JXB Advance Access originally published online on March 28, 2008
Journal of Experimental Botany 2008 59(6):1241-1252; doi:10.1093/jxb/ern031
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RESEARCH PAPER |
Cloning and characterization of the UDP-glucose:anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian
1Iwate Biotechnology Research Center, 22-174-4, Narita, Kitakami, Iwate 024-0003, Japan
2Graduate School of Agriculture, Iwate University, 3-18-8, Ueda, Morioka, Iwate 020-8550, Japan
To whom correspondence should be addressed. E-mail address: mnishiha{at}ibrc.or.jp
Received 29 November 2007; Revised 11 January 2008 Accepted 21 January 2008
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Abstract |
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Blue-flowered gentian (Gentiana triflora) is known to accumulate gentiodelphin, a unique polyacylated delphinidin-type anthocyanin, in the petals. Almost all of the structural genes involved in gentiodelphin biosynthesis have been isolated, but an important gene encoding UDP-glucose:anthocyanin 5-O-glucosyltransferase (5GT) remained to be identified. In this study, an attempt was made to isolate and characterize gentian 5GT, which is responsible for glucosylation of anthocyanidin 3-glucoside. A PCR-based cloning strategy identified seven 5GT candidates from gentian flowers. Among them, the deduced amino acid sequence of the 5GT gene from gentian petal cDNA, designated Gt5GT7, exhibited 36.0–41.7% identities with those of 5GTs from other plant species, and phylogenic analysis also suggested that Gt5GT7 belongs to the 5GT subfamily. The expression analysis showed that Gt5GT7 transcripts were detected predominantly in petals and weakly in filaments but not in leaves, stems, and other floral organs. In addition, increased levels of Gt5GT7 transcripts in petals coincided with flower development, a pattern identical to that of 5GT enzymatic activity as determined by in vitro assay using petal crude proteins. The substrate specificity of Gt5GT7 was analysed in vitro using the recombinant enzyme produced by Escherichia coli. Gt5GT7 could transfer a glucosyl moiety to anthocyanidin 3-glycosides but not to other flavonoid compounds. Delphinidin 3-glucoside, the precursor of gentiodelphin, was the best substrate among several anthocyanidin 3-glycosides tested. Heterologous expression of Gt5GT7 in tobacco plants led to additional accumulation of cyanidin 3-rutinoside-5-glucoside, confirming that Gt5GT7 has a valid enzymatic activity in planta.
Key words: Anthocynian 5-O-glucosyltransferase, flower colour, gentian, gentiodelphin, 5GT, transgenic tobacco
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary dataReferences |
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Anthocyanins are broadly distributed in flowering plants and are well known as natural pigments contributing to various colours, ranging from orange to pink, red, and purple. Over the past decades, their biosynthesis and regulation have been well studied at the molecular level (Mol et al., 1998; Grotewold, 2006). Anthocyanins are extremely diverse in nature, comprising more than 500 molecular species (Andersen and Jordheim, 2006), and enable plants to display various attractive flower colours. Regardless of such high diversity of anthocyanins, anthocyanidin aglycones usually consist of only six basic structures, namely pelargonidin, cyanidin, peonidin, petunidin, delphinidin, and malvidin (Strack and Wray, 1992). The diversity of anthocyanins mostly reflects modifications of common aglycones such as by hydroxylation, glycosylation, methylation, and acylation. Although the aglycone biosynthetic pathway is strongly conserved among higher plants, the later steps of aglycone modification are considered to be unique in different plant species.
Among enzymes that modify anthocyanins, glycosyltransferase (GT) plays an important role in the diversity of flower coloration and catalyses a regiospecific glycosyl transfer from UDP-sugar to the hydroxyl moiety of anthocyanins (Gachon et al., 2005). Glycosylation is a widespread modification of plant secondary metabolites. It is involved in various functions, including the regulation of plant hormones and detoxification (Gachon et al., 2005). From a chemical point of view, sugar conjugation results in both increased stability and water solubility. In the case of anthocyanin, the glycosylated form is considered to be easily transferred from the cytoplasmic production site to the vacuole. In most cases in higher plants, anthocyanidins are first modified at the 3-moeity by UDP-glucose:anthocyanidin 3-O-glucosyltransferase (3GT), except in rare instances of rose (Ogata et al., 2005). The genes encoding 3GTs have been isolated and well characterized in maize (Zea mays; Schiefelbein et al., 1985), snapdragon (Antirrhinum majus; Martin et al., 1991), grape (Vitis vinifera; Sparvoli et al., 1994), and gentian (Gentiana triflora; Tanaka et al., 1996). In petunia, the gene encoding UDP-rhamnose anthocyanidin 3-O-glucoside rhamnosyltransferase (RT) has been isolated (Brugliera et al., 1994; Kroon et al., 1994). The gene encoding UDP-glucose:anthocyanidin 3-O-glucoside glucosyltransferase (Dunsky) has been isolated from Japanese morning glory (Ipomoea nil; Morita et al., 2005). Arabidopsis contains 99 glycosyltransferase homologues, and their molecular phylogenic relationship has been analysed (Li et al., 2001). Two putative glycosyltransferase genes (At5g17050 and At4g14090) were confirmed to encode flavonoid 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively, based on comprehensive analysis of the metabolome and transcriptome of Arabidopsis overexpressing the PAP1 gene encoding a MYB transcriptional factor (Tohge et al., 2005).
Gentian (Gentiana triflora) has brilliant blue-coloured flowers that accumulate a unique anthocyanin called gentiodelphin (delphinidin 3-O-glucosyl-5-O-caffeoyl-glucosyl-3'-O-caffeoyl-glucoside) in their petals (Goto et al., 1982). Almost all genes encoding enzymes involved in gentiodelphin biosynthesis have been identified and characterized (Tanaka et al., 1996; Fujiwara et al., 1998; Kobayashi et al., 1998; Fukuchi-Mizutani et al., 2003; Nakatsuka et al., 2005). However, only the gene encoding anthocyanin 5-O-glucosyltransferase (5GT) has not yet been identified from gentian. Several studies reported the isolation of 5GT genes involved in glucosylation at the 5-position of anthocyanidin. In perilla and verbena, 5GT genes were first isolated by the differential display technique (Yamazaki et al., 1999). Thereafter, 5GT genes were also isolated from petunia, torenia, and iris by the screening of cDNA libraries (Yamazaki et al., 2002; Fukuchi-Mizutani et al., 2003; Imayama et al., 2004).
Here the isolation of the 5GT gene from gentian flowers using a degenerate PCR technique is reported. The substrate specificity analyses using recombinant protein indicated that gentian 5GT (Gt5GT7) was able to catalyse glucosylation of the 5-moiety of a broader range of anthocyanidin-3-glycosides compared with 5GTs isolated from other plants. Interestingly, in transgenic tobacco plants overexpressing Gt5GT7 cDNA, de novo 5-moeity glucosylation of original cyanidin-3-rutinoside and a slight change of flower colour were observed, due to the additional formation of cyanidin 3-O-rutinoside-5-O-glucoside. Therefore, Gt5GT7 might be a useful tool to modify flower colour in several floricultural plants, and the possibility for molecular engineering of flower colour using Gt5GT7 is also discussed.
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Materials and methods |
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Plant materials
Gentiana triflora cv. Maciry was grown in an experimental field at the Iwate Agriculture Research Center. Petals at four different flower development stages, as defined by Nakatsuka et al. (2005), and leaf and stem samples were collected and stored at –80 °C until use.
Enzyme assay with crude protein from gentian petals
One gram of gentian petals at each of four different flower developmental stages was ground in liquid nitrogen with a pestle and mortar. The powdered tissues were suspended in 5 ml of extraction buffer A [0.1 M potassium phosphate (pH 8.5), 1 mM DTT, 0.1 mM (p-aminophenyl) methylsulphony fluoride, 10% (v/v) glycerol, 1 mM UDP-glucose, and protease inhibitor cocktail (Sigma-Aldrich, MO, USA)] supplemented with 500 mg of DOWEX (Muromach Technos, Tokyo, Japan). After centrifuging, the supernatant was purified through a Millex-GV (Millipore) and desalting column PD-10 (GE Healthcare, Chalfont St Giles, Bucks, UK). Protein concentrations were measured as described by Bradford (1976).
Gentian 5GT activity was measured using Cya3G and UDP-glucose as substrates. The reaction mixture (total volume 50 µl) consisted of 0.1 M potassium phosphate (pH 8.5), 1.25 µg of Cya3G, 2 mM UDP-glucose, and 100 µg of crude protein for each petal. The mixture was incubated for 30 min at 30 °C. The reaction was stopped by adding an equal volume of methanol containing 1% (v/v) hydrochloric acid. After centrifuging, the supernatant was subjected to HPLC analysis.
HPLC analysis of anthocyanins was performed with a reverse phase column Asahipack ODA-50 4E (4.6x250 mm; Showa-Denko, Tokyo, Japan) with a gradient elution of 10–60% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid for 30 min, followed by an isocratic elution of 60% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid for 2 min at a flow rate of 0.6 ml min–1 at 40 °C with a monitoring absorbance of 500 nm. Anthocyanins were identified by comparing the retention time and spectral patterns of standard compounds. All flavonoid compounds used in this study were purchased from Extrasynthese (France) and Plantech (UK).
Cloning of 5GT cDNA from gentian
The total RNA was isolated from the gentian petals at flower developmental stage 3, which showed the highest 5GT activity for cyanidin 3-glucoside. Gentian petal cDNA was synthesized from the total RNA by RNA PCR (AMV) kit version 2.1 (Takara-bio, Tokyo, Japan).
Degenerate primers were designed from the conserved amino acid sequences of 5GTs isolated from perilla (AB013597 [GenBank] ; Yamazaki et al., 1999), verbena (AB013598 [GenBank] ; Yamazaki et al., 1999), petunia (AB027455 [GenBank] ; Yamazaki et al., 2002), and torenia (AB076698 [GenBank] ) (Table S1 in Supplementary data available at JXB online). The PCR mixture (total volume 25 µl) contained 1 µl of first-strand cDNA, 1x Ex buffer, 200 µM dNTPs, 5 µM each primer, 1.25 units of Ex Taq polymerase (Takara-bio). The reaction conditions consisted of pre-heating at 94 °C for 90 s, 35 cycles at 95 °C for 20 s, 40 °C or 45 °C for 40 s and 72 °C for 1 min, and extension at 72 °C for 10 min. The amplified fragments were subcloned into the pCR4 TA TOPO cloning vector (Invitrogen, Carlsbad, CA, USA) and subjected to sequence analysis using the Big-Dye terminal cycle sequencing kit version 1.1 and the ABI PRISM 3100 DNA sequencer (Applied Biosystems Japan, Tokyo).
To obtain full-length cDNA of Gt5GT7, 3'- and 5'-rapid amplification of cDNA ends (RACE) technology was performed with the GeneRacer kit (Invitrogen) using the primers described in Table S1 in Supplementary data available at JXB online. The amplified fragments were subcloned into the pCR4 TA TOPO cloning vector and subjected to sequence analysis as described above. The full-length cDNA sequence of Gt5GT genes was assembled into 3'- and 5'-RACE sequences using the GENETYX-MAC software version 12.0 (GENETYX, Tokyo). The nucleotide sequence was translated to the respective deduced amino acid sequences using DNASIS version 3.6 (HITACHI, Tokyo, Japan) and compared with other sequences in the NCBI (National Center for Biotechnology Information) database using the BLAST search tool. A phylogenic tree was produced using ClustalW (Thompson et al., 1994) and TreeView version 1.6.6 (Page, 1996).
Expression analysis of Gt5GT candidate genes
Expression analysis of Gt5GTs in gentian plants was performed using total RNA isolated using plant RNA reagents (Invitrogen) from petals at the four different flower developmental stages defined above, from other reproductive tissue (pistil, filament, anther, and sepal) at stage 3, and from mature leaves and stems.
For screening the 5GT candidates among putative Gt5GT genes, quantitative reverse transcription–PCR (qRT-PCR) was performed on an ABI PRISM 7700 system (Applied Biosystems Japan) and QuantiTect SYBR Green PCR (QIAGEN, Valencia, CA, USA). cDNAs were synthesized from total RNA with genomic DNA removed using QuantiTect Reverse Transcription (QIAGEN) according to the manufacturer's instructions. Reaction mixtures (total volume 20 µl) consisted of the following components: 1x master mix, 0.5 µM each primer, and 1 µl of cDNA template. Primer sets used for qRT-PCR analysis are shown in Table S1 in Supplementary data available at JXB online. Cycle conditions were 95 °C for 15 min, and then 95 °C for 15 s and 60 °C for 1 min by 40 cycles. After the cycle programs, a melting curve analysis was performed to verify the specificity and identity of qRT-PCR products. Expression levels of each gene were calibrated with expression of the 18S RNA (UBQ) gene and indicated as relative values based on maximum expression levels.
To investigate further the detailed expression profiles of the gentian 5GT candidate (Gt5GT7), northern blot analysis was carried out using total RNA from several tissues in gentian. The total RNA (5 µg) was separated on a 1.25% MOPS–agarose gel and transferred to Hybond-N+ membranes (GE Healthcare). Probes were prepared with a PCR-DIG Probe Synthesis Kit (Roche Diagnostics) using primer sets as described in Table S1 in Supplementary data available at JXB online. Membranes were hybridized in high-SDS hybridization buffer at 50 °C overnight. After hybridization, membranes were washed twice in 2x SSC and 0.1% SDS for 15 min at 68 °C, and twice in 0.1x SSC and 0.1% SDS for 15 min at 65 °C. Detection was performed using a DIG Nucleic Acid Detection Kit (Roche Diagnostics).
Heterologous expression of Gt5GT7 in Escherichia coli
The open reading frame (ORF) of Gt5GT7 was cloned into a pMAL-c2x vector (New England BioLabs, lpswich, MA, USA) to produce a recombinant fusion protein with maltose-binding protein at the N-terminal. The resulting vector was designated as pMAL-Gt5GT7. The plasmid was transformed into E. coli strain Rosetta (DE3) pLysS (Novagen, San Diego, CA, USA). The cells were incubated in 1.0 l of induction medium (containing 10 g l–1 tryptone, 5 g l–1 yeast extract, 5 g l–1 NaCl, 2 g l–1 glucose, 100 mg l–1 ampicillin) at 28 °C until A600 reached 0.6. After addition of IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 1 mM, the cells were further cultured for 4 h at 28 °C, then collected and resuspended in column buffer [20 mM TRIS–HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA and 10 mM β-mercaptoethanol]. Following disruption of the cells by sonication, the soluble solution was loaded on an amylose resin column, then eluted and fractionated with column buffer supplemented with 10 mM maltose. The high-concentration fractions were collected and purified through a desalting column PD-10 (GE Healthcare). Protein concentrations were measured as described above.
Heterologous expression of Gt5GT7 in transgenic tobacco plants
For expression in tobacco plants, the ORF of Gt5GT7 was inserted under the control of an Agrobacterium rhizogenes rolC promoter (Sugaya and Uchimiya, 1992) in a binary vector harbouring the bialaphos herbicide-resistance gene (bar) to produce pSMABR–rolCproGt5GT7, which is a derivative of the binary vector pSMAB704 (Nishihara et al., 2006). The rolC promoter can be used for flower colour alteration in tobacco and gentian plants (Nakatsuka et al., 2007b, 2008). The constructs were then transformed into A. tumefaciens strain EHA101 by electroporation (MicroPulser; Bio-Rad, Tokyo, Japan). Tobacco plants (Nicotiana tabacum cv. SR-1) aseptically grown from seeds for about 1 month were transformed via an A. tumefaciens-mediated leaf disc procedure (Horsch et al., 1985) and selected using 5 µg ml–1 bialaphos (Meiji Seika Co., Tokyo, Japan) as a selection reagent. After rooting and acclimatization, regenerated plants were grown in a greenhouse and self-pollinated seeds were obtained. T1 transgenic plant lines selected on 5 µg ml–1 bialaphos-containing medium were transferred to soil and used for further analyses.
HPLC analysis and mass spectrometric characterization of anthocyanins in petal extracts from transgenic tobacco plants
Anthocyanins were extracted from the petals of Gt5GT7-expressing transgenic and wild-type tobacco plants with methanol containing 1% (v/v) hydrochloric acid. HPLC analysis was carried out as described above.
The extract was diluted 1:10 with methanol containing formic acid prior to analysis. The supernatant was applied to an HPLC/ESI-MS system comprising an Agilent G1956B mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and an Agilent HPLC 1200 series (Agilent Technologies). HPLC was carried out on a ZORBAX SB-C18 column (3.5 µm; 4.6 x 75 mm; Agilent Technologies) at a flow rate of 0.2 ml min–1 with an elution gradient of solvent A [1% (v/v) formic acid] and solvent B (acetonitrile) and the following elution profile (0 min 5% A, 30 min 50% A) using linear gradients between the time points. Mass spectra were acquired from 600 m/z to 800 m/z in positive ion mode. System control, data acquisition, data analysis, and MSD data evaluation were performed using Agilent CHEMSTATION software (Agilent Technologies).
Measurement of colour parameters
Chromaticities of petals from Gt5GT7-expressing and wild-type tobacco plants were measured with a spectrophotometer (NR-1, Nippon denshoku, Tokyo, Japan). These were expressed by the CIELAB (CIE 1976) method: L*, a*, and b* values indicated lightness, redness, and yellowness, respectively (Robertson, 1977). Colour saturation and hue were expressed as chroma and hue-angle, respectively, which were calculated from the a* and b* values.
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Results and discussion |
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Profile of anthocyanin accumulation and 5GT activity in gentian petals
The composition of anthocyanins accumulating in the petals of gentian (Gentiana triflora cv. Maciry) was determined. High performance liquid chromatography (HPLC) detected a single major peak in the extracts from petals at anthesis (Fig. 1A, peak 1). Because gentiodelphin has been reported to be the major anthocyanin of gentian flowers (Goto et al., 1982), the compound represented by peak 1 in the chromatograph was defined as gentiodelphin. The accumulation of gentiodelphin was coincident with flower development and the maximum level was attained just before anthesis (Fig. 1B), corresponding to the accumulation of delphinidin derivatives as described previously (Nakatsuka et al., 2005). However, no significant changes in other minor peaks were detected throughout flower development (data not shown).
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To investigate the profile of 5GT activity in gentian flowers, crude protein extracts from petals at four developmental stages were incubated with cyanidin 3-glucoside (Cya3G) and UDP-glucose, and then subjected to HPLC analysis (Fig. 1B). The reaction mixture formed an additional peak when crude protein extracts were incubated with both Cya3G and UDP-glucose, but did not form this without UDP-glucose. This product was confirmed as cyanidin 3,5-diglucoside (Cya3,5G) by comparing its spectral absorptance and retention time with those of several cyanidin glucosides. The 5GT activity estimated from the formation of Cya3,5G is shown in Fig. 1B. The 5GT activity for Cya3G was very weak at the early bud stage (S1), then increased coincident with flower development and peaked just before anthesis (S3). On the other hand, the crude protein extracts from gentian leaves did not form Cya3,5G (data not shown), suggesting that 5GT activity for Cya3G was flower-specific. The profile of 5GT activity for Cya3G corresponded well with that of gentiodelphin accumulation in gentian petals (Fig. 1B). Therefore, these results implied that this 5GT activity was related to gentiodelphin biosynthesis in gentian flowers. Petal crude protein extracts exhibited the optimum 5GT activity for Cya3G at pH 8.0–8.5 (data not shown), which is similar to those reported previously for crude 5GT preparations from petunia, common stock, and dahlia (Jonsson et al., 1984; Teusch et al., 1986; Ogata et al., 2001).
Isolation of 5GT candidate genes from gentian petals
Using Cya3G and UDP-glucose as substrates, it was confirmed that gentian petals had 5GT activity, which was classified as UDP-glucose-dependent glucosyltransferase. 5GT genes have been isolated from several plant species using either differential display (Yamazaki et al., 1999) or cDNA library screening (Yamazaki et al., 2002; Imayama et al., 2004). Instead, in this study, a degenerate PCR technique was employed, using primers designed from the deduced amino acid sequences of 5GT genes from dicotyledonous plants including perilla, verbena (Yamazaki et al., 1999), petunia (Yamazaki et al., 2002), and torenia (Fukuchi-Mizutani et al., 2003) (Table S1 in Supplementary data available at JXB online). Putative 5GT fragments were amplified using cDNA from gentian petals just before anthesis (stage 3), which showed the highest 5GT activity (Fig. 1B). By determining the nucleotide sequences of >100 clones, seven independent 5GT partial candidates, designated as Gt5GT1–7, were isolated from gentian petals. To investigate the expression of each putative gentian 5GT gene, qRT-PCR analysis was performed using primers for each Gt5GT (Fig. 2, and Table S1 in Supplementary data available at JXB online). Among seven Gt5GT candidates, Gt5GT7 transcripts were detected preferentially in the flowers and markedly increased according to flower development before peaking at stage 3, which corresponded with the profile of 5GT activity in gentian flowers (Fig. 1B). The other six clones did not show such expression profiles. Hence, the full-length cDNA of Gt5GT7 was isolated by RACE technology. Gt5GT7 cDNA (accession number AB363839) was 1892 bp in length and contained an ORF encoding 504 amino acid residues. The phylogenic tree based on the deduced amino acid sequences of plant glucoyltransferases is shown in Fig. 3. Gt5GT7 was classified into a subfamily of 5GT enzymes for anthocyanin. In phylogenic analyses, glycosyltransferases for anthocyanin were categorized into different subfamilies based on their specificity for sugar moieties and acceptor molecules (Bowles et al., 2005; Gachon et al., 2005). Gt5GT7 exhibited 41.7, 41.3, 36.0, and 41.7% identities with 15GTs of petunia, verbena, perilla, and torenia, respectively (Fig. 4). The Gt5GT7 contained a 44 amino acid consensus signature sequence, the so-called plant secondary product glycosyltransferase (PSPG) motif, which was highly conserved among plant glycosyltransferases (Gachon et al., 2005). Therefore, Gt5GT7 was presumed to encode 5GT in gentian flowers and was subjected to further analyses.
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Expression analysis of Gt5GT7 in gentian plants
To determine the expression profile of Gt5GT7 in gentian in detail, northern blot analysis was performed using total RNA from several tissues. The accumulation of Gt5GT7 transcripts was detected only in the petals and markedly increased just before anthesis, as indicated by qRT-PCR analysis (Fig. 2, and Fig. S1 in Supplementary data available at JXB online). In addition, the expression of Gt5GT7 was also detected weakly in the filaments, which are fused with the petals and have slight pigmentation in the terminal portions (Fig. S1 in Supplementary data). The expression profile of Gt5GT7 corresponded well with that of 5GT activity determined by crude protein extracts. The profile of Gt5GT7 accumulation in gentian petals was similar to that of genes encoding enzymes catalysing the final steps in the gentiodelphin biosynthetic pathway, including the flavanone 3-hydroxylase (F3H), flavonoid 3'5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), 3GT, anthocyanin 5-aromatic acyltransferase (5AT), and UDP-glucose: anthocyanin 3'-O-glucoyltransferase (3'GT) genes (Fujiwara et al., 1998; Fukuchi-Mizutani et al., 2003; Nakatsuka et al., 2005). Therefore, it was suggested that the expression of Gt5GT7 might be controlled by a common transcriptional factor(s) regulating later steps of anthocyanin biosynthesis in gentian flowers.
Southern blot analysis indicated that the gentian genome contains one or two copies of Gt5GT7 (Fig. 5), and that the copy number is less than for other anthocyanin structural genes (Nakatsuka et al., 2005).
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Enzymatic characterization of Gt5GT7 using the recombinant proteins
To examine enzymatic properties of Gt5GT7, first an attempt was made to produce recombinant proteins using heterologous expression systems. The recombinant proteins produced by a bakers’ yeast expression system showed low 5GT activity. After screening several E. coli expression systems, it was found that the recombinant Gt5GT7 retained high activity when it was fused with maltose-binding protein (MBP). Because removal of the MBP-tag from the fused protein resulted in remarkable loss of 5GT activity, enzymatic characterization of Gt5GT7 was performed using MBP-fused protein. Yamazaki et al. (1999) predicted that it would be difficult to purify 5GT protein due to its low and unstable activity. MBP:Gt5GT7 converted Cya3G to Cya3,5G in the presence of UDP-glucose (Fig. 6B) comparable with the 5GT activity assay using crude protein from gentian petals. By contrast, MBP protein purified from bacteria extracts as a vector control did not possess any glucosylation activity (Fig. 6C). The substrate specificities and kinetic parameters of Gt5GT7 were analysed further (Table 1). The analyses of substrate specificities for several flavonoid compounds revealed that MBP:Gt5GT7 had glucosylation activity specific to anthocyanin 3-glycosides. Delphinidin 3-glucoside (Del3G) was the best substrate among the anthocyanin 3-glycosides used in this study. The Km value and Vmax for Del3G were determined to be 29.5 µM and 1.49 nmol min–1 mg protein–1, respectively, when UDP-glucose (2 mM) was used as a sugar donor. On the other hand, those for Cya3G were determined to be 20.9 µM and 0.98 nmol–1 min–1 mg–1 protein, respectively, and relative 5GT activity for Cya3G (66%) was lower than that for Del3G (100%). The petals of pink-flowered gentian plants accumulated gentiocyanin (cyanidin 3-O-glucosyl-5-O-caffeoyl-glucoside) as a major anthocyanin (Hosokawa et al., 1995). Although cultivars derived from G. scabra also accumulate both gentiodelphin and gentiocyanin, gentiodelphin accumulates more abundantly than gentiocyanin in their petals (Hosokawa et al., 1997). These anthoycanin compositions might be regulated partially by the substrate specificity of 5GT activity in addition to the difference of flavonoid 3'-hydroxylase (F3'H) and F3'5'H activities between G. triflora and G. scabra. Pelargonidin 3-glucoside (Pel3G) and malvidin 3-glucoside (Mal3G) that were not naturally accumulated in gentian petals were also converted to pelargonidin 3,5-diglucoside (Pel3,5G) and malvidin 3,5-diglucoside (Mal3,5G); their relative activities were 45% and 35%, respectively, compared with that of Del3G (100%). Cyanidin 3-galactoside (Cya3Gal) and cyanidin 3-rutinoside (Cya3R) were also utilized as substrates of MBP:Gt5GT7 and were converted to cyanidin 3-galactoside-5-glucoside (data not shown) and cyanindin 3-rutinoside-5-glucoside (Fig. S2 in Supplementary data available at JXB online), respectively. No activity against other substrates, including anthocyanidins, flavanones, flavones, and flavonols, was detected. It was also confirmed that MBP:Gt5GT7 could not utilize UDP-galactose as a UDP-sugar donor (data not shown). MBP:Gt5GT7 exhibited the optimum 5GT activity for Cya3G at the same pH (8.0–9.0) as that for crude protein from gentian petals (data not shown).
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Cya3G is indicated to be the best substrate for perilla 5GT, because perilla accumulates malonylshisonin [cyanidin 3-(p-coumaryl)glucoside-5-(malonyl)glucoside] as the major anthocyanin (Yamazaki et al., 1999). On the other hand, petunia and iris 5GTs could utilize delphinidin 3-(p-coumaroyl)-rutinoside and anthocyanin 3-rutinoside, respectively, but never anthocyanin 3-glucosides as substrates (Yabuya et al., 2002; Yamazaki et al., 2002). Therefore, maximum enzymatic activities of 5GTs seem to be optimized for the precursor anthocyanins that accumulated predominantly in each plant. In gentian, gentiodelphin is a major anthocyanin and it is reasonable to hypothesize that Gt5GT7 can catalyse Del3G as the optimal substrate. Although Pel3G, Mal3G, Cya3R, and Cya3Gal are not accumulated in gentian plants, Gt5GT7 is capable of transferring a glucosyl moiety to these anthocyanin 3-glycosides. This latent activity of Gt5GT7 might be useful in order to change the anthocyanin composition and to enhance anthocyanin diversity in other plants. Recently, the crystal structures of some glucosyltransferases, such as triterpene/flavonoid GT UGT71G1 from Medicago truncatula (Shao et al., 2005) and grape VvGT1 (Offen et al., 2006), have been determined, and the data suggest the presence of several key residues that interact with UDP-sugar and the flavonoid backbone. Further investigation of the substrate recognition of plant GTs is required.
Expression of Gt5GT7 and anthocyanin composition in tobacco flowers
To investigate whether Gt5GT7 has a functional activity in vivo, transgenic tobacco plants expressing Gt5GT7 were produced and analysed. Some recent studies have used tobacco as a model plant for studies of flower colour modification (Lloyd et al., 1992; Shimada et al., 1999; Nishihara et al., 2005; Nakatsuka et al., 2006, 2007a, b). Tobacco flowers are known to accumulate anthocyanin without the modification of the 5-moiety in the petals (cyanidin 3-rutinoside; Aharoni et al., 2001), which is the potential substrate of Gt5GT7 (Fig. S2 in Supplementary data available at JXB online). In addition, no Gt5GT7-deficient gentian mutants are presently known. Thus, tobacco plants were used to analyse in vivo Gt5GT7 activity. To this end, the Gt5GT7 ORF driven under the control of the Agrobacterium rhizogenis rolC promoter was transformed into Nicotiana tabacum cv. SR1. Among 11 transgenic tobacco T1 plants, a clone (no. 7) that had strong Gt5GT7 expression was chosen by northern blot analysis (Fig. 7E) and used for further analysis. The flower of this line displayed a slightly deeper pink colour (Fig. 7B) compared with that of the wild type (Fig. 7A). Chromaticity analysis confirmed that the a* value, which indicates negative for green and positive for red, in Gt5GT7-expressing tobacco flowers increased significantly compared with that of wild-type flowers (Table 2), whereas other values, such as L*, b*, chroma, and hue-angle, were less different. HPLC analysis showed that extracts from the flowers of Gt5GT7-expressing tobacco had a novel peak (Fig. 7D), which was absent in wild-type flowers (Fig. 7C). LC-MS analysis established that the [M]+ of the additional peak was 757.2 (Fig. 7F), which was consistent with the molecular weight of cyanidin-3-rutinoside with an additional glucosyl moiety. Therefore, the additional compound produced by overexpression of Gt5GT7 in tobacco flowers was speculated to be cyanidin 3-rutinoside-5-glucoside (Fig. 7F). Some other Gt5GT7-expressing tobacco lines also accumulated cyanidin-3-rutinoside in their petals (data not shown). The production of cyanidin 3-rutinoside-5-glucoside in Gt5GT7-expressing tobacco flowers was speculated either to utilize anthocyanidin 3-rutinoside as a substrate, or to be the result of endogenous RT activity after glucosylation of cyanindin 3-glucoside by Gt5GT7. The RT gene has been identified in petunia but its substrate specificity has not been studied (Brugliera et al., 1994; Kroon et al., 1994), so further study is needed to reveal the order of anthocyanin modification in tobacco flowers. No other additional peaks were detected at 250–550 nm by HPLC analysis. In addition, no other visual changes were observed in the transgenic lines compared with wild-type plants (data not shown). Two transgenic ornamental plants have been reported that exhibit alteration of the glucosylation of anthocyanins. The first reported was transgenic lisianthus, possessing the 3GT gene from Antirrhinum majus, which altered anthocyanin glycosylation and acylation in the petals (Schwinn et al., 1997). More recently, a transgenic petunia transformed with gentian 3'GT and torenia 5GT expression cassettes was reported to produce delphinidin 3-,5-,3'-triglucoside (Fukuchi-Mizutani et al., 2003). Both transgenic plants had a modified anthocyanin complement in their flowers but did not show a significant change in flower colour compared with their host plants, probably due to the low levels of additional production of the anthocyanins. However, Gt5GT7-expressing tobacco plants exhibited slight enhancement of flower pigmentation (Fig. 7B, Table 2) due to alteration of anthocyanin glucosylation. Therefore, Gt5GT7 might be useful for changing anthocyanin modification and, consequently, modification of flower colour in several floricultural plants that have no glucosylation of anthocyanins at the 5 position. The potential use of Gt5GT7 should be investigated in the future. Moreover, down-regulation of Gt5GT7 by RNAi would provide useful information about the function of Gt5GT7 in gentian plants.
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In conclusion, the 5GT gene, which encodes the last structural enzyme (5GT) necessary for gentiodelphin biosynthesis, was successfully isolated from blue-flowered gentian, and flower colour enhancement in transformed tobacco plants was demonstrated. Gt5GT7 from gentian has high glucosylation activity for Del3G, in contrast to 5GTs isolated from other higher plants previously, and therefore is potentially a valuable resource for the genetic engineering of blue flowers.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResults and discussionSupplementary dataReferences |
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Supplementary data in one table and two figures can be found at JXB online.
Supplementary Table S1. Primers used in this study.
Supplementary Figure S1. Expression analysis of Gt5GT7 gene in gentian plants.
Total RNA isolated from petals at four different stages (lanes 1 to 4 correspond to stages S1 to S4, respectively), as defined in the Materials and Methods, pistil (5), filament (6), anther (7), sepal (8), leaf (9) and stem samples (10) in Gentiana triflora cv. Maciry subjected to northern blot analysis. Ethidium bromide-stained ribosomal RNA bands (rRNA) are shown as a control.
Supplementary Figure S2. HPLC analysis of glucosyltransfer reaction for cyanidin 3-rutinoside using recombinant Gt5GT7.
A) Elution profile of standard cyanidin 3-rutinoside (Cya3R) with reverse phase HPLC. Absorbance was measured at 500 nm.
B) The reaction product formed in the presence of MBP:Gt5GT7 purified from E. coli protein extracts. Cya3R5G denotes cyanidin 3-rutinoside-5-glucoside.
C) The reaction product formed in the presence of MBP protein purified from E. coli protein extracts.
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Acknowledgements |
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We thank H Kawamura and J Abe, Iwate Agriculture Research Center, for providing material of the gentian. We thank A Kubota, Y Abe, Y Kakizaki, C Yoshida, H Takahashi, J Kuzuo, R Horikiri, and R Takahashi for technical assistance. This study was supported in part by a Grant-in-Aid for Young Scientists B (no. 18789005) from the Japan Society for the Promotion of Science (JSPS).
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* These authors contributed equally to this work.
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