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1.  Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation 
Journal of Experimental Botany  2012;63(7):2465-2478.
Flavonols, phenylalanine-derived secondary metabolites, have protective and regulatory functions in plants. In Arabidopsis thaliana, they are consecutively glycosylated at their 3-OH and 7-OH groups. UGT78D1 and UGT78D2 are the major flavonol 3-O-glycosyltransferases in Arabidopsis leaves. The ugt78d1 ugt78d2 double mutant, which was strongly compromised in the initial 3-O-glycosylation, showed a severe and specific repression of flavonol biosynthesis, retaining only one-third of the wild-type level. This metabolic phenotype was associated with a repressed transcription of several flavonol biosynthetic genes including the committed step chalcone synthase [(CHS) or TRANSPARENT TESTA 4 (TT4)]. Furthermore, the committed step of the upstream, general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL), was down-regulated in its enzyme activity and in the transcription of the flavonol-related PAL1 and PAL2. However, a complete blocking of flavonoid biosynthesis at CHS released PAL inhibition in a tt4 ugt78d1 ugt78d2 line. PAL activity was even enhanced in the flavonol synthase 1 mutant, which compromises the final formation of flavonol aglycones. The dependence of the PAL feedback inhibition on flavonols was confirmed by chemical complementation of tt4 ugt78d1 ugt78d2 using naringenin, a downstream flavonoid intermediate, which restored the PAL repression. Although aglycones were not analytically detectable, this study provides genetic evidence for a novel, flavonol-dependent feedback inhibition of the flavonol biosynthetic pathway and PAL. It was conditioned by the compromised flavonol-3-O-conjugation and a decrease in flavonol content, yet dependent on a residual, flavonol synthase 1 (FLS1)-related capacity to form flavonol aglycones. Thus, this regulation would not react to a reduced metabolic flux into flavonol biosynthesis, but it might prevent the accumulation of non-glycosylated, toxic flavonols.
doi:10.1093/jxb/err416
PMCID: PMC3346215  PMID: 22249996
Feedback inhibition; flavonoids; flavonols; flavonol synthase; phenylalanine ammonia-lyase; phenylpropanoids; UDP-carbohydrate-dependent glycosyltransferase
2.  Post-Transcriptional Silencing of Flavonol Synthase mRNA in Tobacco Leads to Fruits with Arrested Seed Set 
PLoS ONE  2011;6(12):e28315.
Flavonoids are synthesized by phenylpropanoid pathway. They are known to participate in large number of physiological and biochemical processes in plants. Parthenocarpy and male sterility has earlier been reported by silencing chalcone synthase (CHS) encoding gene. Silencing of CHS has blocked the synthesis of most of useful flavonoids including flavan-3-ols and flavonols. Also, these studies could not identify whether parthenocarpy/male sterility were due to lack of flavan-3-ols or flavonols or both. Flavonol synthase (FLS) is an important enzyme of flavonoid pathway that catalyzes the formation of flavonols. In this article, we propose a novel strategy towards the generation of seedless or less-seeded fruits by downregulation of flavonol biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) through post-transcriptional gene silencing (PTGS) of FLS encoding mRNA. The FLS silenced lines were observed for 20-80% reduction in FLS encoding gene expression and 25–93% reduction in flavonol (quercetin) content. Interestingly, these FLS silenced tobacco lines also showed reduction in their anthocyanidins content. While the content of flavan-3-ols (catechin, epi-catechin and epi-gallocatechin) was found to be increased in FLS silenced lines. The delayed flowering in FLS silenced lines could be due to decrease in level of indole acetic acid (IAA) at apical region of their shoots. Furthermore, the pollen germination was hampered and pollens were unable to produce functional pollen tube in FLS silenced tobacco lines. Pods of FLS silenced lines contained significantly less number of seeds. The in vitro and in vivo studies where 1 µM quercetin was supplied to germination media, documented the restoration of normal pollen germination and pollen tube growth. This finding identified the role of flavonols particularly quercetin in pollen germination as well as in the regulation of plant fertility. Results also suggest a novel approach towards generation of seedless/less-seeded fruits via PTGS of FLS encoding gene in plants.
doi:10.1371/journal.pone.0028315
PMCID: PMC3228754  PMID: 22145036
3.  Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera 
Journal of Experimental Botany  2009;60(3):853-867.
Anthocyanins, flavan-3-ols, and flavonols are the three major classes of flavonoid compounds found in grape berry tissues. Several viticultural practices increase flavonoid content in the fruit, but the underlying genetic mechanisms responsible for these changes have not been completely deciphered. The impact of post-veraison sunlight exposure on anthocyanin and flavonol accumulation in grape berry skin and its relation to the expression of different transcriptional regulators known to be involved in flavonoid synthesis was studied. Treatments consisting of removing or moving aside the basal leaves which shade berry clusters were applied. Shading did not affect sugar accumulation or gene expression of HEXOSE TRANSPORTER 1, although in the leaf removal treatment, these events were retarded during the first weeks of ripening. Flavonols were the most drastically reduced flavonoids following shading and leaf removal treatments, related to the reduced expression of FLAVONOL SYNTHASE 4 and its putative transcriptional regulator MYB12. Anthocyanin accumulation and the expression of CHS2, LDOX, OMT, UFGT, MYBA1, and MYB5a genes were also affected. Other regulatory genes were less affected or not affected at all by these treatments. Non-transcriptional control mechanisms for flavonoid synthesis are also suggested, especially during the initial stages of ripening. Although berries from the leaf removal treatment received more light than shaded fruits, malvidin-3-glucoside and total flavonol content was reduced compared with the treatment without leaf removal. This work reveals that flavonol-related gene expression responds rapidly to field changes in light levels, as shown by the treatment in which shaded fruits were exposed to light in the late stages of ripening. Taken together, this study establishes MYB-specific responsiveness for the effect of sun exposure and sugar transport on flavonoid synthesis.
doi:10.1093/jxb/ern336
PMCID: PMC2652055  PMID: 19129169
bHLH; flavonoids; grape; leaf removal; MYB12; PAR; sugar; source; sink; WDR
4.  Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase, isolated from Solanum lycopersicum 
BMC Plant Biology  2010;10:21.
Background
Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism.
Results
The cloned and sequenced tomato F3'5'H gene was named CYP75A31. The gene was inserted into the pYeDP60 expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested in vitro using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the CYP75A31 gene was also tested in vivo, in various parts of the vegetative tomato plant, along with other key genes of the flavonoid pathway using real-time PCR. A clear response to nitrogen depletion was shown for CYP75A31 and all other genes tested. The content of rutin and kaempferol-3-rutinoside was found to increase as a response to nitrogen depletion in most parts of the plant, however the growth conditions used in this study did not lead to accumulation of anthocyanins.
Conclusions
CYP75A31 (NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the CYP75A31 gene was found to increase in response to nitrogen deprivation, in accordance with other genes in the phenylpropanoid pathway, as expected for a gene involved in flavonoid metabolism.
doi:10.1186/1471-2229-10-21
PMCID: PMC2825239  PMID: 20128892
5.  The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower 
PLoS ONE  2013;8(8):e72364.
Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development.
doi:10.1371/journal.pone.0072364
PMCID: PMC3741151  PMID: 23951318
6.  Transcriptional regulation of flavonoid biosynthesis in nectarine (Prunus persica) by a set of R2R3 MYB transcription factors 
BMC Plant Biology  2013;13:68.
Background
Flavonoids such as anthocyanins, flavonols and proanthocyanidins, play a central role in fruit colour, flavour and health attributes. In peach and nectarine (Prunus persica) these compounds vary during fruit growth and ripening. Flavonoids are produced by a well studied pathway which is transcriptionally regulated by members of the MYB and bHLH transcription factor families. We have isolated nectarine flavonoid regulating genes and examined their expression patterns, which suggests a critical role in the regulation of flavonoid biosynthesis.
Results
In nectarine, expression of the genes encoding enzymes of the flavonoid pathway correlated with the concentration of proanthocyanidins, which strongly increases at mid-development. In contrast, the only gene which showed a similar pattern to anthocyanin concentration was UDP-glucose-flavonoid-3-O-glucosyltransferase (UFGT), which was high at the beginning and end of fruit growth, remaining low during the other developmental stages. Expression of flavonol synthase (FLS1) correlated with flavonol levels, both temporally and in a tissue specific manner. The pattern of UFGT gene expression may be explained by the involvement of different transcription factors, which up-regulate flavonoid biosynthesis (MYB10, MYB123, and bHLH3), or repress (MYB111 and MYB16) the transcription of the biosynthetic genes. The expression of a potential proanthocyanidin-regulating transcription factor, MYBPA1, corresponded with proanthocyanidin levels. Functional assays of these transcription factors were used to test the specificity for flavonoid regulation.
Conclusions
MYB10 positively regulates the promoters of UFGT and dihydroflavonol 4-reductase (DFR) but not leucoanthocyanidin reductase (LAR). In contrast, MYBPA1 trans-activates the promoters of DFR and LAR, but not UFGT. This suggests exclusive roles of anthocyanin regulation by MYB10 and proanthocyanidin regulation by MYBPA1. Further, these transcription factors appeared to be responsive to both developmental and environmental stimuli.
doi:10.1186/1471-2229-13-68
PMCID: PMC3648406  PMID: 23617716
Anthocyanin; Transcriptional regulation; MYB; Peach; Nectarine; Prunus persica; Light
7.  Regiospecific Methylation of a Dietary Flavonoid Scaffold Selectively Enhances IL-1β Production following Toll-like Receptor 2 Stimulation in THP-1 Monocytes* 
The Journal of Biological Chemistry  2013;288(29):21126-21135.
Background: Plant natural products typically contain regiospecific modifications in their side chains that lead to different bioactivities.
Results: Only 3-O-methylated flavonols enhance IL-1β production in THP-1 cells costimulated with the Toll-like receptor 2 agonist Pam3CSK4.
Conclusion: Regiospecific methylation of flavonols controls their bioactivity as immunomodulators.
Significance: This study provides a platform to explore the use of regiospecific-modified natural products as novel immunomodulators.
It is now recognized that innate immunity to intestinal microflora plays a significant role in mediating immune health, and modulation of microbial sensing may underpin the impact of plant natural products in the diet or when used as nutraceuticals. In this context, we have examined five classes of plant-derived flavonoids (flavonols, flavones, flavanones, catechins, and cyanidin) for their ability to regulate cytokine release induced by the Toll-like receptor 2 (TLR2) agonist Pam3CSK4. We found that the flavonols selectively co-stimulated IL-1β secretion but had no impact on the secretion of IL-6. Importantly, this costimulation of TLR2-induced cytokine secretion was dependent on regiospecific methylation of the flavonol scaffold with a rank order of quercetin-3,4′-dimethylether > quercetin-3-methylether > casticin. The mechanism underpinning this costimulation did not involve enhanced inflammasome activation. In contrast, the methylated flavonols enhanced IL-1β gene expression through transcriptional regulation, involving mechanisms that operate downstream of the initial NF-κB and STAT1 activation events. These studies demonstrate an exquisite level of control of scaffold bioactivity by regiospecific methylation, with important implications for understanding how natural products affect innate immunity and for their development as novel immunomodulators for clinical use.
doi:10.1074/jbc.M113.453514
PMCID: PMC3774379  PMID: 23760261
Interleukin; Natural Products; Signaling; Small Molecules; Toll-like Receptors (TLR); IL-1β; Plant Natural Products; TLR Signaling; Methylated Flavonols
8.  Kaempferol 3-O-rhamnoside-7-O-rhamnoside is an endogenous flavonol inhibitor of polar auxin transport in Arabidopsis shoots 
The New Phytologist  2013;201(2):466-475.
Polar auxin transport (PAT) plays key roles in the regulation of plant growth and development. Flavonoids have been implicated in the inhibition of PAT. However, the active flavonoid derivative(s) involved in this process in vivo has not yet been identified. Here, we provide evidence that a specific flavonol bis-glycoside is correlated with shorter plant stature and reduced PAT.Specific flavonoid-biosynthetic or flavonoid-glycosylating steps were genetically blocked in Arabidopsis thaliana. The differential flavonol patterns established were analyzed by high-performance liquid chromatography (HPLC) and related to altered plant stature. PAT was monitored in stem segments using a radioactive [3H]-indole-3-acetic acid tracer.The flavonoid 3-O-glucosyltransferase mutant ugt78d2 exhibited a dwarf stature in addition to its altered flavonol glycoside pattern. This was accompanied by reduced PAT in ugt78d2 shoots. The ugt78d2-dependent growth defects were flavonoid dependent, as they were rescued by genetic blocking of flavonoid biosynthesis. Phenotypic and metabolic analyses of a series of mutants defective at various steps of flavonoid formation narrowed down the potentially active moiety to kaempferol 3-O-rhamnoside-7-O-rhamnoside. Moreover, the level of this compound was negatively correlated with basipetal auxin transport.These results indicate that kaempferol 3-O-rhamnoside-7-O-rhamnoside acts as an endogenous PAT inhibitor in Arabidopsis shoots.
doi:10.1111/nph.12558
PMCID: PMC4260840  PMID: 24251900
Arabidopsis thaliana; flavonol biosynthesis; flavonol glycoside; flavonol glycosyltransferases; plant growth; polar auxin transport
9.  Advanced Knowledge of Three Important Classes of Grape Phenolics: Anthocyanins, Stilbenes and Flavonols 
Grape is qualitatively and quantitatively very rich in polyphenols. In particular, anthocyanins, flavonols and stilbene derivatives play very important roles in plant metabolism, thanks to their peculiar characteristics. Anthocyanins are responsible for the color of red grapes and wines and confer organoleptic characteristics on the wine. They are used for chemotaxonomic studies and to evaluate the polyphenolic ripening stage of grape. They are natural colorants, have antioxidant, antimicrobial and anticarcinogenic activity, exert protective effects on the human cardiovascular system, and are used in the food and pharmaceutical industries. Stilbenes are vine phytoalexins present in grape berries and associated with the beneficial effects of drinking wine. The principal stilbene, resveratrol, is characterized by anticancer, antioxidant, anti-inflammatory and cardioprotective activity. Resveratrol dimers and oligomers also occur in grape, and are synthetized by the vine as active defenses against exogenous attack, or produced by extracellular enzymes released from pathogens in an attempt to eliminate undesirable toxic compounds. Flavonols are a ubiquitous class of flavonoids with photo-protection and copigmentation (together with anthocyanins) functions. The lack of expression of the enzyme flavonoid 3′,5′-hydroxylase in white grapes restricts the presence of these compounds to quercetin, kaempferol and isorhamnetin derivatives, whereas red grapes usually also contain myricetin, laricitrin and syringetin derivatives. In the last ten years, the technological development of analytical instrumentation, particularly mass spectrometry, has led to great improvements and further knowledge of the chemistry of these compounds. In this review, the biosynthesis and biological role of these grape polyphenols are briefly introduced, together with the latest knowledge of their chemistry.
doi:10.3390/ijms141019651
PMCID: PMC3821578  PMID: 24084717
grape; polyphenols; anthocyanins; stilbenes; flavonols
10.  Molecular characterization and functional expression of flavonol 6-hydroxylase 
BMC Plant Biology  2004;4:20.
Background
Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis.
Results
A cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H), was isolated and characterized from Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein. This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity. The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4'-trimethylquercetin as the preferred substrate. The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C. americanum genome.
Conclusions
Recombinant F6H has been functionally expressed and characterized at the molecular level. The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme. The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction.
doi:10.1186/1471-2229-4-20
PMCID: PMC544895  PMID: 15596008
11.  Expression analysis of flavonoid biosynthesis genes during Arabidopsis thaliana silique and seed development with a primary focus on the proanthocyanidin biosynthetic pathway 
BMC Research Notes  2010;3:255.
Background
The coordinated activity of different flavonoid biosynthesis genes in Arabidopsis thaliana results in tissue-specific accumulation of flavonols, anthocyanins and proanthocyanidins (PAs). These compounds possess diverse functions in plants including light-attenuation and oxidative stress protection. Flavonoids accumulate in a stimulus- and/or development-dependent manner in specific parts of the plant. PAs accumulate in the seed coat (testa).
Findings
We describe the biological material and the preparation of total RNA for the AtGenExpress developmental silique and seed series. AtGenExpress ATH1 GeneChip expression data from the different stages were reanalyzed and verified using quantitative real time PCR (qPCR). We observed organ-specific transcript accumulation of specific flavonoid biosynthetic genes consistent with previously published data and our PA compound accumulation data. In addition, we investigated the regulation of PA accumulation in developing A. thaliana seeds by correlating gene expression patterns of specific flavonoid biosynthesis genes with different seed embryonic developmental stages and organs and present two useful marker genes for isolated valve and replum organs, as well as one seed-specific marker.
Conclusions
Potential caveats of array-based expression data are discussed based on comparisons with qPCR data. Results from ATH1 microarray and qPCR experiments revealed a shift in gene activity from general flavonoid biosynthesis at early stages of seed development to PA synthesis at late (mature) stages of embryogenesis. The examined PA accumulation-associated genes, including biosynthetic and regulatory genes, were found to be exclusively expressed in immature seeds. Accumulation of PAs initiates at the early heart stage of silique and seed development. Our findings provide new insights for further studies targeting the PA pathway in seeds.
doi:10.1186/1756-0500-3-255
PMCID: PMC2958888  PMID: 20929528
12.  Isolation and characterization of GtMYBP3 and GtMYBP4, orthologues of R2R3-MYB transcription factors that regulate early flavonoid biosynthesis, in gentian flowers 
Journal of Experimental Botany  2012;63(18):6505-6517.
Flavonoids are one of the major plant pigments for flower colour. Not only coloured anthocyanins, but also co-pigment flavones or flavonols, accumulate in flowers. To study the regulation of early flavonoid biosynthesis, two R2R3-MYB transcription factors, GtMYBP3 and GtMYBP4, were identified from the petals of Japanese gentian (Gentiana triflora). Phylogenetic analysis showed that these two proteins belong to the subgroup 7 clade (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. Gt MYBP3 and Gt MYBP4 transcripts were detected specifically in young petals and correlated with the profiles of flavone accumulation. Transient expression assays showed that GtMYBP3 and GtMYBP4 enhanced the promoter activities of early biosynthetic genes, including flavone synthase II (FNSII) and flavonoid 3′-hydroxylase (F3′H), but not the late biosynthetic gene, flavonoid 3′,5′-hydroxylase (F3′5′H). GtMYBP3 also enhanced the promoter activity of the chalcone synthase (CHS) gene. In transgenic Arabidopsis, overexpression of Gt MYBP3 and Gt MYBP4 activated the expression of endogenous flavonol biosynthesis genes and led to increased flavonol accumulation in seedlings. In transgenic tobacco petals, overexpression of Gt MYBP3 and Gt MYBP4 caused decreased anthocyanin levels, resulting in pale flower colours. Gt MYBP4-expressing transgenic tobacco flowers also showed increased flavonols. As far as is known, this is the first functional characterization of R2R3-MYB transcription factors regulating early flavonoid biosynthesis in petals.
doi:10.1093/jxb/ers306
PMCID: PMC3504500  PMID: 23125348
Early flavonoid biosynthesis; flavone; flower colour; Japanese gentian; R2R3-MYB; transcription factor
13.  Evolution and Expression of Tandem Duplicated Maize Flavonol Synthase Genes 
Flavonoids are specialized compounds widely distributed and with diverse functions throughout the plant kingdom and with several benefits for human health. In particular, flavonols, synthesized by flavonol synthase (FLS), protect plants against UV-B radiation and are essential for male fertility in maize and other plants. We have recently characterized a UV-B inducible ZmFLS1, corresponding to the first to be described in monocot plants. Interestingly, the new assembly of the B73 maize genome revealed the presence of a second putative FLS gene (ZmFLS2), with very high identity with ZmFLS1. ZmFLSs expression was analyzed in different maize tissues, and by combining electrophoretic mobility shift assays and transient expression experiments, we show that both genes are direct targets of anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. ZmFLS expression analyses show higher levels of both transcripts in high altitude landraces than inbred lines, and both genes are regulated by UV-B radiation in all lines analyzed. Moreover, the high sequence conservation of the ZmFLS promoters between maize lines suggests that the differences observed in ZmFLS expression are due to allelic variations in the transcription factors that regulate their activities. Finally, we generated pFLS1::FLS1-RFP transgenic plants and analyzed ZmFLS1 expression in different maize tissues; we found that this enzyme is localized in the ER and the perinuclear region.
doi:10.3389/fpls.2012.00101
PMCID: PMC3360202  PMID: 22654889
UV-B; duplication; grasses; natural variation; maize
14.  Solution structure and function of YndB, an AHSA1 protein from Bacillus subtilis 
Proteins  2010;78(16):3328-3340.
The solution structure of the Bacillus subtilis protein YndB has been solved using NMR in order to investigate proposed biological functions. The YndB structure exhibits the helix-grip fold, which consists of a β-sheet with two small and one long α-helix, forming a hydrophobic cavity that preferentially binds lipid-like molecules. Sequence and structure comparisons to proteins from eukaryotes, prokaryotes, and archaea suggest that YndB is very similar to the eukaryote protein Aha1, which binds to the middle domain of Hsp90 and induces ATPase activity. Based on these similarities, YndB has been classified as a member of the Activator of Hsp90 ATPase homolog-like protein (AHSA1) family with a function that appears to be related to stress response. An in silico screen of a compound library of ~18,500 lipids was used to identify classes of lipids that preferentially bind YndB. The in silico screen identified, in order of affinity, the chalcone/hydroxychalcone, flavanone, and flavone/flavonol classes of lipids, which was further verified by 2D 1H-15N HSQC NMR titration experiments with trans-chalcone, flavanone, flavone, and flavonol. All of these compounds are typically found in plants as precursors to various flavonoid antibiotics and signaling molecules. The sum of the data suggests an involvement of YndB with the stress response of B. subtilis to chalcone-like flavonoids released by plants due to a pathogen infection. The observed binding of chalcone-like molecules by YndB is likely related to the symbiotic relationship between B. subtilis and plants.
doi:10.1002/prot.22840
PMCID: PMC2976784  PMID: 20818668
15.  Functional Characterization of Dihydroflavonol-4-Reductase in Anthocyanin Biosynthesis of Purple Sweet Potato Underlies the Direct Evidence of Anthocyanins Function against Abiotic Stresses 
PLoS ONE  2013;8(11):e78484.
Dihydroflavonol-4-reductase (DFR) is a key enzyme in the catalysis of the stereospecific reduction of dihydroflavonols to leucoanthocyanidins in anthocyanin biosynthesis. In the purple sweet potato (Ipomoea batatas Lam.) cv. Ayamurasaki, expression of the IbDFR gene was strongly associated with anthocyanin accumulation in leaves, stems and roots. Overexpression of the IbDFR in Arabidopsis tt3 mutants fully complemented the pigmentation phenotype of the seed coat, cotyledon and hypocotyl. Downregulation of IbDFR expression in transgenic sweet potato (DFRi) using an RNAi approach dramatically reduced anthocyanin accumulation in young leaves, stems and storage roots. In contrast, the increase of flavonols quercetin-3-O-hexose-hexoside and quercetin-3-O-glucoside in the leaves and roots of DFRi plants is significant. Therefore, the metabolic pathway channeled greater flavonol influx in the DFRi plants when their anthocyanin and proanthocyanidin accumulation were decreased. These plants also displayed reduced antioxidant capacity compared to the wild type. After 24 h of cold treatment and 2 h recovery, the wild-type plants were almost fully restored to the initial phenotype compared to the slower recovery of DFRi plants, in which the levels of electrolyte leakage and hydrogen peroxide accumulation were dramatically increased. These results provide direct evidence of anthocyanins function in the protection against oxidative stress in the sweet potato. The molecular characterization of the IbDFR gene in the sweet potato not only confirms its important roles in flavonoid metabolism but also supports the protective function of anthocyanins of enhanced scavenging of reactive oxygen radicals in plants under stressful conditions.
doi:10.1371/journal.pone.0078484
PMCID: PMC3817210  PMID: 24223813
16.  Transcriptional control of flavonoid biosynthesis 
Plant Signaling & Behavior  2014;9:e27522.
Flavonoids are plant secondary polyphenolic metabolites and fulfil many vital biological functions, offering a valuable metabolic and genetic model for studying transcriptional control of gene expression. Arabidopsis thaliana mainly accumulates 3 types of flavonoids, including flavonols, anthocyanins, and proanthocyanidins (PAs). Flavonoid biosynthesis involves a multitude of well-characterized enzymatic and regulatory proteins. Three R2R3-MYB proteins (MYB11, MYB12, and MYB111) control flavonol biosynthesis via activating the early biosynthetic steps, whereas the production of anthocyanins and PAs requires the MYB-bHLH-WD40 (MBW) complex to activate the late biosynthetic genes. Additional regulators of flavonoid biosynthesis have recently come to light, which interact with R2R3-MYBs or bHLHs to organize or disrupt the formation of the MBW complex, leading to enhanced or compromised flavonoid production. This mini-review gives an overview of how these novel players modulate flavonoid metabolism and thus plant developmental processes and further proposes a fine-tuning mechanism to complete the complex regulatory network controlling flavonoid biosynthesis.
doi:10.4161/psb.27522
PMCID: PMC4091223  PMID: 24393776
flavonoids; MYBL2; SPL9; TT1; TCP3; MBW complexes; Arabidopsis thaliania
17.  Genome-wide identification and characterisation of R2R3-MYB genes in sugar beet (Beta vulgaris) 
BMC Plant Biology  2014;14(1):249.
Background
The R2R3-MYB genes comprise one of the largest transcription factor gene families in plants, playing regulatory roles in plant-specific developmental processes, metabolite accumulation and defense responses. Although genome-wide analysis of this gene family has been carried out in some species, the R2R3-MYB genes in Beta vulgaris ssp. vulgaris (sugar beet) as the first sequenced member of the order Caryophyllales, have not been analysed heretofore.
Results
We present a comprehensive, genome-wide analysis of the MYB genes from Beta vulgaris ssp. vulgaris (sugar beet) which is the first species of the order Caryophyllales with a sequenced genome. A total of 70 R2R3-MYB genes as well as genes encoding three other classes of MYB proteins containing multiple MYB repeats were identified and characterised with respect to structure and chromosomal organisation. Also, organ specific expression patterns were determined from RNA-seq data. The R2R3-MYB genes were functionally categorised which led to the identification of a sugar beet-specific clade with an atypical amino acid composition in the R3 domain, putatively encoding betalain regulators. The functional classification was verified by experimental confirmation of the prediction that the R2R3-MYB gene Bv_iogq encodes a flavonol regulator.
Conclusions
This study provides the first step towards cloning and functional dissection of the role of MYB transcription factor genes in the nutritionally and evolutionarily interesting species B. vulgaris. In addition, it describes the flavonol regulator BvMYB12, being the first sugar beet R2R3-MYB with an experimentally proven function.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0249-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0249-8
PMCID: PMC4180131  PMID: 25249410
Beta vulgaris; Caryophyllales; R2R3-MYB; Transcription factor; Gene family; Flavonol regulator
18.  A flavonoid 3-O-glucoside:2″-O-glucosyltransferase responsible for terminal modification of pollen-specific flavonols in Arabidopsis thaliana 
The Plant Journal  2014;79(5):769-782.
Flavonol 3-O-diglucosides with a 1→2 inter-glycosidic linkage are representative pollen-specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild-type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild-type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP-glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen-specific flavonol structure. Kaempferol and quercetin 3-O-glucosyl-(1→2)-glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild-type plants. Recombinant UGT79B6 protein converted kaempferol 3-O-glucoside to kaempferol 3-O-glucosyl-(1→2)-glucoside. UGT79B6 recognized 3-O-glucosylated/galactosylated anthocyanins/flavonols but not 3,5- or 3,7-diglycosylated flavonoids, and prefers UDP-glucose, indicating that UGT79B6 encodes flavonoid 3-O-glucoside:2″-O-glucosyltransferase. A UGT79B6-GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.
doi:10.1111/tpj.12580
PMCID: PMC4282749  PMID: 24916675
glucosyltransferase; At5g54010; NM_124780; tapetum; pollen; glycosyltransferase; flavonol; flavonoid; Arabidopsis thaliana
19.  Transcriptome sequencing of a chimaera reveals coordinated expression of anthocyanin biosynthetic genes mediating yellow formation in herbaceous peony (Paeonia lactiflora Pall.) 
BMC Genomics  2014;15(1):689.
Background
Herbaceous peony (Paeonia lactiflora Pall.) is a traditional flower in China and a wedding attractive flower in worldwide. In its flower colour, yellow is the rarest which is ten times the price of the other colours. However, the breeding of new yellow P. lactiflora varieties using genetic engineering is severely limited due to the little-known biochemical and molecular mechanisms underlying its characteristic formation.
Results
In this study, two cDNA libraries generated from P. lactiflora chimaera with red outer-petal and yellow inner-petal were sequenced using an Illumina HiSeq™ 2000 platform. 66,179,398 and 65,481,444 total raw reads from red outer-petal and yellow inner-petal cDNA libraries were generated, which were assembled into 61,431 and 70,359 Unigenes with an average length of 628 and 617 nt, respectively. Moreover, 61,408 non-redundant All-unigenes were obtained, with 37,511 All-unigenes (61.08%) annotated in public databases. In addition, 6,345 All-unigenes were differentially expressed between the red outer-petal and yellow inner-petal, with 3,899 up-regulated and 2,446 down-regulated All-unigenes, and the flavonoid metabolic pathway related to colour development was identified using the Kyoto encyclopedia of genes and genomes database (KEGG). Subsequently, the expression patterns of 10 candidate differentially expressed genes (DEGs) involved in the flavonoid metabolic pathway were examined, and flavonoids were qualitatively and quantitatively analysed. Numerous anthoxanthins (flavone and flavonol) and a few anthocyanins were detected in the yellow inner-petal, which were all lower than those in the red outer-petal due to the low expression levels of the phenylalanine ammonialyase gene (PlPAL), flavonol synthase gene (PlFLS), dihydroflavonol 4-reductase gene (PlDFR), anthocyanidin synthase gene (PlANS), anthocyanidin 3-O-glucosyltransferase gene (Pl3GT) and anthocyanidin 5-O-glucosyltransferase gene (Pl5GT).
Conclusion
Transcriptome sequencing (RNA-Seq) analysis based on the high throughput sequencing technology was an efficient approach to identify critical genes in P. lactiflora and other non-model plants. The flavonoid metabolic pathway and glucide metabolic pathway were identified as relatived yellow formation in P. lactiflora, PlPAL, PlFLS, PlDFR, PlANS, Pl3GT and Pl5GT were selected as potential candidates involved in flavonoid metabolic pathway, which inducing inhibition of anthocyanin biosynthesis mediated yellow formation in P. lactiflora. This study could lay a theoretical foundation for breeding new yellow P. lactiflora varieties.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-689) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-689
PMCID: PMC4159507  PMID: 25134523
Anthocyanins; Flower colour; Flavonoids; Herbaceous peony; Transcriptome; Yellow
20.  Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen 
BMC Plant Biology  2010;10:107.
Background
Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments.
Results
A full-length cDNA putatively identified as a F3'5'H (CpF3'5'H) was isolated from cyclamen flower tissue. Amino acid and phylogeny analyses indicated the CpF3'5'H encodes a F3'5'H enzyme. Two cultivars of minicyclamen were transformed via Agrobacterium tumefaciens with an antisense CpF3'5'H construct. Flowers of the transgenic lines showed modified colour and this correlated positively with the loss of endogenous F3'5'H transcript. Changes in observed colour were confirmed by colorimeter measurements, with an overall loss in intensity of colour (C) in the transgenic lines and a shift in hue from purple to red/pink in one cultivar. HPLC analysis showed that delphinidin-derived pigment levels were reduced in transgenic lines relative to control lines while the percentage of cyanidin-derived pigments increased. Total anthocyanin concentration was reduced up to 80% in some transgenic lines and a smaller increase in flavonol concentration was recorded. Differences were also seen in the ratio of flavonol types that accumulated.
Conclusion
To our knowledge this is the first report of genetic modification of the anthocyanin pathway in the commercially important species cyclamen. The effects of suppressing a key enzyme, F3'5'H, were wide ranging, extending from anthocyanins to other branches of the flavonoid pathway. The results illustrate the complexity involved in modifying a biosynthetic pathway with multiple branch points to different end products and provides important information for future flower colour modification experiments in cyclamen.
doi:10.1186/1471-2229-10-107
PMCID: PMC3095274  PMID: 20540805
21.  Proanthocyanidin oxidation of Arabidopsis seeds is altered in mutant of the high-affinity nitrate transporter NRT2.7 
Journal of Experimental Botany  2014;65(3):885-893.
Summary
The seed-specific nitrate transporter AtNRT2.7 is involved in flavonoid accumulation as evidenced by the higher proanthocyanidin content in nrt2.7-2 mutant seeds. As TT10 laccase activity is not modified, the link between NRT2.7 and proanthocyanidin accumulation has yet to be discovered.
NRT2.7 is a seed-specific high-affinity nitrate transporter controlling nitrate content in Arabidopsis mature seeds. The objective of this work was to analyse further the consequences of the nrt2.7 mutation for the seed metabolism. This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation. Indeed, this phenotype resembled those of tt10 mutant seeds defective in the laccase-like enzyme TT10/LAC15, which is involved in the oxidative polymerization of flavonoids such as the proantocyanidins (PAs) (i.e. epicatechin monomers and PA oligomers) and flavonol glycosides. nrt2.7-2 and tt10-2 mutant seeds displayed the same higher accumulation of PAs, but were partially distinct, since flavonol glycoside accumulation was not affected in the nrt2.7-2 seeds. Moreover, measurement of in situ laccase activity excluded a possibility of the nrt2.7-2 mutation affecting the TT10 enzymic activity at the early stage of seed development. Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype. However, the PA-related phenotype of nrt2.7-2 seeds was not strictly correlated to the nitrate content of seeds. No correlation was observed when nitrate was lowered in seeds due to limited nitrate nutrition of plants or to lower nitrate storage capacity in leaves of clca mutants deficient in the vacuolar anionic channel CLCa. All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation.
doi:10.1093/jxb/ert481
PMCID: PMC3924729  PMID: 24532452
Flavonoids; laccase; nitrate; NRT2.7; proanthocyanidins; seeds; transporter; TT1.0.
22.  Cloning and characterization of a glucosyltransferase from Crocus sativus stigmas involved in flavonoid glucosylation 
BMC Plant Biology  2009;9:109.
Background
Flavonol glucosides constitute the second group of secondary metabolites that accumulate in Crocus sativus stigmas. To date there are no reports of functionally characterized flavonoid glucosyltransferases in C. sativus, despite the importance of these compounds as antioxidant agents. Moreover, their bitter taste makes them excellent candidates for consideration as potential organoleptic agents of saffron spice, the dry stigmas of C. sativus.
Results
Using degenerate primers designed to match the plant secondary product glucosyltransferase (PSPG) box we cloned a full length cDNA encoding CsGT45 from C. sativus stigmas. This protein showed homology with flavonoid glucosyltransferases. In vitro reactions showed that CsGT45 catalyses the transfer of glucose from UDP_glucose to kaempferol and quercetin. Kaempferol is the unique flavonol present in C. sativus stigmas and the levels of its glucosides changed during stigma development, and these changes, are correlated with the expression levels of CsGT45 during these developmental stages.
Conclusion
Findings presented here suggest that CsGT45 is an active enzyme that plays a role in the formation of flavonoid glucosides in C. sativus.
doi:10.1186/1471-2229-9-109
PMCID: PMC2736960  PMID: 19695093
23.  Absorption, Conjugation and Efflux of the Flavonoids, Kaempferol and Galangin, Using the Intestinal CACO-2/TC7 Cell Model 
Journal of functional foods  2009;1(1):74-87.
Flavonoids are biologically active compounds in food with potential health effects. We have used the Caco-2 cell monolayer model to study the absorption and metabolism of two flavonols, a class of flavonoids, specifically kaempferol and galangin. Metabolism experiments allowed identification of 5 kaempferol conjugates: 3-, 7- and 4′-glucuronide, a sulphate and a glucurono-sulphate; and 4 galangin conjugates: 3-, 5- and 7-glucuronides, and a sulphate, using specific enzyme hydrolysis, HPLC-MS, and HPLC with post column metal complexation/tandem MS. Transport studies showed that the flavonols were conjugated inside the cells then transported across the monolayer or effluxed back to the apical side. Sulphated conjugates were preferentially effluxed back to the apical side, whereas glucuronides were mostly transported to the basolateral side. For kaempferol, a small amount of the unconjugated aglycone permeated in both directions, indicating some passive diffusion. When kaempferol-3-glucuronide and quercetin7-sulphate were applied to either side of the cells, no permeation in either direction was observed, indicating that conjugates cannot re-cross the cell monolayer. Formation of apical kaempferol-7- and 4′-glucuronides was readily saturated, whereas formation of other conjugates at the apical side and all at the basolateral side increased with increasing concentration of kaempferol, implying different transporters are responsible at the apical and basolateral sides. The results highlight the important but complex metabolic changes occurring in flavonoids during absorption.
doi:10.1016/j.jff.2008.09.011
PMCID: PMC2765672  PMID: 20046888
24.  Isolation and Molecular Characterization of Thirteen R2R3-MYB Transcription Factors from Epimedium sagittatum 
Epimedium sagittatum (Sieb. et Zucc.) Maxim, a popular traditional Chinese medicinal plant, has been widely used for treating sexual dysfunction and osteoporosis in China. The main bioactive components in herba epimedii are prenylated flavonol glycosides, which are end products of a branch of the flavonoid biosynthetic pathway. The MYB transcription factors (TF) act as activators or repressors to regulate the flavonoid pathway. In this study, 13 full-length cDNA clones of R2R3-MYB TFs from E. sagittatum (designated as EsMYB1 to EsMYB13) were isolated and characterized. Sequence similarity and phylogenetic analysis placed nine R2R3-MYB members of E. sagittatum into five subgroups of the Arabidopsis R2R3-MYB family, while four members were not clustered into a defined subgroup. The number and length of introns from Epimedium R2R3-MYB genes varied significantly, but intron positions and phases were well conserved. Expression patterns of Epimedium R2R3-MYB genes in various tissues showed diverse. Finally, it is suggested that five Epimedium R2R3-MYB genes may be involved in regulating the flavonoid pathway and could be used as valuable candidate genes for metabolic engineering studies in future. Sequence information of 13 R2R3-MYB genes discovered here will also provide an entry point into the overview of whole R2R3-MYB family in Epimedium.
doi:10.3390/ijms14010594
PMCID: PMC3565284  PMID: 23271373
Epimedium; medicinal plant; flavonoid pathway; MYB; transcription factor
25.  Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements 
International Journal of Oncology  2014;45(2):831-842.
Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability.
doi:10.3892/ijo.2014.2452
PMCID: PMC4091967  PMID: 24859601
flavonoids; quercetin; flavonol analogs; polyphenolic; polar; hydrophilic; hydrophobic; lipophilic; prostate cancer; DU-145; PC-3; human infant foreskin fibroblasts; anticancer activity; MTT; MTS; tetrazolium dye; Alamar Blue; CTG; crystal violet; Hoechst 33342; propidium iodide; rhodamine 123; trypan blue; colorimetric; cell viability; proliferation; cytotoxicity; EC50; therapeutic agent; Suzuki-Miyaura cross-coupling

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