Potential strong regulation of guard cell phosphoenolpyruvate carboxylase through phosphorylation

In plants, water availability and CO, partial pressure modulate stomatal ap erture. Phosphoenolpyruvate carboxylase (PEPCase) is a major enzyme in the pathway leading to malate synthesis which is, with chloride, the main count erion for potassium accumulated in the guard cell vacuole during stomatal o pening. Whether phosphorylation of PEPCase could be a major event in guard cell regulation was investigated. Antibodies (APS-IgGs) raised to a synthet ic polypeptide of 23 amino acids containing the phosphorylation site (ser-8 ) of the Sorghum PEPCase recognized the guard cell PEPCase from Commelina c ommunis L. at 110 and 120 kDa. The in vitro phosphorylation of the 110 kDa isoform by PKA was 50% inhibited by APS-IgGs demonstrating that the regulat ory phosphorylation site was present and functional in the guard cell enzym e. Phosphorylation by PKA resulted in a 50% increase in the V-max of the en zyme (4.2+/-0.3 compared to 2.8+/-0.4 pmol h(-1) GCP(-1), pH 7.3 and 200 mu M PEP) and a reduction in L-malate inhibition (64% compared to 82% inhibit ion by 1 mM L-malate). In the presence of 1 mM L-malate (pH 7.3) phosphoryl ation of the enzyme by PKA resulted in a 3-fold increase in the V-max. Bind ing of APS-IgGs to the phosphorylation site of the enzyme led to the highes t activity (10.9+/-2.6 pmol h(-1) GCP(-1)) and to an absence of inhibition by 1 mM L-malate at pH 7.3 and 8.0, These changes in the kinetic properties of the enzyme after phosphorylation should have important consequences in terms of stomatal regulation.