Functional characterization of two flavone synthase II members in citrus

Abstract

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 did not affect 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.

Acknowledgements

This research was supported by the China Postdoctoral Science Foundation (2021 M700124 and 2021 M692845), the National Natural Science Foundation of China (32072132), and the Fundamental Research Funds for the Central Universities (K20220104).

Author contributions

C.S., C.Zhao and J.Z. designed the project. J.Z. performed the experiments with assistance from C.Zhao, X.L., Z.L., Q.G., and C.Zhou. J.Z. and C.Zhao analysed the data and drafted the manuscript. Y.L. conducted the molecular docking analysis. Y.W. helped with the LC–MS analysis. L.L. and D.W. provided plant materials for this study. C.S. and J.C. improved the draft. All authors approved this article.

Data availability

The raw data of transcriptome are available at GenBank. The accession numbers are PRJNA923786 and PRJNA924350. Other data presented in the research are all comprised in this article.

Conflict of interest statement

All authors declare that they have no conflict of interest.

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