RESEARCH REPORT: In VivoPhosphorylation of Phosphoenolpyruvate Carboxylase in Guard Cells ofVicia fabaL. Is Enhanced by Fusicoccin and Suppressed by Abscisic Acid

Plants regulate water loss and CO2gain by modulating the aperture sizes of stomata that penetrate the epidermis. Aperture size itself is increased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma. Guard cell phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Regulation of this cytosolic enzyme by effectors is well documented, but additional regulation by posttranslational modification is predicted by the alteration of PEPC kinetics during stomatal opening (FEBS Lett.352, 45–48). In this study, we have investigated whether this alteration is associated with the phosphorylation status of this enzyme. Using sonicated epidermal peels (“isolated” guard cells) preloaded with32PO4, we induced stomatal opening and guard cell malate accumulation by incubation with 5 μmfusicoccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 μmabscisic acid (ABA). The phosphorylation status of PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. PEPC was phosphorylated when stomata were stimulated to open, and phosphorylation was lessened by incubation with ABA. Thus, we conclude that regulation of guard cell PEPCin vivois multifaceted; the effects of regulatory metabolites and the activation status of the enzyme are integrated to control malate synthesis. These results, together with the coincident alteration in the kinetics of the enzyme (FEBS Lett.352, 45–48), constitute the first unequivocal demonstration of regulatory posttranslational modification of a guard cell protein that is specifically implicated in stomatal movements.