Potential strong regulation of guard cell phosphoenolpyruvate carboxylase through phosphorylation

Abstract

In plants, water availability and CO 2 partial pressure modulate stomatal aperture. Phosphoenolpyruvate carboxylase (PEPCase) is a major enzyme in the pathway leading to malate synthesis which is, with chloride, the main counterion for potassium accumulated in the guard cell vacuole during stomatal opening. Whether phosphorylation of PEPCase could be a major event in guard cell regulation was investigated. Antibodies (APS-lgGs) raised to a synthetic polypeptide of 23 amino acids containing the phosphorylation site (ser-8) of the Sorghum PEPCase recognized the guard cell PEPCase from Commelina communis L. at 110 and 120 kDa. The in vitro phosphorylation of the 110 kDa isoform by PKA was 50% inhibited by APS-lgGs demonstrating that the regulatory phosphorylation site was present and functional in the guard cell enzyme. Phosphorylation by PKA resulted in a 50% increase in the V max of the enzyme (4.2±0.3 compared to 2.8±0.4 pmol h -1 GCP -1 , pH 7.3 and 200 μM PEP) and a reduction in L-malate inhibition (64% compared to 82% inhibition by 1 mM L-malate). In the presence of 1 mM L-malate (pH 7.3) phosphorylation of the enzyme by PKA resulted in a 3-fold increase in the V max . Binding of APS-lgGs to the phosphorylation site of the enzyme led to the highest 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.