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Abstract
背景回顾：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 O-methyltransferases (OMTs).
提出问题：However, the specific OMTs that catalyze the systematic O-methylation of hydroxyflavones remain largely unknown.
主要发现：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.
结果1-关键基因鉴定：Our results demonstrated that three homologous genes, CreOMT3, CreOMT4, and CreOMT5, are crucial for PMF biosynthesis in citrus, and their encoded methyltransferases exhibit multisite O-methylation activities for hydroxyflavones, producing seven PMFs in vitro and in vivo.
结果2-功能基因演化：Comparative genomic and syntenic analyses indicated that the tandem CreOMT3, CreOMT4, and CreOMT5 may be duplicated from CreOMT6 and contributes to the genetic basis of PMF biosynthesis in the mandarin group through neofunctionalization.
结果3-蛋白功能变异：We also demonstrated that N17 in CreOMT4 is an essential amino acid residue for C3-, C5-, C6-, and C3′-O-methylation activity and provided a rationale for the functional deficiency of OMT6 to produce PMFs in early-diverging citrus and some domesticated citrus species.
结果4-基因表达变异：A 1,041-bp deletion in the CreOMT4 promoter, which is found in most modern cultivated mandarins, has reduced the PMF content relative to that in wild and early-admixture mandarins.
结论：This study provides a framework for reconstructing PMF biosynthetic pathways, which may facilitate the breeding of citrus fruits with enhanced health benefits.
摘 要
多甲氧基黄酮PMFs是一类在柑橘中特有的代谢物，具较强的抗癌活性。PMFs上多个甲氧基都是来自于一系列羟化酶和O-甲基转移酶催化的甲基化修饰。但是，催化羟基黄酮的O-甲基化的特定OMTs还不清楚。本文中，作者报道了PMFs在野生柑橘以及源自柑橘种质中显著富集，然而在柑橘类早期分化的物种及其近缘种中几乎检测不到PMFs。进一步的研究显示，CreOMT3/4/5三个同源基因对于柑橘中PMF合成至关重要，其编码的甲基转移酶表现出对羟基黄酮多位点的O-甲基化活性，在体内和体外试验中能够产生7种PMFs。比较基因组学和合成分析显示，串联的CreOMT3/4/5可能源自于CreOMT6基因的复制，并通过新功能化作用于柑橘类群中的PMF生物合成。作者还发现CreOMT4中的N17氨基酸残基对于 C3/5/6/3′-O-甲基化活性至关重要，为早期分化的柑橘物种以及一些驯化柑橘物种中OMT6不能合成PMFs的功能缺陷提供了理论依据。在大多数现代栽培柑橘中，CreOMT4基因启动子上缺失了一段长1,041 bp的序列，这明显降低了这些栽培柑橘中PMF的含量。本文的研究为重构PMF生物合成通路提供了一个框架，有助于加速以创制高健康价值水果种质为目标的柑橘育种。
华中农业大学徐娟教授、陈嘉景副教授和徐强教授为本文共同通讯作者，彭昭欣博士为本文第一作者。该研究得到了国家重点研发计划（2023YFD2300600）、湖北洪山实验室基金（2021hszd016）、国家自然科学基金（32272685）以及湖北省自然科学基金重点项目（2021CFA017）的联合资助。
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