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).