Specific Binding of vf14-3-3a Isoform to the Plasma Membrane H+-ATPase in Response to Blue Light and Fusicoccin in Guard Cells of Broad Bean

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Abstract
The plasma membrane H+-ATPase is activated by blue light with concomitant binding of the 14-3-3 protein to the C terminus in guard cells. Because several isoforms of the 14-3-3 protein are expressed in plants, we determined which isoform(s) bound to the H+-ATPase in vivo. Four cDNA clones (vf14-3-3a, vf14-3-3b,vf14-3-3c, and vf14-3-3d) encoding 14-3-3 proteins were isolated from broad bean (Vicia faba) guard cells. Northern analysis revealed that mRNAs encoding vf14-3-3a and vf14-3-3b proteins were expressed predominantly in guard cells. The 14-3-3 protein that bound to the H+-ATPase in guard cells had the same molecular mass as the recombinant vf14-3-3a protein. The H+-ATPase immunoprecipitated from mesophyll cell protoplasts, which had been stimulated by fusicoccin, coprecipitated with the 32.5-kD 14-3-3 protein, although three 14-3-3 isoproteins were found in mesophyll cell protoplasts. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with cyanogen bromide gave the identical migration profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but that of vf14-3-3b gave a different profile. Mass profiling of trypsin-digested 14-3-3 protein bound to the H+-ATPase gave the predicted peptide masses of vf14-3-3a. Far western analysis revealed that the H+-ATPase had a higher affinity for vf14-3-3a than for vf14-3-3b. These results suggest that the 14-3-3 protein that bound to the plasma membrane H+-ATPase in vivo is vf14-3-3a and that it may play a key role in the activation of H+-ATPase in guard cells.