Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (
Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (
STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (
CHSs) several times independently in stilbene-producing plants.
STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large
STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of
STS genes in grapevine. Our reannotation of the
STS and
CHS gene families yielded 48
STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the
STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the
STS gene family revealed that both
STS and
CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among
STS genes. However, we found a few sites under different selection pressures in
CHS and
STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.Plants produce a vast array of secondary metabolites, many of them being restricted to specific groups of plant species. This extraordinary chemical diversity is believed to have evolved from a limited number of ubiquitous biosynthetic pathways through gene duplication followed by functional divergence (
Pichersky and Gang, 2000). The phenylpropanoid pathway, derived from Phe, illustrates perfectly this phenomenon, as it gives rise to a large diversity of phenolic compounds playing key roles in plants, including participation in structural polymers, defense against herbivores and pathogens, protection from abiotic stress, and important functions in plant-pollinator interactions. Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including dicotyledon angiosperms such as grapevine (
Vitis vinifera), peanut (
Arachis hypogaea), and Japanese knotweed (
Fallopia japonica, formerly
Polygonum cuspidatum), monocotyledons like sorghum (
Sorghum bicolor), and gymnosperms such as several
Pinus and
Picea species. In addition to their participation in both constitutive and inducible defense mechanisms in plants, several stilbenes display important pharmacological properties. Since resveratrol (3,5,4′-trihydroxy-trans-stilbene) was postulated to be involved in the health benefits associated with a moderate consumption of red wine (
Renaud and de Lorgeril, 1992), plant stilbenes have received considerable interest. Nowadays, resveratrol ranks among the most extensively studied natural products, and hundreds of studies have shown that it can slow the progression of a wide variety of illnesses, including cancer and cardiovascular disease, as well as extend the life spans of various organisms (
Baur and Sinclair, 2006). Stilbene synthases (
STSs) are characteristic of stilbene-producing plants and catalyze the biosynthesis of the stilbene backbone from three malonyl-CoA and one CoA-ester of a cinnamic acid derivative.
STSs are members of the type III polyketide synthases family, chalcone synthases (
CHSs), which catalyze the first step of flavonoid biosynthesis, being the most ubiquitous polyketide synthase in plants. Both
CHS and
STS use
p-coumaroyl-CoA and malonyl-CoA as substrates and synthesize the same linear tetraketide intermediate. However,
STS uses a specific cyclization mechanism involving a decarboxylation to form the stilbene backbone.
STS proteins share extensive amino acid sequence identity with
CHS, and phylogenetic analysis of the
STS and
CHS gene families has shown that
STS genes may have evolved from
CHS genes several times independently (
Tropf et al., 1994). In most stilbene-producing plants,
STS genes form small families of closely related paralogs. For example, two
STS cDNAs have been cloned from peanut (
Schröder et al., 1988), the genome of Scots pine (
Pinus sylvestris) has been shown to contain a small family of four
STS genes (
Preisig-Müller et al., 1999), and three
STS genes have been characterized in Japanese red pine (
Pinus densiflora;
Kodan et al., 2002). Only one
STS gene has been isolated from Japanese knotweed to date (
Liu et al., 2011), and the sequencing of sorghum genome has shown that
SbSTS1 was the only
STS gene in this plant species (
Yu et al., 2005;
Paterson et al., 2009). Grapevine is a noteworthy exception among stilbene-producing plants, as its genome has been shown to contain a large family of putative
STS genes. Early Southern-blot experiments suggested that the grapevine genome contained more than 20
STS genes (
Sparvoli et al., 1994). Analyses of the first drafts of the grapevine genome sequence confirmed the large size of this multigene family, with an estimated number of
STS genes ranging from 21 to 43 (
Jaillon et al., 2007;
Velasco et al., 2007). However, these relatively low-coverage sequence drafts did not allow a precise analysis of large families of highly similar genes. The more recently released 12× genome sequence of grapevine inbred Pinot Noir cultivar PN40024 offered an improved sequence quality, allowing an accurate analysis of the
STS gene family. In this work, we take advantage of the improved 12× sequence of the grapevine ‘PN40024’ genome to analyze the grapevine
STS gene family. Furthermore, we combine molecular evolution to structural and functional analyses to gain more insight into the significance of the remarkable amplification of the
STS family in grapevine.
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