Expression of the polyphenol oxidase gene <i>MdPPO7</i> is modulated by MdWRKY3 to regulate browning in sliced apple fruit

Browning is a pervasive problem in horticultural products, substantially diminishing the appearance, flavor, and nutritional value of fruit, including important fruits like apple (Malus × domestica Borkh.). In this study, we compared the physiological characteristics of the browning-resistant line 'Rb-18' with the susceptible variety 'Fuji' and found that the polyphenol oxidase (PPO) enzyme activity and phenolic content of 'Rb-18' were significantly lower than those in 'Fuji'. In addition, the PPO enzyme in 'Fuji' showed a stronger affinity for its substrate, catechol, compared to 'Rb-18'. Through transcriptome and RT-qPCR analyses, MdPPO7 expression was identified as contributing to flesh browning after cutting. Subsequent fruit injection and stable genetic transformation of the MdPPO7 gene into apple fruit and calli determined that syringic acid, procyanidin, phloridzin, chlorogenic acid, gallic acid, catechin, and caffeic act as its catalytic substrates in the process involved in browning. Furthermore, luciferase reporter, yeast 1-hybrid, β-glucuronidase reporter assays and ChIP-qPCR analysis demonstrated that a WRKY transcription factor (MdWRKY3) binds to the promoter region of polyphenol oxidase gene (MdPPO7) and positively regulates its expression to promote apple flesh browning. This study provides insights into the molecular regulatory mechanisms of fruit browning in fresh-cut apples and provides a theoretical basis for the generation of high-quality apple germplasm resources.