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).