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Plant Science 168 (2005) 13091318 www.elsevier.

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Temporal expression of avonoid biosynthesis-related genes regulates ower pigmentation in gentian plants$
Takashi Nakatsuka, Masahiro Nishihara *, Keiichiro Mishiba, Saburo Yamamura
Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan Received 26 November 2004; received in revised form 14 January 2005; accepted 14 January 2005 Available online 29 January 2005

Abstract The cDNA clones encoding homologues of avanone 3-hydroxylase ( F3H), anthocyanidin synthase (ANS), avonoid 30 -hydroxylase ( F30 H) and avone synthase II ( FSII) genes were isolated from petals of Gentiana triora. Deduced amino acid sequences exhibited 6080% identities with the corresponding sequences from other dicotyledonous species. Southern blot analysis showed that they were present as multiple copies in the gentian genome, and Northern blot analysis showed that the avonoid biosynthesis-related genes could be classied into three groups by their temporal expression patterns during gentian ower development. The rst included chalcone synthase (CHS) and chalcone isomerase (CHI) expressing during all ower development stages; the second included F30 H and FSII expressing at ower early developmental stages; and the third included F3H, avonoid 30 ,50 -hydroxylase (F30 ,50 H), dihydroavonol 4-reductase (DFR), ANS, UDPglucose:avonoid 3-O-glucosyltransferase (3GT) and anthocyanin 5-aromatic acyltransferase (5AT) expressing at ower late developmental stages. In general, low or undetectable levels of expression were observed in both the leaves and stems. High performance liquid chromatography (HPLC) analysis revealed that avone accumulates from the early ower bud stage, but anthocyanin accumulation peaked at the later ower anthesis stage. A signicant correlation between gene expression and pigment accumulation has been found, indicating that avonoid biosynthesis during gentian ower development is regulated by temporal expression of these genes. # 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Anthocyanin; Flavone; Flavonoid biosynthesis; Gene expression; Gentian (Gentiana triora)

1. Introduction The genus Gentiana comprises more than 400 species, including Gentiana triora and G. scabra, which are popular ornamental plants in Japan and used as cut owers and potted plants [1]. Because Japanese gentian cultivars derived from these species are composed mainly of blue owers, the
Abbreviations: ANS, anthocyanidin synthase; 5AT, anthocyanin 5-aromatic acyltransferase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroavonol 4-reductase; F3H, avanone 3-hydroxylase; F30 H, avonoid 30 -hydroxylase; F30 ,50 H, avonoid 30 ,50 -hydroxylase; FSII, avone synthase II; 3GT, UDP-glucose avonoid 3-glucosyltransferase; HPLC, high performance liquid chromatography $ The nucleotide sequences reported in this paper have been submitted to DDBJ, EMBL and GenBank under accession numbers AB193310 (GtANS), AB193311 (GtF3H-1), AB193312 (GtF3H-2), AB193313 (GtF30 H) and AB193314 (GtFSII). * Corresponding author. Tel.: +81 197 68 2911; fax: +81 197 68 3881. E-mail address: mnishiha@ibrc.or.jp (M. Nishihara).

diversication of ower color is one of the breeding objectives. Thus, we developed an in vitro culture and genetic transformation system for the purpose of producing ower color-modied cultivars of gentian [24]. Flower pigmentation is due to accumulation of plant secondary metabolites such as anthocyanin, carotenoid and betalain within the epidermal cells [5]. Anthocyanin is one of the main plant pigments in owers, and Japanese gentian cultivars also contain anthocyanin derivatives in their ower petals. Anthocyanin of gentian owers has been extensively studied due to the vivid blue color of many cultivars, and anthocyanin modied with two aromatic acyl groups, that is, gentiodelphin (delphinidin 3-O-b-D-glucoside-5-O-(6-Ocaffeoyl- b-D-vglucoside)-30 -(6-O-caffeoyl-b-D-glucoside)), was identied [6]. Furthermore, Hosokawa et al. [7,8] also isolated albireodelphinidin and gentiocyanin from owers of different gentian cultivars. The 30 -O-caffeoyl-grucosyl moiety of gentiodelphin, but not the 5-O-caffeoyl moiety,

0168-9452/$ see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2005.01.009

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is thought to contribute to both blue color development and stabilization by intra-molecular sandwich-type stacking of the caffeoyl moiety with the B-ring [9]. The avonoid biosynthesis pathway is a side branch of the phenylpropanoid biosynthesis pathway, and further branches off to avone, avonol and anthocyanin biosynthesis pathways. Flavonoid biosynthesis has been well characterized in the owers of petunia and snapdragon, and in the kernels of maize, and is therefore one of the most well studied pathways among plant secondary metabolisms [5,10,11]. The putative avonoid biosynthesis pathway in gentians is illustrated in Fig. 1. The rst key step in the biosynthesis of all avonoids is condensation of 4-coumaroyl-CoA with three molecules of malonyl-CoA catalyzed by chalcone synthase (CHS); the chalcone is stereospecically cyclized by the action of chalcone isomerase (CHI). Hydroxylation of avanone at the 3-position by avanone 3-hydroxylase (F3H) leads to the formation of dihydroavonol, which is catalyzed by dihydroavonol 4-reductase (DFR) to leucoanthocyanidin. Anthocyanidin synthase (ANS) catalyzes

the reaction from the colorless leucoanthocyanidin to the colored anthocyanidin, and hydroxylation of the 30 - and 50 -positions of the B-ring is catalyzed by avonoid 30 hydroxlase (F30 H) and avonoid 30 ,50 -hydroxlase (F30 ,50 H), respectively. Hydroxyl groups in the B-ring are known to affect the absorption spectrum of anthocyanin, and especially, F30 ,50 H is important for formation of blue colored delphinidin. Almost all anthocyanidin undergoes several modications, such as glycosylation and acylation, by UDPglucoside:avonoid 3-O-glucosyltransferase (3GT), UDPglucoside:avonoid 30 -O-glucosyltransferase (30 GT) and anthocyanin 5-aromatic acyltransferase (5AT). On the other side of the reaction, avone synthase II (FSII) is a key enzyme for avone biosynthesis; it catalyzes introduction of the double bond between C-2 and C-3 of avanone to produce avone [12]. Some structural genes involved in avonoid biosynthesis have already been isolated from gentians and characterized; namely, CHS [13], CHI (accession number D38168), F30 ,50 H, DFR and 3GT [14], 5AT [15] and 30 GT [16]. However, F3H, ANS, F30 H and FSII remain to be isolated from gentian plants, and no integrative study on the expression of these genes has been performed yet. In this study, we isolated cDNA clones encoding F3H, ANS, F30 H and FSII from the gentian petal cDNA library and revealed the expression patterns of these genes together with the avonoid biosynthesis-related genes previously reported in gentians during ower development. In addition, accumulation levels of avonoid in gentian owers were also determined by high performance liquid chromatography analysis. These data confer a useful piece of information on biosynthesis of ower pigmentation in gentian plants and facilitate our understanding of modication of ower color by genetic engineering. This is the rst systematic research that aims to determine the expression of avonoid biosynthesis-related genes in gentian owers.

2. Materials and methods 2.1. Plant materials The ornamental gentian Gentiana triora cv. Maciry (blue ower cultivar) from which petal, leaf and stem samples were collected was grown in a eld of Iwate Agriculture Research Center, Japan. Petal samples were collected at four different ower developmental stages dened as follows: stage 1, <30 mm long with unpigmented buds; stage 2, 3040 mm long with slightly pigmented buds; stage 3, 4050 mm long with budsjustbeforeanthesis;stage4,fullyopened owers. All samples were stored at 80 8C until use.
Fig. 1. Schematic representation of the avonoid biosynthesis pathway in gentians. The genes enclosed with an open box were isolated from the gentian petal cDNA library in this study. The genes in parenthesis were not analyzed in this study.

2.2. Cloning of gentian F3H, F30 H, ANS and FSII cDNA Total RNA was isolated from 1 g fresh weight of gentian petals, leaves and stems with Fast RNA Kit-GREEN

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(BIO101, CA) then puried by lithium chloride precipitation methods. First strand cDNA was synthesized using total RNA of gentian petals at various ower developmental stages using an RNA PCR Kit (AMV) version 2.1 (TaKaRa, Tokyo) according to the manufacturers protocol. Degenerated primers for the amplication of partial F3H, ANS, F30 H and FSII were designed from consensus amino acid sequences of these genes conserved among other dicotyledonous species. The degenerated primers used in this study were as follows: for F3H, 50 -GCYTGRTGRTCIGCRTTYTTRAA-30 and 50 -TAYCCIAARTGYCCICARCCIGA-30 ; for ANS, 50 -GARTGGGARGAYTAYTTYTTYCA-30 and 50 GGRCAYTTIGGRTARTARTTDAT-30 ; for F30 H, 50 -THAAAGCHTTRCTHTTGAAC-30 and 50 -CTCADHCCIGCACADATYCT-30 ; for FSII, 50 -CCITAYGGIMCITAYTGGAA-30 and 50 -ARCATIGGDATIGGIGGRTG-30 . The reaction mixture in a total volume of 25 ml contained 0.5 ml rst strand cDNA, 400 mM dNTPs, 5 mM of each primer, 1.25 units of Ex Taq polymerase (TaKaRa, Tokyo) and 1 Ex Taq PCR buffer. The reaction conditions were pre-heating at 94 8C for 1.5 min, 35 cycles at 95 8C for 20 s, 55 8C for 40 s and 72 8C for 1 min then extension at 72 8C for 10 min. The amplied fragments for each gene were subcloned into the pCR2.1 TA cloning vector (Invitrogen, CA) and sequenced. Sequence analyses were carried out using a Big-Dye Terminator cycle sequencing Kit version 1.1 and the DNA sequencer model ABI PRISM 377 (Applied Biosystems Japan, Tokyo). Nucleotide sequences were translated to amino acid sequences using GENETYX-MAC version 12.0 (GENETYX, Tokyo) and compared with those in the BLAST network service from the National Center for Biotechnology Information (NCBI) [17]. Phylogenic tree were produced using CLUSTALW [18] and TREEVIEW [19] programs. cDNA library screening of the F3H, F30 H, ANS and FSII genes was carried out with the ECL-Direct system (Amersham Pharmacia, Sweden). Construction of the cDNA library from petals of G. triora cv. Maciry was previously described by Kobayashi et al. [13]. The subcloned fragments of each gene were labeled by the ECL-Direct labeling system (Amersham Pharmacia, Sweden), and used as probes. Approximately 4.0 107 plaques from the cDNA library transferred on Hybond-N+ (Amersham Pharmacia, Sweden) were screened for each probe using an ECL-Direct detection Kit (Amersham Pharmacia, Sweden). pBluescript plasmids (Stratagene, CA) containing cDNA inserts were obtained by in vivo excision according to the manufacturers instructions; they were then sequenced as described above. 50 rapid amplication of cDNA ends (50 RACE) was performed on clones that did not contain fulllength cDNA using 50 -RACE system version 2.0 (Invitrogen, CA) according to the manufacturers instructions. The 50 end fragments of F30 H and FSII were amplied using 50 GGCCTTAGACGAGAAAAGATGG-30 and 50 -AATATTGGGCCGTAGCGTTTGTAG-30 , respectively. These amplied fragments were subcloned and sequenced as described above. The nucleotide sequences reported in this paper have

been submitted to DDBJ, EMBL and GenBank under accession numbers AB193310 (GtANS), AB193311 (GtF3H-1), AB193312 (GtF3H-2), AB193313 (GtF30 H) and AB193314 (GtFSII). 2.3. Southern blot analysis of avonoid biosynthesisrelated genes Total genomic DNA was isolated from 10 g of leaf sample using the CTAB method described by Murray and Thompson [20] with some modications. Ten micrograms of genomic DNA digested by EcoRI and HindIII (TaKaRa, Tokyo) were separated on 0.6% agarose gel then transferred to Nytran N membranes (Schleicher & Schuell, Germany). Probes for avonoid biosynthesis-related genes were prepared with a DIG-High Prime DNA Labeling Kit (Roche Diagnostics, Germany) using the open reading frame (ORF) of each homolog then hybridized in high-SDS hybridization buffer at 42 8C overnight. After hybridization, the membranes were washed in 2 SSC and 0.5% SDS for 5 min at room temperature, and twice in 2 SSC and 0.1% SDS for 15 min at 65 8C, then twice in 0.1 SSC and 0.5% SDS for 30 min at 68 8C. Detection was performed using a DIG Nucleic Acid Detection Kit (Roche Diagnostics, Germany). 2.4. Expression analysis of avonoid biosynthesis-related genes Northern blot analysis of avonoid biosynthesis-related genes in G. triora was accomplished using total RNA isolated from petals at the four different ower developmental stages dened above, and from mature leaves and stems. Five micrograms of total RNA were separated on 1.25% MOPS-agarose gel then transferred to Hybond-N+ membranes (Amersham Pharmacia, Sweden). Probes for the avonoid biosynthesis genes were prepared with a PCR-DIG Probe Synthesis Kit (Roche Diagnostics, Germany) then hybridized in high-SDS hybridization buffer at 50 8C overnight. After hybridization, the membranes were washed twice in 2 SSC and 0.1% SDS for 15 min at 68 8C, and twice in 0.1 SSC and 0.1% SDS for 15 min at 65 8C. Detection was performed as described in the Southern blot analysis section. 2.5. Flavonoid extraction and HPLC analysis For avone analysis, frozen petal, leaf and stem samples (250 mg) were ground in liquid nitrogen then extracted twice with 10 ml methanol. After extraction, the solution was evaporated and the dried samples were re-dissolved in 500 ml methanol. For hydrolysis of avone glucosides, 4.5 ml 10% HCl was added before incubation at 100 8C for 90 min. The hydrolysis solution was extracted twice with ethyl acetate then the ethyl acetate fraction was evaporated and re-dissolved in 500 ml methanol. This solution was ltrated through a 0.22-mm lter (Millipore) and analyzed

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by high performance liquid chromatography (HPLC; D7000 HPLC system manager, HITACHI). HPLC was carried out with a reverse phase column J sphere ODS M80 (4.6 mm 150 mm, YMC) using 50% methanol and 3% acetic acid as the solvent for 20 min at 40 8C at a ow rate of 1.0 ml/min. Flavone was quantied by monitoring the peak area of absorbance at 330 nm, with apigenin and luteolin as the standards. For anthocyanidin analysis, frozen petal, leaf and stem samples (250 mg) were ground in liquid nitrogen then extracted using 1 ml EAA solution (ethanol:acetic acid:water = 10:9:1) at 4 8C overnight. For hydrolysis of anthocyanin, a half volume of 3N HCl was added to the supernatant before incubation at 100 8C for 90 min. The hydrolysis solution was ltrated through a 0.22-mm lter (Millipore) then analyzed by HPLC. HPLC was carried out with the same column used for avone analysis under the conditions described by Bloor [21]. Anthocyanidin was quantied by monitoring the peak area of absorbance at

500 nm, with pelargonidin, cyanindin and delphinidin as the standards.

3. Results 3.1. Cloning of F3H, ANS, F30 H and FSII homologues from gentian plants Homologues of four avonoid biosynthesis-related genes encoding F3H, ANS, F30 H and FSII were successfully isolated from the G. triora cv. Maciry petal cDNA library after screening using probes of each DNA fragment amplied by degenerated primers. The comparison of identities among the homologues isolated in this study and those reported from other species is shown in Table 1. Two F3H homologues designated GtF3H-1 and GtF3H2 were obtained from the cDNA library. GtF3H-1 cDNA (1454 bp) was longer than GtF3H-2 cDNA (1382 bp) due to

Table 1 Comparison of the deduced amino acid sequences of newly isolated structural genes involved in avonoid biosynthesis from gentian owers with the known related protein sequences Species Deduced amino acid sequences Length F3H (avanone 3-hydroxylase) Gentian 1 Gentian 2 Grape Egg plant Citrus Petunia Arabidopsis Common stock Apple Carnation Perilla ANS (anthocyanidin synthase) Gentian Apple Petunia Grape Arabidopsis Common stock Perilla Torenia F30 H (avonoid 30 -hydroxylase) Gentian Petunia Perilla Torenia Common stock Arabidopsis FSII (avone synthase II) Gentian Snapdragon Perilla Torenia Gerbera 365 365 364 358 362 369 358 357 365 365 372 366 357 430 355 356 356 362 375 524 512 523 512 513 513 530 506 506 512 511 Identity (%) 100.0 99.7 81.5 81.4 81.2 81.0 80.0 79.2 78.0 77.8 75.1 100.0 77.8 77.0 77.0 74.5 73.9 73.5 70.8 100.0 69.9 64.5 64.0 63.2 63.2 100.0 60.9 58.8 57.9 57.3 AB193311 AB193312 P41090 AAM48289 BAA36553 Q07353 AAM65101 Q05965 BAB92997 Q05964 BAA19657 AB193310 P51091 P51092 BAC07545 CAD91994 T07972 BAA20143 BAB21477 AB193313 AAD56282 BAB59005 BAB87838 AAG49301 NP_196416 AB193314 BAA84071 BAB59004 BAA84072 AAD39549 This study This study Sparvoli et al. [22] Unpublished Moriguchi et al. [23] Britsch et al. [24] Unpublished Britsch et al. [25] Honda et al. [26] Britsch et al. [25] Gong et al. [27] This study Davies [28] Weiss et al. [29] Kobayashi et al. [30] Abrahams et al. [31] Unpublished Saito et al. [32] Nakajima et al. [33] This study Brugliera et al. [34] Kitada et al. [35] Ueyama et al. [36] Unpublished Unpublished This study Akashi et al. [37] Kitada et al. [35] Akashi et al. [37] Martens and Forkmann [12] Accession numbers References

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different positions of the poly A tail, but the putative open reading frame (ORF) of both cDNAs encoded 365 amino acid residues. These two gentian F3H homologues had 11 different nucleotide sequences, but ten were synonymous substitutions that did not change the amino acid sequence of the protein. Only one nonsynonymous substitution was found, by which a single amino acid residue changed from tyrosine to lysine at the 32nd position. The GtANS cDNA was 1307 bp in length and contained a putative ORF encoding 366 amino acid residues. The phylogenic tree based on the deduced amino acid sequence of plant 2oxoglutarate dependent dioxygenases is shown in Fig. 2. This phylogenic tree demonstrates that GtF3H and GtANS

belong to the corresponding subfamilies as expected, suggesting that our cDNA clones indeed code for the corresponding enzymes. Both GtF3H and GtANS have also conserved the iron binding site at the catalytic center of the iron-containing soluble oxygenases and 2-oxoglutarate binding site. Because no complete cDNA for gentian homologues of F30 H and FSII was isolated by cDNA library screening, we isolated full-length cDNA for both genes by the 50 -RACE method. The full-length cDNA for the gentian F30 H homologue was 1787 bp long and contained a putative

Fig. 2. Phylogenic analysis of 2-oxoglutarate-dependent oxygenases in various plant species calculated on the basis of amino acid sequences. The phylogenic tree was generated using CLUSTALW [18] and TREEVIEW [19] programs. Numerals next to the branches indicate bootstrap values from 1000 replicates. The bar indicates an evolutionary distance of 0.1%. Accession numbers in the GenBank/DDBJ databases are as follows: avanone 3-hydroxylase (F3H): gentian (the present study), Perilla frutescens (BAA19657), petunia (AAC49929), apple (BAB92997), maize (T03385), carnation (Q05964); anthocyanidin synthase (ANS): gentian (the present study), maize A2 (CAA39022), torenia (BAB21477), Perilla frutescens (BAA20143), apple (CAA50498), common stock (AAB82287), grape (BAC07545), petunia (S36233); gibberellin 3 beta-hydroxylase: Arabidopsis thaliana (AAC83647), rice (BAB62154); gibberellin 20-oxidase: Arabidopsis thaliana (AAC39313), rice (AAL87949); hyoscyamine 6 beta-hydroxylase (H6H): belladonna (BAA78340), Hyoscyamus niger (BAA05630).

Fig. 3. Phylogenic analysis of the cytochrome P450 superfamily in various plant species calculated on the basis of amino acid sequences. The phylogenic tree was generated as in Fig. 2. Accession numbers in the GenBank/ DDBJ databases are as follows: avone synthase II (FSII): gentian (the present study), torenia (AB028152), snapdragon (AB028151), Perilla frutescens (AB045592), gerbera (AF156976), china aster (AF188612); avonoid 30 ,50 -hydroxylase (F30 ,50 H): gentian (D85184), petunia (Z22545), eggplant (X70824), lisianthus (U72654), Catharanthus roseus (AJ011862), campanula (D14590); avonoid 30 -hydroxylase (F30 H): gentian (the present study), Perilla frutescens (AB045593), Arabidopsis (AF271651); isoavone synthase (IFS): licorice (AB023636), Cicer arietinum (AJ243804), soybean (AF022462); avonoid 2-hydroxylase (F2H): licorice (AB001380); ferulate-5-hydroxylase (F5H): tomato (AF150881), Arabidopsis thaliana (U38416); cinnamate 4-hydroxylase (C4H): licorice (U87520), parsley (L38898), Arabidopsis thaliana (U71080).

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ORF encoding 524 amino acid residues. The full-length cDNA for gentian FSII homologue was 1836 bp, and contained a putative ORF encoding 530 amino acid residues. The phylogenic tree based on the deduced amino acid sequence of plant cytochrome P450 is shown in Fig. 3. This phylogenic tree demonstrates that GtF30 H and GtFSII belong to the corresponding subfamilies as expected, suggesting that our cDNA clones indeed code for the corresponding enzymes. Both GtF30 H and GtFSII also have typical features, such as heme-binding region and motifs, which are thought to be essential for oxygen binding and activation. 3.2. Southern blot analysis of F3H, ANS, F30 H and FSII genes The genomic information of the avonoid biosynthesisrelated genes cloned in this study were obtained by Southern blot analysis using total genomic DNA of gentians as indicated in Fig. 4. Because the ORF sequences of GtANS and GtFSII contained a single HindIII site and that of GtFSII contained two EcoRI sites, it was suggested that the GtF3H, GtANS, F30 H and FSII homologues were present as multiple copies in the gentian genome. 3.3. Expression analysis of avonoid biosynthesis-related genes The expression levels of 10 avonoid biosynthesisrelated genes (CHS, CHI, F3H, F30 H, F30 ,50 H, DFR, ANS,

Fig. 5. Expression analysis of avonoid biosynthesis-related genes during ower development. (A) Gentian ower developmental stages referred to in Section 2 (bar = 1 cm). (B) Five micrograms of total RNA from petals at stage (S) 14, leaves (Le) and stems (St) were loaded on each lane. Used probes are shown on the left of the panels. Ethidium bromide stained ribosomal RNA bands are shown as a control.

Fig. 4. Southern blot analysis of the avonoid biosynthesis-related genes in gentians. Ten micrograms of genomic DNA were digested with HindIII (H) and EcoRI (E). The blots were then hybridized by each probe with an open reading frame for F3H, ANS, F30 H and FSII as described in Section 2. The DNA size marker is indicated on the left of the panel.

3GT, 5AT and FSII) were investigated in gentian petals at four different developmental stages, and in leaves and stems (Fig. 5). Northern blot analysis indicated that these genes were preferentially expressed in petals compared with leaves and stems, and they were consequently classied into three groups based on their expression patterns. The rst group (group I) includes enzymes that catalyze the early steps of avonoid biosynthesis, CHS and CHI. The expression of group I stayed at a high level from stages 1 to 4 during ower development. The second group (group II) included the enzymes that catalyze the middle steps of avonoid biosynthesis, F30 H and FSII. The expression level of group II was prominent at ower developmental stages 1 and 2 prior to ower bud pigmentation, and declined at

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later stages. The third group (group III) included the enzymes that catalyze the late steps of avonoid biosynthesis, F3H, F30 ,50 H, DFR, ANS, 3GT and 5AT. The expression level of group III was not detected or was detected in small amounts at ower developmental stage 1, but increased with ower development, peaking at stage 3 just before opening of the ower bud. In general, low or undetectable levels of expression were observed in both the leaves and stems.

avonol was detected in gentian owers, leaves or stems (data not shown). Most of the anthocyanidin that accumulated in the gentian petal tissues was in the delphinidin; pelargonidin and cyanidin were barely detected (Fig. 6B). The accumulation of delphinidin drastically increased with ower development, reaching a maximum at stage 3. Leaf and stem tissues contained little or no anthocyanin. The accumulation of anthocyanin coincided well with the visible onset of blue pigmentation in ower petals in stage 2 (see Figs. 5 and 6).

3.4. Analysis of avone and anthocyanin accumulation in gentians 4. Discussion Flavone and anthocyanin contents were determined in petals at four developmental stages, and in leaves and stems by HPLC (Fig. 6). Apigenin and luteolin were detected as the main avone compounds in gentians. They started to accumulate at the early ower developmental stage, and peaked at stage 2 (Fig. 6A). In owers, apigenin accumulations were about three times higher than those of luteolin, whereas an almost equal amount was detected in the leaves. Leaves contained less avone than owers, and stems did not have any detectable levels of avone. No In the present study, we isolated cDNA clones encoding F3H, ANS, F30 H and FSII homologues from the cDNA library of ornamental gentian (G. triora) petals. Sequence comparison showed that they share 6080% identities with each corresponding gene reported previously (Table 1). From the sequence analysis, GtF3H and GtANS were shown to have characteristic motifs conserved in the 2-oxoglutarate-dependent dioxygenase family, including two histidines and one aspartic acid as part of the putative iron-binding site and an arginine residue as a part of the 2-oxoglutarate binding site [38]. The deduced amino acid sequences of GtF30 H and GtFSII gene have several conserved regions of the cytochrome P450 family [39]. They contain the heme-binding domain (FxxGxRxCxG), including a cysteine residue that heme binds to in the active site. They also contain a proline rich region (PPxP), which acts as a hinge required for optimal orientation of the enzyme with regards to the membrane. Another consensus sequence (A/G)Gx(D/E)T(T/S), which forms a threonine-containing binding pocket for oxygen molecules required in catalysis, is also conserved. Furthermore, the Nterminal of GtF30 H is expected to contain a membraneanchored hydrophobic region according to the PSORT program [40] for the prediction of protein localization sites in cells. It has also been hypothesized that membrane-bound P450 monooxygenases, such as F30 H and cinnamate 4hydroxylase, recruit other enzymes of general phenylpropanoid and avonoid metabolism into membrane-associated complexes [41] These ndings strongly suggest that our isolated genes are homologues of the four corresponding genes. Moreover, our preliminary results showed that GtF30 H and GtFSII have enzymatic activity in transgenic tobacco plants (data not shown). Experimental analyses on enzymatic activities in vitro also would provide further characterization of these genes. Southern blot analysis suggested that multiple copies of the F3H, ANS, F30 H and FSII homologues are present in the gentian genome (Fig. 4). It was also conrmed that the other six genes studied here consist of at least two copies (data not shown). These results might be due to the fact that cultivated gentian plants are heterozygous, and have been bred by cross-pollination. However, we could only isolate a

Fig. 6. Flavone and anthocyanidin accumulation levels during ower development. The amount of avone (A) and anthocyanidin (B) in petals at the four developmental stages (S1, S2, S3 and S4), and in leaves (leaf) and stems (stem) were determined by HPLC analysis. Values indicate the average of three replicates for each sample.

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single isoform of cDNA for each gene, except for the F3H gene, by screening the gentian ower cDNA library. This might reect the fact that some homologues of these genes are not or only weakly expressed in owers. It has also been reported that avonoid biosynthesis-related genes comprise a multi gene family in other plant species [42]. Since most structural genes involved in avonoid biosynthesis are now available as a result of previous work as well as the results of this study, we attempted to determine their expression proles to elucidate how they are expressed during and how they regulate avonoid biosynthesis in gentian plants. Although carotenoid biosynthesis-related genes have been characterized in another gentian species (Gentiana lutea), and been shown to result in accumulation of yellow carotenoid pigments in the owers [43,44], the overall expression pattern of avonoid biosynthesis-related genes in gentians remains unknown. More recently, Noda et al. [45] reported that avonoid biosynthesis-related genes coordinately regulate avonol and anthocyanin biosynthesis in lisianthus plants that belong to the family Gentianaceae. Such analysis is important in understanding the molecular mechanism of pigment formation in addition to structural analysis of natural pigment compounds in owers. Based on gene expression analysis, the 10 avonoid biosynthesis-related genes studied here were classied into three groups, which could be roughly dened as early, middle or late stage enzymes involved in avonoid biosynthesis. Group I included CHS and CHI, common enzymes necessary for biosynthesis of both avone and anthocyanin. The enzymes of group II included F30 H, which catalyzes both avone and anthocyanin biosynthesis, and FSII, which catalyzes avone biosynthesis, while on the contrary, group III included F3H, F30 ,50 H, DFR, ANS, 3GT and 5AT, which are concerned with anthocyanin biosynthesis and modication. Fujiwara et al. [15] also suggested that F30 ,50 H, DFR, 3GT and 5AT genes from gentian owers, classied as group III in this study, increased during ower development and decreased with maturation of the ower. In the case of petunias, avonoid biosynthesisrelated genes were classied into two groups, those that catalyze avonol and anthocyanin biosynthesis, respectively [46]. The expression of CHS and DFR were increased during ower development corresponding to anthocyanin pigmentation, whereas that of F30 H and FLS were maximal at early stage of ower development [34]. In the ower tube of snapdragons, these genes were also divided into two groups, one involving both CHS and CHI and another involving F3H, DFR, ANS and UFGT. This might reect the fact that the rst two enzymes (CHS and CHI) of the pathway are precursors for avone as well as anthocyanin biosynthesis [47]. From this point of view, our classication by gene expression pattern is somehow similar to that in snapdragons, though expression of CHS and CHI continued with ower development in gentian owers. This classication might explain why these gene expression proles are correlated with the accumulation of avone and anthocyanin

during ower development (Figs. 5 and 6). Gene expression analysis revealed that F3H gene expression could not be detected at ower developmental stage 1, but then drastically increased with development and pigmentation of the owers. On the other hand, high expression levels of FSII were seen until ower developmental stage 2, but mRNA levels decreased with petal pigmentation. It has also been reported that the expression of FSII was high at the early ower developmental stages in gerbera [12] and torenia plants [36]. The enzymes of F3H and FSII are situated at the branching point for avone and avonoid biosynthesis (Fig. 1). Therefore, it is suggested that temporal regulation of group II and III gene expression controls the biosynthesis of avone and anthocyanin in gentians. Namely, the switching of gene expression from FSII to F3H is considered critical for petal pigmentation by changing the biosynthesis pathway from avone to anthocyanin. This might be to avoid antagonism of avone and anthocyanin biosynthesis. Consequently it will enable enough accumulation of both pigments in gentian ower petals at the anthesis stage. Most owering plants mainly accumulate either avone or avonol pigments in their petals. For example, gentian, snapdragon and torenia plants accumulate avone rather than avonol, while petunia and tobacco plants accumulate avonol. In snapdragons, avone and anthocyanin biosynthesis are regulated by gene expression levels controlled by Delila, which encodes basic helixloophelix transcriptional factor [47]. Previous observations suggest that avone might act as a yellow ower pigment by copigmentation, and also as an inducer of nodulation and a signal compound in different insectplant interactions [12]. In transgenic torenia plants with suppressed FSII expression, accumulation of both avone and anthocyanin decreased in the petals, and the ower color changed from blue to pale blue [36]. Thus, it is likely that avone contributes to the stability of anthocyanin by copigmentation of avone and anthocyanin in gentian plants. In conclusion, the temporal expression of avonoid biosynthesis-related genes during ower development was conrmed in this study. Especially, switching of FSII and F3H seems to be important for the coexistence of avone and anthocyanin in the production of blue ower pigmentation in gentian plants. We are currently conducting transformation studies that up- or down-regulate FSII and F3H in gentians to provide more insight into this phenomenon. Although the regulation mechanisms of expression of these genes are still unknown, sequence and expression data will provide a substantial basis for further elucidation of ower pigmentation in gentians and the production of genetically engineered ower color-modied gentians.

Acknowledgments We thank Katsuo Kodama, Yoshimi Naito, Hiromi Kawamura, Masashi Odajima and Michiko Kuzumaki in Iwate Agriculture Research Center for providing gentian

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cultivars. We also thank Dr. Toshio Aoki (Nihon University, Japan) for technical advice on avonoid analysis by HPLC.

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