title: Two key polymorphisms in a newly discovered allele of the Vitis vinifera TPS24 gene are responsible for the production of the rotundone precursor α-guaiene.
abstract: Rotundone was initially identified as a grape-derived compound responsible for the peppery aroma of Shiraz wine varieties. It has subsequently been found in black and white pepper and several other spices. Because of its potent aroma, the molecular basis for rotundone formation is of particular relevance to grape and wine scientists and industry. We have identified and functionally characterized in planta a sesquiterpene synthase, VvGuaS, from developing grape berries, and have demonstrated that it produces the precursor of rotundone, α-guaiene, as its main product. The VvGuaS enzyme is a novel allele of the sesquiterpene synthase gene, VvTPS24, which has previously been reported to encode VvPNSeInt, an enzyme that produces a variety of selinene-type sesquiterpenes. This newly discovered VvTPS24 allele encodes an enzyme 99.5% identical to VvPNSeInt, with the differences comprising just 6 out of the 561 amino acid residues. Molecular modelling of the enzymes revealed that two of these residues, T414 and V530, are located in the active site of VvGuaS within 4 Å of the binding-site of the substrate, farnesyl pyrophosphate. Mutation of these two residues of VvGuaS into the corresponding polymorphisms in VvPNSeInt results in a complete functional conversion of one enzyme into the other, while mutation of each residue individually produces an intermediate change in the product profile. We have therefore demonstrated that VvGuaS, an enzyme responsible for production of the rotundone precursor, α-guaiene, is encoded by a novel allele of the previously characterized grapevine gene VvTPS24 and that two specific polymorphisms are responsible for functional differences between VvTPS24 alleles.
authors.affiliation: Plant Biochemistry Laboratory, Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, DK-1871 Frederiksberg C, Denmark Wine Science, School of Agriculture, Food and Wine, University of Adelaide, Urrbrae SA 5064, Australia.
authors.forename: Damian Paul
authors.initials: DP
authors.lastname: Drew
authors.affiliation: Plant Biochemistry Laboratory, Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, DK-1871 Frederiksberg C, Denmark.
authors.forename: Trine Bundgaard
authors.initials: TB
authors.lastname: Andersen
authors.affiliation: Wine Science, School of Agriculture, Food and Wine, University of Adelaide, Urrbrae SA 5064, Australia.
authors.forename: Crystal
authors.initials: C
authors.lastname: Sweetman
authors.affiliation: Plant Biochemistry Laboratory, Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, DK-1871 Frederiksberg C, Denmark.
authors.forename: Birger Lindberg
authors.initials: BL
authors.lastname: Møller
authors.affiliation: Wine Science, School of Agriculture, Food and Wine, University of Adelaide, Urrbrae SA 5064, Australia.
authors.forename: Christopher
authors.initials: C
authors.lastname: Ford
authors.affiliation: Plant Biochemistry Laboratory, Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, DK-1871 Frederiksberg C, Denmark hts@plen.ku.dk.
authors.forename: Henrik Toft
authors.identifier: http://orcid.org/0000-0003-3070-807X
authors.initials: HT
authors.lastname: Simonsen
mesh_terms: D000483:Alleles; D052176:Azulenes / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D017343:Genes, Plant; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011110:Polymorphism, Genetic; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D045789:Sesquiterpenes, Guaiane / Q000737:chemistry / Q000378:metabolism*; D040681:Structural Homology, Protein; D027843:Vitis / Q000235:genetics*; D055549:Volatile Organic Compounds / Q000032:analysis
chemical_list: D052176:Azulenes; D010940:Plant Proteins; D012717:Sesquiterpenes; D045789:Sesquiterpenes, Guaiane; D055549:Volatile Organic Compounds; C530290:rotundone; C019408:guaiene
keywords: Pinot Noir; Shiraz; rotundone; sesquiterpene synthase; sesquiterpenoids; wine aroma.