4.2 Regulation of the TCA cycle and associated metabolic
pathways in guard cells
The regulation of plant TCA cycle enzymes is strongly dependent on light
quality and quantity (Nunes-Nesi et al. 2013). Several lines of
evidence point to restricted metabolic fluxes through the TCA cycle in
illuminated leaves (Tcherkez et al. 2009; Gauthier et al.2010; Daloso et al. 2015b; Abadie et al. 2017;
Florez-Sarasa et al. 2019), in which different non-cyclic TCA
flux modes may contribute to maintain the oxidative phosphorylation
system (OxPHOS) (Rocha et al. 2010; Sweetlove et al.2010). The GABA shunt has been shown to be an important alternative
pathway for the synthesis of succinate, the substrate of the complex II
of OxPHOS (Nunes-Nesi et al. 2007b; Studart-Guimaraes et
al. 2007). GABA can be synthesized in the cytosol by glutamate
decarboxylase or in the mitochondria by GABA transaminases, in which
glutamate, 2-oxoglutarate or pyruvate serve as substrates (Bouché &
Fromm 2004). Thus, GABA synthesis is closely associated with the TCA
cycle, representing a hub for the C:N metabolic network (Fait et
al. 2008). Additionally, recent evidence suggests that GABA is an
important modulator of stomatal movements, acting as negative regulator
of stomatal opening (Xu et al. 2021). Our previous data
highlighted that the carbon derived from 13C-sucrose
is incorporated into GABA, but to a lesser extent than into Gln
(Medeiros et al. 2018b). Here, GABA was degraded in a
light-independent manner, which may supply OxPHOS with substrate by
supporting succinate synthesis in mitochondria. However, no13C-enrichment in GABA was observed. In parallel,
increased 13C-enrichment in Glu in illuminated guard
cells was observed. Therefore, while Glu and Gln seems to be important
sinks of the carbons derived from CO2 assimilation
mediated by both RuBisCO and PEPc, the metabolic flux from Glu/Gln to
GABA may be restricted during the dark-to-light transition as a
mechanism to allow stomatal opening. This idea is supported by the fact
that GABA is a negative regulator of ALTM9 (aluminium-activated malate
transporter 9) (Xu et al. 2021; Siqueira et al. 2021), a
key vacuolar anion uptake channel activated during stomatal opening (De
Angeli et al. 2013; Medeiros et al. 2018a).
Illumination increased the
metabolic fluxes throughout the TCA cycle and associated pathways in
guard cells. This idea is supported by the higher F13C
observed in malate, succinate, pyruvate and Glu in the light, when
compared to dark-exposed guard cells (Figure 6). Furthermore, increased
R13C in lactate and aspartate was only observed in the
light. It is noteworthy that these results were obtained in guard cells
with no K+ in the medium, given that the presence of
this ion strongly increased the 13C-enrichment in TCA
cycle metabolites, especially in fumarate and malate (Daloso et
al. 2015a). Thus, one would expect that the light and dark metabolic
differences may be higher in vivo , given that light stimulates
the influx of potassium to guard cells (Hills et al. 2012; Wanget al. 2014b c). Surprisingly, the relative content and the
R13C in fumarate increased substantially under both
dark and light conditions. The light-induced13C-enrichment in fumarate resembles previous13C-feeding experiments using13C-HCO3 (Daloso et al. 2015a,
2016b; Robaina-Estévez et al. 2017). This corroborates the facts
that fumarate is the major organic acid accumulated in the light
(Pracharoenwattana et al. 2010) and that plants with higher
fumarate accumulation have higher g s (Nunes-Nesiet al. 2007a; Araújo et al. 2011b; Medeiros et al.2016, 2017). Furthermore, fumarate emerged as an important hub for the
guard cell metabolic network during dark-to-light transition, in
agreement with previous observations in guard cells (Freire et
al. 2021). However, no evidence has hitherto indicated that fumarate is
neither the main organic acid accumulated nor can act as osmolyte under
dark conditions (Gauthier et al. 2010; Araújo et al.2011a; Cheung et al. 2014; Tan & Cheung 2020). Thus, whether the
accumulation of fumarate in dark-exposed guard cells is a mechanism to
sustain g sn and/or to store carbon skeletons for
the following light period remains unclear. Whilst the characterization
of the dynamic of g sn in plants with altered
fumarate accumulation may be sufficient to understand whether fumarate
acts as osmolyte in the dark, testing the second hypothesis will require
more sophisticated metabolic experiments to determine the pattern and
the subcellular accumulation of fumarate in guard cells during the diel
cycle.
Our results suggest that previously stored, non-labelled organic acids
are used to support the metabolic requirements of guard cell metabolism
in the light, given that the 13C-enrichment in citrate
and malate is lower than in metabolites of the following steps of the
pathway such as fumarate, succinate and Glu. Genome scale metabolic
modelling suggests that citrate is the main organic acid accumulated in
leaf vacuoles during the night period, which is released and used as
substrate for Glu synthesis in the light (Cheung et al. 2014). A
similar model build specifically for guard cell metabolism predicted
that malate accumulates at high rate in the vacuole of guard cells,
especially when K+ accumulation was restricted by the
model (Tan & Cheung 2020). Taken together, modelling and13C-labelling results indicate the importance of
previously stored organic acids to support the TCA cycle and Glu
synthesis in illuminated guard cells, resembling the mechanism of TCA
cycle regulation observed in leaves (Sweetlove et al. 2010;
Nunes-Nesi et al. 2013; da Fonseca-Pereira et al. 2021).