Inhibition of Blue Light-Dependent H+ Pumping by Abscisic Acid through Hydrogen Peroxide-Induced Dephosphorylation of the Plasma Membrane H+-ATPase in Guard Cell Protoplasts

Abstract

Blue light (BL)-dependent H⁺ pumping by guard cells, which drives stomatal opening, is inhibited by abscisic acid (ABA). We investigated this response with respect to the activity of plasma membrane H⁺-ATPase using Vicia guard cell protoplasts. ATP hydrolysis by the plasma membrane H⁺-ATPase, phosphorylation of the H⁺-ATPase, and the binding of 14-3-3 protein to the H⁺-ATPase stimulated by BL were inhibited by ABA at 10 μm. All of these responses were similarly inhibited by hydrogen peroxide (H₂O₂) at 1 mm. The ABA-induced inhibitions of BL-dependent H⁺ pumping and phosphorylation of the H⁺-ATPase were partially restored by ascorbate, an intracellular H₂O₂ scavenger. A single-cell analysis of the cytosolic H₂O₂ using 2′,7′-dichlorofluorescin revealed that H₂O₂ was generated by ABA in guard cell protoplasts. We also indicated that H⁺ pumping induced by fusicoccin and the binding of 14-3-3 protein to the H⁺-ATPase were inhibited slightly (approximately 20%) by both ABA and H₂O₂. By contrast, H₂O₂ at 1 mm did not affect H⁺ pumping by the H⁺-ATPase in microsomal membranes. From these results, we concluded that inhibition of BL-dependent H⁺ pumping by ABA was due to a decrease in the phosphorylation levels of H⁺-ATPase and that H₂O₂ might be involved in this response. Moreover, there are at least two inhibition sites by ABA in the BL signaling pathway of guard cells.