Abstract
Flavones predominantly accumulate as O- and C-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 C-glycosylation) and another involving the direct catalysis of flavanones to flavones for O-glycosylation. However, FNSII has not yet been characterized in kumquats. In this study, we identified two kumquat FNSII genes (FcFNSII-1 and FcFNSII-2), based on transcriptome and bioinformatics analysis. Data from in vivo and in vitro assays showed that FcFNSII-2 directly synthesized apigenin and acacetin from naringenin and isosakuranetin, respectively, whereas FcFNSII-1 showed no detectable catalytic activities with flavanones. In agreement, transient overexpression of FcFNSII-2 in kumquat peels significantly enhanced the transcription of structural genes of the flavonoid-biosynthesis pathway and the accumulation of several O-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 O-glycosyl flavone metabolon that channels flavanones for O-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.
Highlights
Flavones, one of the largest subclasses of flavonoids, perform various physiological roles in plants, such as participating in responses to biotic and abiotic stresses (Morimoto et al, 1998; Du et al, 2010a,b; Kong et al, 2010; Jiang et al, 2016; Righini et al, 2019)
Based on transcriptome analysis of “Hongkong” kumquat (F. hindsii), generated from 13 different tissues from five organs (Zhu et al, 2019), two flavone synthase II (FNSII) homologs were identified. Their expression was positively correlated with the expression levels of upstream genes in the flavonoid-biosynthesis pathway, such as CHS1, CHS2, CHI, and chalcone isomerase-like (CHIL) (Pearson’s correlation coefficient; between FcFNSII-1 and flavonoid biosynthetic genes was >0.7 and between FcFNSII-2 and flavonoid biosynthetic genes >0.6) (Figure 2A)
We characterized a kumquat type II flavone synthases (FNSs) (FcFNSII-2) that catalyzes the direct conversion of flavanones to flavones
Summary
One of the largest subclasses of flavonoids, perform various physiological roles in plants, such as participating in responses to biotic and abiotic stresses (Morimoto et al, 1998; Du et al, 2010a,b; Kong et al, 2010; Jiang et al, 2016; Righini et al, 2019). Flavonoid biosynthesis in higher plants initiates from the stepwise condensation of p-coumaroyl-coenzyme A (CoA) with three malonyl-CoAs in a reaction catalyzed by chalcone synthase (CHS). This is followed by chalcone cyclization into naringenin by chalcone isomerase (CHI) (Winkel-Shirley, 2001). Licorice CYP93B1 (Akashi et al, 1998), maize CYP93G5 (Morohashi et al, 2012), Medicago truncatula CYP93B10/11 (Zhang et al, 2007), rice CYP92G2 (Du et al, 2010a), and sorghum CYP93G3 (Du et al, 2010b) function as flavanone 2-hydroxylases (F2Hs) by catalyzing the conversion of flavanones into 2-hydroxyflavanones for C-glycosylation
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