Proteomics Reveals The Role Of Exogenous NO During Response Of 'Zhonghuashoutao' Peach Fruit To Monilinia Fructicola

Peach(Prunus persica L.Batsch)are typically climacteric fruits and highly susceptible to various pathogens,leading to huge losses.Brown rot,caused by fungus Monilinia fructicola,is one of the most important and serious diseases in postharvest peach fruits.Recently,induction of plant innate resistance to pathogens by biotic or abiotic factors has been becoming a promising approach for controlling postharvest diseases.Among them,the application of nitric oxide(NO)in controlling diseases of postharvest fruits has attracted more attention.The former study of disease resistance of peach fruits induced by NO mainly focused on physiological and biochemical characters and its mechanisms of action have not been fully understood.In this study,'Zhonghuashoutao' peach fruit was used as materials.The effects of exogenous NO on the control of brown rot and defense-related enzyme activities and signals were investigated.The main results are as follows:(1)Peach fruits were pretreated with 15?M NO and deionized water,respectively.Lesion area expansion and resistance correlated index were investigated.The results showed that 15?M NO treatment effectively inhibited brown rot caused by M.fructicola in peach fruits,resulting in significantly lower disease incidence and smaller lesion diameter.In addition,NO treatment significantly increased the activities of PPO,POD and promoted the accumulation of endogenous NO and JA,which contributed to stimulating defense responses of peach fruit and elevate resistance to disease.(2)We applied a TMT-based quantitative proteomic analysis of experimental samples at 12 hpi and 24 hpi.We obtained a total of 5779 proteins,among which 5139 proteins were quantified.The fold-change cutoff was set when proteins with quantitative ratios above 1.3 or below 0.77 were deemed differentially expressed proteins(DEPs).Among the quantified proteins,576 differentially expressed proteins were identified at 12 hpi,of which 420(73%)displayed an increase,and 156(27%)a decrease in abundance.Among the 114 differentially expressed proteins at 24 hpi,73(64%)showed increased abundance,while 41(36%)showed decreased.The number of differentially expressed proteins at 24 hpi decreased,especially for the up-accumulated proteins,suggesting that M.fructicola-response in peach fruit was attenuated at this time point.Given this,a time of 12 hpi was deemed as optimal to comprehensively explore NO-induced responses of peach fruit to M.fructicola invasion.(3)Subcellular localizations analysis reveals most of the proteins were located in the chloroplast(270 proteins)and cytosol(114 proteins).Other proteins were found in nuclear(88 proteins),plasma membrane(35 proteins),mitochondria(21 proteins),extracellular(18 proteins),vacuolar membrane(12 proteins),cytoskeleton(9 proteins),endoplasmic reticulum(5 proteins),golgi apparatus(2 proteins),peroxisome(1 protein)and cytosol,nuclear(1 protein).GO and KEGG enrichment-based analyses,and manual searching jointly were performed to classify the obtained DEPs at 12 hpi,which involved in signal perception and signal transduction(17%),ROS homeostasis and defense-related proteins(22%),transcription and post-transcriptional regulation(13%),carbohydrate and energy metabolism(24%),amino acid and lipid metabolism(24%).(4)To assess whether alteration of gene transcription correlated with protein level changes,we used qRT-PCR to analyse the genes expression of eight DEPs at 12 hpi.Six out of the eight genes under this analysis showed a positive correlation between transcript and protein levels.This result partially validated our proteomic data,showing the reliability of the proteomics results in our study.The results showed that 15?M NO treatment induced the defense response against M.fructicola of postharvest 'Zhonghuashoutao' peach fruit,which is a complicated process involving many organelles and signaling pathways.First,stress-related signal perception,signal transduction and transcription and post-transcriptional regulation were activated during the early responses of peach fruit to pathogen attack.The second strategy involved the upregulation of proteins leading to ROS homeostasis,which has synergy effect with M.fructicola-responsive proteins.Third,rearrangements of several metabolic pathways led to stress adaptation of peach fruit.The research results contributed to understand the defense mechanisms induced by NO in postharvest 'Zhonghuashoutao' peach fruit and provide the theoretical basis for the further research of key signaling pathways and proteins.