Regulation By HNO On Glyceraldehyde-3-phosphate Dehydrogenase In Postharvest Nectarine Fruit

Nitroxyl(HNO)is a single electron reduced congener of nitric oxide(NO),has received extensive attention due to its unique biological activities.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)is a key enzyme in the glycolysis pathway,mainly catalyzing the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate,and it also regulates the expression of histone genes and telomere structure by acting on nuclear t RNA transport,DNA replication and repair.In this study,the nectarine fruit was used as the experimental material,and treated with different concentrations of HNO donor solution.The effects of different concentrations of HNO on the preservation of nectarine fruits during storage were explored,and the effects of HNO on GAPDH and the contents of related substances before and after glycolysis were investigated.The effect of different concentrations of HNO on GAPDH protein was explored by fluorescence spectrophotometry.The reaction between HNO and GAPDH was simulated using Gaussian software,and then the interaction mechanism between HNO and GAPDH was discussed.The conclusions are as follows:(1)HNO treatment inhibited the water loss of nectarine fruit,delayed the increase of the fruit weight loss rate,maintained the high firmness of the fruit,and delayed the increasing of the color difference and browning degree,which had a certain inhibitory effect on the oxidative browning of the fruit.The HNO treatment also delayed the change of the soluble solids content of the nectarine fruit,maintained a higher soluble solid content and soluble protein content in later storage period.During storage,HNO treatment maintained a high content of ascorbic acid in fruit,which was beneficial to maintain the storage quality of nectarine fruit.Among them,10?mol·L^-1 HNO donor solution treatment is best for fruit.(2)HNO treatment down-regulated the expression of nectarine fruit GAPDH gene,inhibited the enzymatic activity of GAPDH,delayed the decomposition of glucose,and indirectly affected the changes of sucrose and fructose contents.It maintained the high sucrose and glucose content in nectarine fruit,so it could effectively maintain the higher quality and delay the aging of nectarine fruit.HNO had a certain inhibitory effect on the enzyme activity of pyruvate dehydrogenase complex(PDHC),leading to slow decarboxylation of pyruvic acid and high pyruvate content,which may affect other metabolic speed to slow down,so as to better maintain nutrients in fruit.(3)HNO causeed the GAPDH protein solution in fluorescence quenching,and the results was consistent with the characteristics of static quenching,indicated that GAPDH protein was combined with HNO to form a complex,resulting in fluorescence quenching of GAPDH protein.Through Scatchrd model analysis,it indicated that HNO and GAPDH mainly formed in 1:1-type complex.(4)There are 12 cysteine residues outside GAPDH protein,which may reacted with HNO to form HNO-Cys.According to the analysis of the orbital energy,bond energy and NBO charge,the HNO-Cys240 was most susceptible to generate and the product was relatively stable,so the HNO first react with Cys240 in the nectarine GAPDH,leading to changes in the protein structure.The above results show that 10?mol·L^-1 HNO donor solution treatment has a fresh-keeping effect on nectarine fruit.The HNO generates 1:1 composite by binding to Cys240 in the GAPDH protein,which changes the protein structure to inhibit GAPDH enzyme.This leads to higher sucrose content in fruit,thereby maintaining the higher nutrient content in the fruit,which is beneficial to maintain the storage quality of the fruit.