Study On Mechanism And Regulation Of Postharvest Blueberry Fruit Softening Based On Phosphoproteomic

Blueberry(Vaccinium spp.)from the Ericaceae family,is deeply loved by consumers for its unique flavor and nutritional value.Blueberry fruits are rich in anthocyanins,flavonols,vitamins,phenolic acids,minerals and other bioactive substances,which have anti-oxidant,antibacterial,anti-cancer and immunity-enhancing effects.Blueberry fruits are harvested during the hot and rainy summer.Because of the higher field heat and poor storability of the fruits,quality deterioration is prone to occur during the post-harvest shelf life,which affects the quality and commercial value of the fruits.Softening is the main quality deterioration phenomenon in the ripening and senescence process of blueberry fruits after harvest.Given that,it is important to study the softening mechanism of postharvest blueberry fruits and explore the corresponding regulation techniques.In this study,firstly,through the observation and analysis of blueberry fruit morphology and cell level,the occurrence of blueberry fruit softening during normal temperature shelf life is studied,and the differential phosphorylation protein after blueberry fruit softening is analyzed through phosphoproteomic,and bioinformatics analysis is performed.Exploring the metabolic pathways involved in differentially phosphorylated proteins,and on this basis,focus on sugar metabolism and plant hormone ethylene synthesis and signal transduction pathways,explore the effect of sugar metabolism and plant hormone ethylene on blueberry fruit softening and the mechanism of action.Based on the abovementioned mechanism research,explore the regulation technique of controlling postharvest blueberry fruit softening.The research results will provide a basis for solving the quality deterioration of blueberry fruits after harvest.And the results of this study will provide a reference for the study of fruit softening mechanism of other berries.The main results are as follows:The firmness of blueberry fruit decreased rapidly during shelf life at 20°C,and the softening phenomenon was obvious at the 8th day of shelf life.The surface morphology,cell structure and water distribution of blueberry fruit were changed after softening.After softening,the flesh of blueberry fruit became turbid and shallow,the degree of cell contraction and wrinkle was obvious,the intercellular gap increased,plasmolysis occurred,the chloroplast was damaged seriously,the fruit water content decreased,and the free water gradually converted into the bound water.According to the changes of fruit firmness and cell structure,the phosphoproteomic sequencing of blueberry fruits with obvious softening phenomenon on the harvest day and at the 8th day of shelf-life was carried out,and a total of 5092 peptide segments were identified,located on 2530 prebioblast proteins.The number of proteins with phosphorylation modification was 2282,and a total of 5635 phosphorylation points were identified,of which 5099 were accurately quantified,of which 742 phosphorylated proteins showed significant differences.GO classification showed that these differential phosphorylated proteins were mainly involved in “cellular process”,“metabolic process”,“response to stimulus”,and “binding”.KEGG metabolic pathway analysis revealed that “metabolic pathways”,“plant-pathogen interactions”,“protein processing in endoplasmic reticulum”,“biosynthesis of secondary metabolites”,“splicesome”,“starch and sucrose metabolism”,“glycolysis”,and “plant hormone signal transduction” pathways enriched a large number of differential phosphorylated proteins.Based on the results of phosphoproteomic analysis,the carbohydrate metabolism pathway was focused on.By verifying the components and content of important substances,key enzyme activity and related gene expression levels in the process of sucrose metabolism and pectin degradation in the process of blueberry fruit softening after harvesting,the effect of sugar metabolism on blueberry fruit softening is further explored.The results showed that,with the softening of the fruit,the content of sucrose in the fruit was significantly less,the content of glucose and fructose increased,the activity of SPS enzyme decreased significantly,the activity of AI enzyme increased significantly,the expression of Vc SPS was significantly up-regulated,while the phosphorylation level of SPS protein increased during the shelf life at room temperature,indicating that sucrose continued to convert to glucose and fructose during the softening process of blueberry fruit.The WSP content in blueberry fruit increased significantly,while the content of SCSP was less,the expression of Vc PE gene was down-regulated,which The Vc PE gene significantly reduced the expression during the normal temperature shelf,and over-expression of Vc PE blueberry fruit was better at maintaining the firmness of the fruit than compared with blueberries in control group.These indicated that the pectin material had been transformed in the process of fruit softening.Exogenous sugar treatment inhibited the decrease of fruit sucrose content,and by the 8th day of shelf,its content was 4.3 times that of the control fruit.The upward trend of Vc SPS expression and the downward trend of Vc PE expression have been significantly inhibited.This further verifies that sugar metabolism plays an important role in the softening of blueberry fruit.Phosphoproteomic analysis shows that,a large number of differentially phosphorylated proteins is enriched in ethylene metabolic pathway.Therefore,through exogenous ethylene treatment and 1-MCP treatment,the effect of ethylene on postharvest blueberry fruit softening was further verified,and the results showed that exogenous ethylene treatment accelerated the decrease of fruit firmness,aggravated the decrease of fruit sucrose content and the increase of fructose and glucose content.Compared with the control,SS and AI activities were increased significantly.And the activities of SPS and NI were reduced significantly.The activities of PG,PE,?-gal and Cx all increased in different degrees.Sucrose metabolism-related genes Vc SPS,Vc SS1,Vc INV9 and Vc CWINV1 was significantly up-regulated,while Vc NIN2 was downregulated to a certain extent in treated blueberry fruits.The pectin hydrolysis-related genes Vc PG and Vc BG6 were significantly up-regulated,while the expression of Vc PE and Vc GAL1 was significantly down-regulated.1-MCP inhibited the decrease of blueberry fruit firmness effectively,the changes of sucrose,fructose and glucose content in the fruit were significantly reduced,and the decrease of SPS activity and the increase of SS activity were alleviated.At the same time,the activities of PG,PE and ?-gal were all Significantly lower than the control fruit.At the late stage of shelf life,the expression levels of Vc SS1 and Vc CWINV1 in fruits treated with 1-MCP were higher than the control fruits significantly,while the expression levels of Vc PG and Vc BG6 were inhibited significantly,and the change range of the expression levels of Vc SPS,Vc INV9,Vc NIN2,Vc PE and Vc GAL1 was reduced.The above results indicate that ethylene is closely related to the sugar metabolism and pectin conversion of blueberries,which can affect the fruit softening process.Based on the above research results,the regulatory effect of ethylene absorbent treatment on the softening of blueberry fruit after harvesting is further discussed.The results showed that ethylene absorbent could inhibit the decrease of the firmness of blueberry fruit effectively compared with control group,which made the firmness of fruit 1.5 times that of the control group at the 8th day of shelf life.The activity of PG and ?-gal was significant inhibited in the early shelf life.Compared with the control fruits,the ethylene production and ACS and ACO activities in ethylene absorbent treated fruits were significantly reduced.In addition,ethylene absorbent treatment inhibited the weight loss and decay of the fruit significantly,and alleviated the change in the SSC content of the fruit.It can be seen that the ethylene absorbent can delay the softening of blueberry fruits effectively and maintain the quality of blueberries during shelf life.