SAUR proteins and PP2C.D phosphatases regulate H+-ATPases and K+ channels to control stomatal movements.

Activation of plasma membrane (PM) H+-ATPase activity is crucial in guard cells to promote light-stimulated stomatal opening, and in growing organs to promote cell expansion. In growing organs, SMALL AUXIN UP RNA (SAUR) proteins inhibit the PP2C.D2, PP2C.D5, and PP2C.D6 (PP2C.D2/5/6) phosphatases, thereby preventing dephosphorylation of the penultimate phosphothreonine of PM H+-ATPases and trapping them in the activated state to promote cell expansion. To elucidate whether SAUR-PP2C.D regulatory modules also affect reversible cell expansion, we examined stomatal apertures and conductances of Arabidopsis thaliana plants with altered SAUR or PP2C.D activity. Here, we report that the pp2c.d2/5/6 triple knockout mutant plants and plant lines overexpressing SAUR fusion proteins exhibit enhanced stomatal apertures and conductances. Reciprocally, saur56 saur60 double mutants, lacking two SAUR genes normally expressed in guard cells, displayed reduced apertures and conductances, as did plants overexpressing PP2C.D5. Although altered PM H+-ATPase activity contributes to these stomatal phenotypes, voltage clamp analysis showed significant changes also in K+ channel gating in lines with altered SAUR and PP2C.D function. Together, our findings demonstrate that SAUR and PP2C.D proteins act antagonistically to facilitate stomatal movements through a concerted targeting of both ATP-dependent H+ pumping and channel-mediated K+ transport.

Rapid structural changes and acidification of guard cell vacuoles during stomatal closure require phosphatidylinositol 3,5-bisphosphate.

Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)-induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H(+)-ATPase vha-a2 vha-a3 and vacuolar H(+)-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P2, which is essential for vacuolar acidification and convolution.