Physiological Mechanism Of Flesh Browning In ‘Fuji’ Apple Fruit During Cold Storage

Apple（Malus domestica Borkh.）is one of the most important fruits in China.In order to maintain its fresh quality and achieve full time and space supply,a significant portion of apple fruit will be refrigerated at a temperature of around 0℃for several months.However,long-term cold storage or inappropriate temperatures often lead to flesh browning,fruit flavor and nutrition losses,and greatly affecting apple sales and consumer trust.In this experiment,transcriptome sequencing and analysis were performed on the browning tissues and normal tissues of‘Fuji’apple during cold storage,to screenthe metabolic pathways that may be potentially related to flesh browning.In addition,the metabolites and enzymes in these browning related pathways were also detected in order to reveal the physiological mechanism of flesh browning in‘Fuji’apple during cold storage.The main results of the study were as follows:（1）Compared with normal fruit（Control）tissue and non-browning（FN）tissue of browning fruit,the quality indicators such as soluble solids,titratable acids,and antioxidant capacity in fruit browning（FB）tissue were at the lowest level,while the relative electrical conductivity was the highest in FB tissue.Based on KEGG enrichment analysis and WGCNA,it was found that metabolic pathways such as sugar metabolism,energy metabolism,phenolic metabolism,plant hormones,and MAPK signaling were closely related to flesh browning.（2）Compared with Control and FN tissues,the activities of amylase and sucrose phosphate synthase in FB tissue decreased significantly,while the activities of sucrose invertase increased,accompanied by lower soluble sugars（especially sucrose）content in FB tissue;In addition,the energy charge level and the activities of enzymes related to energy metabolism(succinate dehydrogenase,cytochrome C oxidase,Ca²⁺-ATPase,and H⁺-ATPase)in FB tissue were the lowest among the three treatments,indicating that the aerobic respiration process in FB tissue was significantly inhibited.Therefore,the disorder of glucose metabolism and energy metabolism may be an important cause of fresh browning.（3）Compared with Control and FN tissues,the activities of lipoxygenase,polyphenol oxidase,and peroxidase,as well as the contents of active oxygen species（ROS）and malondialdehyde in FB tissues were at the highest level;The activities of catalase and ascorbic acid peroxidase and the content of antioxidant substances（total phenols,total flavonoids,ascorbic acid,glutathione,etc.）were the lowest;In browning fruit,compared to FN tissue,the phenylalanine ammonia lyase and p-coumaric acid:Co A ligase activities in FB tissue were increased along with the upregulation of gene expression in the phenylpropane pathway.Meanwhile,the contents of most phenolic acids in FB tissue were significantly increased compared with those in FN tissue.（4）Compared with Control and FN tissues,the expression of genes related to cold stress such as CBF and CRPK1,genes related to Ca²⁺signal transduction such as CNGC and Ca M/CML,genes related to ROS metabolism such as RBOH and OX1,and genes related to defense such as WRKY29 were significantly upregulated in FB tissue;It is speculated that cold stress activates the Ca²⁺signal transduction pathway in apples and induces the cold resistance of fruits through ROS and MAPK cascade reactions.In addition,the transcription levels of AOS and OPR involved in JA biosynthesis,as well as JAZs,the key genes for JA signal transduction pathways,were also upregulated in FB tissue.In summary,the flesh browning of apple fruit during cold storage was related to sugar metabolism,energy metabolism,phenolic metabolism,and ROS metabolism;Plant hormones,Ca²⁺,and MAPK signals play important roles in regulating the development of flesh browning.