Functional Annotation, Genome Organization and Phylogeny of the Grapevine (Vitis vinifera) Terpene Synthase Gene Family Based on Genome Assembly, FLcDNA Cloning, and Enzyme Assays

Abstract

BackgroundTerpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions.ResultsWe present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevine genome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63 VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture and chromosomal location. A dense cluster of 45 VvTPS is localized on chromosome 18. Extensive FLcDNA cloning, gene synthesis, and protein expression enabled functional characterization of 39 VvTPS; this is the largest number of functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functions and/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene family showed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthology with TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functions independent of speciation.ConclusionsThe highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism in grapevine. We provide a detailed experimental functional annotation of 39 members of this important gene family in grapevine and comprehensive information about gene structure and phylogeny for the entire currently known VvTPS gene family.

Biosynthesis of volatile terpenes that accumulate in the secretory cavities of young leaves of Japanese pepper (<i>Zanthoxylum piperitum</i>): Isolation and functional characterization of monoterpene and sesquiterpene synthase genes

“…The deduced amino acid sequences of ZpTPS1 and ZpTPS2 were 50% and 73% identical to that of sesquiterpene synthases from V. vinifera that produce β-caryophyllene (VvCARS; Martin et al 2010) and from Citrus jambhiri that produce δ-elemene (CjELES; Uji et al 2015), respectively, whereas that for ZpTPS3 displayed highest similarity to monoterpene limonene synthase of C. limon (ClLIMS; Lücker et al 2002) with 65% identity at the amino acid level. The ORFs for ZpTPS1 and ZpTPS2 encoded proteins 554 and 557 amino acids in length with predicted molecular masses of 64.4 and 64.7 kDa, respectively.…”

“…To identify putative Z. piperitum TPS genes, we performed a tBLASTn search against the Z. piperitum Expressed Sequence Tags (EST) database using the protein sequence of Vitis vinifera β-caryophyllene synthase (VvCARS; GenBank accession number ADR74221; Martin et al 2010) as the protein query, which returned five contigs. To remove those contigs that had an insignificant match to TPS genes, we chose only full-length genes with more than 45% homologous identity to VvCARS, which resulted in the identification of two contigs that encoded open reading frames of sesquiterpene synthases, named as ZpTPS1 (accession number LC198039) and ZpTPS2 (accession number LC198040).…”

“…Using the protein sequence of V. vinifera β-caryophyllene synthase (Martin et al 2010) or C. limon limonene synthase (Lücker et al 2002) as a query, a BLAST analysis of the EST database of Japanese pepper fruit skin revealed two sesquiterpene synthase-like sequences, named as ZpTPS1 and ZpTPS2, and one monoterpene synthase-like sequence, named as ZpTPS3. The deduced amino acid sequences of ZpTPS1 and ZpTPS2 were 50% and 73% identical to that of sesquiterpene synthases from V. vinifera that produce β-caryophyllene (VvCARS; Martin et al 2010) and from Citrus jambhiri that produce δ-elemene (CjELES; Uji et al 2015), respectively, whereas that for ZpTPS3 displayed highest similarity to monoterpene limonene synthase of C. limon (ClLIMS; Lücker et al 2002) with 65% identity at the amino acid level.…”

The Grapevine Expression Atlas Reveals a Deep Transcriptome Shift Driving the Entire Plant into a Maturation Program

“…An S-plot (Wiklund et al, 2008) was then used to select putative biomarkers within the first (positive biomarkers) and last (negative biomarkers) percentiles (Figures 4B and 4C;see Supplemental Data Set 3 online). Positive biomarkers representing the flowers and stamens included transcripts corresponding to enzymes in the monoterpenoid and sesquiterpenoid biosynthesis pathways (e.g., enzymes that synthesize germacrene, cadinene, terpineol, pinene and myrcene, which are prominent components of floral scents) (Martin et al, 2010). There were also eight pectinesterase and seven polygalacturonase transcripts encoding cell wall-modifying enzymes involved in flower abscission (van Doorn and Stead, 1997) and pollen tube elongation (Bosch and Hepler, 2005).…”

Origins of Grape and Wine Aroma. Part 1. Chemical Components and Viticultural Impacts

“…These precursors are formed either through the cytosolic mevalonic-acid (MVA) pathway from three molecules of acetyl-CoA (Newman and Chappell 1999) or through the plastidial 2-C-methylerythritol-4-phosphate (MEP) pathway from pyruvate and glyceraldehyde-3-phosphate (Rohmer 1999). Monoterpenes are subsequently formed from 2E-geranyl diphosphate (GPP) and sesquiterpenes are formed from 6E-farnesyl diphosphate (FPP) through the action of terpene synthases (TPS) (Lücker et al 2004, Martin et al 2010. The Vitis vinifera terpene synthase (VvTPS) gene family contains the largest number of functionally characterized TPS for any species reported to date (Martin et al 2010).…”