The Transcription Factor VvMYB5b Contributes to the Regulation of Anthocyanin and Proanthocyanidin Biosynthesis in Developing Grape Berries    

Abstract

Among the dramatic changes occurring during grape berry (Vitis vinifera) development, those affecting the flavonoid pathway have provoked a number of investigations in the last 10 years. In addition to producing several compounds involved in the protection of the berry and the dissemination of the seeds, final products of this pathway also play a critical role in berry and wine quality. In this article, we describe the cloning and functional characterization of VvMYB5b, a cDNA isolated from a grape berry (V. vinifera ‘Cabernet Sauvignon’) library. VvMYB5b encodes a protein belonging to the R2R3-MYB family of transcription factors and displays significant similarity with VvMYB5a, another MYB factor recently shown to regulate flavonoid synthesis in grapevine. The ability of VvMYB5a and VvMYB5b to activate the grapevine promoters of several structural genes of the flavonoid pathway was confirmed by transient expression of the corresponding cDNAs in grape cells. Overexpression of VvMYB5b in tobacco (Nicotiana tabacum) leads to an up-regulation of genes encoding enzymes of the flavonoid pathway and results in the accumulation of anthocyanin- and proanthocyanidin-derived compounds. The ability of VvMYB5b to regulate particularly the anthocyanin and the proanthocyanidin pathways is discussed in relation to other recently characterized MYB transcription factors in grapevine. Taken together, data presented in this article give insight into the transcriptional mechanisms associated with the regulation of the flavonoid pathway throughout grape berry development.

The phenylpropanoid biosynthetic pathway leads to the synthesis of numerous compounds that play a key role in many physiological and biochemical processes in plants (Kutchan, 2005). Some of these compounds, belonging to the flavonoid family, contribute to the pigmentation of flowers, fruit, seeds, and leaves, but are also involved in other plant processes, including the signaling between plants and microbes (Hahlbrock and Scheel, 1989; Koes et al., 2005), the male fertility of some species, and plant defense in the form of antimicrobial agents and feeding deterrents (Dixon et al., 1996). In grapevine (Vitis vinifera), flavonoids such as flavonols and catechins protect the plant against UV radiation, whereas others, such as anthocyanins, help attract seed-dispersal agents. Flavonoid compounds have a critical role in the quality of wine by contributing to the bitterness and astringency of wine (proanthocyanidins [PAs]) or color (anthocyanins) and are also known to have nutrient and health benefits for humans. For example, grape seed PA extract has a beneficial effect on physical health, especially affecting the bones (Kamitani et al., 2004), the heart (Bagchi et al., 2000), and the immune system (Lin et al., 2002). As a consequence, moderate consumption of red wine may reduce risk of cardiovascular disease and cancer (Klatsky, 2002).

In plants, the regulation of the phenylpropanoid biosynthetic pathway has been extensively studied, and particularly the control of the anthocyanin pathway is well characterized (Broun, 2005; Koes et al., 2005; Ramsay and Glover, 2005). Three regulatory gene families encoding basic helix-loop-helix (bHLH), WDR (WD40 repeats), and MYB-like proteins appear to be directly involved and have been extensively characterized in various species such as maize (Zea mays; Hernandez et al., 2004), snapdragon (Antirrhinum majus; Schwinn et al., 2006), petunia (Petunia hybrida; Spelt et al., 2000), and Ipomoea (Morita et al., 2006). In fruit crops, characterization studies of MYB factors revealed their role in the regulation of the anthocyanin pathway in strawberry (Fragaria × ananassa; Aharoni et al., 2001) and tomato (Solanun lycopersicum; Mathews et al., 2003). Likewise, in apple (Malus domestica), a MYB transcription factor was recently characterized and its functions provided new evidence of the conserved mechanism related to the regulation of the flavonoid pathway within the plant kingdom (Takos et al., 2006; Ban et al., 2007; Espley et al., 2007). In Arabidopsis (Arabidopsis thaliana), it was shown that a combinatorial control through the action of three different families of regulatory factors (MYB, bHLH, and WDR proteins) modulates the expression of the structural Banyuls (BAN) gene encoding anthocyanidin reductase, which is directly involved in proanthocyanidin (PA) biosynthesis (Baudry et al., 2004, 2006; Lepiniec et al., 2006). In grape berries, even though the identification of structural genes associated with the flavonoid pathway has been undertaken (Boss et al., 1996a; Kennedy et al., 2000; Downey et al., 2003a, 2003b; Bogs et al., 2005), little is known about the transcriptional regulation of the structural genes involved in flavonoid biosynthesis throughout berry development. Recent studies in grapevine have pointed out the key role of the VvMYBA1 and VvMYBA2 transcription factors that regulate specifically the expression of the UDP-Glc:flavonoid 3-O-glucosyltransferase (UFGT) gene, which encodes an enzyme responsible for conversion of anthocyanidins to anthocyanins (Kobayashi et al., 2002, 2004; Walker et al., 2007). Another R2R3-MYB protein has also been recently identified as a key regulator of PA synthesis in berry skin and seeds (Bogs et al., 2007). However, the remaining regulatory genes, controlling the expression of genes that encode enzymes located upstream of UFGT, still remain to be identified. The coordinate expression of some structural genes in berry suggests the contribution of at least two distinct regulatory complexes involved in the early and late steps of berry development, respectively (Boss et al., 1996a). In our group, we have recently identified a MYB gene named VvMYB5a associated with the regulation of the flavonoid pathway during the early phase of berry development (Deluc et al., 2006). Identification of transcriptional regulators involved in the later steps of berry development (i.e. berry ripening) is required to understand the coordinate regulatory mechanisms of this biosynthetic pathway throughout berry development.

In this article, we describe the functional characterization of a MYB transcription factor named VvMYB5b isolated from a cDNA library of Cabernet Sauvignon grape berries harvested at the onset of ripening, termed the veraison stage. Transcript analysis throughout berry development indicated that the VvMYB5b gene is expressed preferentially during berry ripening. Ectopic expression of VvMYB5b in tobacco (Nicotiana tabacum) plants enhanced the expression levels of most flavonoid structural genes and results in accumulation of anthocyanidin- and PA-derived compounds in reproductive tissues. In grape cells, VvMYB5b is able to activate several promoters of structural genes involved not only in the common steps of the flavonoid pathway but also in some specific branches such as PA synthesis. Experiments performed with the VvMYB5a protein (Deluc et al., 2006) lead to similar results indicating a possible functional relationship between those two MYB transcription factors. The putative biological functions of VvMYB5a and VvMYB5b are discussed according to their expression patterns during berry development and to the functional characterization data in homologous and heterologous systems. Taken together, data presented in this article represent an important step toward understanding the role of R2R3-MYB transcription factors in the regulatory mechanisms of the flavonoid biosynthetic pathway in developing grape berries.

RESULTS

VvMYB5b Sequence Analysis

A cDNA clone named VvMYB5b, encoding a putative R2R3-MYB protein, was isolated by PCR from a grape berry cDNA library. The open reading frame (ORF) encodes a protein of 311 amino acid residues with a predicted mass of 34 kD. The deduced amino acid sequence contains near its amino-terminal extremity the R2R3 imperfect repeats (DNA binding domain) involved in binding to target DNA sequences and highly conserved within MYB proteins (Fig. 1  

Figure 1.

Protein sequence alignment of VvMYB5b and other R2R3-MYB transcription factors from various plant species. Identical residues are shown in black, conserved residues in dark gray, and similar residues in light gray. The lines above the alignment locate the different domains and motifs within the proteins. The DNA binding domain corresponds to the R2R3-MYB repeats. The ID domain, involved in the interaction with bHLH proteins, and the C1 and C3 motifs are in bold and italics. GenBank accession numbers are as follows (in parentheses): VvMYB5b (AAX51291), VvMYB5a (AAS68190), BNLGHi233 (AAK19611), PH4 (AAY51377), AtMYB5 (AAC49311), OsMYB4 (BAA23340), VvMYBPA1 (CAJ90831), and VvMYBA1 (BAD18977).

). A phylogenetic analysis (Fig. 2  

Figure 2.

Phylogenetic relationships between VvMYB5b and R2R3-MYB transcription factors from grape and other plant species. Phylogenetic and evolutionary analyses were performed using the neighbor-joining method by the MEGA version 4 program (Kumar et al., 2004). The scale bar represents the number of substitutions per site and the numbers next to the nodes are bootstrap values from 1,000 replicates. The putative regulatory functions of the different R2R3-MYB proteins in the control of secondary metabolite biosynthesis or other biological processes are indicated. GenBank accession numbers are as follows (in parentheses): VvMYBA1 (BAD18977), VvMYBA2 (BAD18978), VvMYBA3 (BAD18979), LeANT1 (AAQ55181), TT2 (CAC40021), AtMYB75 (AAG42001), PH4 (AAY51377), AmRosea1 (ABB83826), AmVenosa (ABB83828), MdMYB10 (ABB84753), ZmMYBPL (AAB67720), ZmMYBC1 (AAA33482), VvMYBPA1 (CAJ90831), PmMBF1 (AAA82943), OSMYB4 (BAA23340), AtMYB5 (AAC49311), BNLGHi233 (AAK19611), VvMYB5b (AAX51291), VvMYB5a (AAS68190), FaMYB1 (AAK84064), AtMYB7 (AAA98762), HOS10 (AAF20989), HvMYB5 (CAA50221), TFMYB6 (AAS19480), GhMYB1 (AAN28270), LeTHM27 (CAA64614), TaMYB1 (AAT37167), and ZmMYB38 (BAD34380).

) of 28 plant MYB proteins associated with different functions indicated that VvMYB5b belongs to the same cluster as VvMYB5a, another R2R3-MYB protein recently characterized in grapevine (Deluc et al., 2006). This small cluster contains six MYB proteins involved in the control of various physiological and developmental processes like the phenylpropanoid pathway in grapevine for VvMYB5a and the chilling tolerance in rice (Oryza sativa) for OSMYB4 (Vannini et al., 2004). AtMYB5 may be involved in cell fate determination and trichome development in Arabidopsis (Li et al., 1996), whereas PH4 activates vacuolar acidification in petunia (Quattrocchio et al., 2006). The last member of this cluster, the BNLGHi233 protein from cotton (Gossypium hirsutum), has not yet been characterized. The other MYB transcription factors from grapevine, like VvMYBA1 and VvMYBA2 (Kobayashi et al., 2002, 2004; Walker et al., 2007) or VvMYBPA1 (Bogs et al., 2007), clearly belong to other clusters, indicating a possible functional divergence between these proteins and VvMYB5a and VvMYB5b. Regarding the sequence similarity, the VvMYB5b protein sequence displays 65% and 56% overall similarity to its nearest neighbors, the VvMYB5a and BNLGHi233 proteins, respectively. These overall levels of similarity appear relevant if we consider that R2R3-MYB proteins usually exhibit significant similarities only within the amino-terminal region where the DNA binding domain is located (Fig. 1; Kranz et al., 1998; Stracke et al., 2001).

The alignment of the VvMYB5b protein sequence with other known R2R3-MYB regulators from various plant species reveals the appearance of conserved peptidic motifs in the C-terminal regions of the proteins (Fig. 1). Apart from the very well-conserved DNA binding domain, referred to as the R2R3 domain, three distinct motifs were found within the analyzed sequences. The first one, [D/E]Lx₂[R/K]x₃Lx₆Lx₃R, is involved in the interaction with bHLH proteins and is present in the majority of the R2R3-MYB proteins (ID domain; Fig. 1; Grotewold et al., 2000; Stracke et al., 2001) The second conserved motif, called C1 (Lx₃GIDPxTHKPL) and initially described by Kranz et al. (1998) in the proteins of the MYB subgroup 4, is found in the six proteins belonging to the VvMYB5b cluster identified by phylogenetic analysis (Fig. 2). Another conserved motif, tentatively named C3 (DDxF[S/P]SFL[N/D]SLIN[E/D]), appears only in the sequences of the six members of the VvMYB5b cluster and is not found in other MYB proteins characterized so far. In conclusion, sequence analysis data indicate that VvMYB5b belongs to the R2R3-MYB family of transcription factors and, more precisely, to a small group of MYB proteins characterized by the presence of two specific conserved motifs, but involved in various physiological or developmental processes.

The Expression of VvMYB5b Is Highest in Berries after Veraison

Reverse transcription (RT)-PCR methods were used to study the expression of VvMYB5b in various tissues of Cabernet Sauvignon and Shiraz (Fig. 3  

Figure 3.

VvMYB5b expression in grapevine tissues. A, VvMYB5b expression in developing Cabernet Sauvignon berries. Southern blots of semiquantitative RT-PCR products were blotted onto a nylon membrane and hybridized with the radiolabeled VvMYB5b 3′-UTR probe. Elongation factor EF1-γ was used as a quantitative control. B, VvMYB5b expression in vegetative tissues of Cabernet Sauvignon vines. Southern blots of semiquantitative RT-PCR products were blotted onto a nylon membrane and hybridized with the radiolabeled VvMYB5b 3′-UTR probe. Elongation factor EF1-γ was used as a quantitative control. C to E, Expression of VvMYB5b in tissues of developing berries from the Shiraz cultivar. Numbers correspond to weeks relative to the onset of ripening (veraison). VvMYB5b expression was analyzed in whole berries from 10 to 6 weeks before veraison and specifically in seed and skin tissues from 6 weeks before up to 8 weeks after veraison. Data are means ± ses of three real-time PCR experiments.

). Semiquantitative methods were used to examine expression in Cabernet Sauvignon berries before and after veraison. To avoid cross-hybridization with other members of the MYB gene family, the 3′-untranslated region (UTR) of the VvMYB5b cDNA was used as a specific radiolabeled probe. In whole berries (Fig. 3A), the VvMYB5b gene appears expressed throughout development. The decrease of VvMYB5b expression level observed at the veraison stage is followed by strong accumulation of the transcripts 2 weeks after veraison. After this large increase, VvMYB5b remained expressed at a high level up to 6 weeks after veraison.

VvMYB5b expression was also investigated in skin and seeds of berries from the Shiraz cultivar using real-time quantitative PCR. During the early stages of berry development (10–6 weeks before veraison), VvMYB5b expression level remained low in the berry with a slight transitory increase 9 weeks before the onset of ripening (Fig. 3B). Between 6 and 4 weeks before veraison, it was possible to separate skin and seed from the developing berries and thus to analyze VvMYB5b transcript abundance in both tissues (Fig. 3, C and D). Generally, VvMYB5b expression was higher in skin than in seeds throughout berry development, except at the veraison stage where the expression was very low in both tissues. In seeds, VvMYB5b was always expressed at lower levels than in skins with a maximal level 2 weeks before veraison (Fig. 3C). In the skin (Fig. 3D), the VvMYB5b expression pattern resembles the one observed in Cabernet Sauvignon berries with a decrease of gene expression at the veraison stage followed by a continuous increase of transcript abundance up to the mature stages (6–8 weeks after veraison).

In summary, the VvMYB5b gene is expressed in grape berries throughout development and also in vegetative tissues of grapevine (Fig. 3E), where expression was detected in young and old leaves and roots. In berries, expression is high in the skin tissues with a first phase of gene activation observed before veraison and a second one, more pronounced, beginning just after veraison. It is interesting to note that the VvMYB5b expression pattern during berry development was very similar in Cabernet Sauvignon and Shiraz berries grown in France and Australia, respectively. This finding indicates that the regulatory mechanisms of gene expression may be conserved in two Vitis cultivars exposed to different environmental conditions.

VvMYB5b and VvMYB5a Activate Promoters of Grapevine Flavonoid Genes

To identify the target genes of VvMYB5b and its closest homolog VvMYB5a in grape berry, we analyzed the ability of these proteins to activate the promoters of grapevine genes encoding enzymes of the flavonoid pathway using a transient expression assay previously described by Bogs et al. (2007). Among the promoters tested, the grape chalcone isomerase (VvCHI), flavonoid 3′5′ hydroxylase (VvF3′5′H), and anthocyanidin synthase (VvANS) promoters control genes of the general flavonoid pathway, whereas VvUFGT is specifically involved in anthocyanin synthesis and the grape anthocyanidin reductase (VvANR) and leucoanthocyanidin reductase (VvLAR1) genes encode the enzymes leading to the synthesis of PAs. The ability of VvMYB5b and VvMYB5a to activate a heterologous promoter was also analyzed using the Arabidopsis ANR promoter (AtBAN). Figure 4  

Figure 4.

Activation of flavonoid gene promoters by VvMYB5b and VvMYB5a. Control indicates the activity of the respective promoter in the absence of a MYB factor. Each transfection contained the 35S:EGL3 construct encoding the bHLH protein EGL3 (GenBank accession no. NM20235) from Arabidopsis and as internal control the Renilla luciferase plasmid pRLuc. The normalized luciferase activity was calculated as the ratio between the firefly and the Renilla luciferase activity. Each column represents the mean value of three independent experiments with error bars indicating ses. Abbreviations are as follows: VvUFGT, V. vinifera UDP-Glc:flavonoid 3-O-glucosyltransferase; VvANR, V. vinifera anthocyanidin reductase; AtBAN, A. thaliana Banyuls; VvLAR1, V. vinifera leucoanthocyanidin reductase 1; VvCHI, V. vinifera chalcone isomerase; VvF3′5′H, V. vinifera flavonoid 3′5′ hydroxylase; and VvANS, V. vinifera anthocyanidin synthase.

summarizes the results obtained with the two MYB proteins and indicates that VvMYB5b is able to activate the VvLAR1 (15-fold), VvANS (14-fold), and VvCHI (approximately 4-fold) promoters. Significant inductions of the VvANR (8-fold) and VvF3′5′H (12-fold) promoters were also detected. These findings clearly indicate that VvMYB5b can enhance the expression of structural genes from the common steps of the flavonoid pathway together with the expression of specific genes associated with the PA biosynthetic pathway such as VvLAR1 and VvANR.

Our results also indicate that the VvMYB5a protein is able to activate the VvLAR1 (27-fold), VvANS (12.5-fold), VvF3′5′H (12-fold), and VvCHI (7-fold) promoters (Fig. 4). However, neither VvMYB5a nor VvMYB5b appears to play a key role in the control of the VvUFGT promoter where the level of activation was very low. In contrast, both VvMYB5a and VvMYB5b appeared able to activate the Arabidopsis AtBAN promoter with almost the same efficiency (between 4- and 5-fold). All promoter assays included a construct expressing AtEGL3 (ENHANCER OF GLABRA3) in the transient expression system, which encodes a bHLH protein involved in flavonoid pathway regulation in Arabidopsis (Ramsay et al., 2003). Similar to other VvMYB regulators, VvMYB5a and VvMYB5b were not able to induce promoter activities significantly without expression of AtEGL3 in our transient assay (data not shown; Bogs et al., 2007; Walker et al., 2007).

Taken together, promoter activation data indicate that VvMYB5a and VvMYB5b are both able to activate the expression of genes encoding enzymes of the general flavonoid pathway involved in synthesis of anthocyanins, PAs, and flavonols (e.g. VvCHI, VvF3′5′H, and VvANS). In addition, VvMYB5b can activate the expression of the two genes specifically involved in PA biosynthesis (e.g. VvLAR1 and VvANR), whereas VvMYB5a appears only implicated in the control of VvLAR1 expression.

Accumulation of Anthocyanin- and PA-Derived Compounds in Transgenic Tobacco Flowers Expressing VvMYB5b

Significant changes in the pigmentation of flowers were observed in tobacco plants constitutively expressing VvMYB5b. The strongest modifications were found in stamens where the amount of pigmentation was much greater in transgenic stamens at various developmental stages (Fig. 5, B and C  

Figure 5.

Phenotypic analysis of transgenic tobacco flowers overexpressing VvMYB5b. Flowers of transgenic plants (B and C) showed an increased pigmentation in petal and stamen epidermal cells compared to control flowers (A). DMACA staining of petals (E) and stamen (G) cells from transgenic flowers shows condensed tannin accumulation when compared to control lines (D and F). H and I, Delay in pollen release observed in anthers at developmental stages 10, 11, and 12 (from left to right, according to Koltunow et al. [1990]) from VvMYB5b sense lines (I) compared to control plant anthers (H). J and K, Cross sections of anthers at stage 10 from control (J) and VvMYB5b-overexpressing tobacco plants (K). Boxed areas are magnified in L and M, respectively. Arrows indicate endothecial cells developing complete (L) or incomplete (M) lignified fibers. Bars = 500 μm (J and K) and 75 μm (L and M).

) when compared to control plants (Fig. 5A). Transgenic petals also appeared deeper pink than for control flowers. This enhanced reddish pigmentation of petals resulted from an unusual distribution of reddish and uncolored cells in the epidermal layer (Fig. 5, B and C). Quantification of anthocyanin content was achieved by spectrophotometry (535 nm) in stamens and petals (Table I  

Table I.

Total anthocyanin content (mg/g DW) of stamens and petal extracts from control and transgenic plants

Quantitative determination of total anthocyanin content in tobacco flowers has been performed by spectrophotometry at 535 nm. A purified keracyanin standard of was used as a control. Data are the mean of three replicates ± sd. DW, Dry weight tissue; N.D., not detected.

Sample	Stamens	Petals
VvMYB5b line A	2.74 ± 0.08	2.87 ± 0.05a
VvMYB5b line B	2.48 ± 0.07	2.98 ± 0.09a
VvMYB5b line C	2.35 ± 0.09	3.03 ± 0.13a
Control	N.D.	1.48 ± 0.26

a

Significantly different from wild type (P < 0.001; Student's t test).

). An accumulation of total anthocyanins was detected in transgenic stamens (2.52 ± 0.08 mg/g of dry weight), whereas no significant level of anthocyanins was detected in control stamens. In petals, the total anthocyanin content in transgenic lines was nearly twice (2.96 ± 0.09 mg/g of dry weight) the amount measured in petals of control plants (1.48 ± 0.26 mg/g of dry weight). Like for the VvMYB5a transcription factor (Deluc et al., 2006), HPLC analysis indicates that the increase of anthocyanins in transgenic petals and stamens is linked to the accumulation of keracyanin (cyanidin-3-rhamnoglucoside), which is the main anthocyanin compound in tobacco (data not shown).

Additional analyses of the transgenic plants also revealed modifications of the PA metabolic pathway. Accumulation of condensed tannins in petals and stamens was detected using dimethylaminocinnamaldehyde (DMACA) staining (Xie et al., 2003). A blue coloration, linked to the presence of PA, was observed in the epidermal cell layers of petals and anther tips of transgenic flowers (Fig. 5, E and G), but not in control line flowers (Fig. 5, D and F). HPLC analysis and quantification confirmed a strong accumulation of free monomer units of epicatechin-derived compound in transgenic petals (10.51 ± 0.09 mg/g of fresh weight) compared to control petals (1.48 ± 0.26 mg/g of fresh weight; Table II  

Table II.

Epicatechin and catechin content (mg/g FW) of petal extracts from control and transgenic plants

Quantitative determination of catechin and epicatechin content in tobacco flowers has been performed by HPLC. The eluate was monitored at 280 nm. Purified standards of catechin and epicatechin were used as controls. Data are the mean of two replicates ± sd. FW, Fresh weight tissue; N.D., not detected.

Sample	Catechins	Epicatechins
VvMYB5b line A	N.D.	9.65 ± 0.05a
VvMYB5b line B	N.D.	10.84 ± 0.09a
VvMYB5b line C	N.D.	11.05 ± 0.13a
Control	N.D.	1.48 ± 0.26

a

Significantly different from the control (P < 0.005; Student's t test).

). Taken together, these results indicated a strong accumulation of anthocyanins and PAs in flowers of plants overexpressing VvMYB5b and appeared similar to those obtained with the VvMYB5a cDNA (Deluc et al., 2006), suggesting similar regulatory functions in tobacco for both MYB factors. As previously observed with VvMYB5a (Deluc et al., 2006), a delay in anther dehiscence was observed. Whereas the pollen was completely released 12 weeks after bloom in control plants (Fig. 5H), the anthers were still closed in VvMYB5b transgenic sense lines (Fig. 5I). Cross section analyses of the anthers from VvMYB5b sense lines revealed changes in the lignification network of the endothecial cell wall, responsible of the stomium breaking and leading to the pollen release. In transformed plants, fewer endothecial cells developed intact lignified fibers. Most of the time, they were incomplete as observed in Figure 5M compared to the control that exhibited complete fibers covering the entire radial walls (Fig. 5L).

VvMYB5b Affects the Expression of Flavonoid Biosynthetic Genes in Transgenic Tobacco Flowers

Expression analysis performed on RNA extracted from petals and stamens of three independent transgenic lines of tobacco expressing the VvMYB5b cDNA indicated that VvMYB5b activated the expression of several structural genes associated with the flavonoid pathway (Fig. 6, A and B  

Figure 6.

Analysis of flavonoid gene expression in flowers of transgenic tobacco overexpressing VvMYB5b. A, Transcripts for four flavonoid biosynthetic genes were detected by semiquantitative RT-PCR in petals and stamens from three VvMYB5b independent lines (1–3) and compared to control lines (Ct). VvMYB5b indicates the transgene expression level. UBI was used as a quantitative control. Abbreviations are as follows: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3 hydroxylase; and F3′5′H, flavonoid 3′5′ hydroxylase. B, Northern-blot analysis of anthocyanidin synthase (ANS) gene expression in petals and stamens from control (Ct) and VvMYB5b lines. Total RNA was extracted from tissues harvested at the opening of flowers. Experiments were repeated with three VvMYB5b and control lines with similar results.

). In petals and stamens of transgenic plants, expression of the chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3 hydroxylase (F3H), and anthocyanidin synthase (ANS) genes was higher than that in control flowers (Fig. 6, A and B). In addition, dihydroflavonol reductase (DFR) gene expression appeared differentially regulated in transgenic petals and stamens indicating the appearance of additional regulatory mechanisms. These results indicate the involvement of VvMYB5b in the regulation of flavonoid genes expression in tobacco flowers. However, this regulatory action may vary depending on the tissue studied as suggested by the contrasting expression of the DFR gene in transgenic flowers.

DISCUSSION

VvMYB5b Encodes a New Member of the R2R3-MYB Family in Grapevine

During their development, grape berries accumulate many different products of the flavonoid pathway. Although there is a separation in timing during which PAs, flavonols, or anthocyanins are synthesized, tissues such as the skin contain relatively large quantities of these compounds at ripeness. The regulatory mechanisms involved in the control of the complex branching pathway that leads to the synthesis of these various flavonoid compounds remain unclear. In this article, we present the identification and the characterization of the grape VvMYB5b transcriptional regulator and describe one of the missing parts of this regulatory puzzle. Phylogenetic analyses indicate that the VvMYB5b protein is clearly distinct from the VvMYBA factors and belongs to a small cluster that includes the recently characterized VvMYB5a gene (Deluc et al., 2006). The presence, in this small cluster, of the Arabidopsis AtMYB5 protein may be interesting if we consider that AtMYB5 appears as a single isolated MYB gene in Arabidopsis and does not belong to any identified cluster of R2R3-MYB proteins (Kranz et al., 1998; Stracke et al., 2001). According to the recent release of the grape genome (http://www.genoscope.cns.fr/externe/English/Projets/Projet_ML/index.html), VvMYB5a and b localize to chromosomes 8 and 6, respectively, and appear to be under the control of different cis-regulatory elements as indicated by their almost opposite expression patterns in developing berries. Thus, VvMYB5a and VvMYB5b may represent paralogous genes that have originated from a duplication event followed by genomic dispersion. Taken together, data presented in this article indicate that the spatiotemporal expression of VvMYB5b, combined with the action of specific regulators like VvMYBA1 and VvMYBPA1, controls the biosynthesis of both anthocyanins and PAs throughout grape berry development (Fig. 7  

Figure 7.

Summary of the possible implication of R2R3-MYB transcription factors in the regulatory mechanisms of the flavonoid pathway during grape berry development. The roles of VvMYBPA1, VvMYB5a, VvMYB5b, VvMYBA1, and VvMYBA2 were assigned according to gene expression levels during berry development together with functional characterization data presented in this article for VvMYB5b and VvMYB5a, in Bogs et al. (2007) for VvMYBPA1, in Deluc et al. (2006) for VvMYB5a, and in Walker et al. (2007) for the VvMYBA genes. Gene expression levels throughout berry development were determined by quantitative RT-PCR and are presented in Figure 3 (VvMYB5b) and in Supplemental Figure S1 for the other genes. The same RNA samples extracted from Shiraz berries collected at various developmental stages (see “Materials and Methods”) were used to analyze the expression level of each gene. According to these data, VvMYBPA1, VvMYB5a, and VvMYB5b appear particularly involved in the regulation of PA synthesis before veraison in skin and seed tissues. At veraison, PA synthesis is complete and anthocyanin synthesis begins in skin cells where UFGT gene expression is specifically regulated by VvMYBA1 and VvMYBA2, whereas genes encoding enzymes of the general flavonoid pathway required for anthocyanidin synthesis appear to be regulated by VvMYB5b. Abbreviations are as follows: PAL, Phe ammonia lyase; C4H, cinnamate 4 hydroxylase; 4CL, 4 coumarate CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3 hydroxylase; F3′5′H, flavonoid 3′5′ hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; and UFGT, UDP-Glc:flavonoid 3-O-glucosyltransferase.

). Moreover, taking into account the findings of Deluc et al. (2006), our results suggest that VvMYB5b and VvMYB5a encode functionally related proteins and that their unique roles in the control of the flavonoid pathway in grape berry may be entirely due to differences in their cis-regulatory sequences, as already demonstrated in Arabidopsis for the WER and GL1 paralogous MYB genes (Lee and Schiefelbein, 2001).

Implication of VvMYB5b in the Regulatory Mechanisms of Anthocyanin Biosynthesis

Promoter activation experiments in grape cells indicated that VvMYB5b is able to activate several genes of the general pathway, but not UFGT, the last enzyme of the anthocyanin biosynthetic pathway. However, the recent findings of Walker et al. (2007), combined with previous work from Kobayashi et al. (2002, 2004), clearly indicate the role of the two MYB genes VvMYBA1 and VvMYBA2 in the specific control of UFGT gene expression in ripening grape berries. Interestingly, both MYBA factors are not functional in white grapes and UFGT is not expressed, whereas the genes encoding enzymes of the general flavonoid pathway (e.g. CHS, DFR, F3H, ANS), which produce anthocyanin precursors (i.e. anthocyanidin) are still expressed in white grapes after veraison (Boss et al., 1996b; Walker et al., 2007). These observations suggest the presence of an additional regulator controlling the expression of genes encoding enzymes of the general flavonoid pathway when anthocyanins are synthesized. Therefore, according to its high expression in skin after veraison and its ability to activate the promoters of general flavonoid genes in grape cells, we postulate that VvMYB5b acts together with VvMYBA1 and VvMYBA2 to regulate anthocyanin biosynthesis in ripening grape berries (Fig. 7).

VvMYB5b Participates in the Regulation of PA Biosynthesis in Developing Grape Berries

In grape, PA synthesis is complete by veraison, after which expression of genes encoding ANR and LAR declines and then remains very low during berry ripening (Davies and Robinson, 2000; Downey et al., 2003a, 2003b; Bogs et al., 2005). Catechins, synthesized by LAR, are the predominant flavan-3-ols found in skin, whereas epicatechin, synthesized by ANR, are the main flavan-3-ols present in grape seeds (Bogs et al., 2005). To date, the R2R3-MYB protein VvMYBPA1 is the only specific regulator of PA synthesis identified in grapevine (Bogs et al., 2007). However, data presented in this article indicate that VvMYB5b may also be involved in the regulatory mechanisms of PA biosynthesis. Expression of VvMYB5b was observed in flowers and in the early steps of berry development in both seed and skin tissues. Combined with its ability to activate the VvLAR1 and, to a lesser extent, the VvANR promoters, this finding suggests that VvMYB5b could play a part in the regulation of both catechin and epicatechin biosynthesis occurring during the early stages of berry development. However, because VvMYB5b appeared expressed at a higher level in young berry skin compared to seed and activated preferentially the VvLAR1 promoter, its main function in PA biosynthesis regulation seems to be the control of catechin synthesis in skin cells.

In this article, we also provide additional data regarding the biological function of VvMYB5a, an R2R2-MYB transcription factor expressed only during the first phase of berry development (i.e. before veraison; Deluc et al., 2006). The similarities observed between VvMYB5a and VvMYB5b include the appearance of the same phenotypic changes in transgenic tobacco plants. In both cases, activation of flavonoid structural gene expression accompanied by the accumulation of anthocyanin and PA-derived compounds was observed in transgenic flowers. The analysis of promoter activation by transient expression of VvMYB5a in grape cells showed that this transcription factor is able to induce promoters of the general flavonoid pathway genes (e.g. VvANS, VvF3′5′H, or VvCHI), of the catechin-specific gene VvLAR1, but not of the VvANR gene. Thus, these new functional characterization data indicate that VvMYB5a appears also involved in the regulation of PA biosynthesis and especially in the control of catechin synthesis in skin cells.

In summary, VvMYBPA1, VvMYB5a, and VvMYB5b appear able to regulate PA biosynthesis in developing grape berries and their expression patterns in different berry tissues combined with their target gene specificities could contribute to the different levels and catechin/epicatechin composition of PAs detected in skins and seeds (Fig. 7; Downey et al., 2003a; Bogs et al., 2005).

Evidence for Additional Regulators of the Flavonoid Biosynthetic Pathway in Grape Berry

In some cases, gene expression patterns do not always correlate exactly with physiological events or metabolite biosynthetic activities unravel by functional characterization approaches. As an example, in this article, promoter activation assays indicated the ability for VvMYB5b to activate VvLAR and VvANR promoters. However, VvMYB5b is expressed in berries after veraison when PA biosynthesis appears to be complete and both VvANR and VvLAR expression remains very low (Bogs et al., 2005). A similar situation was observed by Bogs et al. (2007) with the VvMYBPA1 gene. The discrepancy of the ability for VvMYB5b or VvMYBPA1 to activate VvLAR and VvANR promoters while they are expressed after veraison when anthocyanins begin accumulating and PA biosynthesis is complete is unclear. Differences between transcriptional regulators and target gene expression imply the effect of additional regulatory mechanisms. In the case of PA synthesis, one possibility is the expression in berries after veraison of a strong negative regulator that could act by direct repression or by competition with activators on binding motifs of the VvLAR and VvANR promoters and thus prevent transcription. Repression of phenylpropanoid biosynthesis by MYB transcription factors has already been demonstrated (Jin et al., 2000; Aharoni et al., 2001). In strawberry, the FaMYB1 protein represses flavonoid gene transcription and anthocyanin synthesis at the latter stages of fruit maturation (Aharoni et al., 2001). FaMYB1-related genes are likely to be present in grape and may have similar effects on PA biosynthesis and thus may refine the control of the timing of PA accumulation during berry ripening.

An alternative hypothesis related to the decline of PA gene expression after veraison may be the absence of an interacting protein partner for the MYB proteins. It is now well established that the transcriptional regulators for flavonoid biosynthesis include members of the R2R3-MYB family, the bHLH family, and the WDR proteins. Combinations between these three regulatory factors determine the set of target genes to be expressed (Springob et al., 2003; Broun, 2005; Koes et al., 2005; Ramsay and Glover, 2005), but the precise function of each component of the MYB-bHLH-WDR complex remains elusive. In Arabidopsis, the WDR protein TTG1 may stabilize the interaction between the TT2 MYB factor and the bHLH protein TT8 (Baudry et al., 2004; Lepiniec et al., 2006) and thus promote the transcriptional activation of flavonoid structural genes. More recently, the results of Hernandez et al. (2007) indicate that, in maize, the binding of the C1 MYB factor to specific cis-regulatory regions is essential for the recruitment of the bHLH protein R, which may subsequently act as a docking platform for additional factors like the WDR protein PAC1 (Carey et al., 2004). Interestingly, other proteins like RIF1, an EMSY-related factor involved in chromatin functions, also appear to interact with the bHLH protein R and thus represent a new class of proteins implicated in the regulatory mechanisms of gene expression by MYB transcription factors (Hernandez et al., 2007). In this article, modifications in PAs, anthocyanins, and even lignins were only observed in reproductive parts of transgenic tobacco overexpressing VvMYB5b, suggesting interaction with tissue-specific partners. For the grapevine MYB proteins VvMYB5a, VvMYB5b, and VvMYBPA1, the presence of a bHLH protein was essential to activate gene expression in our promoter experiments, indicating the necessity of an interacting partner for both proteins. Thus, the lack of a specific interacting partner after veraison in skin cells might explain the absence of VvANR and VvLAR expression even in the presence of MYB regulators. This hypothesis implies that MYB factors and their interacting proteins not only cooperate in directing tissue-specific production of flavonoid compounds, but also that a particular interaction may control a specific branch of the pathway like PA biosynthesis (Hartmann et al., 2005; Park et al., 2007). In any case, future work will also have to integrate the important role of bHLHs, WDRs, and EMSY-related proteins in the control and the specificity of the mechanisms of gene expression involving MYB transcription factors.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Berries from grape (Vitis vinifera ‘Cabernet Sauvignon’) plants from Domaine du Grand Parc (INRA) were sampled at 2-week intervals during the 2002 and 2003 growing seasons. Grapevine tissues of Shiraz grape were collected from a commercial vineyard (Adelaide, Australia) 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 by Downey et al. (2003a). Berry development stages were chosen according to criteria including size, titers of soluble sugars, and softening and color of the berries (Boss et al., 1996a; Downey et al., 2003a). Berries for RNA extraction were sampled and immediately frozen in liquid nitrogen before storage at −80°C pending further analysis. Separate skin and flesh samples were obtained by peeling fresh berries just before freezing in liquid nitrogen.

Control and transgenic tobacco (Nicotiana tabacum ‘Xanthii’) seeds were sterilized in 70% ethanol for 2 min, incubated in 2.5% potassium hypochlorite solution for 10 min, and finally washed three times in sterile water. After cold treatment at 4°C for 48 h, seeds were germinated in tissue culture conditions at 23°C under a 16-h-light/8-h-dark regime on Murashige and Skoog medium (Murashige and Skoog, 1962) containing 3% (w/w) Suc and 100 μg/mL kanamycin for transgenic plants. Tobacco plants were finally transferred to soil and grown in individual pots in a greenhouse.

Cloning of VvMYB5b cDNA and Plant Transformation

A cDNA library from grape Cabernet Sauvignon berries (veraison stage) was constructed using the Smart cDNA library construction kit (CLONTECH) and used as template for PCR cloning. Two degenerate oligonucleotides from plant MYB cDNA conserved sequences, 5′-GANRTMAARAAYTAYTGGAACWCN-3′ in forward and 5′-NGTGTTCCARTARTTYTTKAYNTC-3′ in reverse orientation, were used in combination with the T7 and 5′ primers located in the pTriplex vector (CLONTECH). PCR products were subsequently ligated to pGemTeasy (Promega) and the insert DNA was sequenced on both strands (Genome Express). Specific oligonucleotides were then defined within the 5′ and 3′ noncoding regions of sequenced cDNA and used to amplify the complete full-length cDNA. For plant transformation, the complete coding sequence of VvMYB5b cDNA was amplified with a specific forward primer designed to introduce an XbaI restriction site (5′-GGGGTCTAGAGAGAAAGAAGAA-3′) and a reverse primer designed to introduce a SacI restriction site (5′-TTAACTATAGAGCTCATTGCA-3′). The XbaI/SacI fragment was ligated to the binary vector pGiBin19 (provided by Dr. D. Inzé, Ghent, Belgium) between the 35S promoter of cauliflower mosaic virus and the nopaline synthase (nos) poly(A) addition site, creating the pGiBin19-VvMYB5b plasmid. The construct was introduced into the Agrobacterium tumefaciens LB4401 strain. Leaf disc transformation and regeneration of transgenic plants were performed as previously described by Horsch et al. (1986). Transformed tobacco plants were selected using kanamycin (100 μg/mL) as a selective marker. Fifteen independent lines from the T₂ progeny of transgenic plants showing no phenotypic variations in their broad aspect were used for further analysis.

RNA Extraction and Analysis

Different methods of RNA extraction were used depending on the plant material. Total RNA was isolated from the various Shiraz berry tissues as described in Downey et al. (2003b). For Cabernet Sauvignon berry tissues, RNA extraction was carried out according to Asif et al. (2000). RNAs from control and transformed tobacco leaves were extracted using RNeasy plant mini kits (Qiagen) according to the manufacturer's instructions. Finally, total RNAs from tobacco flowers were extracted as described by Verwoerd et al. (1989). All RNA samples were treated with Rnase-free Dnase I (Promega), followed by phenol/chloroform extraction and ethanol (95%) precipitation. No DNA contamination was detected by PCR amplification. For semiquantitative RT-PCR assays, gene-specific primers were preferentially chosen in the 3′-UTR regions of the target mRNAs as described by Deluc et al. (2006; Table III  

Table III.

Primers for RT-PCR analysis

Genes	Accession No.	Sense Primers	Antisense Primers
NtCHS	AF311783	5′-CCTTTGGGAATTTCTGATTGG	5′-TCCCACAATATAAGCCCAAGC
NtF3H	AF036169	5′-AGCTAGAGACTACTCCAGGTG	5′-AACCGTGATCCAAGTTTTGCCA
NtUBI	U66264	5′-TCCAGGACAAGGAGGGTAT	5′-GAGACCTCAAGTAGACAAAGC
VvMYB5b3′-UTR	AY899404	5′-GATGACAGCCGGTGTTCTTT	5′-AACAACACAACCACATACA
VvEF1-γ	AF176496	5′-GCGGGCAAGAGATACCTCAA	5′-TCAATCTGTCTAGGAAAGGAAG

; see Supplemental Fig. S1). To estimate the transcript amounts for the VvMYB5b transgene in tobacco, we used a forward specific primer of VvMYB5b (5′-GATAAGCGGTTCTGACAGC-3′) in combination with a reverse primer designed from the 3′ transcribed region of the nos terminator (5′-TCATCGCAAGACCGGCAACA-3′). Specific primers for tobacco CHI and DFR were designed from cDNA clones generously provided by Dr. Cathie Martin (John Innes Centre), to whom requests may be addressed. In all cases, cloning and sequencing of the RT-PCR products confirmed the amplification specificity. The ANS cDNA probe from petunia (Petunia hybrida) was provided by Dr. Asaph Aharoni (Plant Research International).

For RT-PCR analysis, 1 μg of total RNA was reverse transcribed with oligo(dT)_12-18 in a 20-μL reaction mixture using the Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. After heat inactivation of the reaction mixture, PCR was performed using 1 μL of the first-strand cDNA sample with 25 pmol of the primers shown in Table I in a 50-μL reaction. For all experiments presented in this article, 15 cycles of PCR amplification were performed, except for the detection of VvMYB5b transgene in tobacco (Fig. 5, A, B, and D), where 20 cycles were performed. RT-PCR products were separated on agarose gels and analyzed by DNA gel-blot hybridization using random-primed ³²P-gene-specific probes. RNA gel blots were prepared and hybridized according to standard protocols (Sambrook et al., 1989). Equal loading of RNA samples and uniform transfer onto membranes were confirmed by ethidium bromide staining of the RNA gels and hybridization with a ³²P-labeled ubiquitin probe. After hybridization at 42°C, membranes were washed at room temperature three times for 20 min each in 1× SSC and 0.1% SDS, followed by one wash in 0.2× SSC and 0.1% SDS at 65°C for 15 min before exposure to a phosphor imager screen (Molecular Imager FX; Bio-Rad).

Expression analysis by real-time PCR was performed according to Bogs et al. (2007). The primers VvMYB5BF (5′-GGTGTTCTTTAATTTGGCTTCA-3′) and VvMYB5BR (5′-CACAACAACACAACCACATACA-3′) were used to amplify a 143-bp PCR product from the 3′-UTR of the VvMYB5b cDNA (AY899404). The primers MYB5AF (5′-CATGTCTCCCTGAAAATGATGA-3′) and MYB5AR (5′-TGCAAGGATCCATTTCACATAC-3′) were used to amplify a 179-bp PCR product from the 3′-UTR of the VvMYB5a cDNA (AY555190). Data were normalized according to the VvUbiquitin1 (TC53702; The Institute for Genomic Research database, VvGi5) gene expression level, which is relatively stable throughout berry development (Downey et al., 2003b; Bogs et al., 2005). All samples were measured in triplicate.

HPLC Analyses and Quantification of Anthocyanin- and PA-Derived Compounds

HPLC analyses and quantification of total anthocyanins and PA-derived compounds from petal limbs and stamens harvested from transgenic and control tobacco were performed as described in Deluc et al. (2006). The histochemical staining of condensed tannins in epidermal cell layers in tobacco flowers was conducted according to Porter (1989) by staining tissues in a solution of ethanol: 6 m HCl (1:1) containing 0.1% (w/v) DMACA (Sigma-Aldrich) for 3 to 6 min, then by washing three times with water.

Transient Transfection Experiments and Dual-Luciferase Assay

As described in Bogs et al. (2007), a transient assay was applied using a cell suspension of a Chardonnay petiole callus culture and the dual-luciferase system. Gold particles were coated with a mixture of DNA constructs (150 ng of the respective plasmid, giving a total plasmid concentration of 750 ng/shot) by the method described in Ramsay et al. (2003) and used to bombard Chardonnay cells at a helium pressure of 350 kPa within a vacuum of 75 kPa and a distance of 14 cm (Torregrossa et al., 2002). For the dual-luciferase assay, each bombardment contained a positive control of 3 ng of the Renilla luciferase plasmid pRluc (Horstmann et al., 2004). Cells were harvested 48 h after transfection and lysed by grinding on ice in 150 μL of passive lysis buffer (Promega). After centrifugation of the lysates for 2 min at 500g, measurement of the luciferase activities was performed with a dual-luciferase reporter assay system (Promega) by sequential addition of 25 μL of LARII and Stop & Glo to 10 μL of the lysate supernatant. Light emission was measured with a TD-20/20 luminometer (Turner Design) and the relative luciferase activity was calculated as the ratio between the firefly and the Renilla (control) luciferase activity. All transfection experiments were performed in triplicate and each set of promoter experiment was repeated with similar relative ratios to the respective control. Cloning of all promoter luciferase and bHLH constructs used in the transfection experiments of this study is described by Bogs et al. (2007). For transient expression of VvMYB5b, the ORF was amplified by PCR from Shiraz grape cDNA using PfuTurbo polymerase (Stratagene) and the primers Myb5bartF (5′-ATGCTCGAGGGGACGAGAGAGAAAGAAGAAA-3′) and Myb5bartR (5′-AGCTCTAGAATATCTCATTGCAGGGTGTTGA-3′). The VvMYB5a ORF was amplified using the primers MYB5aartF (5′-TAGCTCGAGAAGCCAGAGGGATGAGAAATC-3′) and MYB5aartR (5′-TAGTCTAGATGATTCATCATTTTCAGGGAGA-3′). The generated PCR fragments were purified, digested with XhoI and XbaI, and cloned in the vector pART7 (Gleave, 1992).

Sequence Analyses

Comparison and analysis of the VvMYB5b sequence were conducted with the advanced BLAST program (Altschul et al., 1990) at the National Center for Biotechnological Information (http//www.ncbi.nlm.nih.gov). Multiple sequence alignments were performed using the ClustalW (version 1.83.1) program (Ramu et al., 2003) and the edition of aligned sequence was performed using GeneDoc software (version 1.6). Phylogenic analyses were achieved using MEGA package software (Kumar et al., 2004; version 3.2.1).

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Figure S1. Quantitative RT-PCR analysis of the relative transcript levels of the VvMYB5a, VvMYBPA1, and VvMYBA genes in Shiraz berries over complete berry development.

ACKNOWLEDGMENTS

We are grateful to Dr. Cathie Martin and Dr. Asaph Aharoni for providing some of the cDNA probes used in this work and to Dr. Laurence Geny for PA analysis.

Author notes

1

This work was supported by grants from the “Conseil Interprofessionnel du Vin de Bordeaux,” The Grape and Wine Research and Development Corporation and the Cooperative Research Centre for Viticulture, and the GABI-Future program of the German Ministry of Education and Research.

2

This article is dedicated to the memory of our friend and colleague, Professor Saïd Hamdi.

3

These authors contributed equally to the article.

4

Present address: Department of Biochemistry, University of Nevada, 1664 North Virginia St., Reno, NV 89557.

5

Present address: Heidelberger Institut für Pflanzenwissenschaften, 69120 Heidelberg, Germany.

^*

Corresponding author; e-mail .

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: François Barrieu ().

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