1. Structural, functional and evolutionary analysis of the unusually large stilbene synthase gene family in grapevine (Vitis vinifera) Claire Parage 1*, Raquel Tavares 2,6*, Stéphane Réty 3, Raymonde Baltenweck 1, Anne Poutaraud 1, Lauriane Renault 1, Dimitri Heintz 4, Raphaël Lugan 4, Gabriel Marais 2,6, Sébastien Aubourg 5 and Philippe Hugueney 1 * first co-authors 1 INRA UMR 1131 Colmar France, 2 CNRS UMR 5558 Villeurbanne France, 3 CNRS UMR 8015 Paris France, 4 CNRS UPR 2357 Strasbourg France, 5 INRA UMR 1165/CNRS ERL 8196 Evry France, 6 IGC Oeiras Portugal Abstract Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to being involved in defence mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are probably involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STS), which catalyze stilbene backbone biosynthesis, seem to have evolved from chalcone synthases (CHS) several times independently in stilbene-producing plants. STS genes usually form small families including 2 to 5 relatively similar copies. By contrast, the sequence of grapevine reference genome (PN40024, Jaillon et al., Nature 2007) has revealed an unusually large STS gene family. Here we combine molecular evolution, structural and functional analyses to investigate further the high STS copy number in grapevine. Our re-annotation of the STS and CHS gene families using the grapevine updated 12X version yielded to 48 STS genes including at least 32 potentially functional copies. Our functional analysis of 9 genes representing most of the STS gene family diversity clearly indicated that these genes do indeed encode for proteins with stilbene synthase activity. Our phylogenetic dN/dS analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection. We did not find evidence for strong selection for new protein functions among STS copies. Very different expression patterns in UV stress conditions among STS copies were not found either. However, we found a few sites under different selection pressures in CHS and STS and used a 3D model of the grapevine STS protein to have clues on how they might have affected STS evolution. One such amino acid with increased dN/dS (positive selection) in early grapevine STS evolution was found close to the STS catalytic domain, which is consistent with this amino acid having contributed to the emergence of STS activity in grapevine. Other amino acids located at the STS protein periphery showed interesting dN/dS patterns and could be involved in interaction between STS and other proteins. Altogether, our results suggest that the amplification of the STS gene family in grapevine is associated to only slight protein functional diversification among copies. Selection for increased dosage or expression patterns diversification in non-stress conditions (not tested here) may explain why the STS family has become so large in grapevine. Figure 4: Mapping of evolutionary contrasted amino acid sites on the 3D model of STS protein The Vitis STS10 protein was modelled using the structure of STS from Pinus sylvestris as a template (Austin et al., Chem Biol 2004). Amino acids under positive and negative selection are highlighted in red and blue, respectively. P 269 is coloured in purple and indicates early positive selection in the Vitis STS family. The position of the resveratrol product is indicated. Table 1: Amino acid sites under contrasted selection pressures in CHS/STS in Vitis vinifera. A codeml analysis (Yang, MBE 2007) revealed very low global dN/dS in both STS and CHS (not shown). Contrasted amino acid positions detected using Fitmodel (Guindon et al., PNAS 2004) are indicated in bold letters. Sites under positive selection are indicated in red, sites evolving neutrally in black and sites under a negative selection pressure are indicated in blue. P 269 is coloured in purple to indicate positive selection on the basal branch of the Vitis STS family (see Figure 3). The STS sequences cells are highlighted in a darker background. Psyl = Pinus sylvestris; Vv = Vitis vinifera. Discussion Two hypotheses can be put forward for explaining the large STS gene family size in grapevine compared to other stillbene-producing plants. First, functional diversification among STS copies could explain why the family has become so large. In this case, the STS copies should show evidence for positive selection at protein level and/or diversified expression patterns, which would indicate the evolution of new functions among STS genes. The dN/dS analysis does not support an important neofunctionalization at the protein level. STS and CHS genes show very similar global dN/dS<1 and very few amino acid sites have distinct evolution in STS and CHS. Only one amino acid site was found under widespread positive selection within the STS gene family (site 230). This site is located at the STS protein periphery possibly involved in protein-protein interaction. The expression data from our work (in UV stress conditions, not shown) revealed very similar expression patterns among STS genes, but a quantitative expression analysis in normal conditions will be needed to test the neofunctionalization hypothesis for expression patterns. Another possibility is that the STS gene family has undergone selection for increased dosage. Increased dosage can be obtained through the evolution of enhancers that will increase expression levels but also by simply duplicating a gene over and over. In this case, no or vey little functional diversification and similar expression patterns would be expected among STS. This hypothesis predicts some dosage effects should be observed for STS in stress responses and other pathways in which they are involved. It also predicts that STS dosage should be unusually elevated in grapevine compared to other stilbene-producing plants. Phenylpropanoid pathway coumaroyl-coA malonyl-coA Chalcone synthase Stilbene synthase Flavones, flavonones, anthocyanes Ubiquitous biosynthesis pathway in all plants Resveratrol, viniferin, piceid, pterostilbene In few plants only Arachis Vitis Sorghum Figure 1: Flavonoids and stilbenes biosynthesis in plants Stilbene synthase (STS) is characteristic of stilbene-producing plants and catalyzes the biosynthesis of the stilbene backbone. Most stilbene-producing plant species possess a few STS genes, whereas Vitis vinifera includes more than 40 putative STS genes. FLAVONOIDS STILBENES Figure 2: Genomic organization of the grapevine STS gene family Out of 48 STS genes, 32 encode potentially full- length proteins, 11 genes correspond to pseudogenes and 5 genes are still partial. Gene structure is highly conserved with 2 coding exons and 1 intron. The conservation level inside the VvSTS protein family is very high, ranging from 90.3% identity (between VvSTS36 and VvSTS19 proteins) to 99.7% (i.e only one different residue, between VvSTS15 and VvSTS21, and between VvSTS41 and VvSTS45 proteins). Chromosome 10 STS cluster (14216000-14307000) VvSTS1VvSTS2 VvSTS3 VvSTS4 VvSTS5VvSTS6 Full STS gene Partial STS gene STS pseudogene Unsequenced gap Other pseudogene Transposable element VvSTS30 VvSTS7VvSTS8 VvSTS9 VvSTS10 VvSTS11 VvSTS12VvSTS13 VvSTS14 VvSTS15 VvSTS16 VvSTS17 VvSTS18 VvSTS19VvSTS20 VvSTS21VvSTS22 VvSTS23 VvSTS24 VvSTS25 VvSTS26VvSTS27VvSTS28 VvSTS29 VvSTS31VvSTS32VvSTS33 VvSTS34 VvSTS35VvSTS36 VvSTS37 VvSTS38VvSTS39VvSTS40 VvSTS41 VvSTS42VvSTS43 VvSTS44 VvSTS45 VvSTS46VvSTS47 VvSTS48 Chromosome 16 STS cluster (16239000-16712000) Figure 3: Phylogenetic analysis of the CHS/STS family in plants ML tree based on the coding sequences alignment of 74 CHS/STS sequences from 8 different species, including the 32 full-length Vitis STS genes. Proteins for which the STS activity was validated in planta (using Agrobacterium-mediated transformation of Nicotiana benthamiana, not shown) are indicated with *. Chromosome 10 STS cluster Chromosome 16 STS cluster Other species STS Ah: Arachis hypogaea At: Arabidopsis thaliana Sb: Sorghum bicolor Psyl: Pinus sylvestris Pstr: Pinus strobus Pn: Psilotum nudum Pt: Populus trichocarpa Vv: Vitis vinifera