Integrative analysis of transcriptome and metabolome reveals key enzyme genes involved in flavonoid biosynthesis in Citrus aurantium L.

Aurantii Fructus is defined as the dried unripe fruit of Citrus aurantium L. and its cultivated varieties (Rutaceae family), typically harvested from late June to early July in China. Its primary bioactive components are flavonoids, along with volatile oils, alkaloids, and other pharmacologically active compounds. Although its pharmacological effects have been systematically investigated, the molecular mechanisms of its key flavonoids remain poorly understood. In this study, samples collected at eight harvesting periods were analyzed using ultra-high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry. A total of 20 compounds were identified, including 14 flavonoids, and the dynamic accumulation patterns of eight key flavonoid components were elucidated. Transcriptome sequencing generated 20.98 Gb of raw data, which, after alignment to the citrus genome, assembly, and correction, yielded 137,610 high-confidence consensus sequences with a 99.85% gene annotation rate. A comprehensive reconstruction of the flavonoid biosynthetic pathway identified 35 key enzyme-encoding genes showing distinct spatiotemporal expression patterns. Furthermore, in this study, the specific functions of two key enzymes in the modification of C. aurantium L. flavonoids were experimentally validated through enzyme activity assays. O-methyltransferase was demonstrated to catalyze the conversion of naringenin to isosakuranetin, while glycosyltransferase was confirmed to mediate the biosynthesis of hesperetin-7-O-glucoside. Based on integrated metabolomic and transcriptomic analyses, a spatiotemporal regulatory network model for C. aurantium L. flavonoid components was constructed. This model not only provides a theoretical foundation for elucidating the regulatory mechanisms of flavonoid biosynthesis but also offers strategic insights for enhancing the content of pharmacologically active flavonoid components through targeted regulation.