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 used 13C-HCO3 labelling of 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 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 into 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 collectively suggest 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.

Recent results suggest that metabolism-mediated stomatal closure mechanisms are important to regulate differentially the stomatal speediness between ferns and angiosperms. However, evidence directly linking mesophyll metabolism and the slower stomatal conductance (gs) in ferns is missing. Here we investigated the effect of exogenous application of abscisic acid (ABA), sucrose and mannitol on gs kinetics and carried out a metabolic fingerprinting analysis of ferns and angiosperms leaves harvested throughout a diel course. Ferns stomata did not respond to ABA in the time period analysed. No differences in the relative decrease in gs was observed between ferns and the angiosperm following provision of sucrose or mannitol. However, ferns have slower gs responses to these compounds than angiosperms. Metabolomics analysis highlights that ferns have higher accumulation of secondary rather than primary metabolites throughout the diel course, with the opposite being observed in angiosperms. Our results indicate that metabolism-mediated stomatal closure mechanism is conserved among ferns and angiosperms and that the slower stomatal closure in ferns is associated to a reduced capacity to respond to mesophyll-derived sucrose and to a higher carbon allocation toward secondary metabolism, which likely modulates both photosynthesis-stomatal movements and growth-stress tolerance trade-offs.

The understanding of the dynamics of stomatal movements has increased substantially through genetic manipulation of plant metabolism either at the whole plant level or specifically in guard cells. However, the regulation of stomatal speediness remains not completely elucidated. Here we shown that reduced expression of guard cell sucrose synthase 2 (NtSUS2) of Nicotiana tabacum L. altered the topology and the connectivity of the guard cell metabolic network and the accumulation of metabolites positively correlated with stomatal speediness during dark-to-light transition. This leads to a slower light-induced stomatal opening, lower steady-state stomatal conductance and a strong reduction (up to 44%) in daily whole plant transpiration in the transgenics, when compared to wild type plants. Furthermore, the transgenic lines transpired more or have lower reduction in whole plant transpiration under short water deficit periods, indicating a higher effective use of water under this condition. Our results collectively suggest that the regulation of stomatal movement and speediness involve a complex modulation of the guard cell metabolic network, in which NtSUS2 has an important role. The results are discussed on the role of guard cell metabolism for the regulation of both stomatal speediness and whole plant transpiration.