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

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 phosphoenolpyruvate 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 [(32)P]orthophosphate ((32)Pi); then stomata were incubated with fusicoccin (FC), which activates the guard-cell plasma membrane H(+)-ATPase. [(32)P]PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and auto radiography. In -FC controls, stomatal size, guard-cell malate, and [(32)P]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 similar to80% 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. (C) 2002 Elsevier Science (USA). All rights reserved.