Integrating guard cell metabolomics data with stomatal speediness parameters
We next created a correlation-based network by combining guard cell metabolite profiling data with stomatal speediness parameters (Sl max and t50%). We further integrated this with the previous multivariate analysis in order to obtain a systemic view of the physiological and metabolic alterations induced by NtSUS2 that could potentially modulate stomatal speediness. Sixteen metabolites were found to be positively and negatively correlated to Sl max and t50%, respectively (Figure 8A). Five of those metabolites (Ser, Ala, citrate, aconitate and mannitol) are also present in the VIP scores and S-plots of PLS-DA and orthoPLS-DA models carried out using relative guard cell metabolic changes during dark-to-light transition (Figure 8A). These results indicate that these metabolites are great contributors to the discrimination between the genotypes observed in both PLS-DA and orthoPLS-DA models and are positively correlated with the speed of light-induced stomatal opening. By contrast, trehalose and adipic acid were negatively and positively correlated to Sl max and t50%, respectively (Figure 8A), indicating a negative correlation with the light-induced stomatal opening speediness. Interestingly, the metabolites that are positively and negatively related to the speed of light-induced stomatal opening have respectively lower and higher relative metabolic changes in both transgenic lines when compared to the WT during dark-to-light transition (Figure 8B). Comparing the dark-to-light transition in each genotype, it is interesting to note that aconitate increased significantly only in the WT, while adipic acid increase in all genotypes and mannitol decreased in L13 (Figure 8C). The higher accumulation of aconitate in WT and the lower relative changes in citrate observed in L3 further suggest that sucrose breakdown is important to feed the C6-branch of the TCA cycle. Collectively, our results suggest that NtSUS2 is important for guard cell metabolism and, by consequence, for the regulation of light-induced stomatal opening and WPT.