Arabidopsis Thaliana Expresses a Second Functional Flavonol Synthase

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FEBS Letters 583 (2009) 1981–1986

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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 Philipps-Universität Marburg, Institut für Pharmazeutische Biologie, Deutschhausstrasse 17A, D-35037 Marburg/Lahn, Germany b Bielefeld University, Faculty of Biology, Chair of Genome Research, Universitaetsstrasse 27, D-33615 Bielefeld, Germany c Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D-35037 Marburg/Lahn, Germany

article info abstract

Article history: Arabidopsis thaliana L. produces flavonoid pigments, i.e. flavonols, anthocyanidins and proanthocy- Received 18 February 2009 anidins, from dihydroflavonol substrates. A small family of putative flavonol synthase (FLS) genes Revised 27 April 2009 had been recognized in Arabidopsis, and functional activity was attributed only to FLS1. Neverthe- Accepted 4 May 2009 less, other FLS activities must be present, because A. thaliana fls1 mutants still accumulate signifi- Available online 10 May 2009 cant amounts of flavonols. The recombinant FLSs and leucoanthocyanidin dioxygenase (LDOX) Edited by Ulf-Ingo Flügge 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 Keywords: Flavonoid biosynthesis first time that LDOX is capable of catalyzing the in planta formation of flavonols. Flavonol synthase Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Leucoanthocyanidin dioxygenase Arabidopsis thaliana

1. Introduction enzyme revealed that FLS is bifunctional, capable of oxidizing flavanones as well as dihydroflavonols to the respective flavonols Flavonoids have been shown to carry out significant functions (Fig. 1) [11]. This bifunctionality has also been shown for the clo- for the growth and reproduction of plants, and also appear to be sely related leucoanthocyanidin dioxygenase (LDOX; syn. anthocy- essential in adaptation to specific ecological niches. Beyond pig- anidin synthase, ANS) [12,13], however, LDOX is considered to mentation and protection against ultraviolet light, these functions catalyze in planta the conversion of leucoanthocyanidins to antho- include, signaling interactions with insects or microbes, and roles cyanidins (Fig. 1) [14,15]. as feeding deterrents and phytoalexins [1]. Arabidopsis thaliana L. The side activities of FLS and LDOX raised the possibility that (mouse ear cress, Brassicaceae) accumulates predominantly flavo- these enzymes provide an alternative source of flavonols in vivo nol glucosides, as well as anthocyanins and proanthocyanidins (PA) in mutant lines lacking flavanone 3b-hydroxylase (FHT; syn. F3H) [2–7], which all derive from dihydroflavonols at a branch point of or FLS activities. The Arabidopsis genome contains a small family the anthocyanin/PA pathway (Fig. 1). The formation of flavonols of FLS genes [2,16] with only FLS1 being functionally expressed depends on flavonol synthase (FLS), and the first FLS activity re- [17]. FLS2-6 were considered to be inactive [18,19]. However, some ported from irradiated parsley cells had been characterized unexplained flavonol and anthocyanin accumulation was reported in vitro as a soluble 2-oxoglutarate-dependent dioxygenase (2- for mutant lines of FHT (transparent testa6, tt6) and FLS1 of A. tha- ODD) [8]. The oxidation reaction introducing the C-2/C-3 double liana, whereas mutant lines of CHS (tt4) lack all flavonoid com- bond (Fig. 1) was considered to be specific for dihydroflavonol sub- pounds at all [3,7,19,20 and refs. therein; Ric de Vos, personal strates. Putative FLS cDNAs were subsequently cloned from Petunia communication]. These observations suggest the presence of addi- hybrida Hort. (Solanaceae) and other plants, and expressed in yeast tional FLS and/or LDOX side activities. First support for this [9]. Moreover, a Citrus sp. FLS expressed in Escherichia coli was hypothesis was recently provided by the biochemical characteriza- characterized in great detail [10]. Examination of the recombinant tion 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 mul- Abbreviations: FLS, flavonol synthase; LDOX, leucoanthocyanidin dioxygenase; ti-functionality) of FLS and LDOX would require a redrawing of 2-ODD, 2-oxoglutarate dependent dioxygenases; PA, proanthocyanidins; TLC, thin- the metabolic grid considering, in particular, the biosynthesis in layer chromatography * Corresponding author. Fax: +49 6421 2825366. mutants or plants accumulating only trace amounts of flavonoids. E-mail address: [email protected] (S. Martens). 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 1982 A. Preuß et al. / FEBS Letters 583 (2009) 1981–1986

R1 OH

HO O

flavanones OH O

FHT R1 flavonols R1 OH OH FLS HO O HO O

dihydroflavonols OH OH OH O OH O

DFR anthocyanidins cis-flavan-3-ols R1 R1 R1 OH OH OH

HO O LDOX HO O ANR HO O + flavan-3,4-diols OH OH OH OH OH ANS OH OH

FGT ???

anthocyanins proanthocyanidins

Fig. 1. Schematic outline of the pathway to major flavonoid products in Arabidopsis thaliana. FHT, flavanone 3b-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4- reductase; LDOX, leucoanthocyanidin dioxygenase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FGT, flavonoid glycosyltransferases. 2-Oxoglutarate- dependent 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 (pDONR201) and subcloned into yeast (pYES-DEST52) and bacte- flavonoid accumulation are conducted, because alternate activities rial (pDEST14) expression vector by Gateway Technology (Invitro- could significantly affect the results. gen, Groningen, The Netherlands). Expression constructs were Both FLS4 and FLS6 had been identified as pseudogenes because verified by restriction analysis and DNA sequencing. of their considerably truncated C-termini [18,19]. The potential activities of FLS2, 3 and 5, however, deserve reinvestigation and 2.4. Heterologous expression were therefore studied in vitro. Furthermore, the role of LDOX in flavonol formation was examined. These studies revealed a second Competent Saccharomyces cereviseae cells of the strain INV Sc1 active A. thaliana FLS and a novel in situ activity of LDOX [18]. (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 2. Material and methods previously [22,23]. Competent E. coli BL21-A1 cells (Invitrogen) were transformed with the respective FLSx- or LDOX-pDEST14 2.1. Chemicals constructs and subsequently subcultured to a density of

OD600 = 0.4 in Luria–Bertani medium containing ampicillin All flavonoids used in these studies were obtained from Trans- (100 lg/ml). Arabinose solution (20%) was added for the induction MIT GmbH Projektbereich Flavonoidforschung (Giessen, Germany). of protein expression, and the bacteria were harvested following Radiolabeled substrates were synthesised as described in [22]. an additional 4 h of growth at 37 °C and stored frozen at À70 °C. The recombinant FLS1 and putative FLS proteins were partially 2.2. Crude protein preparation from plant tissue purified from crude bacterial extracts by a modified procedure de- vised for Petunia FHT as described elsewhere [24]. The purification Plant tissue from seedlings and adult plants of A. thaliana wild- was monitored by enzyme assay and SDS–PAGE [11,25]. type (Col-0) and mutant lines (fls1-2, ldox/fls1-2) were used. Solu- ble protein preparations were prepared as described previously 2.5. Enzyme assays with recombinant proteins [22,23]. The crude protein extract were used for biochemical characterization in a standard 2-ODD assay [22,23]. FLS activity was assayed as described previously [23]. Flavo- noids were isolated from the mixture by repeated extraction with 2.3. Construction of expression vectors ethyl acetate (2 Â 100 ll), analyzed and identified by thin-layer co- chromatography with authentic standards on cellulose (Merck, The cDNAs containing coding sequences of putative FLSs and Darmstadt, Germany) in solvent system CAW (chloroform:acetic LDOX from A. thaliana were cloned into pENTRY vector acid:water; 50:45:5, by vol.) or 30% acetic acid. A. Preuß et al. / FEBS Letters 583 (2009) 1981–1986 1983

2.6. Bioconversion A FLS1 FLS2 FLS3 FLS5 LDOX

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 dihydroflavo- DHK nols (dihydrokaempferol, dihydroquercetin, dihydrotamarixitin, dihydrorobinitin and dihydroisorhamnetin) as substrates after induction of protein expression [26]. The cultures were incubated Km 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; B 30:3:10, by vol.) and by HPLC.

2.7. HPLC DHQ

Reaction products were extracted with ethyl acetate (2 Â Qu 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]. 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 2.8. SDS–PAGE and immunoassays (30–80%) and size-exclusion chromatography were incubated with (A) [14C]dihydrokaempferol, DHK (42.4 pmol; 55 mCi/mmol) or (B) [14C]dihydroqu- The protein composition of the samples was examined by SDS– ercetin, DHQ (42.4 pmol; 55 mCi/mmol). The conversion to kaempferol (Km) and PAGE [26] utilizing the SDS-7 marker for mass calibration (Sigma, quercetin (Qu), respectively, was examined by autoradiography after extraction of the incubation mixtures with ethyl acetate and ascending cellulose thin-layer Deisenhofen, Germany). Proteins in the SDS gel were stained with chromatography (TLC) separation of the extracts in the CAW solvent system. 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 and three genes involved in flavonoid compartmentation [7]. A sin- were visualized after immunodetection by staining using a mixture gle LDOX gene and a family of six FLS genes were identified in Ara- of 100 ll 5-bromo-4-chloro-3-indolylphosphate (BCIP, 50 mg/ml bidopsis, but only FLS1 has been proposed to yield a functional FLS DMF) and 300 ll nitro-blue tetrazolium (NBT, 75 mg/ml 70% [17]. In experiments employing A. thaliana wild-type and mutant DMF) in 1 M tricine buffer pH 8.8 (10 ml) containing 1 mM MgCl2. lines, the role of LDOX or FLSs for flavonol accumulation was inves- tigated. Standard enzyme assays with naringenin or dihydroka- 2.9. Homology modeling empferol as substrate clearly revealed FHT and FLS activities in wild-type extracts, converting both substrates to kaempferol, Homology modeling was performed with the translated poly- whereas extracts of the fls1-2 single mutant and the ldox/fls1-2 peptides of FLS1 and the putative isogenes FLS3 and FLS5. The mod- double mutant lacked these activities (data not shown). However, els were generated by the WEB-based SWISS-MODEL server as extracts of all three genotypes contained flavonols, albeit at greatly described previously [30]. different levels [18]. This can be explained only by residual FLS activity contributed by either one or more of the FLS2-6 polypep- 3. Results and discussion tides or by LDOX side activity. Recombinant LDOX from Arabidopsis had been shown to exert FLS activity [13,15,32,33], although the A. thalíana has been employed as a model plant for multiple bio- enzyme was unstable during isolation and in vitro assays [14]. chemical and genetic studies of secondary metabolism including Accordingly, LDOX activity has never been detected in native Ara- the flavonoid pathway [31]. Flavonols (quercetin, kaempferol, bidopsis extracts. Nevertheless, an A. thaliana ldox/fls1-2 double isorhamnetin glucosides) and the corresponding flavan-3-ols or mutant revealed a significant reduction in flavonol level (less than dihydroflavonols (Fig. 1), as well as proanthocyanidins and antho- 0.00005% of the wild-type level [18]) as compared to the wild-type cyanins were reported from wild-type plants [7]. Seventeen genes and the fls1-2 mutant (ca. 5% of wild-type level as deduced from essential for this pathway have been identified, including eight [19]) suggesting some FLS activity of LDOX under in situ conditions. structural (e.g. FLS, LDOX) and six regulatory genes (e.g. TT2, TT8), Alternatively, low FLS activity of either one of the other putative

kDa M SDS-PAGE Western blotanalysis 45

36 FLS1 FLS2 FLS5 FLS3 FLS5 FLS1 FLS2 FLS3

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]. 1984 A. Preuß et al. / FEBS Letters 583 (2009) 1981–1986

30 min, or which may be due to improper stability under experi- mental 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 un- 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 der the conditions of the assays (Fig. 3). These results suggested in FLS1) affects the assembly of 149His which participates in substrate binding. The that the FLS activity of LDOX might contribute to the formation calculation also revealed that proper binding of naringenin to FLS5 appears highly of flavonols in the Arabidopsis fls1-2 mutant, which is supported unlikely (not shown). by the additional drop of flavonol contents observed in the ldox/ fls1-2 double mutant [18]. Nevertheless, the small but significant FLS polypeptides in crude extracts might have escaped the detec- residual flavonol content observed in the ldox/fls1-2 mutant [18] tion in the in vitro assays which were commonly run for only 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 1985

Complementary to the in vitro assays, the bioconversion of line, but final proof requires the analysis of the respective triple mu- various flavanones (naringenin, eriodictyol) and dihydroflavo- tant (ldox/fls1-2/fls3). While the FLS activity of LDOX and FLS3 may be nols (dihydrokaempferol, dihydroquercetin, dihydromyricetin, relevant only in fls1 mutant lines, all three dioxygenases are capable dihydrotamarixetin, dihydrorobinitin and dihydroisorhamnetin) to a variable extent of bypassing an FHT block. These and previous by yeast transformants expressing recombinant FLSs or LDOX findings [18,19] let us presume that the Arabidopsis FLS gene family was examined, with the assumption that the stability of enzymes resulted from recent gene duplication events and that a pseudogen- was improved under these assay conditions and that the assays isation/mutation process currently eliminates ‘‘unnecessary” genes could be carried out for extended times. The cultures were rou- and their protein functions. Alternatively, FLS3 may have acquired tinely incubated for 12 h in the presence of 5 mg of either one of another function while preserving some FLS activity. the potential substrates [26]. The FLS1 transformant accepted all dihydroflavonols and flavanones to a variable degree, and formed Acknowledgments the corresponding flavonols without detectable intermediates (data not shown). At a considerably lower rate, the same applied Funding by EU Project FLAVO (FOOD CT-2004-513960) and the to the LDOX-transformant. In no case did the transformants har- Deutsche Forschungsgemeinschaft is gratefully acknowledged. bouring one of the other FLS constructs or the empty expression Authors would like to thank Dr. Peter for critical reading of the vector show a significant conversion of the flavonoid substrates. manuscript. This provides strong evidence of FLS activity for the LDOX transformant, although further corroboration by kinetic data was not References possible since steady state conditions in the biotransformation system are unlikely. [1] Harborne, J.B. and Williams, C.A. (2000) Advances in flavonoid research since Analogous to FLS4 and FLS6 which represent truncated pseudo- 1992. Phytochemistry 55, 481–504. genes [18,19], and based on the truncated C-terminus and lack of [2] Pelletier, M.K., Murrell, J.R. and Shirley, B.W. (1997) Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis. appropriate iron and 2-oxoglutarate binding sites, FLS2 likely en- Plant Physiol. 113, 1437–1445. coded a non-functional polypeptide. This assumption was con- [3] Pelletier, M.K., Burbulis, I.E. and Winkel-Shirley, B. (1999) Disruption of firmed by expression of FLS2. Likewise, recombinant FLS5 also specific flavonoid genes enhance the accumulation of flavonoid enzymes and lacked enzyme activity. It was shown previously that marginal end-products in Arabidopsis seedlings. Plant Mol. Biol. 40, 45–54. [4] Veit, M. and Pauli, G.F. (1999) Major flavonoids from Arabidopsis thaliana changes in the binding sites of substrate, ferrous iron and oxoglu- leaves. J. Nat. Prod. 62, 1301–1303. tarate cosubstrate by 2-oxoglutarate-dependent dioxygenases (2- [5] Peer, W.A., Brown, D.E., Tague, B.W., Muday, G.K., Taiz, L. and Murphy, A.S. ODDs) may strongly affect the substrate and/or product specificity (2001) Flavonoid accumulation patterns of transparent testa mutants of 219 Arabidopsis. Plant Physiol. 126, 536–548. [32]. Sequence alignments with FLS1 revealed that Arg/Lys and [6] Broun, P. 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(FNSI) from parsley [30]. Models of the LDOX-naringenin (2brt) [9] Holton, T.A., Brugliera, F. and Tanaka, Y. (1993) Cloning and expression of [33] and LDOX-dihydroquercetin complexes [32] were employed flavonol synthase from Petunia hybrida. Plant J. 4, 1003–1010. [10] Wellmann, F., Lukacin, R., Moriguchi, T., Britsch, L., Schiltz, E. and Matern, U. as templates for homology modeling of FLS1 and comparison with (2002) Functional expression and mutational analysis of flavonol synthase FLS3 and FLS5, because FLS oxidizes the same substrates [12,22] from Citrus unshiu. Eur. J. Biochem. 269, 4134–4142. and the conserved binding sites for 2-oxoglutarate (298Arg in [11] Lukacˇin, R., Wellmann, F., Britsch, L., Martens, S. and Matern, U. (2003) LDOX) and iron (232His, 288His and 234Asp in LDOX) are distributed Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase. Phytochemistry 62, 287–292. 319 251 with an analogous spacing in the FLS polypeptides ( Arg, His, [12] Prescott, A.G., Stamford, N.P.J., Wheeler, G. and Firmin, J.L. (2002) In vitro 309His and 253Asp in FLS 1 and FLS3). The model endorses the role properties of recombinant flavonol synthase from Arabidopsis thaliana. of 327Glu in substrate binding and the lack of activity of FLS5 (not Phytochemistry 60, 589–593. [13] Turnbull, J.J., Nakajima, J-I., Welford, R.W.D., Yamazaki, M., Saito, K. and shown). LDOX was proposed to bind naringenin through p-stack- Schofield, C.J. (2004) Mechanistic studies on three 2-oxoglutarate-dependent ing of the substrate A-ring with 304Phe [32] which corresponds to oxygenases of flavonoid biosynthesis. J. Biol. Chem. 279, 1206–1216. 325Phe in FLS1 and FLS3 (Fig. 4). FLS3 differs from FLS1, however, [14] Saito, K., Kobayashi, M., Gong, Z., Tanaka, Y. and Yamazaki, M. 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(2007) Differential regulation of closley related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the tended bioconversion studies (48 h in the presence of 20 mg sub- Arabidopsis thaliana seedlings. Plant J. 50, 660–677. strate/50 ml) using the respective yeast transformant. Under the [17] Wisman, E., Hartmann, U., Sagasser, M., Baumann, E., Palme, K., Hahlbrock, K., assay conditions, the conversion of both dihydrokaempferol and Saedler, H. and Weisshaar, B. (1998) Knock-out mutants from an En-1 dihydroquercetin to kaempferol and quercetin, respectively, was mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes. Proc. Natl. Acad. Sci. USA 95, 12432–12437. demonstrated by TLC and HPLC co-chromatographies (Fig. 5). Non- [18] Stracke, R., De Vos, R.C., Bartelniewoehner, L., Ishihara, H., Sagasser, M., induced yeast transformants or cells harbouring an empty vector Martens, S. and Weisshaar, B. 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