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

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Blue light (BL)-dependent H⁺ pumping by guard cells, which drives stomatal opening, is inhibited by phytohormone abscisic acid (ABA). We investigated this response in respect with activity of the plasma membrane H⁺-ATPase, and hydrogen peroxide (H₂O₂) generated by ABA in Vicia guard-cell protoplasts (GCPs). BL-dependent phosphorylation of the H⁺-ATPase and subsequent binding of a 14-3-3 proteins were inhibited by ABA at 10 μM or H₂O₂ at 1mM in similar degrees. Inhibitions of BL-dependent H⁺-pumping and phosphorylation of the H⁺-ATPase by ABA in GCPs were partially reversed by ascorbic acid. A single-cell analysis revealed that H₂O₂ is generated by ABA in GCPs mainly from guard-cell chloroplasts. Fusicoccin-dependent H⁺ pumping and binding of a 14-3-3 protein to the H⁺-ATPase were inhibited slightly by both ABA and H₂O₂. These findings suggest that H₂O₂ may be involved in ABA-inhibition of BL-dependent H⁺-pumping through decrease in phosphorylation levels of the H⁺-ATPase.