3.3 Reduction of P-containing glycolytic intermediates in barley and other plant species under Al stress
We first conducted metabolomics of the root tips of XZ29 and XZ9 under 6-72 h of Al treatment and identified a total of 52 primary metabolites (Table S6). The temporal changes of these metabolites were measured and analyzed with K-means clustering (Figure 4a). Most of the metabolites in Clusters 1, 2 and 3 showed a gradual decrease during 24-72 h of Al treatment. Interestingly, most of these metabolites belong to the glycolysis pathway and its branches. The decrease in the five P-containing glycolytic intermediates in Cluster 1 (glucose-6-phosphate, Glu-6-P; fructose-6-phosphate, Fru-6-P; 3-phosphoglyceric acid, 3-PGA; 2-phosphoglyceric acid, 2-PGA; phosphoenolpyruvate, PEP) were larger and occurred earlier in XZ9 than in XZ29 (Figure 4a, 4b). Many of the metabolites downstream of glycolysis (inositol, galactinol, galactose, serine, cysteine, leucine, isoleucine, threonine and valine) showed similar patterns in XZ29 and XZ9, but the relative changes were greater in XZ9 than for XZ29, especially at 72 h (Figures 4b, S7). Moreover, highly significant correlations were detected between the glycolytic intermediates and their downstream metabolites (Figure S8). In order to understand the transcriptional regulation of the glycolysis pathway in barley in response to Al stress, we examined the expression of key genes in this pathway in XZ29 and XZ9 under 0.5h – 24h of Al treatments. The up-regulations of Al-induced expression of most glycolysis pathway genes were found earlier in XZ9 (6 h) than in XZ29 (12 h) (Figure 4b). The opposing trends in gene expression and metabolite levels indicated that the inhibition of glycolysis during Al stress may not occur at the transcriptional level.
To further investigate whether the response of glycolytic intermediates to Al stress is evolutionarily conserved in land plants, we performed additional metabolome analysis in rhizoids of a liverwort (Marchantia polymorpha ) and roots of a gymnosperm (Ginkgo biloba ), a eudicot (Medicago truncatula ), and two monocots (Zea mays and Triticum aestivum ). Al-induced reduction of Glu-6-P and Fru-6-P were observed in roots of the examined plant species, except for the rhizoids of liverwort (Figure 4c). Moreover, Al-induced changes in sucrose, inositol, and threonine were detected in all tested plant species (Figure 4c), indicating an early evolution of these metabolites in Al response of land plants.