Apoplastic sugars regulate stomatal opening and closure through integrated osmotic and metabolic mechanisms

The role of sucrose in stomatal regulation has been debated for more than a century, yet its underlying mechanism remains incompletely understood. Recent evidence indicates that apoplastic sucrose acts as a mesophyll-derived signal promoting stomatal opening in Arabidopsis, yet whether this mechanism operates in other species and under fluctuating environmental conditions, how sugars contribute to stomatal closure, and to what extent these responses depend on sucrose uptake and metabolism within guard cells (GCs) remain unresolved. Here, we carried out stomatal, metabolomics, and modeling analyses in leaves, the leaf apoplast, and GCs from tobacco wild-type and antisense lines for the GC SUCROSE TRANSPORTER 1 (NtSUT1). Daily variation in stomatal conductance (gs) was associated with the accumulation of sugars and organic acids in the apoplast. Stomatal opening and closure were associated with low and high concentrations of apoplastic sucrose over the diel course, respectively. Sugar feeding accelerated stomatal opening and closure in dark and light-adapted leaves, respectively, both in a concentration-dependent manner. NtSUT1 displayed alterations in apoplast and GC metabolite profiles, which resulted in lower gs and impaired sucrose-induced stomatal closure, compared with tobacco wild-type. Our findings suggest that apoplastic sugars and organic acids are associated with diurnal gs dynamics and that sugars can accelerate both stomatal opening and closure through an osmotic mechanism in the apoplast, and are also dependent on sucrose import and degradation within GCs. These results extend current models of stomatal regulation by demonstrating that sugars regulate both stomatal opening and closure through integrated osmotic and metabolic mechanisms.