Protease Inhibitor-Dependent Inhibition of Light-Induced Stomatal Opening

Stomata in the epidermis of plants play essential roles in the regulation of photosynthesis and transpiration. Stomata open in response to blue light (BL) by phosphorylation-dependent activation of the plasma membrane (PM) H⁺-ATPase in guard cells. Under water stress, the plant hormone abscisic acid (ABA) promotes stomatal closure via the ABA-signaling pathway to reduce water loss. We established a chemical screening method to identify compounds that affect stomatal movements in Commelina benghalensis. We performed chemical screening using a protease inhibitor (PI) library of 130 inhibitors to identify inhibitors of stomatal movement. We discovered 17 PIs that inhibited light-induced stomatal opening by more than 50%. Further analysis of the top three inhibitors (PI1, PI2, and PI3; inhibitors of ubiquitin-specific protease 1, membrane type-1 matrix metalloproteinase, and matrix metalloproteinase-2, respectively) revealed that these inhibitors suppressed BL-induced phosphorylation of the PM H⁺-ATPase but had no effect on the activity of phototropins or ABA-dependent responses. The results suggest that these PIs suppress BL-induced stomatal opening at least in part by inhibiting PM H⁺-ATPase activity but not the ABA-signaling pathway. The targets of PI1, PI2, and PI3 were predicted by bioinformatics analyses, which provided insight into factors involved in BL-induced stomatal opening.