IN-VIVO PHOSPHORYLATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE IN GUARD-CELLS OF VICIA-FABA L IS ENHANCED BY FUSICOCCIN AND SUPPRESSED BY ABSCISIC-ACID

Plants regulate water loss and CO2 gain by modulating the aperture siz es of stomata that penetrate the epidermis. Aperture size itself is in creased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma, Guard cell phosphoenolpyru vate carboxylase (PEPC, EC 4.1.1.31), which catalyzes the regulated st ep leading to malate synthesis, is crucial for charge and pH maintenan ce during osmolyte accumulation. Regulation of this cytosolic enzyme b y effecters is well documented, but additional regulation by posttrans lational modification is predicted by the alteration of PEPC kinetics during stomatal opening (FEES Lett. 352, 45-48), In this study, we hav e investigated whether this alteration is associated with the phosphor ylation status of this enzyme. Using sonicated epidermal peels (''isol ated'' guard cells) preloaded with (PO4)-P-32, We induced stomatal ope ning and guard cell malate accumulation by incubation with 5 mu M fusi coccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 mu M abscisic acid (ABA). The phosphorylati on status of PEPC was assessed by immunoprecipitation, electrophoresis , immunoblotting, and autoradiography, PEPC was phosphorylated when st omata were stimulated to open, and phosphorylation was lessened by inc ubation with ABA. Thus, we conclude that regulation of guard cell PEPC in vivo is multifaceted; the effects of regulatory metabolites and th e activation status of the enzyme are integrated to control malate syn thesis. These results, together with the coincident alteration in the kinetics of the enzyme (FEBS Lett. 352, 45-48), constitute the first u nequivocal demonstration of regulatory posttransitional modification o f a guard cell protein that is specifically implicated in stomatal mov ements. (C) 1997 Academic Press