Two Caffeoyl-CoA O-methyltransferase-like enzyme are involved in the biosynthesis of polymethoxyflavones in Citrus reticulata 'Chachiensis'.

Polymethoxyflavones (PMFs) are abundant in citrus plants and exhibit remarkable biological activities in the inhibition of the occurrence and development of various tumours. O-methyltransferase (OMTs)-mediated methylation plays a crucial role in the biosynthesis of PMFs. However, the OMTs catalyzing the methylation of hydroxyflavonoids in citrus have not been fully identified. In this study, we identified two novel CCoAOMT-like enzymes in Citrus reticulata 'Chachiensis', designated CrcCCoAOMT7-1 and CrcCCoAOMT7-2. The transient overexpression and virus-induced gene silencing experiments further confirmed the essential role of CrcCCoAOMT7-1/7-2 in the regulation of PMF synthesis in citrus. Furthermore, in vitro functional analysis showed that CrcCCoAOMT7-1 preferentially catalyzes flavones, while CrcCCoAOMT7-2 exhibits a preference for flavanones and quercetin (flavonol). The two enzymes methylate the 3'-, 4'-, 3-, 6-, 8-OH sites and the 3'-, 4'-, 6-, 7-, 8-OH sites with hydroxyflavonoids, respectively, covering nearly all methylation sites during the biosynthesis of PMFs. Functional diversification of CCoAOMTs expended our understanding of PMFs biosynthesis modes in citrus. This study enriches the PMF biosynthetic pathway in citrus and lays the foundation for the selection and breeding of high-quality Citrus reticulata 'Chachiensis' mandarins, as well as the synthesis of natural anticancer PMFs through genetic engineering.