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Ulrich Matern - One of the best experts on this subject based on the ideXlab platform.
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multifunctional flavonoid dioxygenases flavonol and anthocyanin biosynthesis in arabidopsis thaliana l
Phytochemistry, 2010Co-Authors: Stefan Martens, Anja Preuss, Ulrich MaternAbstract:Flavonols and conditionally also anthocyanins, aside from Flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are "multifunctional" enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed.
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Arabidopsis thaliana expresses a second functional flavonol synthase
FEBS letters, 2009Co-Authors: Anja Preuss, Ulrich Matern, Ralf Stracke, Bernd Weisshaar, Alexander Hillebrecht, Stefan MartensAbstract:Arabidopsis thaliana L. produces flavonoid pigments, i.e. Flavonols, anthocyanidins and proanthocyanidins, from dihydroflavonol substrates. A small family of putative flavonol synthase (FLS) genes had been recognized in Arabidopsis, and functional activity was attributed only to FLS1. Nevertheless, other FLS activities must be present, because A. thalianafls1 mutants still accumulate significant amounts of Flavonols. The recombinant FLSs and leucoanthocyanidin dioxygenase (LDOX) proteins were therefore examined for their enzyme activities, which led to the identification of FLS3 as a second active FLS. This enzyme is therefore likely responsible for the formation of Flavonols in the ldox/fls1-2 double mutant. These double mutant and biochemical data demonstrate for the first time that LDOX is capable of catalyzing the in planta formation of Flavonols.
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flavonol synthase from citrus unshiu is a bifunctional dioxygenase
Phytochemistry, 2003Co-Authors: Richard Lukacin, Frank Wellmann, Lothar Britsch, Stefan Martens, Ulrich MaternAbstract:Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2R,3R)-dihydroFlavonols, i.e. dihydrokaempferol, to the corresponding Flavonols (kaempferol). Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3b-hydroxylase and flavonol synthase activities to the enzyme. In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2R)-naringenin as substrate. The product which was not converted any further on repeated incubations was identified by 1 H NMR and CD spectroscopies as ()-trans-dihydrokaempferol. The data demonstrate that Citrus flavonol synthase encompasses an additional non
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functional expression and mutational analysis of flavonol synthase from citrus unshiu
FEBS Journal, 2002Co-Authors: Frank Wellmann, Lothar Britsch, Richard Lukacin, Takaya Moriguchi, Emile Schiltz, Ulrich MaternAbstract:Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII/2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m/z 37888 ± 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N-terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5–6 with dihydroquercetin as substrate and a temperature optimum at about 37 °C. Km values of 272, 11 and 36 µm were determined for dihydroquercetin, FeII and 2-oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (Km 45 µm). Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30–60% similarity were apparent with other dioxygenases. Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for FeII-coordination, an arginine (Arg287) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality. Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity. Alternatively, the substitution of Pro207 by glycine hardly affected the activity. The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.
Alan Crozier - One of the best experts on this subject based on the ideXlab platform.
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Antioxidant Flavonols from fruits, vegetables and beverages: measurements and bioavailability.
Biological research, 2000Co-Authors: Alan Crozier, Azlina Abdul Aziz, Jennifer Burns, Amanda J. Stewart, Helena S Rabiasz, Gareth I. Jenkins, Christine A. Edwards, Michael E. J. LeanAbstract:Flavonols are polyphenolic secondary plant metabolites that are present in varying levels in commonly consumed fruits, vegetables and beverages. Flavonols have long held an interest for nutritionists, which has increased following a Dutch study in the early 1990s showing that dietary intake of Flavonols was inversely correlated with the incidence of coronary heart disease. The main factors that have hindered workers in the field of flavonol research are (i) the accurate measurement of these compounds in foods and biological samples, and (ii) a dearth of information on their absorption and metabolism. This review aims to highlight the work of the authors in attempting to clarify the situation. The sensitive and selective HPLC procedure to identify and quantify common Flavonols and their sugar conjugates is described. In addition, the results of an on-going screening program into the flavonol content of common produce and beverages are presented. The bioavailability of dietary Flavonols is discussed with reference to an intervention study with onions, as well as pilot studies with tea, red wine and cherry tomatoes. It is concluded that Flavonols are absorbable and accumulate in plasma and that consuming high flavonol-containing varieties of fruits and vegetables and particular types of beverages could increase their circulatory levels
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Occurrence of Flavonols in tomatoes and tomato-based products
Journal of agricultural and food chemistry, 2000Co-Authors: Amanda J. Stewart, Gareth I. Jenkins, Michael E. J. Lean, Sophie Bozonnet, William Mullen, Alan CrozierAbstract:The flavonol contents of 20 varieties of tomato fruit were investigated in relation to variety, size, season, and country of origin. Ten commonly consumed tomato-based food products were also assessed. Free and conjugated Flavonols were identified and quantified using reversed-phase HPLC. Ninety-eight percent of Flavonols detected in tomatoes were found to occur in the skin. Tomatoes contained, primarily as conjugates, quercetin and kaempferol. The main quercetin conjugate was identified as rutin (quercetin 3-rhamnosylglucoside) by LC-MS. The total flavonol content of the different varieties of tomato that were analyzed varied from 1.3 to 22.2 microgram/g of fresh weight (fw). Smaller cherry tomato fruits originating from warm sunny climates, such as Spain and Israel, were found to contain the highest concentration of Flavonols. Among the tomato-based products investigated, tomato juice and tomato puree were rich in Flavonols, containing 14-16 microgram/mL and 70 microgram/g fw, respectively. In contrast to fresh tomatoes, most tomato-based products contained significant amounts of free Flavonols.
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dietary Flavonols protect diabetic human lymphocytes against oxidative damage to dna
Diabetes, 1999Co-Authors: M E J Lean, Jennifer Burns, Mostafa Noroozi, I Kelly, Dinesh Talwar, Naveed Sattar, Alan CrozierAbstract:Diabetic patients have reduced antioxidant defenses and suffer from an increased risk of free radical-mediated diseases such as coronary heart disease. Epidemiological evidence has suggested that antioxidant dietary flavonoids may protect against heart disease, but a biological effect has yet to be demonstrated directly in humans. In this study, 10 stable type 2 diabetic patients were treated for 2 weeks on a low-flavonol diet and for 2 weeks on the same diet supplemented with 76-110 mg of Flavonols (mostly quercetin) provided by 400 g of onions (and tomato sauce) and six cups of tea daily. Freshly collected lymphocytes were subjected to standard oxidative challenge with hydrogen peroxide, and DNA damage was measured by single-cell gel electrophoresis. Fasting plasma flavonol concentrations (measured by high-performance liquid chromatography) were 5.6 +/- 2.9 ng/ml on the low-flavonol diet and increased 12-fold to 72.1 +/- 15.8 ng/ml on the high-flavonol diet (P < 0.001). Oxidative damage to lymphocyte DNA was 220 +/- 12 on an arbitrary scale of 0-400 U on the low-flavonol diet and 192 +/- 14 on the high-flavonol diet (P = 0.037). This decrease was not accounted for by any change in the measurements of diabetic control (fasting plasma glucose or fructosamine) or by any change in the plasma levels of known antioxidants, including vitamin C, carotenoids, alpha-tocopherol, urate, albumin, and bilirubin. In conclusion, we have shown a biological effect of potential medical importance that appears to be associated with the absorption of dietary Flavonols.
Gloria K Muday - One of the best experts on this subject based on the ideXlab platform.
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Flavonols control pollen tube growth and integrity by regulating ros homeostasis during high temperature stress
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Joelle K Muhlemann, Trenton L B Younts, Gloria K MudayAbstract:Plant reproduction requires long-distance growth of a pollen tube to fertilize the female gametophyte. Prior reports suggested that mutations altering synthesis of flavonoids, plant specialized metabolites that include Flavonols and anthocyanins, impair pollen development in several species, but the mechanism by which Flavonols enhanced fertility was not defined. Here, we used genetic approaches to demonstrate that Flavonols enhanced pollen development by reducing the abundance of reactive oxygen species (ROS). We further showed that Flavonols reduced high-temperature stress-induced ROS accumulation and inhibition of pollen tube growth. The anthocyanin reduced (are) tomato mutant had reduced flavonol accumulation in pollen grains and tubes. This mutant produced fewer pollen grains and had impaired pollen viability, germination, tube growth, and tube integrity, resulting in reduced seed set. Consistent with Flavonols acting as ROS scavengers, are had elevated levels of ROS. The pollen viability, tube growth and integrity defects, and ROS accumulation in are were reversed by genetic complementation. Inhibition of ROS synthesis or scavenging of excess ROS with an exogenous antioxidant treatment also reversed the are phenotypes, indicating that Flavonols function by reducing ROS levels. Heat stress resulted in increased ROS in pollen tubes and inhibited tube growth, with more pronounced effects in the are mutant that could be rescued by antioxidant treatment. These results are consistent with increased ROS inhibiting pollen tube growth and with Flavonols preventing ROS from reaching damaging levels. These results reveal that flavonol metabolites regulate plant sexual reproduction at both normal and elevated temperatures by maintaining ROS homeostasis.
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abscisic acid induced reactive oxygen species are modulated by Flavonols to control stomata aperture
Plant Physiology, 2017Co-Authors: Justin M Watkins, Jordan M Chapman, Gloria K MudayAbstract:Abscisic acid (ABA) increases reactive oxygen species (ROS) in guard cells to close Arabidopsis (Arabidopsis thaliana) stomata. In tomato (Solanum lycopersicum), we find that ABA-increased ROS is followed by stomatal closure and that both responses are blocked by inhibitors of ROS-producing respiratory burst oxidase enzymes. ABA-induced ROS sensor fluorescence accumulates in the nucleus, chloroplasts, and endomembranes. The accumulation of flavonol antioxidants in guard cells, but not surrounding pavement cells, was visualized by confocal microscopy using a flavonol-specific fluorescent dye. Decreased Flavonols in guard cells in the anthocyanin reduced (are) mutant and elevated levels in the anthocyanin without (aw) mutant were quantified by confocal microscopy and in leaf extracts by mass spectrometry. Consistent with Flavonols acting as antioxidants, higher levels of ROS were detected in guard cells of the tomato are mutant and lower levels were detected in aw both at homeostasis and after treatment with ABA. These results demonstrate the inverse relationship between Flavonols and ROS. Guard cells of are show greater ABA-induced closure than the wild type, reduced light-dependent guard cell opening, and reduced water loss, with aw having opposite responses. Ethylene treatment of wild-type tomato plants increased flavonol accumulation in guard cells; however, no flavonol increases were observed in Neverripe (Nr), an ethylene receptor mutant. Consistent with lower levels of ROS due to elevated Flavonols, ethylene treatments decreased ABA-induced stomatal closure in the wild type, but not Nr, with ethylene responses attenuated in the are mutant. Together, these results are consistent with Flavonols dampening the ABA-dependent ROS burst that drives stomatal closure and facilitating stomatal opening to modulate leaf gas exchange.
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ethylene induced flavonol accumulation in guard cells suppresses reactive oxygen species and moderates stomatal aperture
Plant Physiology, 2014Co-Authors: Justin M Watkins, Paul J Hechler, Gloria K MudayAbstract:Guard cell swelling controls the aperture of stomata, pores that facilitate gas exchange and water loss from leaves. The hormone abscisic acid (ABA) has a central role in regulation of stomatal closure through synthesis of second messengers, which include reactive oxygen species (ROS). ROS accumulation must be minimized by antioxidants to keep concentrations from reaching damaging levels within the cell. Flavonols are plant metabolites that have been implicated as antioxidants; however, their antioxidant activity in planta has been debated. Flavonols accumulate in guard cells of Arabidopsis thaliana, but not surrounding pavement cells, as visualized with a flavonol-specific dye. The expression of a reporter driven by the promoter of CHALCONE SYNTHASE, a gene encoding a flavonol biosynthetic enzyme, in guard cells, but not pavement cells, suggests guard cell-specific flavonoid synthesis. Increased levels of ROS were detected using a fluorescent ROS sensor in guard cells of transparent testa4-2, which has a null mutation in CHALCONE SYNTHASE and therefore synthesizes no flavonol antioxidants. Guard cells of transparent testa4-2 show more rapid ABA-induced closure than the wild type, suggesting that Flavonols may dampen the ABA-dependent ROS burst that drives stomatal closing. The levels of Flavonols are positively regulated in guard cells by ethylene treatment in the wild type, but not in the ethylene-insensitive2-5 mutant. In addition, in both ethylene-overproducing1 and ethylene-treated wild-type plants, elevated Flavonols lead to decreasing ROS and slower ABA-mediated stomatal closure. These results are consistent with Flavonols suppressing ROS accumulation and decreasing the rate of ABA-dependent stomatal closure, with ethylene-induced increases in guard cell Flavonols modulating these responses.
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auxin and ethylene induce flavonol accumulation through distinct transcriptional networks
Plant Physiology, 2011Co-Authors: Daniel R Lewis, Melissa V Ramirez, Nathan D Miller, Prashanthi Vallabhaneni, Keith W Ray, Richard F Helm, Brenda S J Winkel, Gloria K MudayAbstract:Auxin and ethylene are key regulators of plant growth and development, and thus the transcriptional networks that mediate responses to these hormones have been the subject of intense research. This study dissected the hormonal cross talk regulating the synthesis of Flavonols and examined their impact on root growth and development. We analyzed the effects of auxin and an ethylene precursor on roots of wild-type and hormone-insensitive Arabidopsis (Arabidopsis thaliana) mutants at the transcript, protein, and metabolite levels at high spatial and temporal resolution. Indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) differentially increased flavonol pathway transcripts and flavonol accumulation, altering the relative abundance of quercetin and kaempferol. The IAA, but not ACC, response is lost in the transport inhibitor response1 (tir1) auxin receptor mutant, while ACC responses, but not IAA responses, are lost in ethylene insensitive2 (ein2) and ethylene resistant1 (etr1) ethylene signaling mutants. A kinetic analysis identified increases in transcripts encoding the transcriptional regulators MYB12, Transparent Testa Glabra1, and Production of Anthocyanin Pigment after hormone treatments, which preceded increases in transcripts encoding flavonoid biosynthetic enzymes. In addition, myb12 mutants were insensitive to the effects of auxin and ethylene on flavonol metabolism. The equivalent phenotypes for transparent testa4 (tt4), which makes no Flavonols, and tt7, which makes kaempferol but not quercetin, showed that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth. Collectively, these experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. This study also provides new evidence that quercetin is the flavonol that modulates basipetal auxin transport.
Stefan Martens - One of the best experts on this subject based on the ideXlab platform.
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multifunctional flavonoid dioxygenases flavonol and anthocyanin biosynthesis in arabidopsis thaliana l
Phytochemistry, 2010Co-Authors: Stefan Martens, Anja Preuss, Ulrich MaternAbstract:Flavonols and conditionally also anthocyanins, aside from Flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are "multifunctional" enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed.
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Arabidopsis thaliana expresses a second functional flavonol synthase
FEBS letters, 2009Co-Authors: Anja Preuss, Ulrich Matern, Ralf Stracke, Bernd Weisshaar, Alexander Hillebrecht, Stefan MartensAbstract:Arabidopsis thaliana L. produces flavonoid pigments, i.e. Flavonols, anthocyanidins and proanthocyanidins, from dihydroflavonol substrates. A small family of putative flavonol synthase (FLS) genes had been recognized in Arabidopsis, and functional activity was attributed only to FLS1. Nevertheless, other FLS activities must be present, because A. thalianafls1 mutants still accumulate significant amounts of Flavonols. The recombinant FLSs and leucoanthocyanidin dioxygenase (LDOX) proteins were therefore examined for their enzyme activities, which led to the identification of FLS3 as a second active FLS. This enzyme is therefore likely responsible for the formation of Flavonols in the ldox/fls1-2 double mutant. These double mutant and biochemical data demonstrate for the first time that LDOX is capable of catalyzing the in planta formation of Flavonols.
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flavonol synthase from citrus unshiu is a bifunctional dioxygenase
Phytochemistry, 2003Co-Authors: Richard Lukacin, Frank Wellmann, Lothar Britsch, Stefan Martens, Ulrich MaternAbstract:Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2R,3R)-dihydroFlavonols, i.e. dihydrokaempferol, to the corresponding Flavonols (kaempferol). Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3b-hydroxylase and flavonol synthase activities to the enzyme. In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2R)-naringenin as substrate. The product which was not converted any further on repeated incubations was identified by 1 H NMR and CD spectroscopies as ()-trans-dihydrokaempferol. The data demonstrate that Citrus flavonol synthase encompasses an additional non
Ralf Stracke - One of the best experts on this subject based on the ideXlab platform.
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natural variation in flavonol accumulation in arabidopsis is determined by the flavonol glucosyltransferase bglu6
Journal of Experimental Botany, 2016Co-Authors: Hirofumi Ishihara, Bernd Weisshaar, Takayuki Tohge, Prisca Viehover, Alisdair R Fernie, Ralf StrackeAbstract:Flavonols are colourless secondary metabolites, primarily regarded as UV-protection pigments that are deposited in plants in their glycosylated forms. The glycosylation of Flavonols is mainly catalysed by UDP-sugar-dependent glycosyltransferases (UGTs). Although the structures of flavonol glycosides accumulating in Arabidopsis thaliana are known, many genes involved in the flavonol glycosylation pathway are yet to be discovered. The flavonol glycoside profiles of seedlings from 81 naturally occurring A. thaliana accessions were screened using high performance thin layer chromatography. A qualitative variation in flavonol 3-O-gentiobioside 7-O-rhamnoside (F3GG7R) content was identified. Ler × Col-0 recombinant inbred line mapping and whole genome association mapping led to the identification of a glycoside hydrolase family 1-type gene, At1g60270/BGLU6, that encodes a homolog of acyl-glucose-dependent glucosyltransferases involved in the glycosylation of anthocyanins, possibly localized in the cytoplasm, and that is co-expressed with genes linked to phenylpropanoid biosynthesis. A causal single nucleotide polymorphism introducing a premature stop codon in non-producer accessions was found to be absent in the producers. Several other naturally occurring loss-of-function alleles were also identified. Two independent bglu6 T-DNA insertion mutants from the producer accessions showed loss of F3GG7R. Furthermore, bglu6 mutant lines complemented with the genomic Ler BGLU6 gene confirmed that BGLU6 is essential for production of F3GGR7. We have thus identified an accession-specific gene that causes a qualitative difference in flavonol glycoside accumulation in A. thaliana strains. This gene encodes a flavonol 3-O-glucoside: 6″-O-glucosyltransferase that does not belong to the large canonical family of flavonol glycosyltransferases that use UDP-conjugates as the activated sugar donor substrate.
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the grapevine r2r3 myb transcription factor vvmybf1 regulates flavonol synthesis in developing grape berries
Plant Physiology, 2009Co-Authors: Stefan Czemmel, Ralf Stracke, Bernd Weisshaar, Nicole Jane Cordon, Nilangani N Harris, Amanda R Walker, Simon P Robinson, Jochen BogsAbstract:Flavonols are important ultraviolet light protectants in many plants and contribute substantially to the quality and health-promoting effects of fruits and derived plant products. To study the regulation of flavonol synthesis in fruit, we isolated and characterized the grapevine (Vitis vinifera 'Shiraz') R2R3-MYB transcription factor VvMYBF1. Transient reporter assays established VvMYBF1 to be a specific activator of flavonol synthase1 (VvFLS1) and several other promoters of grapevine and Arabidopsis (Arabidopsis thaliana) genes involved in flavonol synthesis. Expression of VvMYBF1 in the Arabidopsis mutant myb12 resulted in complementation of its flavonol-deficient phenotype and confirmed the function of VvMYBF1 as a transcriptional regulator of flavonol synthesis. Transcript analysis of VvMYBF1 throughout grape berry development revealed its expression during flowering and in skins of ripening berries, which correlates with the accumulation of Flavonols and expression of VvFLS1. In addition to its developmental regulation, VvMYBF1 expression was light inducible, implicating VvMYBF1 in the control of VvFLS1 transcription. Sequence analysis of VvMYBF1 and VvFLS1 indicated conserved putative light regulatory units in promoters of both genes from different cultivars. By analysis of the VvMYBF1 amino acid sequence, we identified the previously described SG7 domain and an additional sequence motif conserved in several plant MYB factors. The described motifs have been used to identify MYB transcription factors from other plant species putatively involved in the regulation of flavonol biosynthesis. To our knowledge, this is the first functional characterization of a light-inducible MYB transcription factor controlling flavonol synthesis in fruit.
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Arabidopsis thaliana expresses a second functional flavonol synthase
FEBS letters, 2009Co-Authors: Anja Preuss, Ulrich Matern, Ralf Stracke, Bernd Weisshaar, Alexander Hillebrecht, Stefan MartensAbstract:Arabidopsis thaliana L. produces flavonoid pigments, i.e. Flavonols, anthocyanidins and proanthocyanidins, from dihydroflavonol substrates. A small family of putative flavonol synthase (FLS) genes had been recognized in Arabidopsis, and functional activity was attributed only to FLS1. Nevertheless, other FLS activities must be present, because A. thalianafls1 mutants still accumulate significant amounts of Flavonols. The recombinant FLSs and leucoanthocyanidin dioxygenase (LDOX) proteins were therefore examined for their enzyme activities, which led to the identification of FLS3 as a second active FLS. This enzyme is therefore likely responsible for the formation of Flavonols in the ldox/fls1-2 double mutant. These double mutant and biochemical data demonstrate for the first time that LDOX is capable of catalyzing the in planta formation of Flavonols.