Flavanone 2-hydroxylase Research Articles

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

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.

Genome-Wide Identification and Expression Profiles of 13 Key Structural Gene Families Involved in the Biosynthesis of Rice Flavonoid Scaffolds.

Flavonoids are a class of key polyphenolic secondary metabolites with broad functions in plants, including stress defense, growth, development and reproduction. Oryza sativa L. (rice) is a well-known model plant for monocots, with a wide range of flavonoids, but the key flavonoid biosynthesis-related genes and their molecular features in rice have not been comprehensively and systematically characterized. Here, we identified 85 key structural gene candidates associated with flavonoid biosynthesis in the rice genome. They belong to 13 families potentially encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), leucoanthocyanidin dioxygenase (LDOX), anthocyanidin synthase (ANS), flavone synthase II (FNSII), flavanone 2-hydroxylase (F2H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). Through structural features, motif analyses and phylogenetic relationships, these gene families were further grouped into five distinct lineages and were examined for conservation and divergence. Subsequently, 22 duplication events were identified out of a total of 85 genes, among which seven pairs were derived from segmental duplication events and 15 pairs were from tandem duplications, demonstrating that segmental and tandem duplication events play important roles in the expansion of key flavonoid biosynthesis-related genes in rice. Furthermore, these 85 genes showed spatial and temporal regulation in a tissue-specific manner and differentially responded to abiotic stress (including six hormones and cold and salt treatments). RNA-Seq, microarray analysis and qRT-PCR indicated that these genes might be involved in abiotic stress response, plant growth and development. Our results provide a valuable basis for further functional analysis of the genes involved in the flavonoid biosynthesis pathway in rice.

Flavonoids are indispensable for complete male fertility in rice.

Flavonoids are essential for male fertility in some but not all plant species. In rice (Oryza sativa), the chalcone synthase mutant oschs1 produces flavonoid-depleted pollen and is male sterile. The mutant pollen grains are viable with normal structure, but they display reduced germination rate and pollen-tube length. Analysis of oschs1/+ heterozygous lines shows that pollen flavonoid deposition is a paternal effect and fertility is independent of the haploid genotypes (OsCHS1 or oschs1). To understand which classes of flavonoids are involved in male fertility, we conducted detailed analysis of rice mutants for branch-point enzymes of the downstream flavonoid pathways, including flavanone 3-hydroxylase (OsF3H; flavonol pathway entry enzyme), flavone synthase II (CYP93G1; flavone pathway entry enzyme), and flavanone 2-hydroxylase (CYP93G2; flavone C-glycoside pathway entry enzyme). Rice osf3h and cyp93g1 cyp93g2 CRISPR/Cas9 mutants, and cyp93g1 and cyp93g2 T-DNA insertion mutants showed altered flavonoid profiles in anthers, but only the osf3h and cyp93g1 cyp93g2 mutants displayed reduction in seed yield. Our findings indicate that flavonoids are essential for complete male fertility in rice and a combination of different classes (flavanones, flavonols, flavones, and flavone C-glycosides) appears to be important, as opposed to the essential role played primarily by flavonols that has been previously reported in several plant species.

Indirect and direct routes to C-glycosylated flavones in Saccharomyces cerevisiae

BackgroundC-glycosylated flavones have recently attracted increased attention due to their possible benefits in human health. These biologically active compounds are part of the human diet, and the C-linkage makes them more resistant to hydrolysis and degradation than O-glycosides. In contrast to O-glycosyltransferases, few C-glycosyltransferases (CGTs) have so far been characterized. Two different biosynthetic routes for C-glycosylated flavones have been identified in plants. Depending on the type of C-glycosyltransferase, flavones can be glycosylated either directly or indirectly via C-glycosylation of a 2-hydroxyflavanone intermediate formed by a flavanone 2-hydroxylase (F2H).ResultsIn this study, we reconstructed the pathways in the yeast Saccharomyces cerevisiae, to produce some relevant CGT substrates, either the flavanones naringenin and eriodictyol or the flavones apigenin and luteolin. We then demonstrated two-step indirect glycosylation using combinations of F2H and CGT, to convert 2-hydroxyflavanone intermediates into the 6C-glucoside flavones isovitexin and isoorientin, and the 8C-glucoside flavones vitexin and orientin. Furthermore, we established direct glycosylation of flavones using the recently identified GtUF6CGT1 from Gentiana triflora. The ratio between 6C and 8C glycosylation depended on the CGT used. The indirect route resulted in mixtures, similar to what has been reported for in vitro experiments. In this case, hydroxylation at the flavonoid 3′-position shifted the ratio towards the 8C-glucosylated orientin. The direct flavone glycosylation by GtUF6CGT1, on the other hand, resulted exclusively in 6C-glucosides.ConclusionsThe current study features yeast as a promising host for production of flavone C-glycosides, and it provides a set of tools and strains for identifying and studying CGTs and their mechanisms of C-glycosylation.

Cytochrome P450 93G1 Is a Flavone Synthase II That Channels Flavanones to the Biosynthesis of Tricin O-Linked Conjugates in Rice.

Flavones are a major class of flavonoids with a wide range of physiological functions in plants. They are constitutively accumulated as C-glycosides and O-linked conjugates in vegetative tissues of grasses. It has long been presumed that the two structural modifications of flavones occur through independent metabolic routes. Previously, we reported that cytochrome P450 93G2 (CYP93G2) functions as a flavanone 2-hydroxylase (F2H) that provides 2-hydroxyflavanones for C-glycosylation in rice (Oryza sativa). Flavone C-glycosides are subsequently formed by dehydratase activity on 2-hydroxyflavanone C-glycosides. On the other hand, O-linked modifications were proposed to proceed after the flavone nucleus is generated. In this study, we demonstrate that CYP93G1, the closest homolog of CYP93G2 in rice, is a bona fide flavone synthase II (FNSII) that catalyzes the direct conversion of flavanones to flavones. In recombinant enzyme assays, CYP93G1 desaturated naringenin and eriodictyol to apigenin and luteolin, respectively. Consistently, transgenic expression of CYP93G1 in Arabidopsis (Arabidopsis thaliana) resulted in the accumulation of different flavone O-glycosides, which are not naturally present in cruciferous plants. Metabolite analysis of a rice CYP93G1 insertion mutant further demonstrated the preferential depletion of tricin O-linked flavanolignans and glycosides. By contrast, redirection of metabolic flow to the biosynthesis of flavone C-glycosides was observed. Our findings established that CYP93G1 is a key branch point enzyme channeling flavanones to the biosynthesis of tricin O-linked conjugates in rice. Functional diversification of F2H and FNSII in the cytochrome P450 CYP93G subfamily may represent a lineage-specific event leading to the prevalent cooccurrence of flavone C- and O-linked derivatives in grasses today.

Identification of a Bifunctional Maize C- and O-Glucosyltransferase

Flavonoids accumulate in plant vacuoles usually as O-glycosylated derivatives, but several species can also synthesize flavonoid C-glycosides. Recently, we demonstrated that a flavanone 2-hydroxylase (ZmF2H1, CYP93G5) converts flavanones to the corresponding 2-hydroxy derivatives, which are expected to serve as substrates for C-glycosylation. Here, we isolated a cDNA encoding a UDP-dependent glycosyltransferase (UGT708A6), and its activity was characterized by in vitro and in vivo bioconversion assays. In vitro assays using 2-hydroxyflavanones as substrates and in vivo activity assays in yeast co-expressing ZmF2H1 and UGT708A6 show the formation of the flavones C-glycosides. UGT708A6 can also O-glycosylate flavanones in bioconversion assays in Escherichia coli as well as by in vitro assays with the purified recombinant protein. Thus, UGT708A6 is a bifunctional glycosyltransferase that can produce both C- and O-glycosidated flavonoids, a property not previously described for any other glycosyltransferase.

Metabolic engineering of the flavone-C-glycoside pathway using polyprotein technology

C-Glycosylated flavonoids are biologically active plant natural products linked to dietary health benefits. We have used polyprotein expression technology to reconstruct part of the respective biosynthetic pathway in tobacco and yeast, such that dihydrochalcone and flavanone precursors are directly converted to C-glycosides. The polyprotein system developed facilitated the simple and efficient co-expression of pathway enzymes requiring different sub-cellular localization in both plants and yeast. The pathway to flavone-C-glucosides comprised a flavanone 2-hydroxylase (F2H), co-expressed with a C-glucosyltransferase (CGT). While pathway engineering in tobacco resulted in only minor C-glycoside formation, when fed with the flavanone naringenin, yeast transformed with the F2H-CGT polyprotein construct produced high concentrations of 2-hydroxynaringenin-C-glucoside in the medium. These fermentation products could then be readily chemically converted to the respective flavone-C-glucosides. The efficiency of the biosynthesis was optimal when both the F2H and CGT were obtained from the same species (rice). These results confirm the coupled roles of the F2H and CGT in producing C-glucosides in vivo, with the use of the polyprotein expression system in yeast offering a useful system to optimize the synthesis of these natural products in quantities suitable for dietary studies.

CYP93G2 Is a Flavanone 2-Hydroxylase Required for C-Glycosylflavone Biosynthesis in Rice

C-Glycosylflavones are ubiquitous in the plant kingdom, and many of them have beneficial effects on human health. They are a special group of flavonoid glycosides in which the sugars are C-linked to the flavone skeleton. It has been long presumed that C-glycosylflavones have a different biosynthetic origin from O-glycosylflavonoids. In rice (Oryza sativa), a C-glucosyltransferase (OsCGT) that accepts 2-hydroxyflavanone substrates and a dehydratase activity that selectively converts C-glucosyl-2-hydroxyflavanones to 6C-glucosylflavones were recently described. In this study, we provide in vitro and in planta evidence that the rice P450 CYP93G2 protein encoded by Os06g01250 is a functional flavanone 2-hydroxylase. CYP93G2 is related to the CYP93B subfamily, which consists of dicot flavone synthase II enzymes. In the presence of NADPH, recombinant CYP93G2 converts naringenin and eriodictyol to the corresponding 2-hydroxyflavanones. In addition, CYP93G2 generates 2-hydroxyflavanones, which are modified by O-glycosylation in transgenic Arabidopsis (Arabidopsis thaliana). Coexpression of CYP93G2 and OsCGT in Arabidopsis resulted in the production of C-glucosyl-2-hydroxyflavanones in the dibenzoylmethane tautomeric form. The same structure was reported previously for the in vitro OsCGT reaction products. Thus, CYP93G2 generates 2-hydroxyflavanone substrates from flavanones for C-glucosylation by OsCGT in planta. Furthermore, knocking down Os06g01250 in rice (O. sativa subsp. japonica 'Zhonghua 11') preferentially depleted the accumulation of C-glycosylapigenin, C-glycosylluteolin, and C-glycosylchrysoeriol but did not affect the levels of tricin, which is frequently present as O-glycosides in cereals. Taken together, our work conclusively assigned CYP93G2 as the first enzyme that channels flavanones to C-glycosylflavone biosynthesis in rice.

Cytochrome P450s in flavonoid metabolism

In this review, cytochrome P450s characterized at the molecular level catalyzing aromatic hydroxylations, aliphatic hydroxylations and skeleton formation in the flavonoid metabolism are surveyed. They are involved in the biosynthesis of anthocyanin pigments and condensed tannin (CYP75, flavonoid 3′,5′-hydroxylase and 3′-hydroxylase), flavones [CYP93B, (2S)-flavanone 2-hydroxylase and flavone synthase II], and leguminous isoflavonoid phytoalexins [CYP71D9, flavonoid 6-hydroxylase; CYP81E, isoflavone 2′-hydroxylase and 3′-hydroxylase; CYP93A, 3,9-dihydroxypterocarpan 6a-hydroxylase; CYP93C, 2-hydroxyisoflavanone synthase (IFS)]. Other P450s of the flavonoid metabolism include methylenedioxy bridge forming enzyme, cyclases producing glyceollins, flavonol 6-hydroxylase and 8-dimethylallylnaringenin 2′-hydroxylase. Mechanistic studies on the unusual aryl migration by CYP93C, regulation of IFS expression in plant organs and its biotechnological applications are introduced, and flavonoid metabolisms by non-plant P450s are also briefly discussed.

Molecular cloning and biochemical characterization of a novel cytochrome P450, flavone synthase II, that catalyzes direct conversion of flavanones to flavones.

Cytochrome P450 cDNAs, AFNS2 and TFNS5, were isolated from snapdragon and torenia petal cDNA libraries, respectively, based on the sequence homology with licorice CYP93B1 cDNA encoding (2S)-flavanone 2-hydroxylase. They were expressed in yeast and identified to encode flavone synthase II catalyzing direct conversion of flavanones to flavones probably via 2-hydroxyflavanones.

Induction of isoflavonoid and retrochalcone branches of the flavonoid pathway in cultured Glycyrrhiza echinata cells treated with yeast extract.

Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4'-dihydroxyflavone). From these cells, putative full-length cDNAs encoding cytochrome P450s, (2S)-flavanone 2-hydroxylase and isoflavone 2'-hydroxylase, were cloned.

Identification of a cytochrome P450 cDNA encoding (2 S)-flavanone 2-hydroxylase of licorice ( Glycyrrhiza echinata L.; Fabaceae) which represents licodione synthase and flavone synthase II

The microsome of insect cells expressing CYP Ge-5 (CYP93B1), a cytochrome P450 cDNA of licorice ( Glycyrrhiza echinata L.), catalyzed the formation of [ 14C]licodione and [ 14C]-2-hydroxynaringenin from (2 S)-[ 14C]liquiritigenin and (2 S)-[ 14C]naringenin, respectively. On acid treatment, the products were converted to 14C-labeled 7,4′-dihydroxyflavone and apigenin. Eriodictyol was also converted to luteolin by the reaction with the microsome of yeast expressing CYP93B1 and subsequent acid treatment. CYP93B1 was thus shown to encode (2 S)-flavanone 2-hydroxylase, which has previously been designated to licodione synthase and flavone synthase II depending on the substrates employed.