Functional analysis of the xyloglucan endotransglycosylase/hydrolase gene MdXTH2 in apple fruit firmness formation

• MdXTH2 is a key gene influencing apple fruit firmness, identified through transcriptomic and metabolomic analyses. • Overexpression of MdXTH2 increased fruit firmness, delayed fruit ripening, and inhibited calli growth in apple and tomato. • MdNAC72 directly binds to the MdXTH2 promoter and represses its transcription, linking fruit firmness with ripening regulation. Apple fruit firmness is a crucial index for measuring the internal quality of apples, which influences palatability, storage capacity and transportability. The primary cause of reduced firmness during fruit development is the hydrolysis of cell wall polysaccharides. Xyloglucan endotransglycosylase/hydrolase (XTH) is a key enzyme involved in the depolymerization of cell wall polysaccharides, but the mechanism of its involvement in the formation of fruit firmness remains unclear. Here, we identified the gene MdXTH2 by integrating metabolomic and transcriptomic data, and analyzed its function and molecular mechanism in the formation of apple fruit firmness. The results showed downward trends in both fruit firmness and cell wall components throughout fruit development. The contents of cell wall material, cellulose, and hemicellulose in various apple varieties exhibited significant positive correlations with firmness, with total correlation coefficients of 0.862, 0.884, and 0.891, respectively. Overexpression of MdXTH2 significantly increased fruit firmness in apple and tomato, inhibited fruit ripening, and significantly suppressed the growth of calli. The upstream transcription factor MdNAC72 of the MdXTH2 gene can promote the expression of fruit ripening-related genes. Furthermore, dual-luciferase, yeast one-hybrid, and electrophoretic mobility shift assay (EMSA) demonstrated that MdNAC72 down-regulates the transcription of MdXTH2 by binding to its promoter. In summary, the results of this study provide a strategy for examining fruit quality regulation and a theoretical basis for breeding apple varieties with moderate firmness through genetic improvement.