RESUMO
Oxidative signalling by ROS has been demonstrated to play a role in seed dormancy alleviation, but the detailed molecular mechanisms underlying this process remain largely unknown. Here, we show dynamic differences in redox-sensitive proteome upon wheat seed dormancy release. Using thiol-specific fluorescent labelling, solubility-based protein fractionation, 2-D IEF PAGE, and MS analysis in conjunction with wheat EST sequence libraries, proteins with reversible oxidoreductive changes were characterized. Altogether, 193 reactive Cys were found in 79 unique proteins responding differentially in dormant, non-dormant, abscisic, or gibberellic acid-treated seed protein extracts from RL4137, a wheat cultivar with extreme dormancy. The identified proteins included groups that are redox-, stress-, and pathogen-responsive, involved in protein synthesis and storage, are enzymes of carbohydrate metabolism, proteases, and those involved in transport and signal transduction. Two types of redox response could be detected: (i) a dramatic increase in protein thiol redox state in seeds during imbibition and hormonal treatment; (ii) higher antioxidant capacity related to sensing of a threshold redox potential and balancing the existing redox pools, in dry dormant versus non-dormant seeds. These results highlight occurrence of the antioxidant defence mechanisms required for the protection of seed during a dormancy stage.
Assuntos
Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Sementes/fisiologia , Triticum/metabolismo , Ácido Abscísico/farmacologia , Eletroforese em Gel Bidimensional , Etiquetas de Sequências Expressas , Biblioteca Gênica , Germinação/efeitos dos fármacos , Germinação/fisiologia , Giberelinas/farmacologia , Focalização Isoelétrica , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Dormência de Plantas/efeitos dos fármacos , Dormência de Plantas/genética , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteoma/análise , Proteoma/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Espectrometria de Fluorescência , Compostos de Sulfidrila/metabolismo , Triticum/efeitos dos fármacos , Triticum/genéticaRESUMO
The thiol redox-sensitive and the total proteome in harvest-ripe grains of closely related genotypes of wheat (Triticum aestivum L.), with either a dormant or a non-dormant phenotype, were investigated using hybrid lines of spring wheat double haploid population segregating transgressively, to gain further insight into seed dormancy controlling events. Redox signalling by reactive oxygen species has been shown to play a role in seed dormancy alleviation. Thiol-disulfide proteins are of particular importance in the context of redox-dependent regulation as a central and flexible mechanism to control metabolic and developmental activities of the cells. Here we describe functional proteomic profiling of reversible oxidoreductive changes and characterize in vivo intrinsic reactivity of cysteine residues using thiol-specific fluorescent labelling, solubility-based protein fractionation, two-dimensional electrophoresis, and mass spectrometry analysis in conjunction with wheat EST sequence libraries. Quantitative differences between genotypes were found for 106 spots containing 64 unique proteins. Forty seven unique proteins displayed distinctive abundance pattern, and among them 31 proteins contained 78 unique redox active cysteines. Seventeen unique proteins with 19 reactive modified cysteines were found to have differential post-translational thiol redox modification. The results provide an insight into the alteration of thiol-redox profiles in proteins that function in major processes in seeds and include groups of redox- and stress-responsive, genetic information processing and cell cycle control, transport and storage proteins, enzymes of carbohydrate metabolism, proteases and their inhibitors.