RESUMO
The plasma membrane regulates biological processes such as ion transport, signal transduction, endocytosis, and cell differentiation/proliferation. To understand the functional characteristics and organ specificity of plasma membranes, plasma membrane protein fractions from rice root, etiolated leaf, green leaf, developing leaf sheath, and flower were analyzed by proteomics. Among the proteins identified, 511 were commonly accumulated in the five organs, whereas 270, 132, 359, 146, and 149 proteins were specifically accumulated in the root, etiolated leaf, green leaf, developing leaf sheath, and developing flower, respectively. The principle component analysis revealed that the functions of the plasma membrane in the root was different from those of green and etiolated leaves and that the plasma membrane protein composition of the leaf sheath was similar to that of the flower, but not that of the green leaf. Functional classification revealed that the root plasma membrane has more transport-related proteins than the leaf plasma membrane. Furthermore, the leaf sheath and flower plasma membranes were found to be richer in proteins involved in signaling and cell function than the green leaf plasma membrane. To validate the proteomics data, immunoblot analysis was carried out, focusing on four heterotrimeric G protein subunits, Gα, Gß, Gγ1, and Gγ2. All subunits could be detected by both methods and, in particular, Gγ1 and Gγ2 required concentration by immunoprecipitation for mass spectrometry detection.
Assuntos
Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/biossíntese , Raízes de Plantas/metabolismo , Flores/genética , Oryza/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , ProteômicaRESUMO
Heterotrimeric G proteins are important molecules for regulating plant architecture and transmitting external signals to intracellular target proteins in higher plants and mammals. The rice genome contains one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical ß subunit gene (RGB1), and five γ subunit genes (tentatively named RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/OsGGC3, and RGG5/OsGGC2). RGG1 encodes the canonical γ subunit; RGG2 encodes the plant-specific type of γ subunit with additional amino acid residues at the N-terminus; and the remaining three γ subunit genes encode the atypical γ subunits with cysteine abundance at the C-terminus. We aimed to identify the RGG3/GS3/Mi/OsGGC1 gene product, Gγ3, in rice tissues using the anti-Gγ3 domain antibody. We also analyzed the truncated protein, Gγ3∆Cys, in the RGG3/GS3/Mi/OsGGC1 mutant, Mi, using the anti-Gγ3 domain antibody. Based on nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the immunoprecipitated Gγ3 candidates were confirmed to be Gγ3. Similar to α (Gα) and ß subunits (Gß), Gγ3 was enriched in the plasma membrane fraction, and accumulated in the flower tissues. As RGG3/GS3/Mi/OsGGC1 mutants show the characteristic phenotype in flowers and consequently in seeds, the tissues that accumulated Gγ3 corresponded to the abnormal tissues observed in RGG3/GS3/Mi/OsGGC1 mutants.
Assuntos
Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Membrana Celular/metabolismo , Flores/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Oryza/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Sementes/metabolismoRESUMO
Heterotrimeric G proteins are the molecule switch that transmits information from external signals to intracellular target proteins in mammals and yeast cells. In higher plants, heterotrimeric G proteins regulate plant architecture. Rice harbors one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical ß-subunit gene (RGB1), and five γ-subunit genes (tentatively designated RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/qPE9-1/OsGGC3, and RGG5/OsGGC2) as components of the heterotrimeric G protein complex. Among the five γ-subunit genes, RGG1 encodes the canonical γ-subunit, RGG2 encodes a plant-specific type of γ-subunit with additional amino acid residues at the N-terminus, and the remaining three γ-subunit genes encode atypical γ-subunits with cysteine-rich C-termini. We characterized the RGG4/DEP1/DN1/qPE9-1/OsGGC3 gene product Gγ4 in the wild type (WT) and truncated protein Gγ4∆Cys in the RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutant, Dn1-1, as littele information regarding the native Gγ4 and Gγ4∆Cys proteins is currently available. Based on liquid chromatography-tandem mass spectrometry analysis, immunoprecipitated Gγ4 candidates were confirmed as actual Gγ4. Similar to α-(Gα) and ß-subunits (Gß), Gγ4 was enriched in the plasma membrane fraction and accumulated in the developing leaf sheath. As RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants exhibited dwarfism, tissues that accumulated Gγ4 corresponded to the abnormal tissues observed in RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants.