Dynamic phosphorylation of RopGEF2 by MPK3/6 kinases fine-tunes diurnal stomatal movements in Arabidopsis

Light triggers diurnal dynamics in stomatal apertures. To balance carbon dioxide uptake for photosynthesis against water loss through transpiration, plants employ mechanisms to prevent excessive stomatal opening. However, the dynamic regulatory mechanism governing stomatal apertures in diurnal stomatal movements remains poorly understood. Here, we report that ROP (RHO OF PLANTS) GUANINE NUCLEOTIDE EXCHANGE FACTOR 2 (RopGEF2), a negative regulator of light-induced stomatal opening, exhibits dynamic phosphorylation in Arabidopsis (Arabidopsis thaliana) guard cells along with changes in stomatal apertures under white light (WL). MITOGEN-ACTIVATED PROTEIN KINASE 3/6 (MPK3/6) directly phosphorylate RopGEF2 at residues Ser18 and Ser352 both in vitro and in vivo. The phosphorylation of RopGEF2 at Ser18 prevents its WL-triggered degradation, whereas its phosphorylation at Ser352 enhances its interaction with and GEF activity toward ROP2. This, in turn, dynamically restricts excessive stomatal opening and water loss in the daytime of the diurnal cycle. Furthermore, both the activation and function of the MPK3/6-RopGEF2 module are dependent on the photoreceptor phyB. Taken together, our findings indicate that the light-triggered phosphorylation of RopGEF2 by MPK3/6 kinases fine-tunes stomatal apertures during diurnal stomatal movements, thereby optimizing water use efficiency.