FEBS Letters 583 (2009) 1981–1986
journal homepage: www.FEBSLetters.org
Arabidopsis thaliana expresses a second functional flavonol synthase Anja Preuß a, Ralf Stracke b, Bernd Weisshaar b, Alexander Hillebrecht c, Ulrich Matern a, Stefan Martens a,* a b c
Philipps-Universität Marburg, Institut für Pharmazeutische Biologie, Deutschhausstrasse 17A, D-35037 Marburg/Lahn, Germany Bielefeld University, Faculty of Biology, Chair of Genome Research, Universitaetsstrasse 27, D-33615 Bielefeld, Germany Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg/Lahn, Germany
a r t i c l e
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Article history: Received 18 February 2009 Revised 27 April 2009 Accepted 4 May 2009 Available online 10 May 2009 Edited by Ulf-Ingo Flügge Keywords: Flavonoid biosynthesis Flavonol synthase Leucoanthocyanidin dioxygenase Arabidopsis thaliana
a b s t r a c t 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. thaliana fls1 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. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Flavonoids have been shown to carry out significant functions for the growth and reproduction of plants, and also appear to be essential in adaptation to specific ecological niches. Beyond pigmentation and protection against ultraviolet light, these functions include, signaling interactions with insects or microbes, and roles as feeding deterrents and phytoalexins [1]. Arabidopsis thaliana L. (mouse ear cress, Brassicaceae) accumulates predominantly flavonol glucosides, as well as anthocyanins and proanthocyanidins (PA) [2–7], which all derive from dihydroflavonols at a branch point of the anthocyanin/PA pathway (Fig. 1). The formation of flavonols depends on flavonol synthase (FLS), and the first FLS activity reported from irradiated parsley cells had been characterized in vitro as a soluble 2-oxoglutarate-dependent dioxygenase (2ODD) [8]. The oxidation reaction introducing the C-2/C-3 double bond (Fig. 1) was considered to be specific for dihydroflavonol substrates. Putative FLS cDNAs were subsequently cloned from Petunia hybrida Hort. (Solanaceae) and other plants, and expressed in yeast [9]. Moreover, a Citrus sp. FLS expressed in Escherichia coli was characterized in great detail [10]. Examination of the recombinant
Abbreviations: FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase; 2-ODD, 2-oxoglutarate dependent dioxygenases; PA, proanthocyanidins; TLC, thinlayer chromatography * Corresponding author. Fax: +49 6421 2825366. E-mail address:
[email protected] (S. Martens).
enzyme revealed that FLS is bifunctional, capable of oxidizing flavanones as well as dihydroflavonols to the respective flavonols (Fig. 1) [11]. This bifunctionality has also been shown for the closely related leucoanthocyanidin dioxygenase (LDOX; syn. anthocyanidin synthase, ANS) [12,13], however, LDOX is considered to catalyze in planta the conversion of leucoanthocyanidins to anthocyanidins (Fig. 1) [14,15]. The side activities of FLS and LDOX raised the possibility that these enzymes provide an alternative source of flavonols in vivo in mutant lines lacking flavanone 3b-hydroxylase (FHT; syn. F3H) or FLS activities. The Arabidopsis genome contains a small family of FLS genes [2,16] with only FLS1 being functionally expressed [17]. FLS2-6 were considered to be inactive [18,19]. However, some unexplained flavonol and anthocyanin accumulation was reported for mutant lines of FHT (transparent testa6, tt6) and FLS1 of A. thaliana, whereas mutant lines of CHS (tt4) lack all flavonoid compounds at all [3,7,19,20 and refs. therein; Ric de Vos, personal communication]. These observations suggest the presence of additional FLS and/or LDOX side activities. First support for this hypothesis was recently provided by the biochemical characterization of FHT from A. thaliana mutant line tt6 [20], by overexpression of LDOX in rice plants [21], and from metabolomic and genetic analyses in A. thaliana [18]. Notably, such side activities (or multi-functionality) of FLS and LDOX would require a redrawing of the metabolic grid considering, in particular, the biosynthesis in mutants or plants accumulating only trace amounts of flavonoids. Moreover, the impact of side activities needs to be considered
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.05.006
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A. Preuß et al. / FEBS Letters 583 (2009) 1981–1986 R1 OH O
HO
flavanones
OH
O
FHT
flavonols
R1 OH
OH
FLS
O
HO
O
HO
OH
OH
dihydroflavonols
OH
R1
O
OH
DFR
O
anthocyanidins
cis-flavan-3-ols R1
R1
LDOX
O
HO
flavan-3,4-diols
OH OH
OH
R1 OH
OH
ANS
HO
OH
ANR
O
+
HO
O
OH OH
FGT
anthocyanins
OH OH
???
proanthocyanidins
Fig. 1. Schematic outline of the pathway to major flavonoid products in Arabidopsis thaliana. FHT, flavanone 3b-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4reductase; LDOX, leucoanthocyanidin dioxygenase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FGT, flavonoid glycosyltransferases. 2-Oxoglutaratedependent dioxygenases are bold-printed. Flavanones: naringenin (R1 = H), eriodictyol (R1 = OH). Dihydroflavonols: dihydrokaempferol (R1 = H), dihydroquercetin (R1 = OH). Flavonols: kaempferol (R1 = H), quercetin (R1 = OH). Flavan-3,4-diols: leucopelargonidin (R1 = H), leucocyanidin (R1 = OH). Anthocyanidins: pelargonidin (R1 = H), cyanidin (R1 = OH). cis-Flavan-3-ols: epiafzelechin (R1 = H), epicatechin (R1 = OH).
when metabolic engineering experiments aiming to modulate the flavonoid accumulation are conducted, because alternate activities could significantly affect the results. Both FLS4 and FLS6 had been identified as pseudogenes because of their considerably truncated C-termini [18,19]. The potential activities of FLS2, 3 and 5, however, deserve reinvestigation and were therefore studied in vitro. Furthermore, the role of LDOX in flavonol formation was examined. These studies revealed a second active A. thaliana FLS and a novel in situ activity of LDOX [18].
2. Material and methods 2.1. Chemicals All flavonoids used in these studies were obtained from TransMIT GmbH Projektbereich Flavonoidforschung (Giessen, Germany). Radiolabeled substrates were synthesised as described in [22]. 2.2. Crude protein preparation from plant tissue Plant tissue from seedlings and adult plants of A. thaliana wildtype (Col-0) and mutant lines (fls1-2, ldox/fls1-2) were used. Soluble protein preparations were prepared as described previously [22,23]. The crude protein extract were used for biochemical characterization in a standard 2-ODD assay [22,23]. 2.3. Construction of expression vectors The cDNAs containing coding sequences of putative FLSs and LDOX from A. thaliana were cloned into pENTRY vector
(pDONR201) and subcloned into yeast (pYES-DEST52) and bacterial (pDEST14) expression vector by Gateway Technology (Invitrogen, Groningen, The Netherlands). Expression constructs were verified by restriction analysis and DNA sequencing. 2.4. Heterologous expression Competent Saccharomyces cereviseae cells of the strain INV Sc1 (S.c EasyComp Transformation Kit, Invitrogen) were transformed with the FLSx- or LDOX-pYES-DEST52 constructs, respectively. Expression, cell lysis and protein isolation was done as described previously [22,23]. Competent E. coli BL21-A1 cells (Invitrogen) were transformed with the respective FLSx- or LDOX-pDEST14 constructs and subsequently subcultured to a density of OD600 = 0.4 in Luria–Bertani medium containing ampicillin (100 lg/ml). Arabinose solution (20%) was added for the induction of protein expression, and the bacteria were harvested following an additional 4 h of growth at 37 °C and stored frozen at 70 °C. The recombinant FLS1 and putative FLS proteins were partially purified from crude bacterial extracts by a modified procedure devised for Petunia FHT as described elsewhere [24]. The purification was monitored by enzyme assay and SDS–PAGE [11,25]. 2.5. Enzyme assays with recombinant proteins FLS activity was assayed as described previously [23]. Flavonoids were isolated from the mixture by repeated extraction with ethyl acetate (2 100 ll), analyzed and identified by thin-layer cochromatography with authentic standards on cellulose (Merck, Darmstadt, Germany) in solvent system CAW (chloroform:acetic acid:water; 50:45:5, by vol.) or 30% acetic acid.
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2.6. Bioconversion
A
Bioconversion experiments were carried out with yeast (50 ml) expressing FLS or LDOX proteins by adding up to 20 mg of either one of various flavanones (naringenin, eriodictyol) or dihydroflavonols (dihydrokaempferol, dihydroquercetin, dihydrotamarixitin, dihydrorobinitin and dihydroisorhamnetin) as substrates after induction of protein expression [26]. The cultures were incubated for 12 h (standard) or up to 2 days (extended) at 30 °C. Empty vector transformants and glucose non-induced cells served as controls. Extraction of flavonoids was performed with 2 vol. ethyl acetate. Analysis of the reaction mixture was done by thin-layer chromatography (TLC) on cellulose in solvent system CAW, 30% acetic acid or Forestal (acetic acid:hydrochloric acid:water; 30:3:10, by vol.) and by HPLC.
FLS1
FLS2
FLS3
FLS5
LDOX
DHK
Km
B DHQ
2.7. HPLC Reaction products were extracted with ethyl acetate (2 200 ll) from culture aliquots (1 ml), vacuum-dried and redissolved in 150 ll methanol for HPLC analysis. Reversed-phase HPLC was carried out on Nucleodure Sphinx C18-column (5 lm; Machery and Nagel, Düren, Germany) as described [27]. 2.8. SDS–PAGE and immunoassays The protein composition of the samples was examined by SDS– PAGE [26] utilizing the SDS-7 marker for mass calibration (Sigma, Deisenhofen, Germany). Proteins in the SDS gel were stained with Coomassie brilliant blue. Western blotting was carried out as described [28] using a Citrus unshiu Marc. (Rutaceae) FLS polyclonal rabbit antiserum [29]. Proteins bound to the nitrocellulose filters were visualized after immunodetection by staining using a mixture of 100 ll 5-bromo-4-chloro-3-indolylphosphate (BCIP, 50 mg/ml DMF) and 300 ll nitro-blue tetrazolium (NBT, 75 mg/ml 70% DMF) in 1 M tricine buffer pH 8.8 (10 ml) containing 1 mM MgCl2. 2.9. Homology modeling Homology modeling was performed with the translated polypeptides of FLS1 and the putative isogenes FLS3 and FLS5. The models were generated by the WEB-based SWISS-MODEL server as described previously [30]. 3. Results and discussion A. thalíana has been employed as a model plant for multiple biochemical and genetic studies of secondary metabolism including the flavonoid pathway [31]. Flavonols (quercetin, kaempferol, isorhamnetin glucosides) and the corresponding flavan-3-ols or dihydroflavonols (Fig. 1), as well as proanthocyanidins and anthocyanins were reported from wild-type plants [7]. Seventeen genes essential for this pathway have been identified, including eight structural (e.g. FLS, LDOX) and six regulatory genes (e.g. TT2, TT8),
and three genes involved in flavonoid compartmentation [7]. A single LDOX gene and a family of six FLS genes were identified in Arabidopsis, but only FLS1 has been proposed to yield a functional FLS [17]. In experiments employing A. thaliana wild-type and mutant lines, the role of LDOX or FLSs for flavonol accumulation was investigated. Standard enzyme assays with naringenin or dihydrokaempferol as substrate clearly revealed FHT and FLS activities in wild-type extracts, converting both substrates to kaempferol, whereas extracts of the fls1-2 single mutant and the ldox/fls1-2 double mutant lacked these activities (data not shown). However, extracts of all three genotypes contained flavonols, albeit at greatly different levels [18]. This can be explained only by residual FLS activity contributed by either one or more of the FLS2-6 polypeptides or by LDOX side activity. Recombinant LDOX from Arabidopsis had been shown to exert FLS activity [13,15,32,33], although the enzyme was unstable during isolation and in vitro assays [14]. Accordingly, LDOX activity has never been detected in native Arabidopsis extracts. Nevertheless, an A. thaliana ldox/fls1-2 double mutant revealed a significant reduction in flavonol level (less than 0.00005% of the wild-type level [18]) as compared to the wild-type and the fls1-2 mutant (ca. 5% of wild-type level as deduced from [19]) suggesting some FLS activity of LDOX under in situ conditions. Alternatively, low FLS activity of either one of the other putative
FLS5
FLS3
FLS2
FLS1
Western blotanalysis
FLS5
FLS3
FLS2
36
Fig. 3. FLS activity assays of extracts from E. coli expressing either one of the FLSs or LDOX from A. thaliana. Enzyme fractions after ammonium sulphate precipitation (30–80%) and size-exclusion chromatography were incubated with (A) [14C]dihydrokaempferol, DHK (42.4 pmol; 55 mCi/mmol) or (B) [14C]dihydroquercetin, DHQ (42.4 pmol; 55 mCi/mmol). The conversion to kaempferol (Km) and quercetin (Qu), respectively, was examined by autoradiography after extraction of the incubation mixtures with ethyl acetate and ascending cellulose thin-layer chromatography (TLC) separation of the extracts in the CAW solvent system.
SDS-PAGE
M
FLS1
kDa 45
Qu
Fig. 2. Immunoblotting of recombinant A. thaliana FLS-like polypeptides. Crude extracts of recombinant E. coli expressing either one of the FLSs were fractionated by ammonium sulphate (30–80%) and size-exclusion chromatography prior to SDS–PAGE separation (left) in 5% stacking and 12.5% separation gels. Proteins were visualized by Coomassie Brilliant Blue staining. FLS-like polypeptides were localized by Western blotting (right) employing anti-FLS antiserum [28].
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Fig. 4. Docking of naringenin into the active sites of FLS1 (yellow) and FLS3 (green) of A. thaliana. The model complex suggests that 136Tyr of FLS3 (in place of 136Leu as in FLS1) affects the assembly of 149His which participates in substrate binding. The calculation also revealed that proper binding of naringenin to FLS5 appears highly unlikely (not shown).
FLS polypeptides in crude extracts might have escaped the detection in the in vitro assays which were commonly run for only
30 min, or which may be due to improper stability under experimental conditions [22]. Arabidopsis FLS6 and FLS4 were recently identified as pseudogenes or non-functional copies [18,19]. Therefore, only FLS1, FLS2, FLS3 and FLS5, as well as LDOX, were cloned and expressed in bacterial or yeast cells. The expression of all polypeptides was confirmed by Western blotting using anti-FLS antiserum (Fig. 2). The FLS activity of the recombinant polypeptides was examined in standard assays conducted with crude extracts or employing the respective fraction after size-exclusion chromatography. FLS1 efficiently converted dihydrokaempferol or dihydroquercetin to kaempferol and quercetin, respectively, with a clear bias towards dihydrokaempferol (Km/Qu ratio 1:0,7; Fig. 3), which is consistent with previous findings [12]. LDOX also oxidized these substrates to the corresponding flavonols, albeit to a lower extent (Km/Qu ratio 0,7:1), whereas the other putative FLSs did not yield a product under the conditions of the assays (Fig. 3). These results suggested that the FLS activity of LDOX might contribute to the formation of flavonols in the Arabidopsis fls1-2 mutant, which is supported by the additional drop of flavonol contents observed in the ldox/ fls1-2 double mutant [18]. Nevertheless, the small but significant residual flavonol content observed in the ldox/fls1-2 mutant [18] required further consideration.
Fig. 5. Bioconversion of dihydrokaempferol or dihydroquercetin by FLS1, FLS3 and LDOX expressed in yeast cells. Incubation of yeast transformants (50 ml culture) were conducted for 48 h in the presence of 20 mg of the substrate. The cultures were extracted subsequently with ethyl acetate, and the extracts were separated by cellulose TLC in CAW (A and B) or by HPLC (C). (A) Yeast cells expressing FLS3 converted DHQ (lane 2) or DHK (lane 4) to kaempferol (Km) and quercetin (Qu), respectively, under these conditions, whereas naringenin (lane 1) or eriodictyol (lane 3) did not serve as substrate. (B) The conversion rate of DHQ to quercetin observed with the yeast transformant expressing FLS3 (lane 3) was lower than that shown by the LDOX transformant (lane 2) and much lower than that by the FLS1 transformant (lane 1). (C) The relative mobility of the bioconversion product on HPLC separation confirmed the identity as quercetin (bottom) by comparison with authentic dihydroquercetin (top) and quercetin (middle).
A. Preuß et al. / FEBS Letters 583 (2009) 1981–1986
Complementary to the in vitro assays, the bioconversion of various flavanones (naringenin, eriodictyol) and dihydroflavonols (dihydrokaempferol, dihydroquercetin, dihydromyricetin, dihydrotamarixetin, dihydrorobinitin and dihydroisorhamnetin) by yeast transformants expressing recombinant FLSs or LDOX was examined, with the assumption that the stability of enzymes was improved under these assay conditions and that the assays could be carried out for extended times. The cultures were routinely incubated for 12 h in the presence of 5 mg of either one of the potential substrates [26]. The FLS1 transformant accepted all dihydroflavonols and flavanones to a variable degree, and formed the corresponding flavonols without detectable intermediates (data not shown). At a considerably lower rate, the same applied to the LDOX-transformant. In no case did the transformants harbouring one of the other FLS constructs or the empty expression vector show a significant conversion of the flavonoid substrates. This provides strong evidence of FLS activity for the LDOX transformant, although further corroboration by kinetic data was not possible since steady state conditions in the biotransformation system are unlikely. Analogous to FLS4 and FLS6 which represent truncated pseudogenes [18,19], and based on the truncated C-terminus and lack of appropriate iron and 2-oxoglutarate binding sites, FLS2 likely encoded a non-functional polypeptide. This assumption was confirmed by expression of FLS2. Likewise, recombinant FLS5 also lacked enzyme activity. It was shown previously that marginal changes in the binding sites of substrate, ferrous iron and oxoglutarate cosubstrate by 2-oxoglutarate-dependent dioxygenases (2ODDs) may strongly affect the substrate and/or product specificity [32]. Sequence alignments with FLS1 revealed that 219Arg/Lys and 327 Ala/Glu exchanges had occurred in FLS5, which presumably affect the hydrogen bonding of the substrate [19]. While these changes explain the inactivity of FLS2 and FLS5 polypeptides, the proposed binding sites appear fully conserved in FLS3. Essential amino acid residues in 2-ODDs may also be identified by homology modeling, as had been demonstrated for FHT and flavone synthase (FNSI) from parsley [30]. Models of the LDOX-naringenin (2brt) [33] and LDOX-dihydroquercetin complexes [32] were employed as templates for homology modeling of FLS1 and comparison with FLS3 and FLS5, because FLS oxidizes the same substrates [12,22] and the conserved binding sites for 2-oxoglutarate (298Arg in LDOX) and iron (232His, 288His and 234Asp in LDOX) are distributed with an analogous spacing in the FLS polypeptides (319Arg, 251His, 309 His and 253Asp in FLS 1 and FLS3). The model endorses the role of 327Glu in substrate binding and the lack of activity of FLS5 (not shown). LDOX was proposed to bind naringenin through p-stacking of the substrate A-ring with 304Phe [32] which corresponds to 325 Phe in FLS1 and FLS3 (Fig. 4). FLS3 differs from FLS1, however, by replacement of 136Leu through 136Tyr, and the model complex predicts that this residue changes the orientation of nearby 149 His involved in substrate binding (Fig. 4). Thus, the co-location of substrate toward the active ferryl species and the kinetic parameters are likely affected. The activity of FLS3 was therefore re-examined through extended bioconversion studies (48 h in the presence of 20 mg substrate/50 ml) using the respective yeast transformant. Under the assay conditions, the conversion of both dihydrokaempferol and dihydroquercetin to kaempferol and quercetin, respectively, was demonstrated by TLC and HPLC co-chromatographies (Fig. 5). Noninduced yeast transformants or cells harbouring an empty vector served as controls and lacked FLS activity (data not shown). The data suggest that FLS3 should be annotated as a second functional gene in A. thaliana, irrespective of the fact that site-directed mutagenesis is necessary to further identify those amino acids relevant for enhanced activity. Furthermore, FLS3 is likely responsible for the formation of residual flavonols in seedlings of the ldox/fls1-2 mutant
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line, but final proof requires the analysis of the respective triple mutant (ldox/fls1-2/fls3). While the FLS activity of LDOX and FLS3 may be relevant only in fls1 mutant lines, all three dioxygenases are capable to a variable extent of bypassing an FHT block. These and previous findings [18,19] let us presume that the Arabidopsis FLS gene family resulted from recent gene duplication events and that a pseudogenisation/mutation process currently eliminates ‘‘unnecessary” genes and their protein functions. Alternatively, FLS3 may have acquired another function while preserving some FLS activity. Acknowledgments Funding by EU Project FLAVO (FOOD CT-2004-513960) and the Deutsche Forschungsgemeinschaft is gratefully acknowledged. Authors would like to thank Dr. Peter for critical reading of the manuscript. References [1] Harborne, J.B. and Williams, C.A. (2000) Advances in flavonoid research since 1992. Phytochemistry 55, 481–504. [2] Pelletier, M.K., Murrell, J.R. and Shirley, B.W. (1997) Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis. Plant Physiol. 113, 1437–1445. [3] Pelletier, M.K., Burbulis, I.E. and Winkel-Shirley, B. (1999) Disruption of specific flavonoid genes enhance the accumulation of flavonoid enzymes and end-products in Arabidopsis seedlings. Plant Mol. Biol. 40, 45–54. [4] Veit, M. and Pauli, G.F. (1999) Major flavonoids from Arabidopsis thaliana leaves. J. Nat. Prod. 62, 1301–1303. [5] Peer, W.A., Brown, D.E., Tague, B.W., Muday, G.K., Taiz, L. and Murphy, A.S. (2001) Flavonoid accumulation patterns of transparent testa mutants of Arabidopsis. Plant Physiol. 126, 536–548. [6] Broun, P. (2005) Transcriptional control of flavonoid biosynthesis: A complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Curr. Opt. Plant Biol. 8, 272–279. [7] Routaboul, J-M., Kerhoas, L., Debeaujon, I., Pourcel, L., Caboche, M., Einhorn, J. and Lepiniec, L. (2006) Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana. Planta 224, 96–107. [8] Britsch, L., Heller, W. and Grisebach, H. (1981) Conversion of flavanone to flavone, dihydroflavonol and flavonol with an enzyme system from cell culture of parsley. Z. Naturforsch. 36, 742–750. [9] Holton, T.A., Brugliera, F. and Tanaka, Y. (1993) Cloning and expression of flavonol synthase from Petunia hybrida. Plant J. 4, 1003–1010. [10] Wellmann, F., Lukacin, R., Moriguchi, T., Britsch, L., Schiltz, E. and Matern, U. (2002) Functional expression and mutational analysis of flavonol synthase from Citrus unshiu. Eur. J. Biochem. 269, 4134–4142. [11] Lukacˇin, R., Wellmann, F., Britsch, L., Martens, S. and Matern, U. (2003) Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase. Phytochemistry 62, 287–292. [12] Prescott, A.G., Stamford, N.P.J., Wheeler, G. and Firmin, J.L. (2002) In vitro properties of recombinant flavonol synthase from Arabidopsis thaliana. Phytochemistry 60, 589–593. [13] Turnbull, J.J., Nakajima, J-I., Welford, R.W.D., Yamazaki, M., Saito, K. and Schofield, C.J. (2004) Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis. J. Biol. Chem. 279, 1206–1216. [14] Saito, K., Kobayashi, M., Gong, Z., Tanaka, Y. and Yamazaki, M. (1999) Direct evidence for anthocyanidin synthase as a 2-oxoglutarate-dependent oxygenase: Molecular cloning and functional expression of cDNA from a red forma of Perilla frutescens. Plant J. 17, 181–189. [15] Welford, R.W.D., Turnbull, J.J., Claridge, T.D.W., Prescott, A.G. and Schofield, C.J. (2001) Evidence for oxidation at C-3 of the flavonoid C-ring during anthocyaninin biosynthesis. Chem. Commun. 182, 8–1829. [16] Stracke, R., Ishihara, H., Barsch, G.H.A., Mehrtens, F., Nieshaus, K. and Weisshaar, B. (2007) Differential regulation of closley related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedlings. Plant J. 50, 660–677. [17] Wisman, E., Hartmann, U., Sagasser, M., Baumann, E., Palme, K., Hahlbrock, K., Saedler, H. and Weisshaar, B. (1998) Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes. Proc. Natl. Acad. Sci. USA 95, 12432–12437. [18] Stracke, R., De Vos, R.C., Bartelniewoehner, L., Ishihara, H., Sagasser, M., Martens, S. and Weisshaar, B. (2009) Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase. Planta 229, 427–445. [19] Owens, D.K., Alerding, A.B., Crosby, K.C., Bandara, A.B., Westwood, J.H. and Winkel, B.S.J. (2008) Functional analysis of a predict flavonol synthase gene family in Arabidopsis. Plant Physiol. 147, 1046–1061. [20] Owens, D.K., Crosby, K.C., Runac, J., Howard, B.A. and Winkel, B.S.J. (2008) Biochemical and genetic characterisation of Arabidopsis flavanone 3bhydroxylase. Plant Physiol. Biochem. 46, 833–843.
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