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.