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).