Phototropins Mediate BL-dependent Activation of the Plasma Membrane H⁺-ATPase in Stomatal Guard Cells from Arabidopsis thaliana

Abstract

Blue light (BL) activates the plasma membrane H⁺-ATPase via phosphorylation of C-terminus with concomitant binding of a 14-3-3 protein, and induces stomatal opening. Recently, we showed that phototropins act as BL-receptors in stomatal opening using Arabidopsis plants. However, BL-responses using Arabidopsis guard-cell protoplasts (AtGCPs) have not been studied. In this study, we investigated BL-dependent activation of the plasma membrane H⁺-ATPase using AtGCPs. BL-dependent H⁺ pumping in AtGCPs reached the maximum 2 min after the start of BL (100 μmol/m²/sec for 30 sec) and sustained for 6 min. BL increased ATP hydrolysis by the H⁺-ATPase. Far Western analysis revealed that a 14-3-3 protein bound to the H⁺-ATPase in response to BL. By contrast, BL-induced H⁺ pumping, activation of the H⁺-ATPase, and binding of the 14-3-3 protein were not found in GCPs from phot1 phot2 double mutant. These results confirm that phototropins mediate BL-dependent activation of the plasma membrane H⁺-ATPase in AtGCPs.