1. Introduction
Guard cell metabolism has been studied for over a century. From the starch-sugar conversion hypothesis to the discovery that inorganic ions such as potassium (K+) modulate guard cell osmotic potential during light-induced stomatal opening (Lloyd 1908; Fischer 1968), the understanding of how guard cell metabolism regulates stomatal movements has evolved dramatically over the decades (Granot & Kelly 2019). Breakdown of sugars, starch and lipids within guard cells are important mechanisms to sustain the speed of light-induced stomatal opening (Antunes et al. 2012; Ni 2012; Daloso et al.2015a, 2016b; Horrer et al. 2016; McLachlan et al. 2016; Medeiros et al. 2018b; Flütsch et al. 2020b). Given the sink characteristics of guard cells (Hite et al. 1993; Ritteet al. 1999), the import of ATP, organic acids and sugars from mesophyll cells contributes to the stomatal opening upon illumination (Hedrich & Marten 1993; Araújo et al. 2011b; Kelly et al.2013; Wang et al. 2014a, 2019; Lugassi et al. 2015; Medeiros et al. 2016; Antunes et al. 2017; Flütschet al. 2020a). Thus, light mediates stomatal opening by both, perception directly in the guard cells (Kinoshita et al. 2001; Wang et al. 2010; Ando & Kinoshita 2018) and indirectly through signals derived from mesophyll cells (Mott 2009; Fujita et al.2019; Flütsch & Santelia 2021). For the latter mode the respiratory activity seems to be of great importance (Nunes-Nesi et al.2007a; Araújo et al. 2011b; Vialet‐Chabrand et al. 2021). Taken together, these works have substantially improved our understanding of light-induced changes in guard cell metabolism. However, light is just one out of several stimuli that affects stomatal aperture and how guard cell metabolism operates in the dark remains unexplored.
As the main energy source for plants, folial illumination activates photosynthesis alongside other mechanisms that sustain plant metabolism (Buchanan 2016). Photosynthetic ATP production and CO2assimilation mediated by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) are pivotal for the functioning of photoautotrophic cells. This is especially true for C3 and C4 plants, in which the vast majority of carbon assimilation occurs in the light period (Hohmann-Marriott & Blankenship 2011; Matthews et al. 2020). Additionally, given the capacity of plant cells to store and transport photoassimilates, the remobilisation of photosynthesis-derived products such as starch and sucrose is an important mechanism that sustains both metabolism and growth during the night period (Sulpice et al. 2014; Apeltet al. 2017; Mengin et al. 2017). By contrast to C3- and C4-plants, a special group of plants that evolved Crassulaceae Acid Metabolism (CAM) can also fix CO2 through the activity of phosphoenol pyruvate carboxylase (PEPc) in the dark (Ranson & Thomas 1960; O’Leary et al. 2011). In these plants, the stomata open in the dark and close in the light. Whilst the stomatal opening in the dark favours the PEPc-mediated CO2 fixation, stomatal closure in the light avoids water loss, leading to the highest water use efficiency (WUE) observed in plants. Curiously, several C3 and C4 plants maintain a substantial transpiration stream during the night period (Caird et al. 2007; Costa et al. 2015). Furthermore, nocturnal stomatal conductance (g sn- the rate of stomatal opening in the night) was positively correlated with relative growth rate in a multi-species meta-analysis (Resco de Dios et al. 2019). However, the regulation ofg sn remains insufficiently understood (Gagoet al. 2020). Given the role of guard cell metabolism in regulating stomatal opening (Daloso et al. 2017; Lawson & Matthews 2020), unveiling how guard cell metabolism operates in the dark will be a prerequisite for a mechanistic understanding ofg sn regulation.
Radiotracer experiments suggest that guard cells can incorporate CO2 in the dark at higher rates than mesophyll cells (Gotow et al. 1988). Furthermore, 13C-labelling results using mesophyll and guard cell protoplasts highlight that guard cells have faster and higher 13C-incorporation into malate under illuminated conditions (Robaina-Estévez et al.2017). These results suggest a high PEPc activity in guard cells under either dark or light conditions. Indeed, recent13C-positional labelling analysis confirmed that guard cells are able to assimilate CO2 in the dark, as evidenced by the increases in the 13C-enrichment in the carbon 4 (4-C) of malate (Lima et al. 2021), which is derived from PEPc activity (Abadie & Tcherkez 2019). High dark CO2 fixation rates are typically found in CAM cell types (Cockburn 1983). However, the idea that guard cells have CAM-like metabolism is not fully supported by transcriptome studies (Wanget al. 2011; Bates et al. 2012; Bauer et al. 2013; Aubry et al. 2016). It remains therefore unclear whether the metabolic photosynthetic mode of guard cells most closely resembles that of C3, C4 or CAM cells. Alternatively, guard cells might not strictly fit into any of these classifications. If this is the case, a specific mode of regulation may be predicted in these cells. This idea is supported by the finding that illumination triggers specific responses observed in guard cells, but not in mesophyll cells, such as the degradation of lipids, starch and sugars and the activation of glycolysis (Hedrich et al. 1985; Zhao & Assmann 2011; Dalosoet al. 2015a, 2016b; Horrer et al. 2016; McLachlanet al. 2016; Robaina-Estévez et al. 2017; Medeiroset al. 2018b; Flütsch et al. 2020b). Additionally, the metabolic fluxes throughout the TCA cycle and associated metabolic pathways seems to be differentially regulated in illuminated guard cells, when compared to mesophyll cells (Daloso et al. 2017; Robaina-Estévez et al. 2017). These observations raise the question how light influences the regulation of key metabolic pathways in guard cells. Here we addressed this question using13C-HCO3 labelling in guard cells subjected to either dark or light conditions. To establish cell type-specific metabolic regulation and obtain an advanced picture of the metabolic flux landscape of guard cells in the light we integrate our current data with those of illuminated Arabidopsis (Arabidopsis thaliana L.) rosettes following provision of13CO2 and with illuminated Arabidopsis guard cells following provision of 13C-sucrose (Szecowka et al. 2013; Medeiros et al. 2018b).