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First published online January 5, 2007; 10.1104/pp.106.093203 Plant Physiology 143:1347-1361 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
The Grapevine Transcription Factor VvMYBPA1 Regulates Proanthocyanidin Synthesis during Fruit Development1,[OA]Commonwealth Scientific and Industrial Research Organization Plant Industry, Glen Osmond, 5064, Australia (J.B., F.W.J., A.M.T., A.R.W., S.P.R.); Cooperative Research Centre for Viticulture, Glen Osmond, 5064, Australia (J.B., A.R.W., S.P.R.); and Heidelberger Institut für Pflanzenwissenschaften, 69120 Heidelberg, Germany (J.B.)
Proanthocyanidins (PAs; or condensed tannins) can protect plants against herbivores, contribute to the taste of many fruits, and act as dietary antioxidants beneficial for human health. We have previously shown that in grapevine (Vitis vinifera) PA synthesis involves both leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR). Here we report the characterization of a grapevine MYB transcription factor VvMYBPA1, which controls expression of PA pathway genes including both LAR and ANR. Expression of VvMYBPA1 in grape berries correlated with PA accumulation during early berry development and in seeds. In a transient assay, VvMYBPA1 activated the promoters of LAR and ANR, as well as the promoters of several of the general flavonoid pathway genes. VvMYBPA1 did not activate the promoter of VvUFGT, which encodes the anthocyanin-specific enzyme UDP-glucose:flavonoid-3-O-glucosyltransferase, suggesting VvMYBPA1 is specific to regulation of PA biosynthesis in grapes. The Arabidopsis (Arabidopsis thaliana) MYB transcription factor TRANSPARENT TESTA2 (TT2) regulates PA synthesis in the seed coat of Arabidopsis. By complementing the PA-deficient seed phenotype of the Arabidopsis tt2 mutant with VvMYBPA1, we confirmed the function of VvMYBPA1 as a transcriptional regulator of PA synthesis. In contrast to ectopic expression of TT2 in Arabidopsis, constitutive expression of VvMYBPA1 resulted in accumulation of PAs in cotyledons, vegetative meristems, leaf hairs, and roots in some of the transgenic seedlings. To our knowledge, this is the first report of a MYB factor that controls genes of the PA pathway in fruit, including both LAR and ANR, and this single MYB factor can induce ectopic PA accumulation in Arabidopsis.
Proanthocyanidins (PAs), also known as condensed tannins, are polyphenolic secondary metabolites synthesized via the flavonoid biosynthetic pathway. They are present in many plants and act in defense against plant diseases and in seed dormancy (Debeaujon et al., 2000  ; Peters and Constabel, 2002). The increase of PAs in important forage crops like alfalfa (Medicago sativa) could protect ruminants against pasture bloat, reduce greenhouse gas, and increase plant disease resistance (Dixon et al., 1996; McMahon et al., 2000). Dietary PAs are present in many fruits and plant products like wine, fruit juices, and teas and contribute to their taste and health benefits. PAs act as potential dietary antioxidants with beneficial effects for human health, including protection against free radical-mediated injury and cardiovascular disease (Bagchi et al., 2000; Middleton et al., 2000; Cos et al., 2004). There is also considerable interest in the PAs found in grapes (Vitis vinifera) because of their importance for the taste and astringency of red and white wine (Glories, 1988). For these reasons, there is a growing interest in metabolic engineering strategies aimed at developing agronomically important food crops and fruits with optimized levels and composition of flavonoids.
The biosynthesis of PAs, anthocyanins, and flavonols share common steps in the flavonoid pathway, and the genetics and biochemistry of this pathway (Fig. 1
) have been characterized in several plant species including Arabidopsis (Arabidopsis thaliana) and grapes (Shirley et al., 1992
In Arabidopsis, the biosynthetic pathway leading to PA accumulation has been characterized by using the transparent testa (tt) and tannin-deficient seed (tds) mutants that fail to accumulate PAs in their seed coat (Shirley et al., 1995 ; Abrahams et al., 2002). Some of the identified TT and TDS loci correspond to enzymes of the general flavonoid pathway and others to enzymes, transporters, and regulators specifically involved in PA accumulation. The structural genes include anthocyanidin reductase (ANR; also called BANYULS) that catalyzes the synthesis of flavan-3-ols such as (–)-epicatechin (Xie et al., 2003), TT19, TT12, and Autoinhibited H+-ATPase isoform 10, which are involved in transport processes of PAs (Debeaujon et al., 2001; Kitamura et al., 2004; Baxter et al., 2005) and TT10 encoding a laccase-type polyphenol oxidase involved in polymerization of flavonoids (Pourcel et al., 2005).
Regulation of flavonoid synthesis occurs mostly via coordinated transcriptional control of the structural genes by the interaction of DNA-binding R2R3 MYB transcription factors, MYC-like basic helix loop helix (bHLH) and WD40 proteins (Mol et al., 1998
The Arabidopsis genes TT8, TT2, and TRANSPARENT TESTA GLABRA1 (TTG1), necessary for PA accumulation in the seed coat, were found to encode bHLH, MYB, and WD40 repeat proteins, respectively. By forming a transcription complex, they regulate the expression of several flavonoid structural genes including ANR and TT12 (Walker et al., 1999
However, the specific regulation of PA synthesis in plant species other than Arabidopsis is not well characterized and until now no MYB factor functionally similar to TT2 has been identified from other plants. Whereas PA synthesis in Arabidopsis is exclusively epicatechin based and limited to the seed coat, many other plants produce both epicatechin- and catechin-based PAs of various amounts and compositions and in a range of different tissues (Dixon et al., 2005 In this article, we isolated and characterized a grapevine gene VvMYBPA1 that encodes a MYB transcription factor that is expressed when PAs accumulate during early grape berry development and in seeds. VvMYBPA1 is able to activate the promoters of both of the PA branch genes VvANR and VvLAR1 but also several of the general flavonoid pathway genes of grapevine. VvMYBPA1 did not activate anthocyanin synthesis, suggesting it may be specific for the PA pathway in grapes. Constitutive expression of this transcription factor in Arabidopsis complemented the PA-deficient seed coat phenotype of the Arabidopsis tt2 mutant and also induced ectopic PA accumulation in other tissues including cotyledons, stems, and roots. This grapevine regulator may provide the potential to alter PA synthesis in fruits and crops by metabolic engineering.
The Grapevine Gene VvMYBPA1 Encodes a MYB Transcription Factor
We identified the Tentative Consensus (TC) sequence TC46393 by searching the grape gene index of the The Institute for Genomic Research (TIGR) expressed sequence tag database (Quackenbush et al., 2000
Taken together, the VvMYBPA1 protein sequence shows the typical features of a plant MYB transcription factor. However, we were not able to detect conserved amino acid homologies between the PA regulators TT2 and VvMYBPA1, which could be used to identify PA-specific MYB regulators from other plant species.
To confirm that VvMYBPA1 is expressed when PAs are accumulating in grape berries, we investigated transcript levels of VvMYBPA1 throughout grape berry (cv Shiraz) development during the season 2000 to 2001 by real-time PCR. VvUbiquitin1 (BN000705) was chosen for normalization of gene expression because it was found to be relatively constant throughout grape berry development (Downey et al., 2003b
Figure 3
shows VvMYBPA1 is expressed in flowers and grapes early in berry development from 10 to 6 weeks before onset of ripening. This early expression of VvMYBPA1 in developing flowers and grape berries correlates with the accumulation of PAs and the expression of the structural genes leucoanthocyanidin dioxygenase (VvLDOX), VvANR, and VvLAR1 that are involved in PA synthesis in grapevine (Bogs et al., 2005
In grape berry skins, transcript levels of VvMYBPA1 were relatively low before véraison, which is the onset of ripening, increased to a maximum 2 weeks after véraison, and then declined to a low level (Fig. 3). The concentration of PAs in skins increased from 5 weeks before véraison, reaching a maximum around the time ripening commenced and then declined during ripening (Bogs et al., 2005 ). In seeds, VvMYBPA1 is expressed before véraison (Fig. 3) when PAs start to accumulate. The expression pattern of VvMYBPA1 in seeds (Fig. 3) correlates with PA synthesis and the expression of VvLAR2 that continued in the seed up until 4 weeks after véraison with a maximum at véraison (Kennedy et al., 2000; Bogs et al., 2005).
To determine which genes of the flavonoid pathway are controlled by VvMYBPA1, we isolated the promoter regions of the genes flavonoid 3',5' hydroxylase (VvF3'5'H1; 1,136 bp; accession no. AM259482), chalcone isomerase (VvCHI; 935 bp; accession no. AM259483), VvANR (1,034 bp; accession no. AM259484), and VvLAR1 (1,342 bp; accession no. AM259481) by genome walking (see experimental procedures). These promoter regions were analyzed using the plant DNA cis-elements (PLACE) database (Higo et al., 1999
To investigate which structural genes of the flavonoid pathway are activated by VvMYBPA1, we established a transient expression method using grape cells grown in suspension culture and the dual-luciferase assay system. In this system, the cotransfection of effectors (transcription factors) and dual-luciferase reporter plasmids allows quantification of promoter activity by measuring firefly (Photinus pyralis) luciferase activity (promoter of interest inserted into pLuc), which is normalized by measuring Renilla reniformis luciferase activity (pRluc; Horstmann et al., 2004
VvMYBPA1 strongly activated the promoters of the genes VvANR (approximately 135-fold), AtANR (approximately 70-fold), and VvLAR1 (approximately 72-fold), showing its ability to induce the PA-specific branch point genes of Arabidopsis and grapevine (Fig. 4, A–C). VvMYBPA1 also induced the promoters of the general flavonoid pathway genes VvCHI (approximately 16-fold), VvF3'5'H1 (approximately 38-fold), and VvLDOX (approximately 125-fold), suggesting it can activate the whole pathway leading to PA synthesis (Fig. 4, D–F). The anthocyanin-specific promoter of VvUFGT was not affected by VvMYBPA1, whereas VvMYBA2 strongly activated (approximately 600-fold) this promoter (Fig. 4G) but did not activate the other genes. In comparison to VvMYBPA1, the activation of the VvF3'5'H1, VvLDOX, VvCHI, VvANR, VvLAR1, and the AtANR promoter by VvMYBA2 was absent or relatively low (Fig. 4). Similar to VvMYBPA1, the Arabidopsis PA regulator TT2 activated the ANR genes of Arabidopsis and grapevine (Fig. 4, A and B) but TT2 was not able to substantially induce the promoters of VvLAR, VvF3'5'H1, VvLDOX, and VvCHI (Fig. 4, C and F). These results suggest VvMYBPA1 is a specific regulator of PA synthesis, potentially regulating the entire general flavonoid pathway and both of the PA branch genes ANR and LAR, leading to PA formation. Similar to other MYB transcription factors, VvMYBPA1 requires a bHLH protein for promoter activation (Fig. 4E; VvMYBPA1/w/o). Therefore, all standard transfections included a construct expressing EGL3 which encodes a bHLH protein involved in flavonoid pathway regulation in Arabidopsis (Ramsay et al., 2003 ; Walker et al., 2007). Figure 4 also shows that using VvMYBPA1 without a bHLH protein in our transfection experiments can induce the VvLDOX promoter up to 3-fold compared to the control without the MYB factor, and similar results (3- to 5-fold inductions) were obtained for other promoters and MYB factors (data not shown). However, in comparison to the 125-fold induction of the VvLDOX promoter by VvMYBPA1 and EGL3 we considered a 3-fold induction as insubstantial and possibly provoked by the large amounts of MYB factor and promoter DNA in the transfection assay.
In Arabidopsis, the bHLH protein TT8 has been shown to interact with TT2 and to be required for PA accumulation in the seed coat (Nesi et al., 2000
The MYB transcription factor TT2 has been shown to regulate PA synthesis in the seed coat of Arabidopsis and the seeds of tt2 mutants appear yellow due to the lack of PAs (Nesi et al., 2001
Only around 20% of the kanamycin-resistant T1 seedlings survived on soil and these were grown on to produce seed. Nine kanamycin-resistant tt2 plants independently transformed with pART27MYBPA1 showed wild-type phenotype and developed brown seeds which stained blue for accumulation of PAs when stained with dimethylaminocinnamaldehyde (DMACA; Fig. 5, A–F
). DMACA is a useful reagent for detection of molecules of the PA pathway because it reacts with both PA monomers and polymers to form a blue chromophore but does not react with anthocyanidin derivatives (Nagel and Glories, 1991
About 80% of the transgenic T1 seedlings showed growth abnormalities with bleaching and necrosis of the first leaves during their development. These plants showed a dwarf phenotype and died about 2 weeks after transferring them to soil. When these seedlings were stained with DMACA we observed accumulation of PAs in cells of cotyledons, hypocotyls, and its apical meristem, roots, trichomes, and basal cells of trichomes indicated by their blue staining (Fig. 7 ). Control plants (tt2 and Col-0) stained with DMACA did not show any blue staining, indicating their inability to accumulate significant amounts of PAs (Fig. 7, A, I, and J).
VvMYBPA1 Encodes a MYB-Type Transcriptional Regulator
In this study, we have identified the transcriptional regulator VvMYBPA1 from grapevine, which has some functional similarity to AtTT2, the only other regulator of PA synthesis isolated so far. Constitutive expression of VvMYBPA1 in Arabidopsis can complement a mutation in tt2 and experimental evidence suggests that VvMYBPA1 regulates PA synthesis during grape berry development. The protein sequence of VvMYBPA1 shows homology to the R2R3 domain of various MYB transcription factors (Fig. 2). However, we were not able to find any of the conserved motifs described by Stracke et al. (2001)
The functional role of VvMYBPA1 in the regulation of PA synthesis in grape berries is supported by its gene expression pattern during grape berry development (Fig. 3). Development of the grape berry occurs in two successive growth phases and the synthesis of flavonols, anthocyanins, and PAs and the expression of different flavonoid pathway genes is temporally separated during berry development. The first phase, from flowering until véraison (the onset of ripening in grapes), coincides with flavonol and PA synthesis and the second phase, starting with the onset of ripening of the berry, coincides with anthocyanin biosynthesis (Robinson and Davies, 2000
As well as activating the promoters of the two PA-specific genes, VvANR and VvLAR, VvMYBPA1 also activated the promoters of general flavonoid pathway genes, whereas the Arabidopsis TT2 regulator appeared to be more specific for ANR in this transient assay (Fig. 4). Anthocyanin and PA biosynthesis share the general flavonoid pathway enzymes until LDOX catalyzes the synthesis of anthocyanidins (Abrahams et al., 2003
In maize, most of the genes involved in anthocyanin synthesis are controlled by the MYB factor C1 interacting with the bHLH protein R (Goff et al., 1992 For the grapevine MYB factors VvMYBA and VvMYBPA1 the interaction with a bHLH protein (EGL3) was essential for their ability to activate gene expression (Fig. 4E). However, as we used EGL3 as bHLH protein in all our promoter experiments, their specificity to control either the anthocyanin-specific gene UFGT or the PA-specific genes ANR and LAR seems not to depend on the identity of the interacting bHLH protein (Fig. 4). The difference in specificity of VvMYBA, VvMYBPA1, and TT2 is likely to be due to their ability to bind to different sites in the promoters of grapevine and Arabidopsis genes. For example, differences of the MYB factors would change their ability to bind the promoters and/or other unidentified factors which are present in the grapevine cell cultures.
Another grapevine MYB, VvMYB5a, recently identified by Deluc et al. (2006)
Control of the pathway in grapevine is thus analogous to that in Arabidopsis, where the MYB transcription factor TT2 controls PA synthesis, whereas PAP1/PAP2 control anthocyanin biosynthesis. However, the Arabidopsis flavonoid MYB factors seem to control different steps in the flavonoid pathway than the similar factors from grapevine. In previous studies it was shown that TT2 controls expression of the flavonoid late biosynthetic genes including DFR, LDOX, AtANR (BAN), and TT12, whereas transcript levels of the flavonoid early biosynthetic genes like CHS, CHI, F3'H, or F3H were not affected by TT2 (Nesi et al., 2001
The complementation of the PA-deficient seed phenotype of the tt2 mutant by VvMYBPA1 indicates that this grapevine transcription factor is capable of substituting for the function of the Arabidopsis PA regulator TT2 (Fig. 5). Although TT2 controls PA synthesis in the Arabidopsis seed coat and its ectopic expression was shown to induce expression of the AtANR promoter, Arabidopsis 70S::TT2 plants failed to accumulate PAs in any tissue other than seeds (Nesi et al., 2001
It would be interesting to determine whether ectopic expression of VvMYBPA1 in plants like alfalfa and clover (Trifolium repens) can induce PA formation in leaves or other tissue, because it is of great interest to engineer PAs in forage crops to reduce the risk of pasture bloat for ruminants (Dixon et al., 2005
Unlike Arabidopsis, grapevine synthesizes PAs of different polymer lengths and composition in leaves, flowers, and in the skin and seeds of the developing fruit (Kennedy et al., 2001
Plant Material
Grapevine (Vitis vinifera) tissues of cultivar Shiraz were collected from a commercial vineyard during the 2000 to 2001 season. Approximately 100 berries from at least 20 bunches were collected at weekly intervals throughout berry development from floral initiation until harvest, as described in Downey et al. (2003a) Arabidopsis (Arabidopsis thaliana) Col-0 and tt2 (SALK_005260) seeds were provided by The Arabidopsis Biological Resource Center.
Total grapevine RNA was isolated from the various plant tissues as described in Downey et al. (2003b)
The ORF of VvMYBPA1 was inserted into the binary vector pART27 for expression of the gene in Arabidopsis. Therefore, the VvMYBPA1 ORF was amplified by PCR from grapevine (cv Shiraz) cDNA (from RNA isolated 10 weeks before véraison) using PfuTurbo polymerase (Stratagene) and the primers MybPAartF (5'-TGAGGTACCGAGAGAGATATGGGCAGAGCAC-3') and MybPAartR (5'-TGAGGATCCTGATCTTTTGGTCTCTCTGCAA-3'). The generated PCR fragment was purified, digested with BamHI and KpnI, and cloned in the vector pART7 (Gleave, 1992
Immature Arabidopsis siliques were finely ground and 80 mg were extracted in 400 µL 70% (v/v) acetone containing 0.1% (w/v) ascorbate for 18 h at room temperature on a rotating wheel in darkness. Samples were centrifuged and 300 µL aliquots of the supernatant were transferred to fresh tubes and vaccum dried at 35°C for 60 min. The pellet was resuspended in 100 µL phloroglucinol buffer (0.25 g ascorbate, 1.25 g phloroglucinol, 215 µL concentrated HCl, 25 mL methanol) and incubated at 50°C for 20 min, then neutralized with 100 µL sodium acetate (200 mM, pH 7.5) for the analysis of PAs. Reverse-phase HPLC was used for analysis of PAs as described by Downey et al. (2003a) The presence of PAs in plant tissue was detected by staining the tissues with DMACA solution (1% DMACA, 1% 6 N HCl in methanol). Dried seeds were stained for 6 to 14 h and seedlings for 10 to 30 min. The tissues were then transferred to distilled water and blue staining of the tissue was visualized with a microscope and documented using a digital camera.
The Universal GenomeWalker kit (CLONTECH) was used to isolate promoter fragments of VvCHI, VvF3'5'H1, VvANR, and VvLAR1. Four libraries of adaptor-ligated genomic fragments were constructed from grapevine (Shiraz) genomic DNA restricted by DraI, EcoRV, PvuII, or StuI endonucleases and generated according to the GenomeWalker protocol. These genomic DNA libraries served as templates for the promoter isolation. Outer and nested gene-specific primers were designed to the 5' ends of the cDNA sequences of VvCHI (accession no. X75963), VvF3'5'H1 (accession no. AJ880356), VvANR (accession no. CAD91911), and VvLAR1 (accession no. AJ865336) and primary and secondary PCRs were performed with the outer adapter primer AP1 and the nested adapter primer AP2, respectively. Primer design and PCR conditions for genome walking were performed according to the manufacturer's instructions. The amplified promoter fragments of the nested PCRs were cloned into pDrive (Qiagen) and sequenced. These DNA sequences were then used to design specific primers for the amplification of the respective promoter from grapevine (Shiraz) genomic DNA using PfuTurbo polymerase (Stratagene). The primers used for these PCR reactions contained restriction sites (in bold) for cloning the promoters into the luciferase reporter vector pLuc (Horstmann et al., 2004 A 1,674-bp DNA fragment of the VvUFGT promoter (accession no. AY955269) was amplified by PCR from the plasmid pART7UFGT:GFP (gift from Paul Boss, Commonwealth Scientific and Industrial Research Organization, Australia) using Pfx polymerase (Invitrogen) with the primers UFGTp2F (5'-ACGGGATCCTCATGCGTCCACCTATTATCAA-3') and UFGTpR (5'-GTACTCGAGGGTTGGAATGGGGGATGTTA-3'). A 1,533-bp DNA fragment of the AtANR promoter was amplified with PfuTurbo polymerase using the primers AtANRf (5'-CGAGGATCCCTGGGAAGACAATCGCTTTA-3') and AtANRr (5'-ATCTCGAGTTGAAATTACAGAGATAGAGATTTAGTTG-3'). A 2,174-bp DNA fragment of the VvLDOX promoter was amplified with PfuTurbo polymerase using the primers LDOXf (5'-CGAGGATCCGTTTGCTTCCATCCCAATCTCACT-3') and LDOXr (5'-TGTCTCGAGAAATATCACTGATCTACTTGTTTTCC-3'). These PCR fragments were gel purified, digested with BamHI and XhoI, and cloned between the respective sites of the vector pLuc. All described grapevine promoters were amplified from grapevine (Shiraz) genomic DNA. For transient expression of TT2 and VvMYBA2 their ORFs were amplified from cDNA by PCR using PfuTurbo polymerase and cloned into the vector pART7, which contains the CaMV 35S constitutive promoter. Therefore, TT2 was amplified using the primers TT2F (5'-AGGTCGACATGGGAAAGAGAGCAACTACTAGTG-3') and TT2R (5'-TACTCGAGTCAACAAGTGAAGTCTCGGAGC-3') from cDNA of Col-0 siliques. The PCR fragment was digested with SalI and XhoI and ligated into pART7, digested with the same enzymes. The ORF of VvMYBA2 was amplified from grapevine postvéraison berry skin cDNA using the primers MybAF (5'-CGCCTCGAGCTCGATGGAGAGCTTAGGAGTTAG-3') and MybAR (5'-CGCTCTAGATAAATCAGATCAAATGATTTACTT-3'). The PCR fragment was digested with XhoI and XbaI and ligated into pART7, digested with the same enzymes. All described PCR fragments were subjected to DNA sequencing before analysis in the transient assay system.
A transient assay was developed using a cell suspension of a Chardonnay petiole callus culture, using the methods detailed in Takos et al. (2006)
Transcript levels of VvMYBPA1 in grapevine were measured by real-time PCR, using SYBR green method on a Rotor-Gene 2000 (version 4.2) real-time cycler (Corbett Research). Each PCR reaction (15 µL) contained: 266 nM primer (each), cDNA (diluted 1:60), and 1x ABsolute QPCR SYBR Green ROX mix (ABgene House). The thermal cycling conditions were 95°C for 15 min followed by 95°C for 30 s, 58°C for 25 s, and 72°C for 25 s for 30 or 35 cycles, followed by a melt cycle from 50°C to 96°C.
The expressed sequence tag clone TC46393 (TIGR database) was used to design the primers MYBPA1F (5'-AGATCAACTGGTTATGCTTGCT-3') and MYBPA1R (5'-AACACAAATGTACATCGCACAC-3') that were used to detect the transcript level of VvMYBPA1 in grapevine and amplified a 190-bp PCR fragment from the 3' untranslated region of the gene. With all cDNAs used the primer set gave a single PCR product that was verified by determining the melt curves for the product at the end of each run, by analysis of the product using gel electrophoresis, and by comparing the DNA sequence of the PCR product with the gene sequence. The efficiency of the primers was tested in preliminary experiments with dilutions of the purified PCR product and maintained an r2 value For detection of the VvMYBPA1 transcript in Arabidopsis (Fig. 7), PCR reactions were performed as described above and analyzed on a 1.5% agarose gel containing ethidium bromide. The primers MYBf (5'-CAACTGACAACTCTCTGGACAA-3') and MYBr (5'-GATCTTTTGGTCTCTCTGCAAC-3') were used to amplify a 146-bp PCR from the 3' translated region of the gene. To determine whether the similar amounts of cDNA were applied to all samples, a 268-bp PCR fragment from the Arabidopsis Actin2 gene (accession no. NM_112764) was amplified with the primers ACT2F (5'-ATTCAGATGCCCAGAAGTCTTGTTCC-3') and ACT2R (5'-ACCACCGATCCAGACACTGTACTTCC-3'). Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AM259485.
We wish to thank to Andrew Turner for help with the luciferase analysis, Eva Decker for the pluc and pRluc vectors, Nicole Cordon for help with HPLC analysis, and Karin Sefton and Debra McDavid for excellent technical assistance. Received November 16, 2006; accepted December 24, 2006; published January 5, 2007.
1 This work was supported by the Australian Government's Cooperative Research Centres Program and the Grape and Wine Research and Development Corporation. 
2 Present address: Heidelberger Institut für Pflanzenwissenschaften, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany. 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.plantphysiol.org) is: Simon P. Robinson (simon.robinson{at}csiro.au).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.093203 * Corresponding author; e-mail simon.robinson{at}csiro.au; fax 61–8–8303–8601.
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ABSTRACT
RESULTS
-hydroxylase; DFR, dihydroflavonol-4-reductase; LDOX, leucoanthocyanidin dioxygenase; FLS, flavonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; and UFGT, UDP-Glc:flavonoid-3-O-glucosyltransferase. The F3'H and F3'5'H enzymes, which hydroxylate flavanones and dihydroflavonols, were omitted to clarify the scheme of the flavonoid pathway (see Bogs et al., 2006
0.98. The expression of genes was normalized to VvUbiquitin1 (TC32075, TIGR database), which transcripts were detected by amplifying a 182-bp product with the primers VvUbiquitin Forward (5'-GTGGTATTATTGAGCCATCCTT-3') and VvUbiquitin Reverse (5'-AACCTCCAATCCAGTCATCTAC-3'). All samples were measured in triplicate. The difference between the cycle threshold (Ct) of the target gene and the Ct of Ubiquitin, 
Ct = CtTarget – CtUbiquitin, was used to obtain the normalized expression of target genes, which corresponds to 2–
