Characterization of a bZIP transcription factor that regulates the phenylpropanoid pathway in grapevine

In grapevine (Vitis vinifera L.) flavonoids accumulate preferentially in the skin and seeds of grapes (Downey et al. 2004) determining the colour but also the final flavour and astringency of red and white wines. Furthermore, flavonoids extracted from grape berries and seeds exhibit strong nutraceutical potential with a broad spectrum of pharmacological and therapeutic effects (Nassiri-Asl and Hosseinzadeh 2009). Although the general flavonoid pathway has been genetically and biochemically elucidated in many plant species and most recently also in grapevine (Hichri et al. 2011), its regulation still remains not completely characterized. In this study we report the characterization of a grapevine bZIP transcription factor involved in flavonoid biosynthesis regulation. According to the ten-classes classification proposed in Arabidopsis, this VvbZIP belongs to the S-group of bZIP. Its expression along fruit development is higher at the flowering stage and is induced by UV-B treatment. The overexpression of VvbZIP in tobacco transgenic lines positively correlates with the flower content of quercetin and cyanidin in the glycosilated form. Furthermore, transient expression assays by bombardment of grapevine suspension cultures, have revealed that VvbZIP activates the flavonoid pathway at different sites and through the interaction with other co-factors

Characterization of a bZIP factor that regulates the flavonoid pathway in grapevine

Flavonoids compose one of the most abundant and important subgroups of secondary metabolites detected so far in higher plants. They are involved in several aspects of plant development and defence. Besides the attraction of pollinators and dispersers to fruits and flowers, flavonoids also protect against a plethora of stresses including pathogen attack, wounding and UV irradiation. Flavonoid content and composition of fruits and plant products have been associated with fruit quality including taste, colour and health-promoting effects (Czemmel et al., 2012). In grapevine (Vitis vinifera L.) flavonoids accumulate preferentially in the skin and seeds of grapes (Downey et al., 2004) determining the colour but also the final flavour and astringency of red and white wines. Furthermore, flavonoids extracted from grape berries and seeds exhibit strong nutraceutical potential with a broad spectrum of pharmacological and therapeutic effects (Nassiri-Asl and Hosseinzadeh, 2009). The synthesis of these compounds is mainly regulated at transcriptional level. Flavonoids accumulate at specific stages and in specific tissues during flower and berry development, as a consequence of the timely expression of the genes necessary for their synthesis. Although the general flavonoid pathway has been genetically and biochemically elucidated also in grapevine, its regulation still remains not completely characterized (Hichri et al., 2011). Similarly knowledge is still lacking about the molecular basis of the varietal variability in terms of quality and quantity of flavonoids. To shed light on this issue a mapping population derived from the cross Syrah x Pinot Noir segregating for flavonoid content was characterized at the metabolic and transcriptional level. The transcriptome analysis of the extreme genotypes of the population has allowed the identification of a set of differentially expressed genes. Among them, a basic leucin zipper transcription factor (VvbZIP) has been selected and further characterized to elucidate its role in regulating flavonoid biosynthesis. A phylogenetic analysis of all the 55 predicted grapevine bZIP factors was performed. According to the ten-classes classification proposed in Arabidopsis, VvbZIP belongs to the S-group of the family. The analysis of the different classes of flavonoids and of VvbZIP expression in Pinot Noir samples collected from flowering to technological maturity (18°Brix), showed a peak of VvbZIP expression as well as of the flavonols quercetin and kaemperol at flowering time. A further evidence of the role of this gene in flavonols regulation came from the analysis of Chardonnay plants exposed to UV-light and monitored over a 3-d time period. The light treatment caused an induction of the VvbZIP, VvFLS1 and VvMYBF1 transcripts within the first 10 h, followed by the accumulation of flavonols at 24 h post treatment. To test the in-planta function, VvbZIP was stably overexpressed into tobacco plants (Nicotiana tabacum). The overexpression of VvbZIP observed in 7 transgenic lines positively correlated with the flower content of quercetin, kaempferol and cyanidin in the glycosilated form suggesting a broader regulatory role of this factor.Luciferase reporter-assays carried out by bombardment of Chardonnay suspension cultures (Czemmel et al., 2009), revealed that VvbZIP binds to the promoters of VvCHS, VvFLS1, VvANR genes activating their transcription, likely through the interaction with other co-factors such as MYBF1 and MYBPA1

A multidisciplinary approach to understand the control of flavonol synthesis in grape berry

Flavonols are flavonoids found in most higher plants, usually in glycosidic forms. They are products of the flavonoid biosynthetic pathway, which also give rise to anthocyanins and to condensed tannins in grapes. In grapevine, they are predominantly synthesized in inflorescence and berry skin, while no detectable levels are found in pulp or seeds (Downey et al., 2003). They act as UV-protectants and as anthocyanin co-pigments in flowers and fruits. For this reason they play a role in defence and pollinators attraction and in conferring stability to the colour of red wines. Although the general flavonol pathway has been genetically and biochemically elucidated in many plant species and most recently also in grapevine (Matus et al., 2009), (Czemmel et al., 2009), its regulation still remains not completely characterized. A population from the cross Syrah x Pinot Noir, segregating for the flavonol content, is being studied to identify the genetic determinants of flavonol accumulation in the berry cells by a biochemical and transcriptional characterization. Individuals of the progeny showing high- and low- flavonol content were selected and characterized at different stages of berry development by a microarray approach. Among the differentially expressed identified genes, we focused our attention on a transcription factor not yet characterized in grapevine. To prove its involvement in the control of flavonoid pathway we identified its targets by a promoter assay approach and we analyzed the phenotype of transgenic tobacco lines overexpressing this gene. Preliminary results will be presented. References Downey et al. .2003. Synthesis of flavonols and expression of flavonol synthase genes in the developing grape berries of Shiraz and Chardonnay (Vitis vinifera L.). Aust J Grape and Wine Res, 9: 110-121 Matus et al. (2009). Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera. J Exp Bot;60(3):853-67 Czemmel et al. (2009). The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiol, 151:1513-30

The Basic Leucin-Zipper factor VvBZIP6 is involved in the regulation of the flavonoid pathway in grapevine

In grapevine (Vitis vinifera L.) flavonoids compose one of the most abundant subgroups of secondary metabolites. They accumulate preferentially in the skin and seeds of grapes and fulfill several important functions: i) they mediate the response to biotic and abiotic stresses(temperature, UV-light, nutrition, water deficit); ii) they provide pigmentation to flowers and fruits, thereby influencing quality and typicity of wines. They have been also associated to the observed health-promoting effects ascribed to grape rich diet including wine. The biosynthesis of these compounds is mainly regulated at the transcriptional level by controlling the genes of the so called general flavonoid pathway which has been genetically and biochemically elucidated in several species including grapevine. Regulation of the structural genes of the pathway is however still not completely elucidated. In this study we report the characterization of one of the 55 predicted grapevine bZIP genes, VvbZIP6, to be involved in flavonoid biosynthesis regulation. Analysis of its expression during Pinot Noir development revealed that this transcript is very abundant at flowering time concomitantly with the peak of the flavonols quercetin and kaemperol. Chardonnay leaves exposed to UV-light showed an induction of VvbZIP6 within the first 10 h, followed by the accumulation of flavonols at 24 h post treatment. The overexpression of VvbZIP6 in tobacco transgenic lines positively correlates with the flower content of flavonol compounds such as quercetin and kampferol but also glycosilated cyanidin and proanthocyanidins pointed towards a role as a general regulator of the grapevine flavonoid pathway. This has been confirmed by luciferase reporter-assays in Chardonnay suspension cultures showing that VvbZIP6 induces the promoters of VvCHS, VvFLS1, VvANR genes likely via interaction with other co-factors

The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis

In grapevine, flavonoids constitute one of the most abundant subgroups of secondary metabolites, influencing the quality, health value, and typicity of wines. Their synthesis in many plant species is mainly regulated at the transcriptional level by modulation of flavonoid pathway genes either by single regulators or by complexes of different regulators. In particular, bZIP and MYB factors interact synergistically in the recognition of light response units present in the promoter of some genes of the pathway, thus mediating light-dependent flavonoid biosynthesis. We recently identified VvibZIPC22, a member of clade C of the grapevine bZIP family, in a quantitative trait locus (QTL) specifically associated with kaemperol content in mature berries. Here, to validate the involvement of this candidate gene in the fine regulation of flavonol biosynthesis, we characterized its function by in vitro and in vivo experiments. A role for this gene in the control of flavonol biosynthesis was indeed confirmed by its highest expression at flowering and during UV light-mediated induction, paralleled by accumulation of the flavonol synthase 1 transcript and flavonol compounds. The overexpression of VvibZIPC22 in tobacco caused a significant increase in several flavonoids in the flower, via induction of general and specific genes of the pathway. In agreement with this evidence, VvibZIPC22 was able to activate the promoters of specific genes of the flavonoid pathway, alone or together with other factors, as revealed by transient reporter assays. These findings, supported by in silico indications, allowed us to propose VvibZIPC22 as a new regulator of flavonoid biosynthesis in grapevin