Studies On Lignification Characteristics Of Chilling Injury And Fruit Cold Resistance Mechanisms In Postharvest Kiwifruit

Chilling injury(CI)is a well-known physiological damage in kiwifruit during low temperature storage.CI is mainly characterized by lignification in the subcutaneous and core tissue,as well as uneven softening in fruits.Such symptoms are susceptible for pathogen invasion during storage,consequently result in rotting and spoiling.This remarkably influences the marketable value of kiwifruit industry.Based on the previous study of cold sensitive evaluation and CI control methods of different kiwifruit cultivars,the chinese cold sensitive cultivar‘Hongyang' and the relatively cold resistant deliciosa ‘Xuxiang' fruit was tested in this study.We investigated the characteristics of lignification in CI of the two cultivars during 0? storage,mainly focused on the microstructure and ultrastructure of tissue cells,the changes of physiological and biochemical indexes,the analysis of tissues widely targeted metabolomics,combined with CBF gene expression pattern analysis,screening the key genes in cold resistance,and anti-cold functional verification.We systematically revealed the CI differences in cultivars and tissues in the aspects of physiological and biochemical and molecular mechanism of cold resistance.Finally,we explored the role of 1-methylcyclopropene(1-MCP),methyl jasmonate(MeJA)and methyl salicylate(MeSA)in controlling postharvest CI.The main findings are:1.There was a significant difference in CI between cultivars and tissues.The cold sensitivity of 'Hongyang' was significantly higher than that of 'Xuxiang',the CI symptoms of 'Hongyang' appeared on 30 d after storage,and the main symptoms were peel browning and lignification of subcutaneous flesh.CI in 'Xuxiang' was observed on the 60 d,the main symptoms were water-soaked appearance of the flesh tissue,browning and lignification in the core tissue.Further analysis of these differences in flesh and core tissues of 'Xuxiang' about cold sensitivity and cold resistance showed that the flesh tissue is more sensitive to low temperature than the core,and the water-soaked symptoms related to the damage of cell membrane structure and cytosolic exosmosis which caused by low temperature.Browning and lignification in the core tissue was caused by accumulation of secondary metabolites.2.Tissue lignification is the typical symptoms of CI in kiwifruit,and mainly occurs in subcutaneous flesh,core tissue,vascular bundle and its surrounding tissues.The lignified cells were mainly observed in parenchyma cell,and were formed by the deposition of lignin particles along the inside of cell wall,resulting in the secondary wall thickening,consequently,lignin fills the whole cell.1-MCP significantly reduced CI incidence and index in 'Hongyang' and 'Xuxiang',decreased the activity of lignin biosynthetic enzymes and the accumulation of lignin in the flesh,therefore mitigated the subcutaneous flesh tissue lignification.However,it caused serious core browning and lignification in 'Xuxiang'.3.Six members of the cold induced transcription factors(i.e.CBF gene family)were successfully cloned from the fruit of 'Xuxiang' by reference to the 'Hongyang' genome database.By analysing the alignment sequence and phylogenetic tree,we found that the cloned six CBF genes had the structural characteristics and conserved sequence region of CBF gene.Evolutionary tree clustering analysis divided the 6 members into two categories,of which AcCBF1 and AcCBF2 were clustered into one branch,and AcCBF3,AcCBF4,AcCBF5 and AcCBF6 were clustered into another branch.4.Through the analysis of the expression characteristics of six CBF genes,we found that these members had significant differences in expression patterns between cultivars and tissues.The expression level of AcCBF1,AcCBF2 andAcCBF4 in 'Xuxiang' was significantly higher than that of 'Hongyang',and the expression of AcCBF2 and AcCBF4 in 'Hongyang' was extremely low.The relative expression of AcCBF3 in 'Hongyang' was slightly higher than that of 'Xuxiang'.There was no significant difference in the expression of AcCBF5 and AcCBF6 between the two cultivars.The relative expression of the AcCBF1 and AcCBF2 gene were significantly higher in 'Xuxiang' than that of other CBF members.By analysing the characteristics of CBF expression in different tissue flesh and core in 'Xuxiang',we observed that the relative expression of all the CBF members in flesh tissue except AcCBF4 was significantly higher than that of core tissue,especially AcCBF2.Based on these results,we infer that AcCBF1 and AcCBF2 are key genes that involved in the cold resistance of kiwifruit.5.MeJA and MeSA treatments significantly reduced the occurrence of CI,prolonged fruit firmness and increased the cell membrane permeability.However,the browning of fruit core was aggravated by MeSA treatment,and the effect of MeJA was better than MeSA on fruit quality.Further analysis of the regulation of MeJA and MeSA(as a signal molecule)on CBF gene expression showed that there was a different pattern of expression regulation by the two chemical substances in different tissues and genes.MeJA increased the expression of AcCBF1 and AcCBF2 in flesh,but generally decreased the expression of AcCBF1,AcCBF2,AcCBF4 and AcCBF5 in core tissue.While MeSA increased the expression of AcCBF1,AcCBF2 and AcCBF5 in flesh and core tissues,and its effect on AcCBF5 was more obvious compared with that of AcCBF4.6.Based on the analysis of protein sequence alignment of CBF gene family members and the expression pattern of cultivars and tissues,it was concluded that the AcCBF1 and AcCBF2 play pivotal role in regulating cold induction and improving the tissue cold resistance.By analysing the function of AcCBF1 and AcCBF2 in cold-resistance and other abiotic stresses,we found that overexpression of AcCBF1 and AcCBF2 in Arabidopsis caused flat growth,delayed flowering,deepened leaf color;significantly improved the resistance of the transgenic plants to freezing and cold temperature,and the adaption to drought and salt stress,thus improved its cold resistance.Meanwhile,cold resistance was significantly different between AcCBF1 and AcCBF2 over-expressed plants,AcCBF2 transgenic plant mortality was 100% at-10? for 6h while AcCBF1 transgenic plant all survived.Therefore,it is confirmed that AcCBF1 has higher effect on regulation of cold resistance than that of AcCBF2.