| Nitric oxide(NO) is a kind of important gas signal molecule, which involves a variety of physiological processes in plants. In order to study the redox mechanism of NO in the mitochondria of fruit and the delaying fruit senescence mechanism of NO, the effect of NO on ascorbic acid-glutathione(As A-GSH) circulatory system in peach fruit was studied by combining with the fruit physiology, the Trx protein expression and the influence of NO on the amount of Trx gene expression was explored, and the nitrosylated protein in mitochondria of peach fruit was detected by using the surface plasma resonance(SPR) technology.In order to characterize the regulation by exogenous NO on antioxidant systems of ascorbate–glutathione metabolism under low temperature stress in peach fruits, peaches were treated with NO(10 ?mol L-1), c-PTIO(5 ?mol L-1) respectively and stored at 0 oC. The contents of malondialdehyde(MDA), ascorbate and glutathione and the activities of protective enzymes, including monodehydroascorbate reductase(MDHAR), dehydroascorbate reductase(DHAR), ascorbate peroxidase(APX) and glutathione reductase(GR) in fruits were determined every week during storage. The results showed that MDA content was only 51% of that of the control at week 2. DPPH? radical scavenging activity and GR activity as 1.3 and 1.7 times high as that of the control. Exogenous NO improved the ratio of As A/DHA in peaches during storage The ratio of As A/DHA in peaches treated with NO was as 4.4, 3.8 and 3.3 times high as that of the control at week 1, 2 and 3, respectively. The results showed that treatment with exogenous NO delayed the increases in the contents of MDA, total ascorbate and As A during storage. The contents of total glutathione and GSH in peaches among treatments increased significantly after week 2. DPPH? radical scavenging activity and the activities of DHAR and GT in peaches were significantly lower at week 1 and then higher after week 2 than that of the control. Exogenous NO inhibited MDHAR activity but increased GR activity before week 2. Exogenous c-PTIO, as NO scavenger, confirmed the regulation by NO on glutathione metabolism in peaches during cold storage. Exogenous NO improved the reducing capacity of ascorbate-glutathione cycle to maintain high antioxidant ability in peaches.According to the known Trx gene of peach fruit in the NCBI, the degenerate primers were designed, then a c DNA encoding Trx protein was isolated from Feicheng peach fruit and recombined. The full-length c DNA of Trx was 664 bp and contained 828 bp of the coding sequence encoding a polypeptide of 153 amino acids. Trx protein was purified and analyzed by SDS-PAGE. The fluorescent quantitation PCR indicated that treatment with NO improved the Trx expression level, and the second week was the highest. The results showed that exogenous NO could regulate the expression level of Feicheng peach fruit during cold storage.The electrochemical impedance spectroscopy and atomic force microscope were used to estimate the formation of self-assembled film in the experiment. This method was based on the specific binding characteristics of biotin–streptavidin, using Biotin-HPDP labeled protein sulfhydryl group as the substrate to detect proteins. The sensor was used to detect bovine serum albumin(BSA), nitrosylated BSA and denitrosylated BSA. The results showed that 90.61% of nitrosylated BSA were reduced, verifying that protein S-nitrosylation is a reversible and effective post-translation modification. This method was successfully applied to detect S-nitrosylated protein in Feicheng peach. Treatment with 15 ?mol L-1 NO caused the maximum wave number change, while the wave number shift showed a downward trend caused by the treatment with 30 ?mol L-1 NO. The results also indicated that the nitrosation functions of protein sulfhydryl groups would be inhibited if NO concentration was high. This method provided a molecular basis for further exploring the mechanism of S-nitrosylation of proteins in plants. |
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