Phosphorylation of two Thr residues in the C-terminal auto-inhibitory domain of plasma membrane H+-ATPase is crucial for light-induced stomatal opening

Plasma membrane H+-ATPase provides the driving force for light-induced stomatal opening. However, the mechanisms underlying the regulation of its activity remain unclear. Here, we showed that the phosphorylation of two Thr residues in the C-terminal autoinhibitory domain is crucial for H+-ATPase activation and stomatal opening. Our phosphoproteome analysis revealed that blue light induces the phosphorylation of Thr-881 within the C-terminal region I domain and penultimate Thr-948 in AUTOINHIBITED H+-ATPASE 1 (AHA1). The site-directed mutagenesis showed that both Thr phosphorylations are essential for H+ pumping and stomatal opening in response to blue light. The phosphorylation of Thr-948 is a prerequisite for the phosphorylation of Thr-881 by blue light. Furthermore, red-light-driven guard cell photosynthesis also caused Thr-881 phosphorylation, possibly contributing to red-light-dependent stomatal opening. Taken together, our findings provide mechanistic insights into H+-ATPase activation that exploits the ion transport across the plasma membrane and light signalling network in guard cells.