AUTHOR=Tian Shulin , Yang Yuyan , Wu Tao , Luo Chuan , Li Xin , Zhao Xijuan , Xi Wanpeng , Liu Xiaogang , Zeng Ming 

TITLE=Functional Characterization of a Flavone Synthase That Participates in a Kumquat Flavone Metabolon

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.826780

DOI=10.3389/fpls.2022.826780

ISSN=1664-462X

ABSTRACT=<p>Flavones predominantly accumulate as <italic>O</italic>- and <italic>C</italic>-glycosides in kumquat plants. Two catalytic mechanisms of flavone synthase II (FNSII) support the biosynthesis of glycosyl flavones, one involving flavanone 2-hydroxylase (which generates 2-hydroxyflavanones for <italic>C</italic>-glycosylation) and another involving the direct catalysis of flavanones to flavones for <italic>O</italic>-glycosylation. However, FNSII has not yet been characterized in kumquats. In this study, we identified two kumquat <italic>FNSII</italic> genes (<italic>FcFNSII-1</italic> and <italic>FcFNSII-2</italic>), based on transcriptome and bioinformatics analysis. Data from <italic>in vivo</italic> and <italic>in vitro</italic> assays showed that FcFNSII-2 directly synthesized apigenin and acacetin from naringenin and isosakuranetin, respectively, whereas <italic>FcFNSII-1</italic> showed no detectable catalytic activities with flavanones. In agreement, transient overexpression of <italic>FcFNSII-2</italic> in kumquat peels significantly enhanced the transcription of structural genes of the flavonoid-biosynthesis pathway and the accumulation of several <italic>O</italic>-glycosyl flavones. Moreover, studying the subcellular localizations of FcFNSII-1 and FcFNSII-2 demonstrated that N-terminal membrane-spanning domains were necessary to ensure endoplasmic reticulum localization and anchoring. Protein–protein interaction analyses, using the split-ubiquitin yeast two-hybrid system and bimolecular fluorescence-complementation assays, revealed that FcFNSII-2 interacted with chalcone synthase 1, chalcone synthase 2, and chalcone isomerase-like proteins. The results provide strong evidence that FcFNSII-2 serves as a nucleation site for an <italic>O</italic>-glycosyl flavone metabolon that channels flavanones for <italic>O</italic>-glycosyl flavone biosynthesis in kumquat fruits. They have implications for guiding genetic engineering efforts aimed at enhancing the composition of bioactive flavonoids in kumquat fruits.</p>