Functional Study Of Xyloglucan Endotransglycosylase/Hydrolase Gene MdXTH2 In Apple (Malus × Domestica Borkh.) Fruit Firmness Formation

As one of the most important indicators of the intrinsic quality of apple fruit,firmness not only affects the palatability of the fruit,but also determines its storability and transportability.The mechanism of firmness formation during fruit development is still unclear and the depolymerization of cell wall polysaccharides is the main reason for the decrease in firmness during fruit development.Xyloglucan endotransglucosylase/hydrolase(XTH),a key enzyme for depolymerizing cell wall polysaccharides,plays an important role in regulating fruit firmness.Therefore,it is important to investigate the changes in fruit firmness and cell wall components during apple development and to explore the key XTH genes affecting fruit firmness,in order to select the best varieties and improve the commercial value of apples.In this study,the 12 apple varieties with different textures were used as materials for the analysis of the changes in fruit firmness and cell wall components during fruit development.We analysed their correlations to determine the main cell wall components affecting fruit firmness.Then,based on the tranome data during fruit development,we screened the key XTH genes that regulate fruit firmness,analyzed the expression characteristics of the candidate gene MdXTH2,and analyzed its important functions in fruit firmness formation.The results of the study are as follows:1.Fruit firmness of 12 varieties is similar at the beginning of development,but the different rates of firmness decline lead to differences in firmness at maturity.The content of cell wall components tended to decrease overall during development.The content of cell wall material,cellulose and hemicellulose were all significantly and positively correlated with firmness in different varieties,with total correlation coefficients of 0.871,0.904 and0.757,respectively,while the correlation between pectin content and firmness differed considerably among varieties.Therefore,the decrease in cellulose and hemicellulose content is the universal reason for the decrease in firmness during fruit development.2.Analysis of XTH gene expression in the developing fruit tranome data targeted MdXTH2 as a candidate gene that may regulate fruit firmness.The expression of MdXTH2 is higher in the fruits of harder firmness,where its protein was localized to the cell membrane.3.MdXTH2 was transiently silenced in apple fruit using virus induced gene silencing(VIGS)technology and its effects on fruit firmness and pulp cells were analysed.The results showed a significant reduction in the content of pectin and cell wall material,and a severe degradation of the cell wall,as well as a significant reduction in fruit firmness.In contrast,transient overexpression of MdXTH2 in apple fruit significantly increased the content of components of cell wall,and inhibited the decrease in fruit firmness.In addition,stable genetic transformation of tomato and ‘Orin’ pulp callus again verified its function.The transgenic tomato overexpressing MdXTH2 showed 20.5% and 35.1% higher firmness than the wild type at the veraison and 7 d after veraison,while the fruit was prevented normal colour change.Overexpression of MdXTH2 retarded the growth of callus,and the content of hemicellulose(0.785 mg/g FW)and covalently bound pectin(0.075 mg/g FW)in the transgenic callus was significantly higher than the wild type,while the expression of gene MdACS3 a relating ethylene synthesis and gene MdPME relating pectin degradation was suppressed.The results suggest that overexpression of MdXTH2 inhibited the decrease in fruit firmness and delayed fruit ripening.