4.1 On the complex interplay among the metabolic pathways that regulate the homeostasis of sugars and organic acids in guard cells
Stomata are important regulators of water use efficiency (WUE) in illuminated leaves (Brodribb et al. 2019). Furthermore, a recent growing body of evidence suggests that nocturnal stomatal conductance (g sn) plays an important role in WUE regulation (Vialet‐Chabrand et al. 2021; McAusland et al. 2021). However, whilst the signalling pathways that regulate stomatal opening in the light have been widely investigated (Inoue & Kinoshita 2017), the mechanisms that regulate g sn remain unknown. Here, we provide insight as to how guard cell metabolism may contribute to the regulation of g sn. Given that the trends observed in both metabolite level and 13C-enrichment are similar between dark-exposed and illuminated guard cells, it could be that similar mechanisms regulate both g sn andg s. This idea is supported by the fact that sucrose breakdown and fumarate synthesis, two mechanisms that support stomatal opening in the light (Daloso et al. 2015a; Medeiroset al. 2016, 2017, 2018b; Granot & Kelly 2019; Flütsch et al. 2020b), were observed under both dark and light conditions. However, it is important to highlight that the metabolic changes were generally more pronounced in illuminated guard cells. Light exposure may contribute to the activation of several other stomatal regulatory mechanisms in guard cells, given the degradation of starch and lipids in illuminated guard cells (Horrer et al. 2016; McLachlan et al. 2016; Flütsch et al. 2020b). Thus, the separation observed in the PCA at 60 min of labelling and the differences in the metabolic networks between dark-exposed and illuminated guard cells could explain the need for more dramatically changes in illuminated guard cells, which would ultimately lead to higher g s values, when compared to g sn. This idea is further supported by the higher degradation rate of sugars observed in the light, which supports findings in which reduced sucrose cleavage capacity of guard cells severely compromises light-induced stomatal opening (Antuneset al. 2012; Ni 2012; Freire et al. 2021).
Reverse genetic studies have indicated that alteration in sugar homeostasis in guard cells affects stomatal behaviour (reviewed in Daloso et al. , 2016a; Flütsch & Santelia, 2021). Additionally, reduced photosynthetic activity in guard cell chloroplasts has been demonstrated to disrupt light-induced stomatal opening (Azoulay-Shemeret al. 2015). Although this can been attributed to changes in the cofactor metabolism of ATP and NADPH (Roelfsema et al. 2006; Wanget al. 2014a), reduced plastidial photosynthetic activity may also compromise sugar homeostasis in guard cells. Indeed, the R13C into sugars was higher in the light, evidencing that the RuBisCO-mediated CO2 assimilation contribute to sugar synthesis in illuminated guard cells. The13C-labelling incorporation into sugars in the dark suggests that gluconeogenesis is active in guard cells. This corroborates the high 13C-enrichment observed in the 3,4-C of glucose under either dark or light conditions (Lima et al. 2021), which are proposed to be the glucose carbons preferentially labelled by gluconeogenesis (Leegood & ap Rees 1978; Beylot et al. 1993). These results highlight that gluconeogenesis may be another metabolic pathway that contributes to sugar homeostasis in guard cells, an elusive source of carbon for sugar synthesis in guard cells that has long been debated (Willmer & Dittrich 1974; Outlaw & Kennedy 1978; Talbott & Zeiger 1998; Zeiger et al. 2002; Outlaw 2003; Vavasseur & Raghavendra 2005; Daloso et al. 2016a).
Relative isotopologue analysis indicates that three13C were incorporated into pyruvate in both dark-exposed and illuminated guard cells, as evidenced by the significant increases in pyruvate m/z 177 after 10 min of exposure to continuous dark or after dark-to-light transition (Supplemental Figure S5). The labelling in pyruvate in the light might occurs by a combination of 13C derived from both RuBisCO and PEPc CO2 assimilation, while the labelling in this metabolite in the dark suggests the activity of phosphoenol pyruvate carboxykinase (PEPCK) and/or malic enzyme (ME), in which labelled OAA and malate would be rapidly converted into PEP and pyruvate, respectively. Additionally, glycolysis and the activity of pyruvate kinase (PK), that converts PEP to pyruvate, could also contribute to pyruvate labelling. Given the labelling observed in sugars in the dark, it seems that the carbon assimilated by PEPc is used to create a substrate cycle between gluconeogenesis and glycolysis, allowing the circulation of carbon between sugars and organic acids without importantly loss of assimilated carbon. Thus, PEPc activity would be important to re-assimilate the CO2 lost by several decarboxylation reactions that occurs in chloroplast, mitochondria and cytosol (Sweetlove et al. 2013). Indeed, previous modelling results suggest that the flux of CO2from the chloroplast to the cytosol is 17-fold higher in guard cells than mesophyll cells and is largely re-assimilated by PEPc in the cytosol (Robaina-Estévez et al. 2017). According to this model, the carbon assimilated by PEPc is transported back to the chloroplast as malate, resulting in a net production of NADPH (Robaina-Estévez et al. 2017). These results collectively suggest that PEPc activity is important for both the carbon re-assimilation and the homeostasis of sugars and organic acids in guard cells. The maintenance of a flux of carbon between sugars and organic acids (gluconeogenesis and glycolysis) could be a mechanism to rapidly provide carbons for starch synthesis or for the TCA cycle and associated pathways during stomatal closure and opening conditions, respectively (Outlaw & Manchester 1979; Medeiroset al. 2018b).