Guard cell metabolic alterations induced by NtSUS2 silencing
We have carried out a metabolite profiling analysis in guard cells of WT, L3 and L13 harvested in the dark and 120 min after the transition to the light. Partial least square discriminant analysis (PLS-DA) and orthogonal PLS-DA (orthoPLS-DA) were performed using the relative metabolic changes observed in each genotype during dark-to-light transition. Both PLS-DA and orthoPLS-DA indicate that reduced guard cellNtSUS2 expression substantially alter the relative metabolic changes during dark-to-light transition, given that both L3 and L13 were clearly separated from WT by the first components (Figures 6A,B; S5A,B). Scatter plots (S-plots) from the orthoPLS-DA and variable importance in projection (VIP) scores of the PLS-DA models demonstrate that several metabolites pertaining to the groups of carbohydrates, amino acids and tricarboxylic acid (TCA) cycle intermediates were responsible for the discrimination observed among the genotypes (Figures 6C,D; S5C,D). Amino acids such as serine and alanine and the TCA cycle intermediates citrate, isocitrate, aconitate and fumarate have increased relative content whilst adipic acid and trehalose have lower relative content in WT, when compared to the transgenic lines after dark-to-light transition (Figures 6C,D; S5C,D).