Unveiling the dark side of guard cell metabolism

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO₂ assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO₂ assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a¹³C-HCO₃ labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO₂ assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the ¹³C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the ¹³C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the ¹³C-enrichment in pyruvate, succinate and glutamate. Only one ¹³C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO₂ assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO₂ assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.