Functional characterization of two flavone synthase II members in citrus.

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 and , respectively, in citrus peel significantly enhanced the accumulation of total PMFs, while virus-induced and genes silencing simultaneously significantly reduced the expression levels of both genes and total PMF content in citrus seedlings. and presented distinct expression patterns in different cultivars as well as different developmental stages. Methyl salicylate (MeSA) treatment reduced the expression as well as the PMFs content in the peel of fruit but did not affect the 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.