Flavonol Synthesis Research Articles

Functional characterization of a HD-ZIP IV transcription factor NtHDG2 in regulating flavonols biosynthesis in Nicotiana tabacum

The HD-ZIP Ⅳ transcription factors have been identified and functional characterized in many plant species. However, no tobacco HD-ZIP IV gene has been isolated, and it is not yet known whether HD-ZIP IV genes are involved in controlling flavonols accumulation in plants. Here, we cloned a HD ZIP gene named NtHDG2 from Nicotiana tabacum, which belongs to the class IV of HD-ZIP family, and the NtHDG2-GFP fusion protein is localized to the nucleus. We further observed that the flavonols contents in the NtHDG2 overexpression leaves increase to 1.9–4.5 folds of that in WT plants, but in the NtHDG2-RNAi plants the flavonols contents reduce to 20.9%–52.7% of that in WT plants. The transcriptions of one regulatory gene NtMYB12, and three structural genes (NtPAL, NtF3′H, NtF3GT), contributing to flavonols biosynthesis, were significantly induced by NtHDG2. However, the transcription level of NtNAC002, a flavonols biosynthesis repressor, was also significantly up-regulated in NtHDG2-overexpression lines, but significantly down-regulated in the RNAi lines, indicating that HDG2 regulates the synthesis of flavonols as a complex regulatory network. Moreover, ectopic expression of NtHDG2 gene promoted the transcription of several AP2/ERF genes, including NtERF1-5, NtERF109, NtDREB1, and NtCIPK11, which participate in regulating root development and resistance to abiotic stresses. Our findings reveal the new function of HD-ZIP IV transcription factors in flavonoids biosynthesis, and indicate that HD-ZIP IV members may play an important role in plant resistance to abiotic stress. The NtHDG2 gene provides a promising target for genetically manipulating to increase the amounts of flavonols in tobacco leaves.

Selective Hydrogen Atom Abstraction from Dihydroflavonol by a Nonheme Iron Center Is the Key Step in the Enzymatic Flavonol Synthesis and Avoids Byproducts.

The plant non-heme iron dioxygenase flavonol synthase performs a regioselective desaturation reaction as part of the biosynthesis of the signaling molecule flavonol that triggers the growing of leaves and flowers. These compounds also have health benefits for humans. Desaturation of aliphatic compounds generally proceeds through two consecutive hydrogen atom abstraction steps from two adjacent carbon atoms and in nature often is performed by a high-valent iron(IV)-oxo species. We show that the order of the hydrogen atom abstraction steps, however, is opposite of those expected from the C-H bond strengths in the substrate and determines the product distributions. As such, flavonol synthase follows a negative catalysis mechanism. Using density functional theory methods on large active-site model complexes, we investigated pathways for desaturation and hydroxylation by an iron(IV)-oxo active-site model. Contrary to thermochemical predictions, we find that the oxidant abstracts the hydrogen atom from the strong C2-H bond rather than the weaker C3-H bond of the substrate first. We analyze the origin of this unexpected selective hydrogen atom abstraction pathway and find that the alternative C3-H hydrogen atom abstraction would be followed by a low-energy and competitive substrate hydroxylation mechanism hence, should give considerable amount of byproducts. Our computational modeling studies show that substrate positioning in flavonol synthase is essential, as it guides the reactivity to a chemo- and regioselective substrate desaturation from the C2-H group, leading to desaturation products efficiently.

Allelopathic Effects of Native Versus Invasive Plants on One Major Invader.

Allelopathy is defined as the effects (stimulatory and inhibitory) of a plant on the development of neighboring plants through the release of secondary compounds. Autoallelophaty is the beneficial or harmful effect of a plant species on itself. The allelopathic potential belonging to a native species could induce a biotic resistance against invasive plants, whereas allelochemicals released by exotic species could favor the establishment of invasive species (invasional meltdown). The aim of our study was to examine the potential allelopathic effect of four plant species on the target species Ludwigia hexapetala using two experiments. In the first experiment, we tested the allelopathic effect of root and leaf leachates of the two congeneric exotic species Ludwigia hexapetala and Ludwigia peploides on L. hexapetala, while in the second experiment, we studied the allelopathic effect of root and leaf leachates of a sympatric exotic species Myriophyllum aquaticum and of one native species Mentha aquatica on L. hexapetala. We measured the stem length to calculate the relative growth rate and four physiological traits (nitrogen balance index and flavonol, chorophyll, anthocyanin indices) of the target plants on a weekly basis. At the end of the experiment, we determined the aboveground and belowground biomass. We also counted the number of lateral branches and measured their lengths. We found that the root leachates of L. peploides and of Myriophyllum aquaticum had stimulated the synthesis of flavonols of L. hexapetala. Leaf leachate of L. hexapetala also stimulated its own flavonol synthesis. Also, the root leachate of L. peploides had stimulated the total biomass and length of lateral branches of L. hexapetala, whereas the production of lateral branches had been stimulated by root leachates of both Ludwigia species and by leaf leachate of Myriophyllum aquaticum. The autoallelopathy of L. hexapetala could explain its invasiveness. Both leachates produced by Mentha aquatica had no effect on the physiological and morphological traits of the invasive L. hexapetala and indicated no biotic resistance in the recipient community. The two invasive plant species Myriophyllum aquaticum and L. peploides could favor the establishment of L. hexapetala. These results suggested an “invasional meltdown.”

Flavonol Profile Is a Reliable Indicator to Assess Canopy Architecture and the Exposure of Red Wine Grapes to Solar Radiation.

Exposure to solar radiation is a determining factor of grape composition. Flavonol synthesis is upregulated by solar radiation leaving a fingerprint on flavonol profile. This study aimed to test the factors affecting flavonol accumulation and profile and their potential as an indicator to assess the overall exposure of red wine grape berry to solar radiation. We performed three experiments to study the response of flavonol accumulation and profile to (1) three different solar radiation exclusion treatments during berry development; (2) canopy porosity and leaf area index (LAI); and (3) spatial variability of water status, vigor and ripening and cultural practices in commercial vineyards. Results showed a strong relationship between global radiation, inverse dormant pruning weights or canopy porosity (inversely proportional to LAI) and % kaempferol or % quercetin. Furthermore, the increase in concentration of the above two flavonols was associated with a reduction of % myricetin. Total flavonol content, % kaempferol, % quercetin, and % myricetin had significant correlations with inverse dormant pruning weights, but these were less sensitive to over-ripening or water deficits. Flavonol profile was associated to site hydrology (wetness index) through changes in vigor, and to LAI; and responded to shoot thinning or fruit-zone leaf removal. These results support the reliability of the flavonol profile as an assessment parameter for studies aiming to discuss canopy architecture or the effect of solar radiation on grapevine berries.

CsMYB60 is a key regulator of flavonols and proanthocyanidans that determine the colour of fruit spines in cucumber.

Spine colour is an important fruit quality trait that influences the commercial value of cucumber (Cucumis sativus). However, little is known about the metabolites and the regulatory mechanisms of their biosynthesis in black spine varieties. In this study, we determined that the pigments of black spines are flavonoids, including flavonols and proanthocyanidins (PAs). We identified CsMYB60 as the best candidate for the previously identified B (Black spine) locus. Expression levels of CsMYB60 and the key genes involved in flavonoid biosynthesis were higher in black-spine inbred lines than that in white-spine lines at different developmental stages. The insertion of a Mutator-like element (CsMULE) in the second intron of CsMYB60 decreased its expression in a white-spine line. Transient overexpression assays indicated that CsMYB60 is a key regulatory gene and Cs4CL is a key structural gene in the pigmentation of black spines. In addition, the DNA methylation level in the CsMYB60 promoter was much lower in the black-spine line compared with white-spine line. The CsMULE insert may decrease the expression level of CsMYB60, causing hindered synthesis of flavonols and PAs in cucumber fruit spines.

Prokaryotic expression and in vitro enzyme activity analysis of flavonol synthase in Chrysanthemum morifolium cv. 'Hangju'

We cloned flavonol synthase gene (named as CmFLS) by RACE from Chrysanthemum morifolium cv. 'Hangju' based on transcriptome database. Sequencing results showed that 1 235 bp sequence was acquired with the largest open reading frame (ORF) of 1 008 bp, which encoded 335 amino acids. The predicted CmFLS encoded protein had an isoelectric point (pI) of 5.41. The phylogenetic tree analysis indicated that CmFLS was highly homologous to other FLSs, which identified from the species of Compositae. The recombinant fusion protein, with a molecular mass of 43 kDa, was successfully expressed by prokaryotic expression system. Meanwhile, Ni-NTA resin was used to purify the recombinant fusion protein, and the Ni-Native Buffer containing 250 mmol·L⁻¹ imidazole was most favorable for elution. The purified recombinant fusion protein was subjected to in vitro catalytic reaction, and then the products were extracted and analyzed by HPLC. The results showed that the recombinant fusion protein CmFLS was able to catalyze the production of quercetin by dihydroquercetin under specific buffer and reaction conditions, which indicated that the functional protein encoded by CmFLS had dioxygenase activity in the biosynthetic pathway of flavonoids biosynthesis in Ch. morifolium cv. 'Hangju'. The above results laid the foundation for further studying on CmFLS, and provided new ideas for the regulation of flavonoids metabolism from the molecular level and the catalytic synthesis of flavonols in vitro.

Low Temperature-Enhanced Flavonol Synthesis Requires Light-Associated Regulatory Components in Arabidopsis thaliana.

Plants are continuously exposed to a myriad of stresses, which lead to the formation of secondary metabolites including flavonoids. Studies suggest that low temperature exposure leads to enhanced flavonoid accumulation in Arabidopsis thaliana. In addition, flavonoid biosynthesis is regulated by light through various regulatory factors. Therefore, plants may possess the capability to integrate light and low temperature signals for survival under freezing conditions. However, the detailed molecular mechanism and the regulatory factors associated with light- and low temperature- responsive flavonoid biosynthesis remain largely unknown. Here, we report a strict requirement for light for the low temperature-enhanced flavonol biosynthesis. Low temperature-induced expression of biosynthetic genes as well as flavonol accumulation was hampered in ELONGATED HYPOCOTYL (hy5) and myb11myb111myb12 triple mutants as compared with the wild type in Arabidopsis. Overexpression of AtHY5 in the hy5 mutant restored induction of gene expression and flavonol accumulation in response to low temperature in light. Metabolite and gene expression analysis also suggests a negative role for CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in accumulation of flavonols in response to low temperature. Overexpression of AtMYB12 enhanced accumulation of flavonols under low temperature in a light-dependent manner. Together, our analysis suggests the requirement for HY5 and flavonol-specific MYB regulatory factors for low temperature-induced flavonol synthesis.

Buckwheat R2R3 MYB transcription factor FeMYBF1 regulates flavonol biosynthesis

Buckwheat (Fagopyrum esculentum) contains high amounts of flavonoids, especially flavonols (e.g., rutin), which are thought to be highly beneficial for human health. Little is known, however, about the regulation of flavonol synthesis in buckwheat. We identified a buckwheat gene encoding an R2R3 MYB transcription factor, and named this gene FeMYBF1. Analysis of the deduced amino acid sequence and phylogenetic analysis suggested that FeMYBF1 encodes an ortholog of the Arabidopsis flavonol regulators AtMYB11, AtMYB12 and AtMYB111. Expression of FeMYBF1 in a flavonol-deficient Arabidopsis triple mutant (myb11 myb12 myb111) restored flavonol synthesis. Constitutive expression of FeMYBF1 driven by the CaMV 35S promoter in Arabidopsis resulted in over-accumulation of flavonol glycosides and upregulation of the expression of AtFLS1. Transient expression assays showed that FeMYBF1 activated the promoter of the Arabidopsis gene encoding AtFLS1, and the promoters of buckwheat genes related to anthocyanin and proanthocyanidin synthesis such as dihydroflavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX) in addition to genes encoding FLS. The results indicate that FeMYBF1 regulates flavonol synthesis and may have a role in synthesis of other flavonoid compounds, and also that buckwheat may have alternative pathway of flavonol synthesis through DFR and LDOX.

Degradation of chlorophyll and synthesis of flavonols during autumn senescence-the story told by individual leaves.

Autumn senescence of deciduous trees is characterized by chlorophyll degradation and flavonoid synthesis. In the present study, chlorophyll and flavonol contents were measured every morning and evening during the whole autumn with a non-destructive method from individual leaves of Sorbus aucuparia, Acer platanoides, Betula pendula and Prunus padus. In most of the studied trees, the chlorophyll content of each individual leaf remained constant until a phase of rapid degradation commenced. The fast phase lasted only ~1 week and ended with abscission. In S. aucuparia, contrary to the other species, the chlorophyll content of leaflets slowly but steadily decreased during the whole autumn, but rapid chlorophyll degradation commenced only prior to leaflet abscission also in this species. An increase in flavonols commonly accompanied the rapid degradation of chlorophyll. The results may suggest that each individual tree leaf retains its photosynthetic activity, reflected by a high chlorophyll content, until a rapid phase of chlorophyll degradation and flavonoid synthesis begins. Therefore, in studies of autumn senescence, leaves whose chlorophyll content is decreasing and leaves with summertime chlorophyll content (i.e. the leaves that have not yet started to degrade chlorophyll) should be treated separately.

Flavonol rhamnosylation indirectly modifies the cell wall defects of RHAMNOSE BIOSYNTHESIS1 mutants by altering rhamnose flux

Rhamnose is required in Arabidopsis thaliana for synthesizing pectic polysaccharides and glycosylating flavonols. RHAMNOSE BIOSYNTHESIS1 (RHM1) encodes a UDP-l-rhamnose synthase, and rhm1 mutants exhibit many developmental defects, including short root hairs, hyponastic cotyledons, and left-handed helically twisted petals and roots. It has been proposed that the hyponastic cotyledons observed in rhm1 mutants are a consequence of abnormal flavonol glycosylation, while the root hair defect is flavonol-independent. We have recently shown that the helical twisting of rhm1 petals results from decreased levels of rhamnose-containing cell wall polymers. In this study, we found that flavonols indirectly modify the rhm1 helical petal phenotype by altering rhamnose flux to the cell wall. Given this finding, we further investigated the relationship between flavonols and the cell wall in rhm1 cotyledons. We show that decreased flavonol rhamnosylation is not responsible for the cotyledon phenotype of rhm1 mutants. Instead, blocking flavonol synthesis or rhamnosylation can suppress rhm1 defects by diverting UDP-l-rhamnose to the synthesis of cell wall polysaccharides. Therefore, rhamnose is required in the cell wall for normal expansion of cotyledon epidermal cells. Our findings suggest a broad role for rhamnose-containing cell wall polysaccharides in the morphogenesis of epidermal cells.

SlMYB12 Regulates Flavonol Synthesis in Three Different Cherry Tomato Varieties

Cherry tomato (Lycopersicon esculentum M.) is considered a healthy fruit worldwide due to its wide range of nutrients. Flavonol, one of the major nutrients in cherry tomato, has antioxidant and cell-modulating properties. In this study, we showed a correlation between the expression of SlMYB12 and flavonol content (R2 = 0.922). To characterize the function of SlMYB12, SlMYB12-overexpressing transgenic tomato plants were generated in three different cherry tomato varieties. Significant increases in flavonol content and flavonol biosynthetic gene expression were identified in SlMYB12-overexpressing plants. Therefore, we suggest that SlMYB12 plays a positive role in the flavonol biosynthesis pathway in cherry tomatoes, which further indicates a potential role as a marker in analyzing flavonol content in different cherry tomato varieties.

Identification of functional flavonol synthase genes from fragrant wild cyclamen (Cyclamen purpurascens)

Cyclamen purpurascens is considered suitable for horticultural breeding of cyclamens because it has an attractive fragrance that is not found in other wild species. To improve the commercial value of cyclamen flowers, this fragrance has been introduced into ornamental cultivars. However, variation in flower color is somewhat limited in these cultivars, and therefore understanding the genetic networks of flower coloration in C. purpurascens is required. We previously isolated DNA fragments of anthocyanin biosynthetic genes from C. purpurascens, broadening our understanding of the biosynthetic pathway of flavonols, which are co-pigments in flower coloration. In this study, we isolated complete open reading frames of flavonol synthase genes from C. purpurascens (CpurFLS1 and CpurFLS2) and analyzed the in planta functions of the genes by molecular complementation assay using the fls mutant of Arabidopsis thaliana. Expression patterns in several organs of C. purpurascens were also determined. The results strongly suggest that the CpurFLS genes participate in flavonol synthesis. We discuss the involvement of these two FLSs in flower coloration in C. purpurascens.

Synthesis of 5-subsituted flavonols via the Algar-Flynn-Oyamada (AFO) reaction: The mechanistic implication

Herein, we report a synthetic method with improved selectivity for 5-substituted flavonols via the Algar-Flynn-Oyamada reaction (AFO), by using of sodium carbonate/hydrogen peroxide A series of 5-substituted flavonols was obtained with moderate to high yields. The mechanism of the AFO reaction was elucidated. LCMS analysis and in situ 1H NMR analysis indicated that the epoxide was involved in the transformation from chalcone to flavonol and/or aurone under alkaline base/peroxide conditions.

Cloning, expression and molecular modeling of the anthocyanidin reductase (FaANR) gene during strawberry fruit development

Introduction - Proanthocyanidins (PAs) are a group of polyphenolic secondary metabolites synthesized in plants via flavonoid pathway. Strawberry (Fragaria x ananassa Duch.) is a rich source of flavonoids and proanthocyanins, which are known to have multiple health benefits. The anthocyanidin reductase (ANR) is an interesting gene to study within the flavonoid biosynthesis pathway, since it diverts the anthocyanin pathway to flavonol synthesis. Materials and methods - The present study describes cloning, semi-quantitative expression analysis and molecular modeling of the strawberry (cv. Sweet Charlie) anthocyanidin reductase (FaANR) gene during the progressive stages of fruit development. Results and discussion - The FaANR gene was 1,020 bp long with an open reading frame encoding a protein of 308 amino acid residues. The estimated molecular mass and isoelectric point of the protein were 32.89 kD and 5.54, respectively. The expression of FaANR was only seen during early stages of fruit development indicating its early involvement in PA accumulation, well before ripening onset. Analysis of the FaANR sequence showed 98% similarity to ANR from diploid strawberry Fragaria vesca. The cladistic analysis indicated that the FaANR was phylogenetically similar to Pyrus and Prunus ANR genes. Protein modeling suggested protein-ligand interactions at active site with NADP binding as the plausible mechanism of action. Conclusion - This is the first report on cloning, expression study and in silico modeling of an anthocyanidin reductase of strawberry. The conditions of in vivo modulation of ANR expression open applied perspectives for commercial production of PAs in strawberry and other berries.

Bioactive compounds and antimicrobial activity of black currant (Ribes nigrum L.) berries and leaves extract obtained by different soil management system

Polyphenols compounds have been found in berries and leaves of black currant (Ribes nigrum L.) are known as agents acting preventively and therapeutically on the human organism. The effect of three soil management system (bare fallow, sawdust mulch and black plastic mulch) on the content of total anthocyanins, anthocyanin glycoside (delphinidin 3-glucoside, delphinidin 3-rutinoside, cyanidin 3-glucoside and cyanidin 3-rutinoside), flavonols (quercetin, myricetin and kaempferol), flavan-3-ols (catechin and epicatechin) and antimicrobial activity of seven black currant cultivars (‘Ben Lomond’, ‘Ben Sarek’, ‘Titania’, ‘Čačanska Crna’, ‘Tisel’, ‘Tiben’ and ‘Tsema’) was investigated.Total anthocyanins content was determined using the single pH and pH differential method. High performance liquid chromatography (HPLC) was employed for the identification of the most abundant metabolites presented in berries and leaves extracts. Microbial properties of extracts were examined using eight selected indicator strains. Soil management systems and cultivars showed highly significant differences in the tested parameters. Berries showed a higher content in anthocyanins, flavonols and flavan-3-ols than leaves. Quercetin was the most abundant flavonol in berries and leaves, and epicatechin the most abundant flavan-3-ol. Comparative research on black currant cultivars and soil management systems suggests an important relationship between cultivars and soil management systems. Soil management systems had an effect on the contents anthocyanins and anthocyanin glycoside in extract leaves, but not in extract berries of black currant cultivars. On the other hand, soil management systems have a significant positive effect on the synthesis and accumulation of flavonols and flavan-3-ols in both berries and leaves. Furthermore, all extracts showed strong antimicrobial activity. These results suggest that berries and leaves of black currant cultivars which growing on different soil management systems may be used as a source of beneficial compounds in food and pharmaceutical industries.

Spontaneous variation regarding grape berry skin color: A comprehensive study of berry development by means of biochemical and molecular markers

Understanding grape berry development and the metabolism of different classes of compounds responsible for traits like berry's color is imperative to control and improve quality aspects of grapes. A colorimetric, biochemical and molecular characterization allowed the comprehensive description of the pigment-related characteristics of nine berry skin color somatic variants, belonging to four different varieties. Although the observed berry skin color variability was not fully explained by MybA locus, the phenolic profiles allowed inferring about specific interferences among the biosynthetic pathways. Data were consistent concerning that grapes showing cyanidin-3-O-glucoside as the major anthocyanin and flavonols with two substituent groups in the lateral B-ring are generally originated by a white ancestor. After retro-mutation, these grapes seem to keep the dysfunction on flavonoid hydroxylases enzymes, which negatively affect the synthesis of both flavonols and anthocyanins with three substituent groups in the lateral B-ring. Overall, the obtained results indicate that the color differences observed between somatic variants are not solely the result of the total amount of compounds synthesized, but rather reflect a different dynamics of the phenolic pathway among the different color variants of the same variety. Chemical compoundsGallic acid (PubChem CID: 370); Caftaric acid (PubChem CID: 6,440,397); Catechin (PubChem CID: 73,160); Epigallocatechin gallate (PubChem CID: 65,064); Quercetin-3-O-galactoside (PubChem CID: 5,281,643); Quercetin-3-O-glucoside (PubChem CID: 25,203,368); Malvidin-3-O-glucoside (PubChem CID: 443,652); Peonidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,849); Malvidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,988); Resveratrol-3-O-glucoside (PubChem CID: 25,579,167).

MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana).

Flavonoids are major polyphenol compounds in plant secondary metabolism. Wild red-fleshed apples (Malus sieversii f. niedzwetzkyana) are an excellent resource because of their much high flavonoid content than cultivated apples. In this work, R6R6, R6R1 and R1R1 genotypes were identified in an F1 segregating population of M.sieversii f. niedzwetzkyana. Significant differences in flavonoid composition and content were detected among the three genotypes by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis. Furthermore, two putative flavonoid-related genes encoding R2R3-MYB transcription factors, designated MYB12 and MYB22, were cloned and characterized. The expression patterns of MYB12 and MYB22 directly correlated with those of leucoanthocyanidin reductase and flavonol synthase, respectively. Their roles in flavonoid biosynthesis were identified by overexpression in apple callus and ectopic expression in Arabidopsis. MYB12 expression in the Arabidopsis TT2 mutant complemented its proanthocyanidin-deficient phenotype. Likewise, MYB22 expression in an Arabidopsis triple mutant complemented its flavonol-deficient phenotype. MYB12 could interact with bHLH3 and bHLH33 and played an essential role in proanthocyanidin synthesis. MYB22 was found to activate flavonol pathways by combining directly with the flavonol synthase promoter. Our findings provide a valuable perspective on flavonoid synthesis and provide a basis for breeding elite functional apples with a high flavonoid content.

A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum.

Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components (BCs) in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from Epimedium sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase) and EsFLS (flavonol synthase), but not the promoters of EsDFRs (dihydroflavonol 4-reductase) and EsANS (anthocyanidin synthase) in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase), NtCHI (chalcone isomerase), NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS) were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived BCs in E. sagittatum. Thus, identification and functional characterization of EsMYBF1 provide insight into understanding the biosynthesis and regulation of the flavonol-derived BCs in Epimedium plants, and also provide an effective tool gene for genetic manipulation to improve the flavonol synthesis.

Light promotes expression of monoterpene and flavonol metabolic genes and enhances flavour of winegrape berries (Vitis viniferaL. cv. Riesling)

Background and Aims Insolation of grape bunches has long been postulated to enhance the flavour of aromatic grape cultivars. This hypothesis was tested by combining gene expression and metabolic analysis of the monoterpene and flavonol synthesis pathways. Methods and Results Grape bunches were shaded or shaded and reilluminated to investigate the influence of light on the monoterpene and flavonol biosynthetic pathways. The expression of terpenoid and flavonol metabolic genes was measured by quantitative polymerase chain reaction under light and shade conditions during the ripening phase and compared to the accumulation of their respective metabolic products. Expression of flavonol synthase and flavonol glycosyltransferase genes was virtually absent in shaded bunches, but expression increased strongly upon reillumination, as did the flavonol content of the berries. The expression of the terpene synthase genes as well as the monoterpene content were greatly reduced in shaded bunches, and then increased upon reillumination. The expression of terpene glycosyltransferases was affected only slightly by light. Conclusions The results of this study show the positive influence of light on monoterpene and flavonol biosynthesis. Significance of the Study Optimising the light exposure of grape berries enables the viticulturist to manipulate grape berry aroma and consequently the composition of wine.

Microwave-assisted one-pot synthesis of some new flavonols by modified Algar–Flynn–Oyamada reaction and their antimicrobial activity

A new series of 3-hydroxy-2-[1-phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-yl]-4H-chromen-4-ones has been synthesized from substituted 2-hydroxyacetophenones and 1-phenyl-3-(thiophen-2-yl)-1H-pyrazole-4-carbaldehyde using NaOH and H2O2 by modified Algar–Flynn–Oyamada reaction under conventional and microwave irradiation conditions. In this method flavonols are synthesized without isolating chalcones in good yields (80–85%). The structures of the compounds were established on the basis of IR, 1H, 13C NMR and mass spectral and elemental analysis data. The synthesized compounds were screened for their antibacterial and antifungal activities.