Protein phosphorylation and 14-3-3 protein in volved in blue-light response of stomata

保卫细胞质膜H^+-ATPase被蓝光激活，伴随着14-3-3蛋白与C末端的结合。由于14-3-3蛋白的几种异构体在植物中表达，我们在体内确定了H^+-ATPase结合的异构体(S)。从蚕豆保卫细胞中分离到4个编码14-3-3蛋白的基因克隆(VF14-3-3a、VF14-3-3b、VF14-3-3c和VF14-3-3d)。Northern分析表明，编码VF14-3-3a和VF14-3-3b蛋白的mRNAs主要在保卫细胞中表达。与保卫细胞H^+-ATPase结合的14-3-3蛋白与重组VF14-3-3a蛋白的相对分子质量相同。在褐霉素刺激的叶肉细胞原生质体中，H+-ATPase免疫共沉淀与32.5 kDa的14-3-3蛋白共沉淀，但在叶肉细胞原生质体中发现3个14-3-3蛋白。结合的14-3-3蛋白和重组的VF14-3-3a与CNBr酶切后得到相同的迁移图谱，而VF14-3-3b的迁移图谱不同。经胰酶消化后与H^+-ATPase结合的14-3-3蛋白进行质量图谱分析，得到了VF14-3-3a的预测多肽质量。Far Western分析表明，H^+-ATPase与VF14-3-3a的亲和力高于与VF14-3-3b的亲和力。这些结果表明，在体内与质膜H^+-ATPase结合的14-3-3蛋白是VF14-3-3a，它可能在保卫细胞H^+-ATPase的激活中起关键作用。

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. Since 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 Vicia 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 (MCPs), which had been stimulated by fusicoccin, co-precipitated with the 32.5-kDa 14-3-3 protein, although three 14-3-3 isoproteins were found in MCPs. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with CNBr gave the identical migration profiles on SDS-PAGE, 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.