Complementary transcriptomic and metabolomics analysis reveal the molecular mechanisms of EGCG3″Me biosynthesis in Camellia sinensis

• High level EGCG3″Me content was detected in JMD but not in FD. • Expression levels of CCoAOMT were consistent with EGCG3″Me content in three tea plant cultivars. • CCoAOMT protein could directly catalyze the biosynthesis of EGCG3″Me. • Thirty eight TFs were found might regulate the biosynthesis of EGCG3″Me. Epigallocatechin-3- O -(3- O -methyl) gallate (EGCG3″Me), naturally occurring in specific tea plant cultivars, is famous for its decent bioavailability and antiallergic activity. To investigate the molecular mechanisms of EGCG3″Me biosynthesis and transcriptional regulation in tea leaves, we combined multi-omics data from three tea plant cultivars (Fuding Dabaicha, FD; Mingke 1 (Jinguanyin), JGY; and Jinmudan, JMD). High-performance liquid chromatography analysis indicated that EGCG3″Me content was highest in JMD (7.78 mg/g), moderate in JGY (3.12 mg/g), and lowest in FD (0 mg/g), which was similar to the results of the non-targeted metabolic analysis. The high level of EGCG3″Me in JMD was concomitant with high levels of monomeric and polymerized catechin derivatives, but low levels of flavonols, flavones, and their glycosides, which was consistent with the expression levels of most catechin/flavonoid biosynthetic genes. Among them, the expression level of the CCoAOMT gene in the three tea plant cultivars was positively associated with the relative content of EGCG3″Me. Further verification revealed that CCoAOMT protein could directly catalyze the formation of EGCG3″Me. We further identified 38 transcription factors from transcriptome as promising regulatory candidates for regulating CCoAOMT gene expression in tea leaves. These findings provide an essential foundation for further research on the biosynthesis and transcriptional regulation of O -methylated catechins in tea leaves.