Neofunctionalization of an <i>OMT</i> cluster dominates polymethoxyflavone biosynthesis associated with the domestication of citrus.

Abstract

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 <i>O</i>-methyltransferases (OMTs). However, the specific <i>OMTs</i> that catalyze the systematic <i>O</i>-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, <i>CreOMT3</i>, <i>CreOMT4</i>, and <i>CreOMT5</i>, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite <i>O</i>-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo. Comparative genomic and syntenic analyses indicated that the tandem <i>CreOMT3</i>, <i>CreOMT4</i>, and <i>CreOMT5</i> may be duplicated from <i>CreOMT6</i> 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'-<i>O</i>-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 <i>CreOMT4</i> 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.