| Peach fruits are harvested in the high temperature and high humidity season.After harvest,they are easily infected by pathogens and rot,causing a lot of losses.Among them,brown rot caused by Monilinia fracticola is one of the most serious diseases.Although low temperature can inhibit the spread of this disease,low temperature often brings serious cold damage problems.Previous studies have found that nitric oxide(NO)can induce peach fruits to improve disease resistance,but the mechanism of induced disease resistance is unclear.In recent years,it has been found that protein nitrosolation is the main form of NO signaling in organisms.S-nitrosoglutathione reductase(GSNOR)participates in signal transduction of NO through the degradation of S-nitrosoglutathione(GSNO),and participates in plant defense responses by regulating S-nitrosothiols(SNOs)and protein nitrosation.However,the role of GSNOR in disease resistance of peach fruits is unclear.Therefore,in this study,"Zhonghuashoutao" was used as experimental material,GSNOR inhibitor(N6022)and NO were used to treat post-harvest peach fruits to explore their effect on the brown rot of peach fruits and its mechanism of action.To investigate the molecular mechanism of GSNOR in the course of disease resistance by nitrosation modification omics analysis after gene transient silencing of GSNOR.The specific results are as follows:(1)Treatment with 60 μM N6022 and 15 μM NO solution significantly reduced the incidence and lesion diameter of peach fruits.They increased the activity of Glutathione reductase(GR)and the gene expression of GR,inhibited the activity of GSNOR and gene expression of GSNOR at 2-4 h.Increased the levels of GSNO,SNOs,reduced glutathione(GSH)and endogenous NO in the cells,and improved the antioxidant capacity of peach fruits.At the same time,the expression of pathogenesis-related gene 1(PR1),nonexpresser of pathogenesis-related gene 1(NPR1)and TGACG-binding factor 1(TGA1)was increased,which was conducive to activating the salicylic acid(SA)signal pathway and promoting the production of systemic acquired resistance(SAR).(2)Using iodo-TMT tag combined with mass spectrometry analysis,it was found that the sulfhydryl group at the 85 th cysteine of GSNOR was nitrosated after 6 h and 12 h of NO treatment in peach fruits.Combining 6 h and 12 h GSNOR activity and GSNOR gene expression analysis,it is speculated that the inhibition of NO on GSNOR activity may be related to S-nitrosation modification of Cys-85 in addition to the early transcriptional regulation.(3)After virus induced gene silencing(VIGS)technology was used to silence GSNOR transiently and inoculated with M.fracticola,it was found that the disease resistance of peach fruits was reduced.The expression of GSNOR and PR1 was significantly reduced,the activity of GSNOR was reduced by about 5 times,and the contents of SNOs and NO were increased by about 4-5 times.High concentrations of SNOs and NO often lead to nitrosation damage,which has a toxic effect on cells,leading to reduced disease resistance.(4)Identification of S-nitrosated protein in peach fruit after transient silence of GSNOR gene by iodo-TMT tag combined with mass spectrometry.A total of 206 differentially expressed proteins were screened after GSNOR silencing,of which 139 were up-regulated and 67 were down-regulated.Two days after M.fracticola was inoculated after GSNOR silencing treatment,a total of 212 differentially expressed proteins were screened,of which 38 were upregulated and 174 were down-regulated.(5)GO annotation,subcellular localization analysis,and KEGG pathway enrichment cluster analysis were performed on the differentially expressed proteins that were screened out.It was found that the differential proteins were mainly located in the chloroplast,cytoplasm,extracellular and nucleus.It involves organic substance metabolic process,cellular metabolic process,primary metabolic process,nitrogen compound metabolic process,response to stress,hydrolase activity,ion binding,transferase activity,oxidoreductase activity and so on.When GSNOR was silenced,the differential proteins were mainly concentrated in the "Carbon fixation in photosynthetic organisms" pathway.When GSNOR was silenced and inoculated with M.fracticola,the differential proteins were mainly involved in the "Proteasome" and "Spliceosome" pathways. |
