Polymethoxylated flavones (PMFs), the main form of flavones in citrus, are derived from the flavone branch of the flavonoid biosynthesis pathway. Flavone synthases (FNSs) are enzymes that catalyze the synthesis of flavones from flavanones. However, the FNS in citrus has not been characterized yet. Here, we identified two type II FNSs, designated CitFNSII-1 and CitFNSII-2, based on phylogenetics and transcriptome analysis. Both recombinant CitFNSII-1 and CitFNSII-2 proteins directly converted naringenin, pinocembrin, and liquiritigenin to the corresponding flavones in yeast. In addition, transient overexpression of CitFNSII-1 and CitFNSII-2 respectively in citrus peel significantly enhanced the accumulation of total PMFs, while virus-induced CitFNSII-1 and CitFNSII-2 genes silencing simultaneously significantly reduced the expression levels of both genes and total PMF content in citrus seedlings. CitFNSII-1 and CitFNSII-2 presented distinct expression patterns in different cultivars as well as different developmental stages. Methyl salicylate (MeSA) treatment reduced the CitFNSII-2 expression as well as the PMFs content in the peel of Citrus sinensis fruit but not affected the CitFNSII-1 expression. These results indicated that both CitFNSII-1 and CitFNSII-2 participated in the flavone biosynthesis in citrus while the regulatory mechanism governing their expression might be specific. Our findings improved the understanding of the PMFs biosynthesis pathway in citrus and laid the foundation for further investigation on flavone synthesis regulation.

中文翻译：

---

柑橘中两个黄酮合酶 II 成员的功能表征

多甲氧基黄酮 (PMF) 是柑橘中黄酮的主要形式，源自类黄酮生物合成途径的黄酮分支。黄酮合酶（FNS）是催化从黄烷酮合成黄酮的酶。然而，柑橘中的 FNS 尚未得到表征。在这里，我们根据系统发育和转录组分析鉴定了两种 II 型 FNS，命名为 CitFNSII-1 和 CitFNSII-2。重组 CitFNSII-1 和 CitFNSII-2 蛋白均直接将柚皮素、松香素和甘草素转化为酵母中相应的黄酮。此外，柑橘皮中CitFNSII-1和CitFNSII-2的瞬时过表达分别显着增强了总PMF的积累，而病毒诱导的CitFNSII-1和CitFNSII-2基因沉默同时显着降低了这两个基因和总PMF的表达水平。柑橘幼苗中的含量。CitFNSII-1和CitFNSII-2在不同品种和不同发育阶段呈现出不同的表达模式。水杨酸甲酯（MeSA）处理降低了柑橘果实中CitFNSII-2的表达以及果皮中PMF的含量，但不影响CitFNSII-1的表达。这些结果表明，CitFNSII-1和CitFNSII-2均参与柑橘中的黄酮生物合成，且其表达的调控机制可能是特定的。我们的研究结果提高了对柑橘PMFs生物合成途径的认识，并为进一步研究黄酮合成调控奠定了基础。