Stomatal closing compounds dissect the signaling pathway for light-induced stomatal opening and confer drought tolerance in plants

Stomata in the plant epidermis open in response to light, but the molecular mechanisms underlying this process remain incompletely understood. In this study, we screened for compounds that affect light-induced stomatal opening and identified two stomatal closing compounds (SCLs), SCL10 and SCL11 (clorgyline). Both compounds completely suppressed light-induced stomatal opening in Commelina benghalensis but did not affect fungal toxin fusicoccin (FC)-induced opening. The half-maximal inhibitory concentrations (IC50) of SCL10 and SCL11 on light-induced opening were 1.40 and 38.25 μM, respectively. Consistently, both compounds fully inhibited blue-light-induced phosphorylation of the penultimate residue, a threonine (Thr948; numbering according to Arabidopsis AHA1) of plasma membrane (PM) H+-ATPase, which is essential for stomatal opening, but did not interfere with FC-induced phosphorylation in Arabidopsis thaliana guard cells. Notably, red- and blue-light-induced phosphorylation of Thr881 in PM H+-ATPase, another key step in stomatal opening, was inhibited by SCL11 but not by SCL10 in guard cells. Additionally, stomatal opening induced by PP242, a compound that promotes stomatal opening partly by suppressing steady-state abscisic acid signaling, was inhibited by SCL11 but not by SCL10. These results imply that SCL10 inhibits blue-light-induced phosphorylation of Thr948 and stomatal opening upstream of the site of action of PP242, whereas SCL11 inhibits the phosphorylation of both Thr948 and Thr881, and acts downstream of the site of action of PP242. Finally, the compounds were sprayed onto rose leaves, and we observed that wilting was suppressed only in leaves sprayed with SCL10, for up to 5 h.