Integrated Metabolomic-Transcriptomic Analyses of Flavonoid Accumulation in Citrus Fruit under Exogenous Melatonin Treatment

The flavonoids in citrus fruits are crucial physiological regulators and natural bioactive products of high pharmaceutical value. Melatonin is a pleiotropic hormone that can regulate plant morphogenesis and stress resistance and alter the accumulation of flavonoids in these processes. However, the direct effect of melatonin on citrus flavonoids remains unclear. In this study, nontargeted metabolomics and transcriptomics were utilized to reveal how exogenous melatonin affects flavonoid biosynthesis in "Bingtangcheng" citrus fruits. The melatonin treatment at 0.1 mmol L-1 significantly increased the contents of seven polymethoxylated flavones (PMFs) and up-regulated a series of flavonoid pathway genes, including 4CL (4-coumaroyl CoA ligase), FNS (flavone synthase), and FHs (flavonoid hydroxylases). Meanwhile, CHS (chalcone synthase) was down-regulated, causing a decrease in the content of most flavonoid glycosides. Pearson correlation analysis obtained 21 transcription factors co-expressed with differentially accumulated flavonoids, among which the AP2/EREBP members were the most numerous. Additionally, circadian rhythm and photosynthesis pathways were enriched in the DEG (differentially expressed gene) analysis, suggesting that melatonin might also mediate changes in the flavonoid biosynthesis pathway by affecting the fruit's circadian rhythm. These results provide valuable information for further exploration of the molecular mechanisms through which melatonin regulates citrus fruit metabolism.