Requirements for activation of the signal-transduction network that leads to regulatory phosphorylation of leaf guard-cell phospho enolpyruvate carboxylase during fusicoccin-stimulated stomatal opening

Abstract

Leaves regulate gas exchange through control of stomata in the epidermis. Stomatal aperture increases when the flanking guard cells accumulate K + or other osmolytes. K + accumulation is stoichiometric with H + extrusion, which is compensated for by phospho enolpyruvate carboxylase (PEPC, EC 4.1.1.31)-mediated malate synthesis. Plant PEPCs are regulated allosterically and by phosphorylation. Aspects of the signal-transduction network that control the PEPC phosphorylation state in guard cells are reported here. Guard cells were preloaded with [ 32P]orthophosphate ( 32Pi); then stomata were incubated with fusicoccin (FC), which activates the guard-cell plasma membrane H +-ATPase. [ 32P]PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. In −FC controls, stomatal size, guard-cell malate, and [ 32P]PEPC were low; maximum values for these parameters were observed in the presence of FC after a 90-min incubation and persisted for an additional 90 min. This high steady-state phosphorylation status resulted from continuous phosphorylation and dephosphorylation, even after the malate-accumulation phase. PEPC phosphorylation was diminished by ∼80% when K + uptake was associated with Cl − uptake and was essentially abolished when stomatal opening was sucrose—rather than K +—dependent. Finally, alkalinization by NH 4 + in the presence of K + did not cause PEPC phosphorylation (as it does in C 4 plants). As discussed, a role for cytoplasmic protons cannot be completely excluded by this result. In summary, activation of the plasma membrane H +-ATPase was essential, but not sufficient, to cause phosphorylation of guard-cell PEPC. Network components downstream of the H +-ATPase influence the phosphorylation state of this PEPC isoform.