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 [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 approximately 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 NH4+ in the presence of K+ did not cause PEPC phosphorylation (as it does in C4 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.
Outlaw
William H
WH
Department of Biological Science, Florida State University, Biology Unit 1 (MC 4370), Chieftan Way, Tallahassee, FL 32306-4370, USA. outlaw@bio.fsu.edu
Du
Zhirong
Z
Xia Meng
Fan
F
Aghoram
Karthik
K
Riddle
Kimberly A
KA
Chollet
Raymond
R
D003593:Cytoplasm / Q000378:metabolism; D006027:Glycosides / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008293:Malates / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D019441:Plant Epidermis / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018515:Plant Leaves / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism; D015398:Signal Transduction; D012965:Sodium Chloride / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology; D031307:Vicia faba / Q000187:drug effects / Q000378:metabolism*
D006027:Glycosides; D008293:Malates; D010710:Phosphates; C007808:fusicoccin; D012965:Sodium Chloride; D013395:Sucrose; C030298:malic acid; D006180:Proton-Translocating ATPases; D010730:Phosphoenolpyruvate Carboxylase; D011188:Potassium