The PpMYB75-PpDFR module reveals the difference between 'SR' and its bud variant 'RMHC' in peach red flesh.

'Red Meat Honey Crisp (RMHC)' has been widely cultivated by growers in recent years due to its early maturity, and red meat type characteristics. As a bud variant of 'Super Red (SR)' peach, red flesh is the most distinctive characteristic of 'Red Meat Honey Crisp (RMHC)'. However, the mechanism of red flesh formation in 'RMHC' remains unclear. In this study, 79 differentially produced metabolites were identified by metabolomics analysis. The anthocyanin content in 'RMHC' was significantly higher than that in 'SR' during the same period, such as cyanidin O-syringic acid and cyanidin 3-O-glucoside. Other flavonoids also increased during the formation of red flesh, including flavonols (6-hydroxykaempferol-7-O-glucoside, hyperin), flavanols (protocatechuic acid, (+)-gallocatechin), and flavonoids (chrysoeriol 5-O-hexoside, tricetin). In addition, transcriptomic analysis and RT-qPCR showed that the expression levels of the flavonoid synthesis pathway transcription factor MYB75 and some structural genes, such as PpDFR, PpCHS, PpC4H, and PpLDOX increased significantly in 'RMHC'. Subcellular localization analysis revealed that MYB75 was localized to the nucleus. Yeast single hybridization assays showed that MYB75 bound to the cis-acting element CCGTTG of the PpDFR promoter region. The MYB75-PpDFR regulatory network was identified to be a key pathway in the reddening of 'RMHC' flesh. Moreover, this is the first study to describe the cause for red meat reddening in 'RMHC' compared to 'SR' peaches using transcriptomics, metabolomics and molecular methods. Our study identified a key transcription factor involved in the regulation of the flavonoid synthetic pathway and contributes to peach breeding-related efforts as well as the identification of genes involved in color formation in other species.