3.5 Glutamate synthesis in illuminated guard cells likely depends on PEPc CO2 assimilation and previously stored citrate
Illumination leads to increased R13C in glutamatem/z 156 (Figure 4). This fragment contains the 2,3,4,5-C of the Glu backbone, which are derived from the TCA cycle (2,3-C) and from pyruvate dehydrogenase (PDH) activity (4,5-C) (Abadie et al.2017). We next investigated the sources of carbon for Glu synthesis in illuminated guard cells by analysing both R13C and RIA data of Glu and metabolites of, or associated to, the TCA cycle. Increased R13C in pyruvate m/z 174 (1,2,3-C) was only observed in the light (Figure 4). However, increases in the M3 isotopologue (m/z 177) of pyruvate was observed at 10 min of labelling in dark-exposed guard cells, indicating the incorporation of three carbons labelled at this time point (Supplemental Figure S5). After 60 min of labelling, the R13C in pyruvate was 2.5-fold higher in the light, when compared to dark-exposed guard cells (Figure 6). No substantial difference was noticed in the intensity of citrate isotopologues of the m/z 273 fragment (1,2,3,4,5-C) in both dark and light samples over time. Although a slight but significant increase in the intensity of m/z 274 was noticed (Supplemental Figure S6), this neither led to an increased R13C over time in the dark nor in the light (Figure 4). Furthermore, no difference in the R13C of m/z 273 between dark-exposed and illuminated guard cells after 60 min of labelling was observed (Figure 6). This fragment contains the 1,2,3,4,5-C of the citrate backbone (Okahashi et al. 2019). Interestingly, the labelling in citrate does not match those observed in Glu. RIA data of the fragmentm/z 156 (2,3,4,5-C) indicates that four 13C were incorporated into Glu after 60 min in the light, as evidenced by the increases in the isotopologues M1-M4 (m/z 157-160) of this fragment (Supplemental Figure S7). This leads to a higher R13C in Glu in the light, when compared to dark-exposed guard cells (Figure 6). These results suggest that the carbon labelled in citrate is probably derived from PEPc activity (Abadie et al. 2017) and that the labelling in citrate is diluted by the incorporation of stored, non-labelled compounds, as previously suggested in leaves (Cheung et al. 2014).
Given that Glu could be synthesized by the activity of both Ala and Asp aminotransferases (AlaAT and AspAT) and degraded toward GABA synthesis, we additionally investigated the 13C-enrichment in these metabolites. An increased R13C in Ala was observed in both conditions, whilst Asp was only labelled in the light, when compared to the time 0 of the experiment (Figure 4). However, no difference in the fractional 13C-enrichment (F13C) of these metabolites between dark and light conditions after 60 min of labelling was observed (Figure 6). No13C-enrichment in GABA m/z 174 was observed (Figure 6). However, assessment of recent data from Arabidopsis guard cells fed with 13C-sucrose (Medeiros et al.2018b), showed an increased F13C in this metabolite during dark-to-light transition (Supplemental Figure S8). Taken together, these results suggest that whilst both AlaAT and AspAT might contribute to the synthesis of Glu, the reactions catalysed by these enzymes do not fully explain the labelling found in Glu. It seems likely that Glu synthesis in illuminated guard cells mostly depends on carbons from both PEPc-mediated CO2 assimilation and previously stored citrate.