The stilbene synthase multigenic family in grapevine: gemome-wide analysis and transcriptional regulation

Plant stilbenes represent a relatively small group of phenylpropanoid compounds characterized by a diphenylethylene backbone and have been detected in only a few unrelated plant species, including pine (Pinaceae), peanut (Fabaceae), sorghum (Poaceae) and grapevine (Vitaceae) (Morales et al., 2000). As with other phenylpropanoids, stilbenes accumulate in response to biotic and abiotic stresses such as infection, wounding, UV-C exposure and treatment with chemicals (Dixon and Paiva 1995). During the last decade stilbenes, and resveratrol in particular, have captured the attention of biology and medicine due to both the biological and medicinal activities of those compounds. Among the biological effects they have been suggested to act as deterrents against animals and insects (Torres et al. 2003), antifungal compounds (Morales et al., 2000; Jeandet et al., 2002), allochemicals (Seigler 2006; Fiorentino et al., 2008) and antioxidants (Privat et al., 2002). In terms of medical applications, they have been shown to have beneficial effects in the treatment of cardiovascular disease, cancer, diabetes and neurodegenerative diseases (Baur et al., 2006). Stilbenes are formed via the phenylalanine/polymalonate route (Hall and Yu, 2008). The last step of the biosynthetic pathway is catalysed by stilbene synthase (STS), which produces resveratrol in a single enzymatic reaction utilizing p-coumaryl-CoA and three malonyl-CoA units as substrates (Schroder and Schroder 1990). Despite the fact that all higher plants are able to accumulate basic compounds like p-coumaroyl CoA or malonyl- CoA through general and ubiquitous enzymes such as phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H) or 4-coumaroyl–CoA ligase (4CL), only a limited group of plants are able to produce resveratrol (and derivatives) through the STS enzyme. This stilbene-producing enzyme belongs to the large CHS type III polyketide synthase family, the main members of which are the chalcone synthases, which shares 75-90% of the amino acid sequence with STSs (Schroder et al., 1988). Both of these enzymes utilize the same substrate, but through different cyclization events lead on one hand to the production of chalcones and flavonoids and, on the other, to the production of resveratrol and stilbenes. Some plant species, such as Pine, Fallopia japonica (syn. Polygonum cuspidatum) and grapevine during several stages of berry development, constitutively accumulate large amounts of stilbenes (Benova et al., 2008). However, most studies concerning stilbene accumulation have been conducted on tissues of peanut and grapevine, where they accumulate in response to various biotic and abiotic stresses, as a result of increased transcription of both STS genes and upstream enzymes in the phenylpropanoid pathway such as PAL and C4H (Lanz et al., 1990; Bais et al., 2000). While little is known about the transcriptional regulation of the stilbene biosynthetic pathway, a number of studies have demonstrated a role for transcription factors in the regulation of other steps of the phenylpropanoid pathway. These regulators include R2R3-MYB transcription factors (TFs), responsible for the regulation of flavonols, lignin and anthocyanin metabolism (Boudet 2007). The R2R3-MYB TF group is the largest MYB TF sub-family in plants (Du et al., 2009) with the grapevine genome estimated to contain 108 R2R3-MYB members (Matus et al., 2008). To date, most R2R3-MYBs have been reported to play a major role in the regulation of secondary metabolism, such as the phenylpropanoid biosynthesis. In grapevine, R2R3-MYB factors have been demonstrated to be involved in the regulation of several steps of the flavonoid biosynthetic pathway: VvMYBA1 and VvMYBA2 are involved in the regulation of anthocyanin biosynthesis. VvMYB5a, VvMYB5b and VvMYBPA1 appear to control general branches of the flavonoid pathway and VvMYB12 regulates the production of flavonols (Bogs et al., 2007; Walker et al., 2007; Deluc et al., 2008). This study involved two principal components: (a) the genome-wide analysis of the whole STS multigenic family in grape and (b) the identification of candidate transcription factors involved in the regulation of the grape stilbene synthase pathway. The first point was achieved firstly by the identification, annotation and phylogenetic study of all members predicted to belong to the STS family and secondarily by transcriptional analysis of the whole STS family in grapevine in response to biotic and abiotic stress conditions and in unstressed healthy tissues at different developmental stages (in collaboration with the University of Verona). The expression of all identified VvSTS members predicted on the 12X V1 grape genome draft was evaluated using mRNA sequencing technology on Pinot noir leaf discs treated by wounding, exposure to UV-C light and downy mildew infection. The analysis was performed using a next generation whole-transcriptome sequencing technology (Illumina) and revealed different sub-groups of VvSTS genes characterized by different degrees of response to the different elicitors. The constitutive expression of VvSTS genes in different tissues was analysed with a different approach, in collaboration with the University of Verona. A grapevine expression atlas obtained using a Nimblegen and Combimatrix microarray technology based on the 12X V1 coverage assembly predictions (kindly provided by the University of Verona) was screened for VvSTS genes providing interesting insights into the transcriptional regulation of VvSTS genes in grapevine. A wide range of grapevine tissues were analysed, including leaf, berry tissues, such as exocarp, endocarp and seed, in vitro roots, rachis, stem, tendril etc. Moreover, tissues were evaluated at different developmental stages such as fruit set, pre-ripening, ripening or withering concerning the berry development or at different temporal stages regarding leaves or other vegetative tissues. Analyses using the grapevine expression atlas confirmed the existence of different VvSTS subgroups characterized by different expression patterns and provided a comprehensive picture of VvSTS expression patterns in planta. Considering the close relationship between the flavonoid and stilbene biosynthetic pathways and the fact that certain key genes within the flavonoid pathway have been shown to regulated by R2R3- MYB transcription factors (Bogs et al. 2007; Walker et al. 2007; Deluc et al. 2008) the mRNAseq data obtained from stresses grape tissues were also examined for the expression MYB TFs that might show similar expression patterns to the VvSTS genes. Of the 108 grape R2R3-MYB factors analysed, two accessions displayed similar expression patterns to the inducible VvSTS genes. These two accessions were previously designated VvMYB14 and VvMYB15 by Matus et al. (2008) based on their homology to the Arabidopsis thaliana MYB14 gene. Validation of the expression data obtained from the mRNAseq analysis, was achieved using a quantitative RT-PCR approach, by screening in more detail the relationship between selected VvSTS and VvMYB14 expression patterns in grape tissues following the application of abiotic and biotic stress treatments. Two highly responsive VvSTS genes were selected for analysis, VvSTS22 and VvSTS36. All treatments confirmed a strong correlation between the expression of VvMYB14 and the two VvSTS genes. To obtain direct evidence of the regulation of VvSTS promoter activity by VvMYB14 a gene reporter assay using a dual luciferase assay system was utilized. Chardonnay cell suspensions transiently expressing VvSTS promoter-luciferase expression constructs showed a statistically significant increase of VvSTS promoter activity when co-transformed with VvMYB14. Finally, to validate the role of VvMYB14 in the regulation of the VvSTS pathway in planta, attempts were made to silence VvMYB14 in a grapevine hairy root system. In a preliminary screening of transformed hairy root lines, those lines showing the highest levels of VvMYB14 silencing were found to show the lowest induction of VvSTS36 in response to wounding, giving a first biological confirmation of real role for VvMYB14 in the regulation of the VvSTS pathway.