The Plant Cell, Vol. 25: 4150–4165, October 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

p-Hydroxybenzoyl-Glucose Is a Zwitter Donor for the Biosynthesis of 7-Polyacylated Anthocyanin in Delphinium

W

Yuzo Nishizaki,a,1 Motoki Yasunaga,a,1 Emi Okamoto,a Mitsutoshi Okamoto,b Yukio Hirose,b Masaatsu Yamaguchi,c Yoshihiro Ozeki,a,2 and Nobuhiro Sasakia,3 a Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan b Department of Agricultural Research, Ehime Research Institute of Agriculture, Forestry and Fisheries, Matsuyama, Ehime 799-2405, Japan c Faculty of Environmental Horticulture, Minami Kyushu University, Miyakonojo, Miyazaki 885-0035, Japan

ORCID IDs: 0000-0002-3002-368X (Y.O.); 0000-0001-7474-9493 (N.S.). The blue color of delphinium (Delphinium grandiflorum) flowers is produced by two 7-polyacylated anthocyanins, violdelphin and cyanodelphin. Violdelphin is derived from the chromophore delphinidin that has been modified at the 7-position by Glc and p-hydroxybenzoic acid (pHBA) molecules. Modification of violdelphin by linear conjugation of Glc and pHBA molecules to a Glc moiety at the 7-position produces cyanodelphin. We recently showed that anthocyanin 7-O-glucosylation in delphinium is catalyzed by the acyl-Glc–dependent anthocyanin glucosyltransferase (AAGT). Here, we sought to answer the question of which enzyme activities are necessary for catalyzing the transfer of Glc and pHBA moieties to 7-glucosylated anthocyanin. We found that these transfers were catalyzed by enzymes that use p-hydroxybenzoyl-Glc (pHBG) as a bifunctional acyl and glucosyl donor. In addition, we determined that violdelphin is synthesized via step-by-step enzymatic reactions catalyzed by two enzymes that use pHBG as an acyl or glucosyl donor. We also isolated a cDNA encoding a protein that has the potential for p-hydroxybenzoylation activity and two AAGT cDNAs that encode a protein capable of adding Glc to delphinidin 3-Orutinoside-7-O-(6-O-[ p-hydroxybenzoyl]-glucoside) to form violdelphin.

INTRODUCTION Delphiniums (Delphinium grandiflorum), with their striking blue flowers, are among the most popular flowers in the world. The blue coloration of delphinium flowers is produced by two anthocyanins, violdelphin and cyanodelphin (Hashimoto et al., 2002) (see Supplemental Figure 1 online). Violdelphin consists of two p-hydroxybenzoic acid (pHBA) molecules linked by a Glc molecule and also conjugated via a second Glc to the 7-position of delphinidin 3-rutinoside [delphinidin 3-O-rutinoside-7-O-(6-O-(4O-(6-O-(p-hydroxybenzoyl)-glucosyl)-oxybenzoyl)-glucoside)] (Kondo et al., 1990). Previously, it was proposed that violdelphin is produced with a single linear chain at the 7-position of the anthocyanin and that a second, branching side chain consisting of a glucosyl-glucosyl-phydroxybenzoyl-glucosyl-p-hydroxybenzoyl moiety is added to form cyanodelphin (delphinidin 3-O-rutinoside-7-O-[3-O-(3-O-(6-O-(4-O(6-O-(p-hydroxybenzoyl)-glucosyl)-oxybenzoyl)-glucosyl)-glucosyl)6-O-(4-O-(6-O-(p-hydroxybenzoyl)-glucosyl)-oxybenzoyl)-glucoside]) (Kondo et al., 1991; Brandt et al., 1993; Hashimoto et al., 2002). 7-Polyacylated anthocyanins (Saito et al., 2007), including violdelphin

1 These

authors contributed equally to this work. correspondence to [email protected]. 3 Current address: Iwate Biotechnology Research Center, Iwate 0240003, Japan. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Yoshihiro Ozeki (ozeky@ cc.tuat.ac.jp). W Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.113.113167 2 Address

and cyanodelphin, often occur in purple-blue flowers; for example, the anthocyanin campanin is present in Campanula medium, cinerarin in cineraria (Senecio cruentus), and platyconin in Chinese bell flower (Platycodon grandiflorum; Yoshida et al., 2009). The aromatic organic acids that bond to the anthocyanin molecule are thought to contribute to the production of the blue color in these flowers. At present, however, the pathway for 7-polyacylation of anthocyanins remains uncertain. It has been suggested that two types of enzyme are needed for polyacyl-anthocyanin biosynthesis, namely, acyltransferases and glucosyltransferases. Two categories of anthocyanin acyltransferase have been identified: acyl-CoA–dependent acyltransferase, and acyl-Glc–dependent acyltransferase. Acyl-CoA–dependent malonyltransferases and hydroxycinnamoyltransferases have been identified in several plant species (Nakayama et al., 2003; D’Auria, 2006). Acyl-Glc–dependent acyltransferases were first identified in a wild tomato species (Lycopersicon pennellii) as the enzymes catalyzing formation of diacyl-Glc (Li et al., 1999) and in Arabidopsis thaliana for the biogenesis of sinapoylmalate and sinapoylcholine (Lehfeldt et al., 2000; Shirley et al., 2001). Recently, acyl-Glc–dependent anthocyanin acyltransferase, which is a Ser-carboxy peptidase-like (SCPL) protein and catalyzes the transfer of p-hydroxycoumaric acid to anthocyanin, was identified in butterfly pea (Clitoria ternatea; Noda et al., 2006). In culture, anthocyanin-producing cells of carrot (Daucus carota) synthesize and accumulate cyanidin 3-O-(299-xylosyl-699-sinapoyl-glucosyl-galactoside) as the major anthocyanin molecule; by contrast, Glehnia littoralis produces cyanidin 3-O-(299-xylosyl699-feruloyl-glucosyl-galactoside). There is no difference between these closely related species in the substrate specificities of their

Biosynthesis of Polyacyl Anthocyanin

acyl-Glc dependent acyltransferases; however, they synthesize and accumulate different species of acyl donors, namely, sinapoylGlc and feruloyl-Glc, respectively (Matsuba et al., 2008). Aliphatic organic acid can also be transferred by SCPL-type acyltransferases using aliphatic acyl-Glc but not acyl-CoA; we previously showed that the malyl residue of anthocyanins in carnation petals is modified by a malyl-Glc–dependent acyltransferase of the SCPL protein subfamily (Abe et al., 2008; Umemoto et al., 2009). With regard to anthocyanin glycosylation, the initial supposition was that all anthocyanin glycosylation reactions were catalyzed by UDP sugar–dependent glycosyltransferases. However, it was recently determined that anthocyanin 7-O-glucosylation in delphinium and agapanthus (Agapanthus africanus) is catalyzed by

4151

acyl-Glc–dependent anthocyanin 7-O-glucosyltransferases (AA7GTs) (Matsuba et al., 2010; Miyahara et al., 2012). These AA7GTs are glucoside hydrolase family 1 (GH1) proteins that contain a transit peptide in their N terminus for movement into the vacuole. The next catalytic step in the generation of violdelphin is thought to be the transfer of a pHBA molecule to the glucosyl moiety at the 7-position of anthocyanin; this catalytic step must occur in the vacuole as the initial glucosylation at the 7-position is catalyzed by a vacuolar enzyme. Further glucosylation at the hydroxyl group of the p-hydroxybenzoyl moiety is also thought to be mediated by a vacuolar glucosyltransferase (i.e., a GH1-type glucosyltransferase). To date, however, it is unclear whether there is transferase activity to anthocyanin 7-glucoside or Glc transfer activity to the hydroxyl group of the

Figure 1. Schematic Representation of the Biosynthetic Pathway for Violdelphin. Dp3R, delphinidin 3-O-rutinoside; Dp3R7G, delphinidin 3-O-rutinoside-7-O-glucoside; Dp3R7BG, delphinidin 3-O-rutinoside-7-O-(6-O-(p-hydroxybenzoyl)glucoside); Dp3R7GBG, delphinidin 3-O-rutinoside-7-O-(6-O-(4-O-(glucosyl)-oxybenzoyl)-glucoside); Dp3R7d(BG), delphinidin 3-O-rutinoside-7-O-(6-O-(4O-(6-O-(p-hydroxybenzoyl)-glucosyl)-oxybenzoyl)-glucoside).

4152

The Plant Cell

p-hydroxybenzoyl moiety on the Glc residue attached to the anthocyanin skeleton. In this study, we show that crude enzyme extracts from delphinium sepals synthesize violdelphin via p-hydroxybenzoyl transfer and glucosyl transfer that uses cyanidin 3,7-diglucoside as an acceptor. Both of these activities use p-hydroxybenzoylGlc (pHBG) as the donor, demonstrating that pHBG acts as a bifunctional donor for p-hydroxybenzoyl transfer and glucosyl transfer. Here, by analogy with the concept of a Zwitterion (a neutral molecule carrying both a positive and a negative charge at different locations within the molecule), we propose that pHBG acts as a Zwitter donor in acylation and glucosylation in modification of anthocyanin in delphinium sepals, to generate the compounds required for blue flowers (Figure 1). We also identified a candidate cDNA that encodes an SCPL protein that mediates transfer of pHBA to Glc moieties; two cDNAs were also obtained, and both cDNAs encode an enzyme that transfers a Glc at the second glucosylation step to the p-hydroxybenzoyl moiety at the 7-position of the anthocyanin. The two cDNAs encode acyl-Glc–dependent anthocyanin 7-O-(6-O-(p-hydroxybenzoyl)-glucoside) glucosyltransferase (AA7BG-GT1 and AA7BG-GT2), which is required for the formation of violdelphin (Figure 1). The contribution of the GH1-type anthocyanin glucosyltransferase and the SCPL acyltransferase to the biosynthetic pathways for anthocyanin pigments in delphinium is described.

RESULTS Anthocyanin p-Hydroxybenzoyltransferase Activity in Crude Extracts from Delphinium Sepals Crude protein extracts were prepared using delphinium sepals from blue flowers (cv Triton Light Blue) and incubated with either pHBG and cyanidin 3,7-O-diglucoside (Cy3,7dG) or with only Cy3,7dG at 30°C for 60 min. After termination of the reactions by addition of phosphoric acid, the reaction mixtures were subjected to an HPLC analysis. An additional peak (P1) on the HPLC chromatogram was detected in the mixture containing both pHBG and Cy3,7dG (Figure 2A). The photodiode array spectrum of the chromatogram obtained from the Cy3,7dG reaction mixture had peaks at 280 and 510 nm (Figure 2B); that of the pHBG and Cy3,7dG mixture showed peaks at 260 and 520 nm (Figure 2C). The peak at 260 nm implied that pHBA was conjugated to the anthocyanin. The molecular ion peak of the product generated by the enzymatic reaction was mass-to-charge ratio (m/z) = 731.1, corresponding to the molecular mass of Cy3,7dG bound to pHBA. To confirm the bonding position of pHBA and the anthocyanin molecule, a large-scale reaction using the crude extract was performed to obtain a purified sample (;12 mg) of the reaction product. The reaction product was analyzed by 1H NMR and 13C NMR spectroscopy using 1H{13C} heteronuclear multiple quantum coherence, 1H{13C} heteronuclear multiple bond

Figure 2. Detection of p-Hydroxybenzoyltransferase Activity in the Crude Extract Prepared from Sepals of Delphinium cv Triton Light Blue. (A) HPLC elution profiles of the products of the reaction performed with the donor molecule and in its absence. Absorbance was measured at 520 nm. mAU, milliabsorbance units. (B) and (C) The photodiode array spectra of Cy3,7dG and P1, respectively.

Biosynthesis of Polyacyl Anthocyanin

coherence (HMBC), totally correlated spectroscopy (TOCSY), H-H correlated spectroscopy, and nuclear Overhauser effect correlated spectroscopy (NOESY) (see Supplemental Table 1 online). The 1H NMR spectrum showed the presence of two Glc moieties (Glc-A and Glc-B) and one p-hydroxybenzoyl moiety (Bz-I) with cyanidin (Figure 3). The anomeric protons of the Glc moieties were assigned at d 5.4 (d, J = 8.02 Hz, H-1 of Glc-A) and d 5.33 (d, J = 6.87 Hz, H-1 of Glc-B), and the substitution positions of the two anomeric protons were confirmed by 1H{13C} HMBC and NOESY as described above (Matsuba et al., 2010). The assignment of the H-6 of Glc-B (d 4.46 and d 4.76) is reliant on the crosspeaks of H-1 and H-6 of Glc-B in the TOCSY spectrum. The chemical shift values of H6 of Glc-B were observed in lower magnetic fields than the values for Cy3,7dG (d 3.79 and d 4.02) (see Supplemental Table 1 online). This suggests that the pHBA is linked to the C-6 of Glc-B, a conclusion supported by the observation of an 1H{13C} HMBC signal between the carboxyl carbon (C-7) of Bz-I (d 168.2) and the H-6 of Glc-B (d 4.46 and 4.76) (two arrowed lines in the structure in Figure 3). From these results, P1 was identified as cyanidin 3-O-glucoside-7-O-(6-O-(p-hydroxybenzoyl)glucoside) (Cy3G7BG), and we designated the enzyme for the reaction as acyl-Glc–dependent anthocyanin 7-O-glucoside acyltransferase (AA7G-AT; Figure 3).

4153

Glucosyl and Acyl Modifications to Cy3G7BG To detect further modification reactions for Cy3G7BG, a crude extract containing purified Cy3G7BG was obtained as described above (Figure 3), and pHBG was analyzed by HPLC after incubation at 30°C for 0.5, 4, or 8 h. Additional peaks were present on the HPLC chromatographs and were designated as peaks P2 and P3 according to their retention times (Figure 4A). The peaks were separated by HPLC and their corresponding fractions collected for mass spectrometry analysis. The molecular ion peak of P2 was observed at m/z = 893.4, corresponding to the molecular mass of Cy3G7BG conjugated with a Glc; the peak for P3 (m/z = 1013.4) corresponded to Cy3G7BG conjugated with a Glc and a pHBA. The photodiode array spectrum of P3 supported the conclusion that the product involved conjugation with Glc and pHBA (Figure 4A) because the lmax of P3 shifted from 520 to 530 nm by bathochromic shift (shift in absorption maximum to a longer wavelength). From consideration of the structure of violdelphin, P2 was thought to be cyanidin 3-O-glucoside-7-O(6-O-(4-O-(glucosyl)-oxybenzoyl)-glucoside) (Cy3G7GBG) and P3 to be cyanidin 3-O-glucoside-7-O-(6-O-(4-O-(6-O-(p-hydroxybenzoyl)glucosyl)-oxybenzoyl)-glucoside) [Cy3G7d(BG)]. These analyses indicated that the complex structure of violdelphin was achieved through consecutive enzymatic reactions in the sepals of delphinium (Figure 4B). We designated the two enzymes for these reactions as acyl-Glc–dependent anthocyanin 7-O-(6-O-(p-hydroxybenzoyl)glucoside) glucosyltransferase (AA7BG-GT) and acyl-Glc–dependent anthocyanin 7-O-(6-O-(4-O-(glucosyl)-oxybenzoyl)-glucoside) acyltransferase (AA7GBG-AT) (Figure 4B).

Isolation of Delphinium SCPL cDNAs from Sepals

Figure 3. Schematic Diagram of the AA7G-AT Reaction. The two-arrowed lines indicate the observation of HMBC correlation signals. Glc-A and -B are identified in the six-membered rings (glucosyl groups), and Bz-I is identified in the benzene ring (p-hydroxybenzoyl group), as indicated by the data in Supplemental Table 1 online.

As delphinium AA7GT acts in vacuoles (Matsuba et al., 2010), the acylation events catalyzed by AA7G-AT and AA7GBG-AT are also likely to occur in the vacuoles. Vacuolar acyl-Glc–dependent anthocyanin acyltransferases are reported to belong to SCPL acyltransferase families (Noda et al., 2006; Fraser et al., 2007; Umemoto et al., 2009). In order to isolate cDNAs that encode AA7G-AT or AA7GBG-AT, we performed degenerate PCR using cDNA prepared from delphinium cv Blue Magic, which has violdelphin as the major anthocyanin (see Supplemental Figure 1B online). The degenerate primers were designed using the conserved amino acid sequences in SCPL acyltransferases from Arabidopsis. In Arabidopsis, these acyltransferases use sinapoylglucose as the acyl donor to generate esters, including sinapoylmalate (sinapoyl-Glc-dependent malate sinapoyltransferase [SMT]), sinapoylcholine (sinapoyl-Glc-dependent choline sinapoyltransferase [SCT]), 1,2-disinapoylglucose (sinapoyl-Glcdependent sinapoylglucose sinapoyltransferase [SST]), and sinapoylated anthocyanins (sinapoyl-Glc-dependent anthocyanin sinapoyltransferase [SAT]) (Lehfeldt et al., 2000; Shirley et al., 2001; Fraser et al., 2007). Three independent and homologous SCPL cDNA fragments were isolated; the full-length cDNAs were obtained using 59- and 39-rapid amplification of cDNA ends methods. These three homologs were designated as Dg SCPL1, Dg SCPL2, and Dg SCPL3. The Wolf PSORT algorithm (http:// www.genscript.com/psort/wolf_psort.html) predicted that these three Dg SCPLs contain a transit peptide of 30 amino acids at

4154

The Plant Cell

Figure 4. Detection of AA7BG-GT and AA7GBG-AT Activities in the Crude Extract Prepared from Sepals of Delphinium cv Triton Light Blue. (A) HPLC analyses of the enzymatic reaction products using Cy3G7BG as the acceptor. Absorbance was measured at 520 nm. The photodiode array spectra on each HPLC chromatogram show Cy3G7BG, P2, and P3 beginning at the top. mAU, milliabsorbance units. (B) Schematic diagram of the AA7BG-GT and AA7GBG-AT reactions. The arrowed line on Cy3G7GBG indicates the observation of HMBC correlation signals. Glc-A, -B, and -C are identified in the six-membered rings (glucoyl groups), and Bz-I is identified in the benzene ring (p-hydroxybenzoyl group), as indicated by the data in Supplemental Table 1 online.

the N terminus that mediates passage into the vacuole. A phylogenetic tree analysis (Figure 5) showed that Dg SCPL1 and Dg SCPL2 are close to SCPL acyl-Glc–dependent acyltransferases of Arabidopsis and Brassica napus (Milkowski et al., 2004), Lp SCPL, which catalyzes the formation of diacyl-Glc (Li et al., 1999), and Avena strigosa (As) SCPL1, which is required for triterpene acylation (Mugford et al., 2009). However, Dg SCPL3 belongs to another clade of SCPL proteins, suggesting that Dg SCPL1 and Dg SCPL2 are stronger candidates for encoding AA7G-AT and/or AA7GBG-AT. In some delphinium cultivars, delphinidin 3-O-rutinoside-7-Oglucoside (Dp3R7G) has been identified as the major anthocyanin (Hashimoto et al., 2002). The delphinium cultivars Ariel White and White Candle use Dp3R7G as the major anthocyanin (see Supplemental Figure 1 online); these cultivars have white sepals

that take on a blue tinge after picking. We speculated that the AA7G-AT reaction might not occur in the sepals of cv Ariel White and cv White Candle flowers. We prepared a crude protein extract from cv Ariel White and cv White Candle sepals and incubated these with pHBG and cyanidin 3-O-glucoside (Cy3G), Cy3,7dG, Cy3G7BG, or Cy3G7GBG at 30°C for 60 min. After termination of the reactions, Cy3,7dG and Cy3G7GBG, the products of the AA7GT and AA7BG-GT reactions, respectively, were present on both HPLC chromatographs; however, neither Cy3G7BG nor Cy3G7d(BG) (the p-hydroxybenzoylated product produced by AA7G-AT and AA7GBG-AT, respectively) were detected (see Supplemental Figure 2 online), suggesting that cv Ariel White and cv White Candle lack AA7G-AT and AA7GBG-AT activities. RT-PCR analysis of SCPL expression in the four delphinium cultivars showed a loss of SCPL2 expression in cv Ariel

Figure 5. A Molecular Phylogenetic Tree of Dg SCPLs, Arabidopsis SCPL Proteins, and AGATs Based on Amino Acid Sequences. AGATs (At SCT, Bn SCT, Lp SCPL, As SCPL1, At SMT, At SAT, and At SST) and Dg SCPLs isolated in this study are indicated in red font. At, Arabidopsis; As, A. strigosa; Bn, B. napus; Dg, D. grandiflorum; Lp, L. pennelli. Bar = 0.1 amino acid substitutions/site.

4156

The Plant Cell

White and cv White Candle (see Supplemental Figure 3A online). Possibly, this failure to obtain amplification products from cv Ariel White and cv White Candle was the result of a deletion or mutation in their SCPL2 primer binding sequences. We therefore performed genomic PCR using the primers DgSCPL2GFwd, which anneals to a region 881 bp upstream from the stop codon on the cDNA, and DgSCPL2GRev, which anneals to a sequence that includes the stop codon (see Supplemental Figure 3C online). All four cultivars yielded an amplification product of ;2.0 kb (see Supplemental Figure 3B online). These fragments were isolated and independently introduced into plasmid vectors followed by transformation into Escherichia coli. A set of eight clones was randomly picked from each transformant derived from cv Triton Light Blue, cv Blue Magic, cv Ariel White, and cv White Candle, and their nucleotide sequences were determined. Three different genomic sequences, SCPL2-1, SCPL2-2, and SCPL2-m, were identified (see Supplemental Figure 3C online). SCPL2-1 had the identical sequence in the open reading frame (ORF) to the corresponding region of SCPL2 cDNA. SCPL2-2 differed in the sequence of four nucleotides in the exon, which did not alter the amino acid sequence, and in 29 nucleotides in the introns compared with SCPL2-1. SCPL2-m had a single nucleotide deletion in the exon that generated a stop codon (see Supplemental Figure 3C online). SCPL2-2 and SCPL2-m were isolated from cv Triton Light Blue (4/8 and 4/8 clones, respectively), SCPL2-1, SCPL2-2, and SCPL2-m were isolated from cv Blue Magic (3/8, 3/8, and 2/8 clones, respectively); however, all eight clones from Ariel White and from White Candle cultivars carried SCPL2-m. We designed a primer set, DgSCPL2QFwd2 and DgSCPL2QRev2, which corresponded to an identical sequence in the ORFs of SCPL2-1, SCPL2-2, and SCPL2-m for use in RT-PCR analysis (see Supplemental Figure 3C online). Quantitative RT-PCR (qRT-PCR) analysis was performed to compare the level of expression of the different SCPLs in sepals at five developmental stages and in the leaves and stems of cv Triton Light Blue (Figure 6B) and at developmental stage 4 of the Blue Magic, Ariel White, and White Candle cultivars. In cv Triton Light Blue, SCPL1 showed relatively low expression in sepals; its highest level of expression was seen in leaves, while SCPL3 expression was relatively high in all sepal stages and in leaves. Expression of both genes was seen in the Blue Magic, Ariel White, and White Candle cultivars (Figure 6A). In cv Triton Light Blue, the highest level of SCPL2 expression was seen in stage 3 sepals; by contrast, no expression was evident in the Ariel White and White Candle cultivars. The expression pattern of SCPL2 was similar to that of other anthocyanin synthesis–related enzymes, such as chalcone synthase (CHS), flavonoid 3959-hydroxylase (F3959H), anthocyanidin synthase (ANS), and AA7GT (Figure 6A). We also investigated AA7G-AT and AA7GBG-AT activities in sepals at developmental stage 4, leaves, and stems in cv Triton Light Blue. Activities of both enzymes could be detected in leaves and stems, but the levels of activity for both were highest in the sepals (see Supplemental Figure 4 online). In summary, the various analyses described above showed that Dg SCPL2 fell into a clade that included other acyl-Glc– dependent acyltransferases, that defects in AA7G-AT and AA7GBGAT activities were correlated with loss of SCPL2 expression, and that expression of SCPL2 was similar to that of other genes encoding

enzymes involved in anthocyanin synthesis. These results indicate that SCPL2 likely encodes an AA7G-AT and/or AA7GBGAT protein. Analyses of the enzyme activities of recombinant proteins might provide biochemical confirmation that the SCPL2 encodes AA7G-AT and/or AA7GBG-AT protein. A recent report on methods of biochemical analyses of recombinant SCPL acyltransferases noted that comprehensive heterologous expression systems, such as transgenic yeasts and plants, should be used to attempt to produce enzymatically active recombinant SCPL acyltransferases for several acyl acceptors other than anthocyanins (Mugford and Milkowski, 2012). However, in previous studies where we attempted to confirm anthocyanin acyl-Glc– dependent acyltransferase activities in E. coli, yeast (Saccharomyces cerevisiae), transgenic Arabidopsis, tobacco (Nicotiana tabacum) cultured cells, and petunia (Petunia hybrida) plants, we failed to obtain evidence of enzymatic activities derived from recombinant proteins (Ozeki et al., 2011). Therefore, we chose to focus our further analyses of the catalytic properties of AA7G-AT and AA7GBG-AT using crude protein extracts prepared from delphinium sepals. Donor Preference of AA7G-AT and AA7GBG-AT Reactions in Crude Protein Extracts To eliminate any AA7BG-GT activities toward Cy3G7BG in the crude extract prepared from delphinium sepals (cv Triton Light Blue), the proteins were separated by ammonium sulfate fractionation. The ammonium sulfate fraction at 30 to 50% was selected for the AA7G-AT and AA7GBG-AT assays that used acyl-Glcs as donors and Cy3,7dG or Cy3G7GBG as acceptors. The optimum pH for AA7G-AT and AA7GBG-AT activity was approximately pH 5.0, and the optimum enzymatic reaction temperature of both enzymes was ;45°C (see Supplemental Figure 5 online). Donor preferences in AA7G-AT and AA7GBG-AT were analyzed using seven different acyl-Glcs. We found that AA7G-AT had a similar preference for sinapoyl-Glc as for pHBG, but showed a higher donor preference for feruloyl-Glc (Table 1). AA7GBG-AT also showed a high preference for feruloyl-Glc, but had a lower preference for sinapoyl-Glc than AA7G-AT (Table 1). Isolation of AA7BG-GT1 and AA7BG-GT2 cDNAs from Delphinium Sepals The glucosyl and p-hydroxybenzoyl modifications of Cy3G7BG in the crude extract (Figure 4) implied that violdelphin might be biosynthesized via step-by-step enzymatic reactions catalyzed by enzymes using pHBG as both an acyl and glucosyl donor in the sepals of delphinium. This possibility led us to speculate that the glucosyltransferase (AA7BG-GT) that catalyzes the modification with Glc to Cy3G7BG might also be a vacuolar acyl-Glcdependent anthocyanin glucosyltransferase (AAGT), similarly to those identified in our previous studies (Matsuba et al., 2010; Miyahara et al., 2012). We performed degenerate primer PCR using primers based on conserved amino acid sequences in AA7GTs isolated from delphinium and agapanthus and in AA5GT from Dianthus caryophyllus (Matsuba et al., 2010; Miyahara et al., 2012). The PCR yielded Dg AA7GT and 10 other independent AAGT homologous cDNA fragments; the full-length cDNAs were

Biosynthesis of Polyacyl Anthocyanin

4157

Figure 6. Expression Profiles of AA7BG-GT and SCPL Transcripts. (A) The expression levels of AA7BG-GT, SCPL, and the anthocyanin biosynthetic genes CHS, F3959H, ANS, and AA7GT. Le, leaves; St, stems; BM, sepals of cv Blue Magic (S4); AW, sepals of cv Ariel White (S4); WC, sepals of cv White Candle (S4). Error bars indicate 6 SD for three biological replicates. ND, not detectable. (B) Sepals at five developmental stages (S1 to S5) of cv Triton Light Blue plants. S4 is the initiation of anthesis, and S5 is 1 or 2 d after anthesis. A detailed description of the developmental stages is given in Methods.

obtained using 59- and 39-rapid amplification of cDNA ends. The predicted amino acids encoded by the full-length cDNAs indicate they encode proteins that belong to the group of plant b-glucosidases (BGLUs) of GH1, similar to AA7GTs and AA5GT. These 10 homologs were designated Dg BGLU1 to Dg BGLU10. To confirm whether these candidate cDNAs have an AA7BG-GT activity for transferring Glc from pHBG to Cy3G7BG, full-length cDNAs were introduced into expression vectors; each recombinant protein was expressed with a His-tag in E. coli. AA7BG-GT activity was detected using Cy3G7BG and pHBG as the substrates for partially purified recombinant proteins. An HPLC analysis of the products of the enzymatic reactions using the recombinant proteins showed that two, BGLU7 and BGLU10, had the ability to attach Glc to Cy3G7BG in a mixture

containing both pHBG and Cy3G7BG (Figure 7). The molecular ion peak was observed at m/z = 893.4, corresponding to P2 (Figure 4A). We analyzed the reaction product of BGLU7 using NMR spectroscopy, which showed the presence of an additional Glc moiety (Glc-C) (see Supplemental Table 1 online). 1H{13C} HMBC analysis showed a cross-peak between H-1 of the Glc-C (d 4.73) and C-4 of Bz-I (d 162.9) in the Cy3G7BG (arrowed line in Figure 4B); this result was confirmed by NOESY, which showed a cross-peak between H-1 of the Glc-C (d 4.73) and H-3 and H-5 of Bz-I (d 6.88). We therefore concluded that the product of BGLU7 and BGLU10 was Cy3G7GBG. We designated BGLU7 and BGLU10 as AA7BG-GT1 and AA7BG-GT2, respectively.

4158

The Plant Cell

Table 1. Acyl Donor Preferences of AA7G-AT and AA7GBG-AT Relative Activity (%) Acyl Donor

AA7G-AT

AA7GBG-AT

p-Hydroxybenzoyl-1-O-b-Glc Vanillyl-1-O-b-Glc Isovanillyl-1-O-b-Glc Vanillyl-1-O-b-Gal p-Coumaroyl-1-O-b-Glc Feruloyl-1-O-b-Glc Sinapoyl-1-O-b-Glc

100.0 6 6.0 7.0 6 0.2 3.0 6 0.03 ND 31.0 6 0.2 170.0 6 0.2 98.0 6 13.0

100.0 6 6.5 12.8 6 0.6 8.0 6 0.4 ND 44.6 6 1.5 137.0 6 3.8 65.8 6 2.4

Analyses of donor preferences were performed with Cy3,7dG, or Cy3G7GBG as the acceptor. Relative activities were calculated by the area of HPLC chromatogram recorded at 520 or 530 nm. All values are means 6 SD of at least three independent determinations. Activities with pHBG of AA7G-AT and AA7GBG-AT were 1.73 pkat mg21 protein and 4.14 pkat mg21 protein, respectively, and were set as 100%. ND, no significant peaks were detected.

Phylogenetic Analysis of AA7BG-GT1 and AA7BG-GT2 Both AA7BG-GT1 and AA7BG-GT2 cDNAs are 1530 bp in length and contain an ORF encoding 510 amino acids residues; the nucleotide sequences of each cDNA showed 87% identity, and the amino acid sequence identity and protein similarity were 82 and 96%, respectively. The first 30 amino acid sequence of each protein was predicted by the Wolf PSORT algorithm to be a transit peptide that mediated passage into the vacuole, suggesting that these enzymes were active in the vacuole. We performed a phylogenetic analysis of Dg AA7BG-GT1, Dg AA7BG-GT2, and the other eight Dg BGLUs with plant GH1s, including AAGTs reported previously. Interestingly, Dg AA7BG-GT1 and Dg AA7BG-GT2 belonged to a different clade than previously reported AAGTs; this clade included Dg AA7GT, Aa AA7GT, Dc AA5GT, and At BGLU10 (Matsuba et al., 2010; Miyahara et al., 2012, 2013) (Figure 8). Although Dg BGLU2, Dg BGLU8, and Dg BGLU9 had high similarities (67, 53, and 55%, respectively) to Dg AA7GT, they did not show AA7BG-GT activity. Expression Analyses of AA7BG-GT1 and AA7BG-GT2 in Delphinium The expression profiles of AA7BG-GT1 and AA7BG-GT2 were examined in the Triton Light Blue, Ariel White, and White Candle cultivars using qRT-PCR analysis. Like other genes involved in anthocyanin synthesis, both AA7BG-GT1 and AA7BG-GT2 showed significant expression in sepals, including sepals of the Ariel White and White Candle cultivars, with the highest level of expression at stage 3; this pattern of expression is similar to that of genes involved in anthocyanin synthesis, such as CHS, F3959H, Dg ANS, and AA7GT (Figure 6B). Therefore, we conclude that AA7BG-GT1 and AA7BG-GT2 were likely to be involved in the 7-polyacylation of anthocyanin and their expression might be regulated by a common transcription factor. Acceptor and Donor Preferences of Recombinant AA7BG-GT1 and AA7BG-GT2 As the level of production of delphinium AA7BG-GT1 and AA7BG-GT2 proteins in E. coli was extremely low compared

with that of AA7GT (Matsuba et al., 2010), making it hard to purify them, we used partially purified recombinant AA7BG-GT1 and AA7BG-GT2 proteins to characterize their enzymatic properties. The optimum temperature for both enzymes was 35°C, and optimum activities occurred at pH 5.5 (see Supplemental Figure 5 online). We analyzed the acceptor preferences of AA7BG-GT1 and AA7BG-GT2 using various acylated anthocyanins as acceptor molecules: cyanidin 3-O-rutinoside-7-O(6-O-(p-hydroxybenzoyl)-glucoside) (Cy3R7BG), delphinidin 3-O-rutinoside-7-O-(6-O-(p-hydroxybenzoyl)-glucoside) (Dp3R7BG), and delphinidin 3-O-rutinoside-7-O-(6-O-(p-coumaroyl)-glucoside) (Dp3R7 coumaroylG). Both AA7BG-GT1 and AA7BG-GT2 showed a preference for Cy3G7BG and Cy3R7BG, the same as Dp3R7BG, which acts as an in vivo acceptor in delphinium. Interestingly, neither enzyme recognized Dp3R7coumaroylG as an acceptor (Table 2). The donor preferences were also analyzed using seven different acyl-Glcs and UDP-Glc with Dp3R7BG as the acceptor. We found that pHBG acted more efficiently as a donor molecule for both enzymes than other acyl-Glcs (Table 2). Subcellular Localization of SCPL2, AA7BG-GT1, and AA7BG-GT2 Proteins To confirm the subcellular locations of SCPL2, AA7BG-GT1, and AA7BG-GT2, constructs with green fluorescent protein (GFP) fused to the first 30–amino acid sequences of each protein and driven by the cauliflower mosaic virus 35S promoter were introduced into onion (Allium cepa) epidermal cells by microprojectile bombardment. In the cells, the transit peptides of SCPL2, AA7BG-GT1, and AA7BG-GT2:GFP exhibited a similar pattern of fluorescence to a Gly-rich protein of Arabidopsis (GRP5) that is normally located in the vacuoles (see Supplemental Figure 6 online) (Mangeon et al., 2009). Accumulation of Acyl-Glc and Cyanodelphin in Delphinium AA7G-AT and AA7GBG-AT recognized various acyl donors in both AT reactions (Table 1). We examined sepals at five developmental stages and leaves and stems (Figure 6B) for evidence of the accumulation of any of these acyl-Glcs; only pHBG was detected, and the acyl-Glc molecules listed in Table 1 were under the detection level on HPLC analysis. We next a performed an HPLC analysis and found that accumulation of pHBG and cyanodelphin were detected only in sepals and that the highest level of the former occurred at S4 to S5; the highest level of cyanodelphin accumulation was also detected in these sepal stages (Figure 9). These results indicate that pHBG is the key substrate for glucosyl and acyl donors and that polyacylation of anthocyanins occurs after the accumulation of sufficient pHBG.

DISCUSSION Violdelphin and cyanodelphin are the major anthocyanins for the purple-blue coloration of delphinium flowers (see Supplemental Figure 1 online) (Hashimoto et al., 2002). Many different polyacylated anthocyanin structures have been identified in a range of species (Yoshida et al., 2009). Intramolecular copigmentation of the anthocyanidin chromophore and the aromatic acyl moieties

Biosynthesis of Polyacyl Anthocyanin

Figure 7. HPLC Analysis of the Reaction Products of AA7BG-GT1 and AA7BG-GT2 Recombinant Proteins. HPLC elution profiles of the products of AA7BG-GT1 and AA7BG-GT2 reactions in the presence or absence of the donor molecule. Absorbance was measured at 520 nm. mAU, milliabsorbance units.

4159

may contribute to the stable blue coloration (Goto and Kondo, 1991; Yoshida et al., 1992; Takeda et al., 1994). Many of the enzymatic reaction steps to synthesize anthocyanin aglycone have been clarified (Tanaka et al., 2008). Various studies have shown that glycosylation at the 3-position of anthocyanidin is commonly catalyzed by UDP sugar–dependent glycosyltransferase (Furtek et al., 1988; Ralston et al., 1988; Brugliera et al., 1994); however, the enzymes involved in glucosylation and acylation of polyacylated anthocyanins, especially at the 7-position of the anthocyanin, have still not been fully elucidated. Recently, we showed that AA7GT catalyzes the glucosyltransfer reaction at the 7-position of anthocyanin in delphinium and agapanthus and that the enzyme uses pHBG as the glucosyl donor (Matsuba et al., 2010; Miyahara et al., 2012). As AA7GT functions in the vacuole, it is likely that the subsequent reactions for acyl and glucosyl transfer after 7-glucosylation also occur there (Matsuba et al., 2010). Vacuolar protein acyltransferases that use acyl-Glc as the acyl donor, termed SCPL proteins, have been identified (Ozeki et al., 2011; Mugford and Milkowski, 2012). The production of Cy3G7GBG and Cy3G7d(BG) after p-hydroxybenzoylation by AA7G-AT and after prolonged incubation (Figures 2 and 4) suggests that pHBG is used as a bifunctional donor to form violdelphin. The postulated order of the modification steps at the anthocyanin 7-position during formation of violdelphin is AA7GT, AA7G-AT, AA7BG-GT, and AA7GBG-AT (Figure 1). Previous reports about the synthesis of cyanodelphin via violdelphin in delphinium suggested this biosynthetic route (Brandt et al., 1993; Hashimoto et al., 2002). These reactions to modify the 7-position on the basic skeleton are catalyzed by acyl-Glc–dependent acyltransferase and glucosyltransferase, which use pHBG as acyl and glucosyl donors, respectively. We therefore propose that the delphinium acyl-Glcs can be termed Zwitter donors, by analogy with the concept of Zwitterions in chemistry (Figure 1). In some species, hydroxycinnamic acids are transferred by SCPL acyltransferases (Noda et al., 2006; Fraser et al., 2007). More recently, hydroxybenzoic acid transfer activity (i.e., galloic acid and benzoic acid) was reported to be catalyzed by SCPL enzymes (Lee et al., 2012; Liu et al., 2012). Since SCPL acyltransferases use acyl-Glc as the acyl donors and are thought to be located in the vacuole (Hause et al., 2002; Fraser et al., 2005), acylation after the 7-glucosylation step might be expected to be catalyzed by an SCPL-type enzyme. Here, three anthocyanin acyl-Glc–dependent acyltransferase (AGAT) candidate cDNAs were isolated by degenerate primer PCR using the amino acid conserved region of SCPL proteins. A Wolf PSORT search predicted that the product of one, SCPL2, contained a putative transit peptide for movement into the vacuole. Indeed, GFP fusion constructs containing the first 30 amino acid sequences caused accumulation of GFP in the vacuoles of onion epidermal cells in a manner similar to that of AA7BG-GT1 and AA7BG-GT2 (see Supplemental Figure 6 online). Our phylogenetic tree analysis (Figure 5) showed that Dg SCPL2 is included in the same clade as other AGATs reported previously and might be a candidate for AA7G-AT and/or AA7GBG-AT proteins. SCPL2 was expressed predominantly in sepals, and its expression profile was very similar to other anthocyanin biosynthetic genes (Figure 6). AA7G-AT and AA7GBG-AT activities in sepals and stems in cv Triton Light Blue were 20-fold and twofold higher than in leaves, respectively

Figure 8. A Molecular Phylogenetic Tree of AAGT and Plant b-Glycosidases of GH1 Based on Amino Acid Sequences. AGATs (Dc AA5GT, Dg AA7GT, Aa AA7GT, At BGLU10, Dg AA7BG-GT1, and Dg AA7BG-GT2) are indicated in red font. Bar = 0.1 amino acid substitutions/site.

Biosynthesis of Polyacyl Anthocyanin

Table 2. Substrate Specificities of Recombinant AA7BG-GT1 and AA7BG-GT2 Relative Activity (%)

Glc acceptor Cyanidin Cy3G Cyanidin 3-O-(6-O-malyl) glucoside Cyanidin 3,5-O-diglucoside Cy3,7dG Cy3G7BG Cy3R7BG Dp3R7BG Dp3R7coumaroylG Dp3R7d(BG) (violdelphin) Glc donor p-Hydroxybenzoyl-1-O-b-Glc Vanillyl-1-O-b-Glc Isovanillyl-1-O-b-Glc p-Coumaroyl-1-O-b-Glc Caffeoyl-1-O-b-Glc Feruloyl-1-O-b-Glc Sinapoyl-1-O-b-Glc UDP-Glc

AA7BG-GT1

AA7BG-GT2

ND ND ND ND ND 97.0 6 0.8 97.0 6 0.4 100.0 6 0.7 ND ND

ND ND ND ND ND 114.4 6 1.8 110.7 6 0.9 100.0 6 3.4 ND ND

100.0 6 0.8 6.9 6 0.4 4.4 6 0.2 53.3 6 0.5 13.6 6 0.2 48.1 6 0.5 14.3 6 0.3 ND

100.0 6 0.01 13.5 6 0.6 8.2 6 0.3 18.3 6 1.5 7.6 6 0.4 15.7 6 0.4 21.8 6 0.8 ND

Analyses of acceptor preferences were performed with pHBG as the Glc donor. Relative activities were calculated using the area of the HPLC chromatogram recording at 520 nm (Cy3G7BG), 523 nm (Cy3R7BG), and 533 nm (Dp3R7BG). Analyses of donor preferences were performed with Dp3R7BG as the acceptor. Relative activities were calculated using the area of the HPLC chromatogram recording at 533 nm. All values are means 6 SD of at least three independent determinations. AA7BG-GT1 and AA7BG-GT2 activities with Cy3G7BG and pHBG gave means of 14.5 and 6.37 pkat mg21 protein, respectively. ND, no significant peaks were detected.

(see Supplemental Figure 4 online); these higher levels showed a similar pattern as the relative expression levels of SCPL2 in sepals, stems, and leaves (Figure 6). SCPL2 transcription was not detected in the Ariel White and White Candle cultivars. AA7G-AT and AA7GBG-AT activities were undetectable in both cultivars, although AA7BG-GT1 and AA7BG-GT2 expression and AA7BG-GT activity were detected (Figure 6; see Supplemental Figure 2 online). These results supported the idea that the depletion of AA7G-AT and AA7GBG-AT in the Ariel White and White Candle cultivars is due to the loss of function of these genes rather than due to lesions in their regulatory genes. Genomic PCR of SCPL2 in the Ariel White and White Candle cultivars gave a single amplicon of SCPL2-m, which has a single nucleotide deletion in an exon that produced a stop codon (see Supplemental Figure 3 online). By contrast, two amplicons (SCPL2-2 and SCPL2-m) were isolated from cv Triton Light Blue, and three amplicons (SCPL2-1, SCPL2-2, and SCPL2-m) were isolated from cv Blue Magic. This genomic sequencing analysis suggests that SCPL2-1 and SCPL2-2 might be allelic, that SCPL2-m is a pseudogene, and that the Ariel White and White Candle cultivars contain a mutant allele of SCPL2. Our results also indicate that SCPL2 cDNA might encode AA7G-AT and/or AA7GBG-AT. However, it is still possible that AA7G-AT is encoded by SCPL2 and that another gene encodes the SCPL protein responsible for the AA7GBG-AT reaction.

4161

We isolated two cDNAs for the AA7BG-GT reaction, which is necessary for polyacylation of the 7-position of anthocyanins in delphinium, and expressed recombinant proteins with enzymatic activity in vitro using E. coli cells. Both enzymes had the ability to transfer a Glc moiety from pHBG to the hydroxybenzoyl group of Dp3R7BG. We also isolated two AAGT cDNAs in our previous study: One cDNA encodes an enzyme for the 5- or 7-O-glucosylation of the anthocyanin skeleton in carnations, delphiniums, or agapanthus (Matsuba et al., 2010; Miyahara et al., 2012); the other cDNA encodes an AAGT that does not catalyze glucosylation of aglycone directly in Arabidopsis but acts on the p-coumaroyl moiety of cyanidin 3-O-[2-O-(2-O(sinapoyl) xylosyl) 6-O-(p-coumaroyl) glucoside]-5-O-(6-O(malonyl) glucoside) (Miyahara et al., 2013). All of the AAGTs belong to plant b-glucosidases of the GH1 group of proteins and are in the same clade (Matsuba et al., 2010; Miyahara et al., 2012, 2013). However, the amino acid sequences of Dg AA7BGGT1 and Dg AA7BG-GT2 belong to a distinct group from those of AAGTs (Figure 8). Thus, Dg AA7BG-GT1 and Dg AA7BG-GT2 might not originate from a common GH1 gene (i.e., Dg AA7BG-GTs and Dg AA7GT might have acquired their glucosyltransferase activity for anthocyanin after the divergence of GH1 proteins). Moreover, Dg AA7BG-GTs might not have arisen by a gene duplication event of a common ancestral gene to Dg AA7GT. This possibility also suggests that the switch from a glycoside hydrolase activity to a glucosyltransferase activity using different Glc acceptor substances might have occurred at a higher frequency in different members of GH1 proteins during evolution. In plants, GH1 proteins are believed to play important roles in many diverse processes, including chemical defense against herbivory, lignification, hydrolysis of cell wall–derived oligosaccharides, and the regulation of active phytohormone levels; these various activities may have been acquired by gene duplications (Xu et al., 2004; Gómez-Anduro et al., 2011). Therefore, it is possible that acyl-Glc–dependent glucosyltransferases may not be limited to anthocyanin glucosyltransferases but also occur in other secondary metabolites in plants. Indeed, rice (Oryza sativa) Os9BGlu31 is a transglucosidase with the capacity to equilibrate

Figure 9. pHBG and Cyanodelphin Accumulation in Delphinium cv Triton Light Blue. Amounts of accumulated pHBG (black bars) and cyanodelphin (gray bars), respectively. S1 to S5, sepals at five developmental stages. Error bars indicate 6 SD for three biological replicates. Le, leaves; St, stems; ND, not detectable; FW, fresh weight.

4162

The Plant Cell

phenylpropanoid, flavonoid, and phytohormone glycoconjugates (Luang et al., 2013). pHBG is believed to be a key substance in violdelphin and cyanodelphin biosynthesis as it is used as both an acyl and glucosyl donor (Figure 1). The question why the acyl moiety of violdelphin and cyanodelphin is composed only of pHBA, whereas AA7G-AT and AA7GBG-AT recognize other acyl donors in vitro (Table 1), might be answered by the results of our analysis of accumulation of different acyl-Glcs in the sepals of delphinium. Our HPLC analysis showed a lack of acyl-Glc species other than pHBG; moreover, pHBG accumulation was detected only in sepals where anthocyanin synthesis occurred. The highest level of accumulation was in S4, just before S5; this is the same time as that of the highest accumulation of cyanodelphin (Figure 9). AA7BG-GT1 and AA7BG-GT2 preferred pHBG as the glucosyl donor (Table 2), whereas AA7G-AT and AA7GBG-AT used feruloyl-Glc more efficiently as the acyl donor than pHBG. The facts that acyl-Glcs other than pHBG could not be detected in delphinium sepals (Figure 9) and that violdelphin synthesized in vivo contains only p-hydroxybenzoyl groups suggest that pHBG is used in vivo by acyltransferase and glucosyltransferase as a bifunctional donor. The Glc ester (i.e., pHBG in delphinium) might be expected to be synthesized by UDP-Glc–dependent glucosyltransferase with pHBA as an acceptor (Leznicki and Bandurski, 1988; Mock and Strack, 1993; Owatworakit et al., 2012; Figure 1). AA7BG-GT1 and AA7BG-GT2 also recognized various acylglucoses as Glc donors. However, with regard to acceptors, both AA7BG-GTs recognized Dp3R7BG but not Dp3R7coumaroylG; the former is acylated by pHBA (C6-C1 skeleton) and the latter by p-coumaric acid (C6-C3 skeleton) at the 7-position Glc attached to the anthocyanidin chromophore (Table 2). The strict acceptor recognition of AA7BG-GTs and pHBG accumulation in sepals might define the anthocyanin molecule as violdelphin in delphiniums. A 10-nm bathochromic shift of the lmax was observed in pHBA-conjugated anthocyanins (Figures 2 and 4). This shift is probably caused by intramolecular copigmentation between the anthocyanidin chromophore and the aromatic ring of pHBA. This intramolecular copigmentation effect might contribute to the occurrence of blue flowers in delphiniums. However, with the exception of the Ariel White and White Candle cultivars, we have been unable to identify other SCPL2 mutants that lack AA7BG-GT activity. It is likely that use of RNA interference (RNAi) stable transformants, in which the levels of SCPL2, AA7BG-GT1, and AA7BG-GT2 transcripts are reduced, will be informative on the roles of these enzymes in vivo. The loss of AA7BG-GT activity should cause the accumulation of Dp3R7BG, which would be detectable as a 10-nm bathochromic shift of the lmax. Therefore, RNAi stable transformants might not only confirm that enzymes encoded by AA7BG-GT1 and AA7BG-GT2 act in vivo as AA7BG-GTs, but might also be of horticultural interest due to the development of novel flower colors. We previously established a system in delphinium in which transgenes can be introduced to generate stable transformants (Hirose et al., 2002). Our future work will be to prepare RNAi stable transformants with reduction of both AA7BG-GT1 and AA7BG-GT2 transcripts using consensus nucleotide sequences as the RNAi target sequence.

METHODS Plant Materials and Chemicals The delphinium (Delphinium grandiflorum) cultivars Triton Light Blue, Ariel White, and White Candle were purchased from a flower market; the two white cultivars were originally established by Miyoshi and Co. and Sakata Seed Co., respectively. D. grandiflorum cv Blue Magic was a gift from the Ehime Research Institute of Agriculture, Forestry, and Fisheries (see Supplemental Figure 1 online). Sepals from five different developmental stages (S1 to S5; shown in Figure 6B), leaves, and stems of cv Triton Light Blue were collected and immediately frozen in liquid nitrogen and stored at – 80°C until use. Five flower developmental stages were selected: S1, S2, and S3 had bud lengths of ;0.5, 1, and 1.5 cm, respectively, and preceded anthesis; S4 was the initiation of anthesis; and S5 was 1 or 2 d after anthesis. Acyl-Glcs, including pHBG, were synthesized using recombinant glucosyltransferase according to the method described previously (Matsuba et al., 2008). Cy3,7dG and cyanidin 3-O-rutinoside-7-O-glucoside were obtained by enzymatic reactions using recombinant AA7GT, as described previously (Matsuba et al., 2010). Cyanodelphin, violdelphin, and Dp3R7G were extracted and purified from cv Triton Light Blue, cv Blue Magic, and cv Ariel White delphinium plants, respectively. Acylated anthocyanins, Cy3R7BG, and Dp3R7BG were obtained by enzymatic reactions using partially purified crude extracts from an ammonium sulfate fraction at 30 to 50% prepared from sepals of cv Triton Light Blue with pHBG and cyanidin 3-O-rutinoside-7-O-glucoside or Dp3R7G as the substrate. In the same way, Dp3R7coumaroylG was synthesized using p-coumaroyl-Glc instead of pHBG. The molecular ion peaks of acylated anthocyanins were observed at m/z = 877.5 [M] + (Cy3R7BG), m/z = 915.2 [M + Na]+ (Dp3R7BG), and m/z = 920.4 [M]+ (Dp3R7coumaroylG), and the results were consistent with the expected structure. Measurement of Cyanodelphin and pHBG Accumulation in Delphinium Total anthocyanin and pHBG were extracted from sepals at five different developmental stages (Figure 6B), leaves, and stems of cv Triton Light Blue using 80% methanol containing 0.1% trifluoroacetic acid. The concentration of total anthocyanin was measured at 540 nm using a spectrophotometer and delphinidin as the standard. The ratio of cyanodelphin to total anthocyanin was calculated by comparison of peak areas using HPLC at 540 nm. pHBG content was estimated at an absorbance of 260 nm using HPLC and calculated by comparison with peak areas using synthetic pHBG as the standard. Protein Extraction and Assay of AA7G-AT and AA7GBG-AT Enzymes To assay the activities of anthocyanin acyltransferases and glucosyltransferases, 1 g of frozen tissue was ground into powder using a mortar and pestle in liquid nitrogen and then placed into 4 mL of extraction buffer (0.1 M potassium phosphate, pH 7.5). Cell debris was removed by centrifugation at 15,000g for 5 min. Proteins were precipitated using 75% ammonium, suspended with extraction buffer, and then desalted using a G-25 spin column (GE Healthcare). In the AA7G-AT and AA7GBG-AT assays, the ammonium sulfate fraction at 30 to 50% was used. Standard enzymatic reactions were performed in a reaction mixture consisting of 3 nmol Cy3,7dG or Cy3G7GBG, 45 nmol pHBG, 2.4 mmol citrate buffer, and 5 to 25 µg of crude protein in a total volume of 30 mL; the mixture was incubated at 30°C for 1 h. Protein concentrations were quantified using a CBB protein assay kit (Thermo Fisher Scientific) with BSA as the standard. Cloning of SCPL and AA7BG-GT cDNAs from Delphinium Total RNA was isolated from delphinium sepals (cv Triton Light Blue) at developmental stage 3 using an RNeasy Plant mini kit (Qiagen).

Biosynthesis of Polyacyl Anthocyanin

First-strand cDNA was synthesized from 500 ng of total RNA using oligo(dT)s and PrimeScript reverse transcriptase (Takara Bio). SCPL cDNA fragments were obtained using the degenerate primer set SCPLdgFwd and SCPLdgRev; AA7BG-GT fragments were obtained using the degenerate primer set AAGTdgFwd and AAGTdgRev (see Supplemental Table 2 online). Degenerate PCR was performed in a 50-mL reaction mixture containing 1 mL of first-strand cDNA, 50 pmol of each primer, 12.5 nmol of deoxynucleotide triphosphates, 53 Prime STAR GXL buffer, and 1.25 units of Prime STAR GXL Polymerase (Takara Bio). The reaction conditions were as follows: 3 min of denaturation at 96°C, then 30 cycles of 10-s denaturation at 96°C, 15-s annealing at 45°C, 20-s extension at 68°C, and a final 1-min extension at 68°C. The amplicons were subcloned into pBluescript SK+ vector. The sequences of the cDNA fragments were determined using a BigDye-Terminator ver.3.1 cycle sequencing kit and an ABI PRISM 3130xl (Applied Biosystems). The 59 and 39 cDNA ends of SCPLs and AA7BG-GTs were amplified with a SMARTer RACE cDNA amplification kit (Clontech Laboratories). Other anthocyanin synthesis genes in Delphinium, namely, chalcone synthase (CHS), flavonoid 3959-hydroxylase (F3959H), anthocyanidin synthase (ANS), and the housekeeping gene actin (Actin) were amplified using the degenerate primer sets CHSdgFwd and CHSdgRev, F3959HdgFwd and F3959HdgRev, ANSdgFwd and ANSdgRev, ActindgFwd and ActindgRev, respectively (see Supplemental Table 2 online). Genomic DNAs were extracted from the sepals of delphinium cultivars using a DNeasy Mini Kit (QIAGEN). Genomic PCR for SCPL2 was performed with genomic DNA prepared from each cultivar as the template using the primers DgSCPL2GFwd and DgSCPL2GRev. Prime STAR GXL DNA Polymerase (Takara Bio) was used for PCR using the following conditions: 3 min of denaturation at 96°C, followed by 37 cycles of 10-s denaturation at 96°C, 15-s annealing at 56°C, and 2-min extension at 68°C. The amplicons were subcloned into pBluescript SK+ vector and the nucleotide sequence of the eight independent clones for each cultivar was determined as described above. Heterologous Production of AA7BG-GT1 and AA7BG-GT2 in Escherichia coli The putative signal peptides of both AA7BG-GT cDNAs, except for the first Met, were deleted by PCR using the following primer sets: for AA7BG-GT1, DgAA7BG-GT1delFwd and DgAA7BG-GT1rtRev; and for AA7BG-GT2, DgAA7BG-GT2delFwd and DgAA7BG-GT2rtRev (see Supplemental Table 2 online). The amplicons were inserted into pTrcHis2 TOPO vector by a topoisomerase reaction using the pTrcHis2 TOPO expression kit (Invitrogen Life Technologies). E. coli strain BL21 (DE3) was used as the host for expression of recombinant AA7BG-GT1 and AA7BG-GT2. E. coli harboring pTrcHis2AA7BG-GT1 or AA7BG-GT2 was inoculated into 600 mL of Luria-Bertani medium containing 50 µg/mL ampicillin and grown at 30°C until an A600 of 0.6 was reached. After the addition of 600 mL of 1 M isopropyl-b-D-thiogalactopyranoside, E. coli was cultured at 16°C for 20 h. The cells were harvested by centrifugation, resuspended in 10 mL of 0.1 M potassium phosphate buffer, pH 7.4, and disrupted by sonication (UD-201; Tomy Seiko). After centrifugation of the lysate at 15,000g for 5 min, the supernatant was partially purified as a His fusion protein using MagneHis Ni-Particle (Promega Biosciences) in accordance with the manufacturer’s protocol. Standard enzymatic reactions were performed in a reaction mixture consisting of 3 nmol Cy3G7BG, 45 nmol pHBG, 2.4 mmol citrate buffer, and 6 to 9 or 8 to 12 µg of partially purified protein (AA7BG-GT1 and AA7BG-GT2, respectively) in a total volume of 30 mL; the mixture was incubated at 35°C for 1 h. Protein concentrations were quantified as described above. qRT-PCR Analysis Total RNA was isolated from sepals at five developmental stages (Figure 6B), leaves and stems from cv Triton Light Blue and from sepals

4163

(S4) of the Blue Magic, Ariel White, and White Candle cultivars. Firststrand cDNAs were synthesized as described above. The primer sets for the amplification were as follows: for CHS, DgCHSQFwd and DgCHSQRev; for F3959H, DgF3959HQFwd and DgF3959HQRev; for ANS, DgANSQFwd and DgANSQRev; for AA7GT, DgAA7GTQFwd and DgAA7GTQRev; for SCPL1, DgSCPL1QFwd and DgSCPL1QRev; for SCPL2, DgSCPL2QFwd and DgSCPL2QRev or DgSCPL2QFwd2 and DgSCPL2QRev2; for SCPL3, DgSCPL3QFwd and DgSCPL3QRev; for AA7BG-GT1, DgAA7BG-GT1QFwd and DgAA7BG-GT1QRev; for AA7BG-GT2, DgAA7BG-GT2QFwd and DgAA7BG-GT2QRev; and for Actin, DgActinQFwd and DgActinQRev. The primer sets were designed using GENETYX Ver.11 Primer3 (Genetyx). qRT-PCR was performed using SYBR Premix Ex Taq (Perfect Real Time; Takara Bio) and the DNA Engine Opticone 2 (Bio-Rad Laboratories). The reaction condition was 2 min at 94°C and then 32 cycles for each pair of primers, consisting of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C. Actin was used as a reference gene. Each assay was repeated at least three times. HPLC Conditions Enzymatic reaction products were separated by an HPLC-photodiode array detector system (LaChrome Elite; Hitachi High-Technologies) equipped with an ODS column (i.d. 4.6 3 250 mm; Wakopak Handy ODS; Wako Pure Chemical Industries) using a linear gradient elution (1 mL min21) of 10 to 26% acetonitrile in 1.5% aqueous phosphoric acid for 20 min or an ODS column (i.d. 4.6 3 50 mm; COSMOSIL 5C18-MS-II; Nacalai Tesque) using a linear gradient elution (1.5 mL min21) of 9 to 20% acetonitrile in 1.5% aqueous phosphoric acid for 10 min. To analyze the enzymatic properties of AA7G-AT, AA7GBG-AT, AA7BG-GT1, and AA7BG-GT2, a short ODS column (i.d. 4.6 3 50 mm; COSMOSIL 5C18-MS-II) and short method (linear gradient elution [1.5 mL min21] of 15 to 80% methanol in 1.5% aqueous phosphoric acid for 5 min) were used as the HPLC conditions. Assignment of Enzymatic Reaction Products The products of the AA7G-AT and AA7BG-GT reactions were separated by flash liquid chromatography (YFLC-AI-580; Yamazen) and reversephase columns (Hi-Flash columns, ODS-S, 50 mm, i.d. 20 3 65 mm, followed by i.d. 26 3 100 mm; Yamazen) using a linear gradient elution (20 mL min21) of 15 to 70% methanol in 0.1% aqueous trifluoroacetic acid for 20 min. The anthocyanin fractions were collected and then the combined anthocyanin fraction was obtained by evaporation. The molecular masses of the enzymatic reaction products were confirmed by electrospray ionization–mass spectrometry (AccuTOF MS, JMST100LC; JEOL). NMR spectra (1H NMR, 13C NMR, 1H{13C} heteronuclear multiple quantum coherence, 1H{13C} HMBC, TOCSY, H-H correlated spectroscopy, and NOESY) were analyzed in methanol-d4-trifluoroacetic acid-d (9:1) on a JNM-ECA-500 spectrometer (JEOL). 1H NMR (500 MHz) and 13C NMR (125 MHz) chemical shifts were referenced to the residual solvent (methanol-d4) signal at dH 3.35 and dC 49.3, respectively. Subcellular Localization of SCPL2, AA7BG-GT1, and AA7BG-GT2 Nucleotide sequences corresponding to the putative transit peptide (30 amino acid sequence) of SCPL2, AA7BG-GT1, and AA7BG-GT2 were prepared by PCR, and these amplicons were introduced into the pDONR/ zeo vector using BP clonase (Invitrogen). These entry vectors for the Gateway System (Invitrogen) were recombined with GFP chimeric expression destination vector pGWB405 (Nakagawa et al., 2009). GFP fusion constructs containing At GRP5 and pSK-GFP plasmid harboring a GFP cDNA driven by cauliflower mosaic virus 35S promoter in pBluescript SK+ vector were prepared as described (Matsuba et al., 2010) and used as the control for onion (Allium cepa) epidermal cells. Transformation of onion epidermal cells was performed as described previously (Matsuba et al., 2010).

4164

The Plant Cell

Phylogenetic Analysis

AUTHOR CONTRIBUTIONS

The amino acid sequences of GH1 and SCPL genes were obtained by GenBank and BLAST searches (see Supplemental Tables 3 and 4 online). The sequences were aligned using ClustalW (Thompson et al., 1994) in the default setting. A full alignment of the sequences used is available as Supplemental Data Sets 1 and 2 online. The phylogenetic tree and bootstrap values were obtained using the neighbor-joining algorithm with the following parameters: Poisson correction, complete deletion, and bootstrap (1000 replicates) in MEGA version 5.05 (Tamura et al., 2007).

Y.N. and M.Yasunaga. contributed equally to the design of all experiments. E.O. isolated and characterized the cDNAs. M.O. and Y.H. prepared the delphinium plants. Y.N., M.Yamaguchi., Y.O., and N.S. wrote the article. Y.O. conducted all experiments. All authors provided comments.

Received April 30, 2013; revised August 24, 2013; accepted October 7, 2013; published October 31, 2013.

Accession Numbers Sequence data from this article can be found in the GenBank/EMBL/DDBJ databases under accession numbers AB811444 (AA7BGGT1), AB811447 (AA7BGGT2), AB811448 (SCPL1), AB811449 (SCPL2), AB811450 (SCPL3), AB819288 (CHS), AB819289 (F3959H), AB819287 (ANS), and AB819286 (Actin). The genomic sequences of SCPL2 can be found under the accession numbers AB844439 (SCPL2-1), AB844440 (SCPL2-2), and AB844441 (SCPL2-m). Accession numbers for sequences used in the phylogenetic analysis can be found in Supplemental Tables 3 and 4 online. Supplemental Data The following materials are available in the online version of this article. Supplemental Figure 1. The Flower Phenotypes of Four Delphinium Cultivars and the Major Anthocyanins Accumulating in Each Cultivar. Supplemental Figure 2. Detection of AA7GT, AA7G-AT, AA7BG-GT, and AA7GBG-AT Activities in Crude Extracts Prepared from Sepals of Delphinium Cultivars Ariel White and White Candle. Supplemental Figure 3. RT-PCR Analysis of SCPLs and Genomic Analysis of SCPL2 in Four Delphinium Cultivars. Supplemental Figure 4. Enzymatic Activities of AA7G-AT and AA7GBG-AT in Sepals, Leaves, and Stems of Delphinium cv Triton Light Blue. Supplemental Figure 5. The Enzymatic Properties of AA7G-AT, AA7GBG-AT, and AA7BG-GTs. Supplemental Figure 6. Subcellular Distributions of the GFP Fusion Proteins Expressed in Onion Epidermal Cells. Supplemental Table 1. 1H and Cy3G7BG, and Cy3G7GBG.

13C

NMR Analyses of Cy3,7dG,

Supplemental Table 2. Primer Sequences Used in This Study. Supplemental Table 3. Accession Numbers for Sequences Used in the Phylogenetic Tree Analysis (Figure 5). Supplemental Table 4. Accession Numbers for Sequences Used in the Phylogenetic Tree Analysis (Figure 8). Supplemental Data Set 1. Protein Sequences Used for Construction of the Phylogenetic Tree (Figure 5). Supplemental Data Set 2. Protein Sequences Used for Construction of the Phylogenetic Tree (Figure 8). Supplemental References 1. Source of References in Supplemental Table 3 and 4.

ACKNOWLEDGMENTS This research was supported by a Grant-in-Aid for Scientific Research (B) (23370016; to Y.O.) and Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (to Y.N.).

REFERENCES Abe, Y., Tera, M., Sasaki, N., Okamura, M., Umemoto, N., Momose, M., Kawahara, N., Kamakura, H., Goda, Y., Nagasawa, K., and Ozeki, Y. (2008). Detection of 1-O-malylglucose:pelargonidin 3-Oglucose-699-O-malyltransferase activity in carnation (Dianthus caryophyllus). Biochem. Biophys. Res. Commun. 373: 473–477. Brandt, K., Kondo, T., Aoki, H., and Goto, T. (1993). Structure and biosynthesis of anthocyanins in flowers of Campanula. Phytochemistry 33: 209–212. Brugliera, F., Holton, T.A., Stevenson, T.W., Farcy, E., Lu, C.Y., and Cornish, E.C. (1994). Isolation and characterization of a cDNA clone corresponding to the Rt locus of Petunia hybrida. Plant J. 5: 81–92. D’Auria, J.C. (2006). Acyltransferases in plants: A good time to be BAHD. Curr. Opin. Plant Biol. 9: 331–340. Fraser, C.M., Rider, L.W., and Chapple, C. (2005). An expression and bioinformatics analysis of the Arabidopsis serine carboxypeptidaselike gene family. Plant Physiol. 138: 1136–1148. Fraser, C.M., Thompson, M.G., Shirley, A.M., Ralph, J., Schoenherr, J.A., Sinlapadech, T., Hall, M.C., and Chapple, C. (2007). Related Arabidopsis serine carboxypeptidase-like sinapoylglucose acyltransferases display distinct but overlapping substrate specificities. Plant Physiol. 144: 1986–1999. Furtek, D., Schiefelbein, J.W., Johnston, F., and Nelson, O.E., Jr.,. (1988). Sequence comparisons of three wild-type Bronze-1 alleles from Zea mays. Plant Mol. Biol. 11: 473–481. Gómez-Anduro, G., Ceniceros-Ojeda, E.A., Casados-Vázquez, L.E., Bencivenni, C., Sierra-Beltrán, A., Murillo-Amador, B., and Tiessen, A. (2011). Genome-wide analysis of the beta-glucosidase gene family in maize (Zea mays L. var B73). Plant Mol. Biol. 77: 159–183. Goto, T., and Kondo, T. (1991). Structure and molecular stacking of anthocyanins-flower color variation. Angew. Chem. Int. Ed. Engl. 30: 17–33. Hashimoto, F., Tanaka, M., Maeda, H., Fukuda, S., Shimizu, K., and Sakata, Y. (2002). Changes in flower coloration and sepal anthocyanins of Cyanic delphinium cultivars during flowering. Biosci. Biotechnol. Biochem. 66: 1652–1659. Hause, B., Meyer, K., Viitanen, P.V., Chapple, C., and Strack, D. (2002). Immunolocalization of 1-O-sinapoylglucose:malate sinapoyltransferase in Arabidopsis thaliana. Planta 215: 26–32. Hirose, Y., Aida, R., and Shibata, M. (2002). Agrobacterium tumefaciensmediated transformation Delphinium spp. Plant Biotechnol. 19: 377–382. Kondo, T., Oki, K., Yoshida, K., and Goto, T. (1990). Structure of violdelphin, an anthocyanin from violet flower of Delphinium hybridum. Chem. Lett. 19: 137–138. Kondo, T., Suzuki, K., Yoshida, K., Oki, K., Ueda, M., Isobe, M., and Goto, T. (1991). Structure of cyanodelphin, a tetra-p-hydroxybenzoated anthocyanin from blue flower of Delphinium hybridum. Chem. Lett. 44: 6375–6378.

Biosynthesis of Polyacyl Anthocyanin

Lee, S., Kaminaga, Y., Cooper, B., Pichersky, E., Dudareva, N., and Chapple, C. (2012). Benzoylation and sinapoylation of glucosinolate R-groups in Arabidopsis. Plant J. 72: 411–422. Lehfeldt, C., Shirley, A.M., Meyer, K., Ruegger, M.O., Cusumano, J. C., Viitanen, P.V., Strack, D., and Chapple, C. (2000). Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism. Plant Cell 12: 1295–1306. Leznicki, A.J., and Bandurski, R.S. (1988). Enzymic synthesis of indole-3-acetyl-1-O-b-D-glucose. I. Partial purification and characterization of the enzyme from Zea mays. Plant Physiol. 88: 1474–1480. Li, A.X., Eannetta, N., Ghangas, G.S., and Steffens, J.C. (1999). Glucose polyester biosynthesis. Purification and characterization of a glucose acyltransferase. Plant Physiol. 121: 453–460. Liu, Y., Gao, L., Liu, L., Yang, Q., Lu, Z., Nie, Z., Wang, Y., and Xia, T. (2012). Purification and characterization of a novel galloyltransferase involved in catechin galloylation in the tea plant (Camellia sinensis). J. Biol. Chem. 287: 44406–44417. Luang, S., et al. (2013). Rice Os9BGlu31 is a transglucosidase with the capacity to equilibrate phenylpropanoid, flavonoid, and phytohormone glycoconjugates. J. Biol. Chem. 288: 10111–10123. Mangeon, A., Magioli, C., Menezes-Salgueiro, A.D., Cardeal, V., de Oliveira, C., Galvão, V.C., Margis, R., Engler, G., and SachettoMartins, G. (2009). AtGRP5, a vacuole-located glycine-rich protein involved in cell elongation. Planta 230: 253–265. Matsuba, Y., Okuda, Y., Abe, Y., Kitamura, Y., Terasaka, K., Mizukami, H., Kamakura, K., Kawahara, N., Goda, Y., Sasaki, N., and Ozeki, Y. (2008). Enzymatic preparation of 1-O-hydroxycinnamoylb-D-glucoses and their application to the study of 1-O-hydroxycinnamoylb-D-glucose-dependent acyltransferase in anthocyanin-producing cultured cells of Daucus carota and Glehnia littoralis. Plant Biotechnol. 25: 369–375. Matsuba, Y., et al. (2010). A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium. Plant Cell 22: 3374–3389. Milkowski, C., Baumert, A., Schmidt, D., Nehlin, L., and Strack, D. (2004). Molecular regulation of sinapate ester metabolism in Brassica napus: Expression of genes, properties of the encoded proteins and correlation of enzyme activities with metabolite accumulation. Plant J. 38: 80–92. Miyahara, T., Sakiyama, R., Ozeki, Y., and Sasaki, N. (2013). Acylglucose-dependent glucosyltransferase catalyzes the final step of anthocyanin formation in Arabidopsis. J. Plant Physiol. 170: 619–624. Miyahara, T., Takahashi, M., Ozeki, Y., and Sasaki, N. (2012). Isolation of an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase from the monocot Agapanthus africanus. J. Plant Physiol. 169: 1321–1326. Mock, H.P., and Strack, D. (1993). Energetics of the uridine 59-diphosphoglucose:hydroxycinnamic acid acyl-glucosyltransferase reaction. Phytochemistry 32: 575–579. Mugford, S.T., and Milkowski, C. (2012). Serine carboxypeptidaselike acyltransferases from plants. Methods Enzymol. 516: 279–297. Mugford, S.T., et al. (2009). A serine carboxypeptidase-like acyltransferase is required for synthesis of antimicrobial compounds and disease resistance in oats. Plant Cell 21: 2473–2484.

4165

Nakagawa, T., Ishiguro, S., and Kimura, T. (2009). Gateway vectors for plant transformation. Plant Biotechnol. 26: 275–284. Nakayama, T., Suzuki, H., and Nishino, T. (2003). Anthocyanin acyltransferases: Specificities, mechanism, phylogenetics, and applications. J. Mol. Catal., B Enzym. 23: 117–132. Noda, N., Kazuma, K., Sasaki, T., Tsugawa, H., and Suzuki, M. (2006). Molecular cloning of 1-O-acylglucose dependent anthocyanin aromatic acyltransferase in ternatin biosynthesis of butterfly pea (Clitoria ternatea). Plant Cell Physiol. 47: S109. Owatworakit, A., et al. (2012). Glycosyltransferases from oat (Avena) implicated in the acylation of avenacins. J. Biol. Chem. 288: 3696–3704. Ozeki, Y., Matsuba, Y., Abe, Y., Umemoto, N., and Sasaki, N. (2011). Pigment biosynthesis I. Anthocyanins. In Plant Metabolism and Biotechnology, H. Ashihara, A. Crozier, and A. Komamine, eds (West Sussex, UK: Wiley), pp. 321–342. Ralston, E.J., English, J.J., and Dooner, H.K. (1988). Sequence of three bronze alleles of maize and correlation with the genetic fine structure. Genetics 119: 185–197. Saito, N., Tatsuzawa, F., Yazaki, Y., Shigihara, A., and Honda, T. (2007). 7-Polyacylated delphinidin 3,7-diglucosides from the blue flowers of Leschenaultia cv. Violet Lena. Phytochemistry 68: 673–679. Shirley, A.M., McMichael, C.M., and Chapple, C. (2001). The sng2 mutant of Arabidopsis is defective in the gene encoding the serine carboxypeptidase-like protein sinapoylglucose:choline sinapoyltransferase. Plant J. 28: 83–94. Takeda, K., Sato, S., Kobayashi, H., Kanaitsuka, Y., Ueno, M., Kinoshita, T., Tazaki, H., and Fujimori, T. (1994). The anthocyanin responsible for purplish blue flower colour of Aconitum chinense. Phytochemistry 36: 613–616. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 1596–1599. Tanaka, Y., Sasaki, N., and Ohmiya, A. (2008). Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J. 54: 733–749. Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680. Umemoto, N., Abe, Y., Cano, E.A., Okamura, M., Sasaki, N., Yoshida, S., and Ozeki, Y. (2009). Carnation serine carboxypeptidaselike acyltransferase is important for anthocyanin malyltransferase activity and formation of anthocyanic vacuolar inclusions. In 5th International Workshop on Anthocyanins 2009 in Japan, pp. 115. Xu, Z., Escamilla-Treviño, L., Zeng, L., Lalgondar, M., Bevan, D., Winkel, B., Mohamed, A., Cheng, C.L., Shih, M.C., Poulton, J., and Esen, A. (2004). Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1. Plant Mol. Biol. 55: 343–367. Yoshida, K., Kondo, T., and Goto, T. (1992). Intramolecular stacking conformation of gentiodelphin, a diacylated anthocyanin from Gentiana makinoi. Tetrahedron 48: 4313–4326. Yoshida, K., Mori, M., and Kondo, T. (2009). Blue flower color development by anthocyanins: From chemical structure to cell physiology. Nat. Prod. Rep. 26: 884–915.

p-Hydroxybenzoyl-Glucose Is a Zwitter Donor for the Biosynthesis of 7-Polyacylated Anthocyanin in Delphinium Yuzo Nishizaki, Motoki Yasunaga, Emi Okamoto, Mitsutoshi Okamoto, Yukio Hirose, Masaatsu Yamaguchi, Yoshihiro Ozeki and Nobuhiro Sasaki Plant Cell 2013;25;4150-4165; originally published online October 31, 2013; DOI 10.1105/tpc.113.113167 This information is current as of March 29, 2015 Supplemental Data

http://www.plantcell.org/content/suppl/2013/10/11/tpc.113.113167.DC1.html

References

This article cites 43 articles, 12 of which can be accessed free at: http://www.plantcell.org/content/25/10/4150.full.html#ref-list-1

Permissions

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs

Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain

CiteTrack Alerts

Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain

Subscription Information

Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm

© American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY