Study On The Mechanism Of Postharvest Fruit Softening In ’Mopan’ Persimmon

Persimmon(Diospyros kaki Thunb.)is one of the main fruit trees originated from China and plays an important role in the economic development in some regions.’Mopan’persimmon is the most widely distributed and high-yielding cultivar in China.However,the fruit of ’Mopan’ persimmon is easy to soften after ripening in postharvest,which results in fruit quality deterioration and even fruit decay,affects fruit storage,transportation,sales and production efficiency,and harmful to the healthy market development of persimmon industry.Up to now,the physiological and biochemical changes and gene regulation mechanisms during the softening process of ’Mopan’persimmon fruit have not been systematically studied.To reveal the softening mechanism of persimmon fruit in postharvest,our study focused on the study of physiological and biochemical changes of the persimmon cultivars ’Mopan’ persimmon(easy to soften after harvest,mainly planted in Hebei province)in postharvest.We studied the changes of fruit physiological indexes and expression changes of softening related genes by the influence of endogenous ethylene in natural softening process of ’Mopan’ persimmon;We studied the physiological indexes and expression changes of softening related genes after exogenous ethylene and 1-MCP treatment of ’Mopan’ persimmon.At the same time,’Yoho’ persimmon,hard to soften after harvest,newly introduced to Hebei province were used as materials to comparative study the transcriptomic and screening of of upstream regulation sequence in cell-wall hydrolysis enzyme gene were carried out to lay a theoretical foundation for further revealing the molecular mechanism and the molecular mechanism of regulating the softening technology in natural softening of ’Mopan’persimmon fruit.Our main results are as follows:1.The natural postharvest softening process under the influence of endogenous ethylene in ’Mopan’ and ’Yoho’ persimmon fruit were studied.The results showed that:in the process of softening,two varieties varied significantly difference in physiological indexes and ripening related gene expressions.As the increasing of storage time,’Mopan’persimmon fruit softened rapidly,the respiratory rate peaked on day 10(2.52 mg/kg/h CO2),the ethylene production peaked on day 12(1.81 ng/kg/s)and the soluble tannin content were decreased.In contrast,’Yoho’ fruit softened slowly,the change in respiratory rate was not significant,the ethylene production peaked on day 15(1.81 ng/kg/s)and the soluble tannin content were unchanged.At the same time,the expression levels of ethylene signal related genes(DkETR1,DkETR2,DkCTR1,DkERS1,DkERF19,DkERF22),de-astringency related genes(DkADH1,DkPDC1,DkPDC2)and cell wall hydrolase related genes(DkPG1,DkXTH2,DkPME1,Dk β-Gall)in the two varieties were different.That means cell wall hydrolases related genes and enzyme activities,astringency related genes and endogenous ethylene play key roles in postharvest ripening of ’Mopan’ and ’Yoho’ fruits during storage.The activities of cell wall enzymes related to the softening of ’Mopan’ fruit was higher than that of ’Yoho’ persimmon fruit.For ’Mopan’persimmon fruit,the effects of enzyme activities on the fruit firmness of the fruit was:Polygalacturonase>Pectin lyase>Pectinesterase,and DkPG1 showed higher expression level than other cell wall hydrolyzed enzymes.2.The flesh of ’Mopan’ persimmon was chosen as the test materials to comparative study postharvest ’Mopan’ persimmon fruit after exogenous ethylene and 1-MCP treatment.The results showed that exogenous ethylene promoted persimmon fruit softening,1-MCP delayed persimmon fruit softening.The skin color of ’Mopan’ fruit changed to orange,the fruit firmness decreased,the respiration rate(3.59 mg/kg/h CO2)and ethylene production(1.96 ng/kg/s)increased,the soluble tannin and soluble solid content decreased.Exogenous ethylene treatment raised the expression of ethylene signal genes(DkETR1,DkETR2 DkCTR1,DkEIL1,DkERF19 and DkERF22),expression of tannins related genes(DkADH1,DkPDC1 and DkPDC2)and cell wall hydrolase related genes(DkPG1,DkXTH2,DkPME1 and Dk β-Gall).The application of 1-MCP effectively delayed the effect of ethylene on a series of physiological changes,including softening,de-astringency,ethylene production(0.96 ng/kg/s)and respiration enhancement(2.07 mg/kg/h CO2)during fruit storage of ’Mopan’ persimmon.The fruits treated with 1-MCP also maintained higher TSS content and lower ripening color,and almost all genes upregulated by ethylene were inhibited by 1-MCP.Moreover,the activities of key enzymes related to softening was significantly negative correlated with fruit firmness after ethylene and 1-MCP treatment.After ethylene and 1-MCP treatment,the activities of polygalacturonase had a relatively greater effect than those of other cell wall enzymes.3.The determination of transcriptome comparative study of ’Mopan’ and ’Yoho’persimmon fruit flesh in natural softening process at day 0,day 4,day 10 and day 20 time points were firstly determined.In the process of natural softening,the two varieties had the highest transcription activity in the early storage and the number of transcription genes were significantly reduced after day 4.’Mopan’ persimmon activated more genetic transcription to soften the flesh rapidly,and in the whole process of softening,the persimmon transcription activities of ’Mopan’ persimmon were stronger than ’Yoho’persimmon.Almost no differentially expressed structural genes and transcriptional regulators in the two cultivars,which indicating that there were significant differences in the molecular mechanism control of fruit firmness.The analysis of the Gene Go enrichment indicated that ’Mopan’ and ’Yoho’ persimmon regulated the nutrient and state changes in the fruit through the catalytic action from the beginning and end of storage.Hydrolytic enzyme was enriched in ’Mopan’ persimmon but not found in ’Yoho’persimmon,which indicates that the hydrolysis enzyme activity has significant impact on the firmness of ’Mopan’ persimmon.At the end of storage,’Mopan’ persimmon enriched with more oxidation reduction process with more rapidly aging and softening,indicating that the oxidation reduction affected the process of aging and softening in ’Mopan’persimmon at the late stage of storage.The KEGG enrichment analysis found that the nitrogen metabolism pathway(in the beginning stage of storage)and lipid metabolism pathway(in the middle stage of storage)were only enriched in ’Yoho’ persimmon,which could cause the difference on fruit softening.4.The transcriptome analysis showed that endogenous ethylene was involved in the natural softening of fruits after harvest and regulated the fruits softening process.ACC synthase(ACS)gene was more important than ACC oxidase(ACO)gene in the control of endogenous ethylene biosynthesis and fruit softening.Some ethylene signaling factor ERF genes had opposite regulatory effects in the two cultivars,which could speculate that these ERF genes may play major roles in the softening of persimmon fruit.Comparing with’Mopan’ fruit,the expression of ACS and several kinds of cell wall degrading genes polygalacturonase,pectin lyase,xyloglucan transferase/hydrolase,pectinesterase,expansion was inhibited at the beginning of fruit storage and increased only at the later storage stage in ’Yoho’ fruit.In the process of fruit ripening,ERF,WRKY,MYB,bHLH and other transcription’ factors were highly active,which may participate in the regulation of fruit softening process.5.The promoter of cell wall degrading genes polygalacturonase DkPG1,which was most related to softening were amplified by genomic walking method in ’Mopan’ fruit,showed no self-activation activity by the detection of yeast one hybrid self-activation and screened as a bait for yeast one hybrid library screening.At last,we obtained 3 protein sequences that interacted with persimmon DkPGl gene in yeast and named DkCBSX3,DkIPCS1and DkNDUFB3.They were predicted to interact with DkPG1 in persimmon softening using the secondary structure analysis,transmembrane domain analysis,hydrophobicity analysis,signal peptide analysis,subcellular localization prediction and protein function prediction of these proteins.