3.4 Fumarate is equally and strongly labelled under both dark and light conditions, but succinate labelling is increased by light exposure
We investigated the R13C in the fragments m/z245 of malate, m/z 245 of fumarate and m/z 247 of succinate, that contain the four carbons (1,2,3,4-C) of these metabolites. Increased R13C in fumarate m/z 245 under both dark and light conditions was observed (Figure 4) and no difference in the R13C of this fragment was noticed after 60 min of labelling between dark and light conditions (Figure 6). By contrast, light exposure leads to higher R13C in succinate m/z 247, when compared to dark-exposed guard cells (Figure 6). Relative isotopologue analysis (RIA) suggests the incorporation of four 13C into succinate under light conditions (Supplemental Figure S4) (see red circles at the Figure 6). No significant 13C-enrichment in malate m/z 245 was observed in either light or dark conditions, when compared to the time 0 of the experiment (Figure 4). However, the R13C-enrichment in malate m/z 245 was higher after 60 min in the light, when compared to guard cells kept in the dark (Figure 6). Intriguingly, the labelling in malate does not match those observed in fumarate and succinate. We have previously shown that only the 4-C of malate was labelled under either dark or light conditions (Lima et al. 2021), which contributes to explain the lack of significant labelling in malate m/z 245. The labelling in malate could be also interpreted by a dilution in the13C-labelling from previously stored, non-labelled malate, which corroborates the role of malate as a counter-ion of potassium (K+) in the vacuole of guard cells (Outlaw & Lowry 1977; Tallman & Zeiger 1988; Talbott & Zeiger 1993).