Functional Characterization of a Flavone Synthase That Participates in a Kumquat Flavone Metabolon.

Flavones predominantly accumulate as - and -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 -glycosylation) and another involving the direct catalysis of flavanones to flavones for -glycosylation. However, FNSII has not yet been characterized in kumquats. In this study, we identified two kumquat genes ( and ), based on transcriptome and bioinformatics analysis. Data from and assays showed that FcFNSII-2 directly synthesized apigenin and acacetin from naringenin and isosakuranetin, respectively, whereas showed no detectable catalytic activities with flavanones. In agreement, transient overexpression of in kumquat peels significantly enhanced the transcription of structural genes of the flavonoid-biosynthesis pathway and the accumulation of several -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 -glycosyl flavone metabolon that channels flavanones for -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.