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).