Neofunctionalization of an cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus.

Polymethoxyflavones (PMFs) are a class of abundant specialized metabolites with remarkable anticancer properties in citrus. Multiple methoxy groups in PMFs are derived from methylation modification catalyzed by a series of hydroxylases and -methyltransferases (OMTs). However, the specific that catalyze the systematic -methylation of hydroxyflavones remain largely unknown. Here, we report that PMFs are highly accumulated in wild mandarins and mandarin-derived accessions, while undetectable in early-diverging citrus species and related species. Our results demonstrated that three homologous genes, , , and , are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite -methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem , , and may be duplicated from and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization. We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3'--methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species. A 1,041-bp deletion in the promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins. This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.