Integration of proteome and targeted redox metabolome profiling of seed dormancy genotype and after-ripening induced changes in wheat.

Drastic redox changes upon imbibition, activation of antioxidant defenses and stress-responsive proteins were previously shown to be involved in seed dormancy maintenance and sprouting resistance. After-ripening induced ROS accumulation modifies redox status and breaks these control mechanisms by altering protein functions through oxidative modifications. We analyze dormancy-imposing processes and alterations in embryo and aleurone tissue-specific proteomes and redox metabolomes using quantitative redox proteomics, fluorescent labeling of redox active cysteines, high resolution 2-DE, LC-MS/MS analysis with site-specific identification and redox metabolite measurements. 2D Gel-based redox proteomic analysis resulted in 378 high confidence protein identifications with at least two-fold change in protein expression and/or redox level between dormant and after-ripened seeds, from which 46 and 59 proteins showed significant disulfide/thiol ratio changes in dormant embryo and aleurone, respectively. Considerable differences were found for embryo diagnostic proteins involved in ascorbate regeneration, membrane trafficking, DNA methylation-dependent gene silencing, ABA and stress-responsive regulation of reproductive development. The level of total glutathione was significantly higher in dormant than in non-dormant and after-ripened embryo tissues, whereas the pool of reduced glutathione was maintained at about the same level upon dormancy release. Therefore, the capacity for GSSG disulfides regeneration decreased dramatically upon after-ripening of dormant embryos and was lower in non-dormant embryos. The level of total ascorbate increased 2-3 fold in embryo upon after-ripening. In dormant embryo the concentration of total and reduced ascorbate were elevated during after-ripening indicating high capacity for ascorbate regeneration. This clearly indicates different roles of ascorbate and glutathione in redox control of dormancy.