other hand, certain
stage-specific responses could be identified as well.
To clarify and visualise the variations between metabolic profiles at
the vegetative and reproductive stages, we performed a chemical
enrichment analysis using ChemRICH (Figure 3; Tables 4 and 5) and
plotted the output by MetaMapp Cytoscape (Figure 4) (Barupal & Fiehn,
2017). Most of the significantly upregulated and downregulated
metabolites (fold-change values ≤ 0.5 and ≥ 1.5, respectively; P ≤ 0.01)
had a wide range of functions including growth stimulation, antifungal
activity, pathogen resistance, energy sources, and secondary signalling
cofactors.