Integrating multi-omics analysis unravels the molecular mechanisms underlying variations in bioactive flavonoids in Citrus reticulata ‘Chachi’ grafted onto different citrus rootstocks

Grafting is the primary means of propagation for citrus, wherein the choice of rootstock significantly influences the phenotypic traits. Nevertheless, the underlying molecular mechanisms by which various rootstocks affect the metabolite profiles of Citrus reticulata 'Chachi' (CRC) fruit peels remain to be clarified. To address the problem, we investigated the metabolomic and transcriptomic profiles of CRC fruit peels grafted onto three different rootstocks. A total of 1276 metabolites from various compound classes were identified, including flavonoids, alkaloids, phenolic acids, lignans, coumarins, and terpenoids. In comparison to homo-grafts, the fruit peels of Poncirus trifoliata rootstock exhibited a significantly higher total flavonoids content, whereas fruit peels from the Citrus limon rootstock demonstrated comparable levels. The differentially expressed genes and metabolites in the CRC fruit peels of different rootstocks were predominantly enriched in the flavonoid biosynthesis process. Moreover, flavonoid biosynthesis-related genes, ranging from upstream (phenylalanine ammonia-lyase) to branch pathway genes (flavone synthase and flavonol synthase), as well as flavonoid-modifying enzymes like O-methyltransferase, demonstrated a significant positive correlation with flavonoid accumulation patterns among the various rootstocks. Additionally, three R2R3-MYB transcription factors involved in flavonoid regulation were found to correlate with flavonoid accumulation patterns. Notably, CrMYB15 was functionally characterized as a key positive regulator of flavonoids. Our study systematically characterized the accumulation patterns of flavonoids and uncovered the underlying regulatory mechanisms in CRC grafted onto distinct rootstocks. These findings provide actionable strategies to enhance medicinal traits of CRC, particularly through rootstock-mediated optimization of bioactive flavonoid profiles.