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Functional Annotation, Genome Organization and Phylogeny of the Grapevine (Vitis vinifera) Terpene Synthase… Martin, Diane M; Aubourg, Sébastien; Schouwey, Marina B; Daviet, Laurent; Schalk, Michel; Toub, Omid; Lund, Steven T; Bohlmann, Jörg Oct 21, 2010

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RESEARCH ARTICLE Open AccessFunctional Annotation, Genome Organization andPhylogeny of the Grapevine (Vitis vinifera)Terpene Synthase Gene Family Based on GenomeAssembly, FLcDNA Cloning, and Enzyme AssaysDiane M Martin1,2†, Sébastien Aubourg3†, Marina B Schouwey4, Laurent Daviet4, Michel Schalk4, Omid Toub 1,2,Steven T Lund2, Jörg Bohlmann1*AbstractBackground: Terpenoids are among the most important constituents of grape flavour and wine bouquet, andserve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearlyhomozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silicoanalysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combinedwith the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finishedwines, prompted a detailed examination of this gene family at the genomic level as well as an investigation intoVvTPS biochemical functions.Results: We present findings from the analysis of the up-dated 12-fold sequencing and assembly of the grapevinegenome that place the number of predicted VvTPS genes at 69 putatively functional VvTPS, 20 partial VvTPS, and 63VvTPS probable pseudogenes. Gene discovery and annotation included information about gene architecture andchromosomal 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 numberof functionally characterized TPS for any species reported to date. Of these enzymes, 23 have unique functionsand/or phylogenetic locations within the plant TPS gene family. Phylogenetic analyses of the TPS gene familyshowed that while most VvTPS form species-specific gene clusters, there are several examples of gene orthologywith TPS of other plant species, representing perhaps more ancient VvTPS, which have maintained functionsindependent of speciation.Conclusions: The highly expanded VvTPS gene family underpins the prominence of terpenoid metabolism ingrapevine. We provide a detailed experimental functional annotation of 39 members of this important gene familyin grapevine and comprehensive information about gene structure and phylogeny for the entire currently knownVvTPS gene family.BackgroundTerpenoids are a large class of metabolites that areinvolved in the fragrance and aroma constituents offlowers and fruits, plant defense, and primary plantmetabolism [2-4]. Although all terpenoids arise from afew structurally simple prenyldiphosphate precursors, anenormous assortment of thousands of possible mole-cules comes to fruition. This chemical diversity of terpe-noid structures is attributed, in large part, to the myriadways of folding and the eventual quenching of reactivecarbocation intermediates in the reaction catalyzed byterpenoid synthases (TPS) [5,6]. The products of TPScan be further modified by other enzymes such a cyto-chrome P450 dependent monooxygenases and varioustransferases.* Correspondence: bohlmann@msl.ubc.ca† Contributed equally1Michael Smith Laboratories, University of British Columbia, 2185 East Mall,Vancouver, B.C, V6T 1Z4, CanadaFull list of author information is available at the end of the articleMartin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226© 2010 Martin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.The initial 8-fold sequencing and assembly of a grape-vine (Vitis vinifera L.) inbred Pinot noir genome(PN40024) lead to the prediction of 89 grapevine TPS(VvTPS) genes, which mirrors a vibrant role for terpe-noid secondary metabolism in grapevine biology [7,8].For example, in wine made from aromatic grape vari-eties, monoterpene alcohols such as linalool, geraniol,and cis-rose oxide impart important floral flavour quali-ties [9]. Sesquiterpenes have also been identified asimportant indicators of grape aroma. Recently, Parkeret al. [10] identified a-ylangene as a sesquiterpene meta-bolite marker associated with peppery aroma and tastein Australian Shiraz grape berries, and the sesquiterpeneketone, rotundone, was found to be the compoundresponsible for this attribute in peppery/spicy AustralianShiraz grapes and wines [11]. Monoterpenes such as1,8-cineol, and sesquiterpenes such as a-humulene,b-caryophyllene as wells as a- and g-muurolene havebeen described in Cabernet Sauvignon pre-veraison ber-ries [12]. Similar profiles of terpene volatiles have alsobeen documented from the headspace above Chardon-nay leaves, flowers, and green berries [13]. The presenceof some terpenes early in berry development and inother parts of the plant may indicate a role in defense.Terpenoid volatiles are released from grapevines follow-ing insect feeding [14] or the application of methyl jas-monate [15].Of all plant species for which genome sequences areavailable, the TPS gene family has been comprehensivelyexplored only in Arabidopsis thaliana, in which 32intact AtTPS genes were identified [16]; functions havebeen established for several of these genes [17-20]. Anumber of TPS genes have also been characterizedagainst the background of the sequenced rice (Oryzasativa) genome [21-26] which has at least 40 TPS-likesequences identified (S. Aubourg unpublished results).For comparison, genome sequence analysis of poplar(Populus trichocarpa) identified 47 TPS genes [27], onlytwo of which have been functionally characterized[28,29]. Prior to the sequencing of a grapevine genome,we reported on the cDNA cloning and product profilesof three VvTPS [30,31] and we detailed the involvementof valencene synthase, a sesquiterpene synthase, in theevolution of grapevine floral scent [32], but a compre-hensive analysis of VvTPS has not been reported thusfar.The importance of terpenoids in grapevine biology andwine flavour and quality motivated a genome-wide inven-tory and functional characterization of the VvTPS genefamily. We present the manually curated annotation ofthe VvTPS gene family from the current 12-fold genomesequence coverage. This work defines 69 putatively func-tional VvTPS, 20 partial VvTPS, and 63 probable VvTPSpseudogenes including VvTPS gene architecture andchromosome localizations. The VvTPS gene family showsextensive gene duplication and in many instances, func-tional diversification across all subfamilies except thoseinvolved in primary metabolism (subfamilies TPS-c andTPS-e). Conclusions regarding diversification are sup-ported by phylogenetic analyses of the VvTPS family andfunctional characterization of heterologously expressedVvTPS proteins.Results and DiscussionGenome-wide identification of TPS genes in Vitis viniferaScreening of the predicted proteome and the six-frames-translated 12-fold genome sequence of V. vinifera withprotein sequences of previously characterized TPS iden-tified 152 loci exhibiting significant similarities withknown TPS (see the Methods section for details). Ourannotation of the 152 TPS-like gene models (AdditionalFile 1) classified them into four types: (i) 53 are com-plete VvTPS genes that contain the expected functionalmotifs and domains [16,33,34] required to render themfunctional; (ii) 16 are complete VvTPS genes but theORFs contain a frameshift or premature stop codoneither due to a point mutation or a possible sequencingerror; (iii) 20 are partial TPS genes disrupted bysequence gaps or located in scaffold extremities; and (iv)63 are obvious pseudogenes disrupted by numerousdeletions, frameshifts and/or stop codons (AdditionalFile 1). After removing the genes of this last type, thenumber of potentially functional VvTPS ranges from aminimum of 53 up to 89 genes. The missing sequencesof the partial genes (group iii) prevented meaningfulsequence alignments and gene classification; therefore,we removed them from our further analysis whichfocused on the 69 VvTPS genes of groups (i) and (ii).The presence of cognate EST and/or cDNA sequencesprovides proof of transcription for 40 (58%) of them(Additional File 1).The relatively high gene sequence and structure con-servation across the plant TPS family [16] allow us to beconfident in the result of the genome-wide VvTPS geneprediction, combining automatic and manual annota-tions. Manual curation and evaluation have substantiallyimproved the identification of VvTPS genes: For exam-ple, out of the 69 VvTPS genes 12 were missed by theautomated pipeline used for annotation of the grapevinegenome [1,35]. Furthermore, intron-exon structures of40 VvTPS genes required manual correction to obtaincomplete and consistent coding sequences. The resultsof the VvTPS gene annotations confirmed a large VvTPSgene family previously predicted from the 8-fold genomeassembly [1] and expand the previous estimation of theVvTPS family size. While a lower estimate of only 35VvTPS genes was reported from the analysis of a secondheterozygous Pinot noir genome sequence [36], theMartin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 2 of 22sequence information available for this genome in NCBIGenBank http://www.ncbi.nlm.nih.gov/genbank alsorevealed about 70 VvTPS. A comparison with otherplant genomes in which TPS genes have been annotatedshowed that the grapevine VvTPS gene family is the lar-gest identified to date. The Arabidopsis thaliana gen-ome contains 32 complete AtTPS genes and eightAtTPS pseudogenes [16], while the rice and poplar gen-omes are predicted to encode 40 to 50 TPS-like genesaccording to [27] and unpublished results (S. Aubourg).Annotation of VvTPS relative to the overall plant TPSgene familyThe 69 candidate VvTPS sequences identified as intactor potentially intact represent five of the seven plantTPS gene subfamilies TPS-a through TPS-g previouslydescribed [16,33,37] (Figure 1; Additional File 1). TheTPS-f subfamily of Clarkia brewerii linalool synthase-like genes and the gymnosperm-specific TPS-d subfam-ily [33], were the only subfamilies missing full-lengthVvTPS members. Although the previous 8-fold grape-vine genome assembly [1] contained one VvTPS-f sub-family member, in the manually curated assembly andannotation of the 12-fold genome sequence, this genewas now fragmented into two partial TPS (AdditionalFile 1).The TPS-a subfamily is substantially extended ingrapevine with 30 VvTPS existing on just two chromo-somes, chromosomes 18 and 19, compared to 22 AtTPSof the TPS-a subfamily in A. thaliana [16]. This subfam-ily typically contains sesquiterpene synthases and possi-bly diterpene synthases of secondary metabolism. Atotal of 19 VvTPS were found in the TPS-b subfamily ofangiosperm monoterpene synthases and these werelocated on at least three chromosomes, chromosomes 8,12 and 13. The TPS-g subfamily, which containssynthases for acyclic monoterpenes of floral scent [37] isgreatly extended in grapevine with 17 VvTPS annotatedcompared to Arabidopsis with just one AtTPS gene inthis subfamily. The chromosomal location of most ofthe VvTPS of the TPS-g subfamily is unknown. Wefound two VvTPS of the TPS-c subfamily and oneVvTPS of the TPS-e subfamily. These two subfamiliescontain TPS genes of primary plant hormone metabo-lism that are not typically represented with multiplegene copies in plant genomes [16,33,38].Chromosomal location of VvTPSThe topological organization of the VvTPS family in thegrapevine genome is characterized by massively tan-demly repeated genes. Of the complete set of 152VvTPS loci identified in this study, 129 (85%) are orga-nized in 13 distinct clusters covering from 2 to 45VvTPS genes or pseudogenes (Additional File 1). Thelargest VvTPS cluster, localized on chromosome 18 andspanning 690 kb, contains 20 complete VvTPS genes (allare members of the TPS-a subfamily), 25 VvTPS pseu-dogenes and numerous traces of Copia-like retrotran-sposons (Figure 2). Although many VvTPS clustertogether, of the 152 VvTPS loci, only 2 VvTPS genesalso localize in the vicinity of other putative terpenoidpathway genes with the same gene orientation: one(VvTPS42) co-localizes with a prenyltranferase gene andthe other (the pseudogene GSVIVT01014893001) with acytochrome P450 gene. Dynamically expanding or con-tracting clusters of closely related genes can evolve asthe result of unequal cross-over, which enriches geneticvariability but limits the divergence through an opposingmechanism of gene conversion as has been shown forplant resistance genes [39,40]. These processes can beintensified by the presence of pseudogenes which contri-bute to the frequency of crossing-over and increase ingene diversity [41]. As previously shown for the Droso-phila melanogaster genome, a high density of repeat ele-ments can also impact the recombination dynamicwithin gene clusters [42]. The genome architecture ofthe VvTPS gene family (i.e., the number, size and natureof VvTPS clusters in the grapevine genome) suggests alarge potential for diversification and variation of terpe-noid metabolism in this species, and may thus accountfor variability of terpenoid profiles among grapevinevarieties and cultivars. The identification of VvTPS geneclusters allows for future work in which resequencing ofthese regions in different varieties and testing for asso-ciations of gene cluster and terpenoid aroma traits canbe undertaken.Intron-exon structure of VvTPS genesIn agreement with highly conserved intron-exon struc-ture of plant TPS genes [16,43,44], all but one of the 66VvTPS genes of the subfamilies TPS-a, TPS-b and TPS-gcontain seven coding exons (Figure 3). The only excep-tion is VvTPS17 (TPS-a) in which the 3’-most exon wasdisrupted by a large and probably recent intron inser-tion. The three genes of the subfamilies TPS-c andTPS-e are characterized by longer sequences (15 and 13exons respectively) as a consequence of the presence ofan additional exon encoding an ancestral 200 aminoacid N-terminal domain of unknown function [16,33,38].Conserved motifs of the V. vinifera TPS protein familyThe grapevine VvTPS protein family is characterized bytwo large domains defined in the PFAM resource [45]:PF01397 corresponds to the N-terminal region andPF03936 corresponds to the C-terminal metal cofactorbinding domain [46]. Just upstream of the PF01397 N-terminal domain, in the region encoded by the firstexon, all VvTPS that putatively function as monoterpeneMartin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 3 of 22Figure 1 Phylogeny and chromosome location of 69 putative intact VvTPS identified as gene models in the 12-fold coverage genomesequence assembly of Vitis vinifera (Pinot noir). Maximum likelihood analysis of the V. vinifera VvTPS gene family. Bootstrap values supportedby ≥ 50% are designated * and those with values ≥ 80% are indicated with ^. Colors indicate chromosome location known at this time. Lightblue = unmapped scaffold, brown = chromosome 13 (unmapped scaffold), dark blue = chromosome 13, orange = chromosome 12, pink =chromosome 8, purple = chromosome 18 (unmapped scaffold), teal = chromosome 19, and red = chromosome 7.Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 4 of 22synthases, also contain the RR(x)8W motif. This motifmay play a role in the initiation of the isomerization-cyclization reaction [47] or act to stabilize the proteinthrough electrostatic interactions [48]; however, TPS insubfamily TPS-g, as well as two VvTPS predicted inTPS-a, and those in TPS-c and TPS-e do not consis-tently contain this motif or they have a highly modifiedversion of it (Figure 3 and 4A). Several of the TPS-gmembers are also truncated with the starting M at posi-tion five of this motif. This may effectively open up thethree dimensional structure of these TPS or it mayaffect subcellular compartmentation of these proteins.Mono- and diterpene synthases typically contain an N-terminal plastidial targeting peptide upstream of theconserved or modified RR(x)8W [33], and such targetingpeptides have been predicted in silico for 21 VvTPS(Figure 3).The C-terminal domain contains two highly conservedaspartate-rich motifs. The first of these, the DDxxDmotif (Figure 3), is involved in the coordination of diva-lent ion(s), water molecules and the stabilization of theactive site [46,49,50]. Only four predicted VvTPS(VvTPS48, VvTPS66 and the two TPS-c proteinsVvTPS67 and VvTPS68) lack the exact DDxxD motifcharacteristic of class I TPS which catalyze reactionsinitiated by cleavage of the diphosphate group of theprenyl diphosphate substrate. The TPS-c proteins arenot expected to have this domain as they do not cleavethe prenyl diphosphate unit; however, they do containthe DXDD sequence critical to the protonation initiatedreaction mechanism of class II TPS [51].A second important motif in the C-terminal domain isthe NSE/DTE motif [52,53]. The reported consensussequence of this motif is (L,V)(V,L,A)(N,D)D(L,I,V)x(S,T)xxxE and a modified version, (L,F)(M,I,S,C,W)(N,D)D(L,M,I)x(S,T,D)xxxE, is found in almost all VvTPS of thesubfamilies TPS-a, -b, and -g (Figure 4B). Three pre-dicted VvTPS are lacking the terminal E. Members ofthe TPS-g subfamily have an altered and highly con-served sequence, LWDDLx(S,T)xxxE.Functional characterization of VvTPS full length cDNAsSince specific functions of TPS genes cannot be accu-rately predicted from sequence analysis alone, it wasimportant to clone and functionally characterize VvTPSfull length (FL)cDNAs. We used the previously pub-lished 8-fold [1] and the new 12-fold genome sequence(GenBank, NCBI project ID 18785) assembly of thePinot noir inbred line for primer design to clone VvTPSFLcDNAs from three grapevine varieties, Pinot noir(PN), Cabernet Sauvignon (CS), and Gewürztraminer(Gw). FLcDNAs were expressed in E. coli using one ofseveral cloning vectors (see Materials and Methods),and recombinant VvTPS proteins were functionallyFigure 2 Genomic organization of a 690 kb multi-gene VvTPScluster. A 690 kb long genomic region of chromosome 18 containsa large cluster of 20 complete VvTPS-a genes (dark green arrows),25 pseudo-TPS-a genes characterized by several deletions,frameshifts and/or stop codons (light green arrows), few traces ofother genes (orange arrows) and numerous vestiges of the Copia-like transposable element (yellow arrows).Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 5 of 22characterized via purified protein or in vivo recombinantE. coli assays with each of the following potential sub-strates, geranyl diphosphate (C10, GPP), (E,E)-farnesyldiphosphate (C15, FPP), and (E,E,E)-geranylgeranyldiphosphate (C20, GGPP). Although a couple of recentstudies identified two tomato TPS which utilize neryldiphosphate or (Z,Z)-farnesyl diphosphate as substrates[54,55], these TPS are members of the TPS-e subfamilyand none of the VvTPS that we characterized belongedto this subfamily. In addition, a screen of the grapevinegenome sequence did not reveal the presence of Z-iso-prenyl diphosphate synthases (unpublished results, M.Chavez, S. Aubourg, and J. Bohlmann), therefore, we didnot include these alternative substrates when assayingfor VvTPS activity. Products were analyzed by GCMSand the majority of VvTPS analyzed produced multipleproducts (Table 1, Table 2, Table 3, and Table 4), whichis a common feature of plant TPS [56,57]. Since manyof the VvTPS FLcDNAs arose from cloning efforts in allthree V. vinifera varieties PN, CS, and Gw, a number ofVvTPS were uncovered as cultivar-specific variants, eachdiffering by only a few amino acids. To ensure thatthese were not unique genes capable of producing a dis-tinct product profile, each variant was functionallyFigure 3 Gene structure and classification of putative intact VvTPS. Exon-intron structures were predicted and manually curated for theputative intact VvTPS gene models. Green arrows and black lines represent at scale protein coding exons and introns, respectively. Theconserved motifs RR(x)8W and DDxxD are represented by yellow and red boxes, respectively. Green circles indicate the prediction of an N-terminal plastidial targeting peptide. Classification into subfamilies is based on phylogenetic analyses supported, where applicable, bycorresponding functional data. Subfamilies TPS-a, TPS-b and TPS-g are characterized by a highly conserved structure of seven exons. The threeVvTPS genes of subfamilies TPS-c and TPS-e have distinct structures with 13 to 15 exons.Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 6 of 22Figure 4 The conserved RR(X8)W and NSE/DTE motifs in VvTPS. A: Alignment of the RR(x)8W motif within the 69 intact VvTPS annotated inthis analysis. B: Alignment of the NSE/DTE motif within the 69 intact VvTPS annotated in this analysis. Dark grey shading indicates amino acidsconserved with ≥ 80% and those shaded with light grey indicate conservation ≥ 60%.Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 7 of 22Table 1 Experimental Functional Annotation of VvTPS Genes of the VvTPS-a SubfamilyFunctional GeneIDFunction Access. # FLcDNA ID Nearest VvTPS genemodelRepresentativeProducts%VvGwECar1 (E)-b-Caryophyllene Syn HM807373 Gw38F6* VvTPS01 (E)-caryophyllene 71VvGwECar2 HM807374 Gw53B1* VvTPS27 a-humulene 23VvGwECar3 HM807375 Gw12001M3* VvTPS02 germacrene D 6VvPNECar1 HM807402 CAN82172^ VvTPS02VvPNECar2 HM807403 CAO16256^ VvTPS13VvGwGerA Germacrene A Syn HQ326230 Gw38F3* VvTPS01 germacrene A 52a-selinene 24selin-11-en-4-a-ol 12VvGwaBer (E)-a-Bergamotene Syn HM807376 Gw56B1* VvTPS10 (E)-a-bergamotene 56Unknown 17Nerolidol 14(E)-b-farnesene 8(Z)-a-farnesene 5VvGwGerD Germacrene D Syn HM807377 Gw64B1* VvTPS07 germacrene D 94VvPNGerD HM807378 PN39M3* VvTPS15 germacrene B 6VvCSaFar (E,E)-a-Farnesene syn HM807379 CS102B7* VvTPS20 (E,E)-a-farnesene 100VvGwgCad g-Cadinene Syn HM807380 Gw330M5* VvTPS08 g-cadinene 83Unknown 17VvPNbCur b-Curcumene syn HM807381 PN62M1* VvTPS30 b-curcumene 22(E)- g-bisabolene 18Iso-italicene 16(-)-a-bisabolol 14Β-bisabolene 7epi-b-santalene 7unknown 5(E)-b-farnesene 3g-curcumene 3unknown 2(Z)-a-bergomotene 1unknown 1VvPNSesq Sesquithujene Syn HM807404 CAO16252^ VvTPS12 (E)-a-bergomotene 1sesquithujene 80(E)-a-bergamotene 4sesquisabinene 8b-bisabolene 4g-bisabolene 2unknown pm 222 1.5b-bisabolol 0.5VvPNaZin a-Zingiberene syn HM807405 CAO16257^ VvTPS14 a-zingiberene 79.5b-sesquiphellandrene 17.5b-bisabolene 3VvPNSeInt Selina-411-diene/Interme deol syn HM807406 CAO39293^ VvTPS24 selina-4,11-diene 34intermedeol 307-epi-a-selinene 15δ-selinene 14a-guaiene 3.5selina-511-diene 2unknown pm 204 1VvPNCuCad Cubebol/δ-Cadinene syn HM807407 CAN76781^ VvTPS26 cubebol 20.5Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 8 of 22characterized. However, only one representative cDNAclone will be described for its function following below,while the additional clones from the other cultivars arelisted in Table 1, Table 2, Table 3, and Table 4.A subset of VvTPS FLcDNAs of the TPS-a subfamily(Table 1) were chemically synthesized and characterizedusing an E. coli strain engineered to produce the FPPsubstrate from mevalonate. Based on previous work[58], an operon encoding the mevalonate lower pathwayof Streptoccoccus pneumoniae was subcloned into a bac-terial expression vector together with the Saccharomycescerevisiae FPP synthase. VvTPS FLcDNAs were addition-ally expressed into this engineered strain and productformation was measured by GCMS in the cultureextract as has been done for the characterization ofother TPS and cytochrome P450 s [59].Nomenclature for functionally characterized VvTPSFLcDNAsWe assigned gene identifiers that include references toboth function and the cultivar (PN, CS, or Gw) fromwhich the gene was isolated (see Functional Gene ID inTable 1, Table 2, Table 3, and Table 4). These func-tional gene identifiers will be used throughout the fol-lowing sections to describe individual genes. Table 1,Table 2, Table 3, and Table 4 provide additional detailedinformation for each FLcDNA regarding clone ID, tissueorigin (see table legend), product profiles with relativequantitative information, as well as identification of theclosest annotated VvTPS gene model reported in thispaper. In some instances multiple cDNAs share thesame functional gene identifier, but are represented asdistinct genes because they occupy unique locationswithin the VvTPS phylogeny. These functional gene IDsare designated with numbers in both the tables andwithin the phylogenetic trees.Functions of VvTPS FLcDNAs of the TPS-a SubfamilyThe majority of VvTPS genes belong to the TPS-a sub-family for which we functionally characterized 13 uniqueFLcDNAs (Table 1). All of the VvTPS-a members werecharacterized as sesquiterpene synthases, and all but oneformed multiple products with FPP as substrate. In sev-eral cases, the product profiles included both terpenoidhydrocarbons and alcohols. As a group, the VvTPS ofthe TPS-a subfamily produce a diverse array of sesqui-terpene products.All five individual VvGwECar and VvPNECar FLcDNAclones produced predominantly (E)-b-caryophyllene. Fourof these clones also produced a-humulene and a smallamount of germacrene D, while one clone (CAN82172)produced only (E)-b-caryophyllene (94%) and a-humulene(6%). The two VvPNECar enzymes characterized in meta-bolically engineered E. coli (clones CAO16256 andCAN82172) showed similar product profiles to VvGwECarclones characterized by in vitro enzyme assays. One of theVvPNECar enzyme (CAO16256) also produced a lowamount ( < 1%) of an unknown sesquiterpene alcohol inaddition to (E)-b-caryophyllene, a-humulene and germa-crene D. The TPS VvGwGerA (Gw38F3) produced primar-ily germacrene A (52%) and a-selinene (24%) and a smallamount of selin-11-en-4-a-ol (12%). The product profile ofVvGwaBer (Gw56B1) consisted of (E)-a-bergamotene(56%), zingiberenol (17%) and nerolidol (14%) as well asTable 1 Experimental Functional Annotation of VvTPS Genes of the VvTPS-a Subfamily (Continued)δ-cadinene 20unknown pm 222 16.5a-cubebene 14a-copaene 13.5a-gurjunene 7.5g-cadinene 3b-cubebene 2.5unknown pm 204 2.5VvPNaHum a-Humulene syn HM807408 CAN64791^ VvTPS11 a-humulene 56hyemalol 37.5(E)-b-caryophyllene 6.5VvPNEb2epi Car (E)-b-Caryophyllene/2-epi-(E)-b-CaryophylleneHM807409 CAO39418^ VvTPS21 (E)-b-caryophyllene 72.52-epi-(E)-b-caryophyllene25Terpenoids produced by individual VvTPS clones when incubated with FPP are listed. FLcDNA clones with redundant functions are listed as well. Clones markedwith an * were characterized by in vitro assays with isolated recombinant VvTPS; clones marked with ^ were characterized in vivo in metabolically engineered E.coli. The tissue specific cDNA used to clone a particular gene is indicated in FLcDNA ID with either B (berry), F (flower), or M (mixed template consisting of stems,berries, flowers, and leaves) when appropriate. FLcDNA ID in bold indicates clone came from cDNA of methyl jasmonate treated tissue. Of the clones labelled as(E)-b-caryophyllene syn all produced an abundance of (E)-b-caryophyllene, with minor components of a-humulene and germacrene d except CAN82172 whichdid not produce germacrene D. Bolded VvTPS gene models indicate proof of transcript not previously known from the available ESTs.Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 9 of 22two minor compounds. Germacrene D was the primaryproduct of VvGwGerD (Gw64B1). VvCSAFar (CS102B7)was the only single-product member of the TPS-a subfam-ily identified here, producing 100% (E,E)-a-farnesene. Twoproducts, g-cadinene (83%) and an unidentified sesquiter-pene (17%), were the only detected products of VvGwgCad(Gw330M5). VvPNbCur (PN62M1) produced b-curcu-mene (22%) (E)-g-bisabolene (18%), iso-italicene) (16%),(-)-a-bisabolol (14%), and at least 9 additional products.The VvPNSesq (CAO16252) generated a sesquiterpeneolefin as major product that was not identified unambigu-ously by GCMS. The product was, therefore, produced inlarger quantity, purified by preparative GC and identifiedby NMR spectroscopy as sesquithujene ((1 S,5S)-2-methyl-5-((R)-6-methylhept-5-en-2-yl)bicyclo[3.1.0]hex-2-ene).Additional reaction products of this TPS included a-berga-motene, sesquisabinene, b- and g-bisabolene, b-bisabololand a trace amount of an unidentified sequiterpene alco-hol. VvPNaZin (CAO16257) is a zingiberene synthase thatalso produced b-sesquiphellandrene and b-bisabolene. Thetwo major products generated by VvPNSeInt (CAO39293)were found to be selina 4,11-diene and intermedeol. Atleast, five other sesquiterpenes including a-guaiene, selina5,11-diene, g-selinene, and 7-epi-a-selinene, were identifiedas reaction products. VvPNCuCAD (CAN76781) encodesa multi-product sesquiterpene synthase capable of produ-cing cubebol, δ-cadinene, a-copaene, a-cubebene and anunknown sesquiterpene alcohol as dominant reactionTable 2 Experimental Functional Annotation of VvTPS Genes of the VvTPS-b SubfamilyFunctional GeneIDFunction Access. # FLcDNA ID Nearest VvTPS genemodelRepresentativeProducts%VvGwaPhe (+)-a-Phellandrene Syn HM807382 Gw74ME VvTPS45 (+)-a-phellandrene 40myrcene 15terpinolene 15a-terpinene 7g-terpinene 6(+)-limonene 5(+)-a-pinene 3(+)-b-phellandrene 3(+)-a-terpineol 3(E)-b-ocimene 2VvPNaPin1 (+)-a-Pinene Syn HM807383 PN20M1 VvTPS44 (+)-a-pinene 47VvPNaPin2 HM807384 PN05S14 VvTPS44 (+)-limonene 25(+)-camphene 9myrcene 6(+)-a-terpineol 5(+)-sabinene 3(3S)-linalool 2(+)-b-pinene 1(+)-a-phellandrene 1(+)-a-thujene 1a-terpinolene 1VvGwbOci (E)-b-Ocimene syn HM807385 Gw22YB2 VvTPS34 (E)-b-ocimene 98VvCSbOci HM807386 CS402F VvTPS35 (Z)-b-ocimene 2VvCSbOciM (E)-b-Ocimene/Myrcene syn HM807387 CS19M VvTPS38 (E)-b-ocimene 53myrcene 42b-pinene 3limonene 2VvGwbOciF (E)-b-Ocimene/(EE)-a-FarneseneSynHM807388 Gw46YB3 VvTPS47 (E)-b-ocimene 100*VvCSbOciF HM807389 CS93F VvTPS47 (E,E)-a-farnesene 100**VvPNRLin (3R)-Linalool syn HM807390 PNTPS09M1 VvTPS31 (3R)-linalool 100Terpenoids produced by individual VvTPS clones when incubated with GPP are listed. Where noted are bifunctional TPS capable of producing products whenincubated with GPP (*) or FPP (**). The tissue specific cDNA used to clone a particular gene is indicated in FLcDNA ID with either B (berry), YB (young berry), F(flower), S (stem) or M (mixed template consisting of stems, berries, flowers and leaves). FLcDNA ID in bold indicates clone came from cDNA of methyl jasmonatetreated tissue. Clones with redundant functions are listed as well. Bolded VvTPS gene models indicate proof of transcript not previously known from the availableESTs.Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 10 of 22products; additional minor products included a-gurjunene,g-cadinene, b-cubebene and an unknown sesquiterpene.The VvPNaHum (CAN64791) produced a-humulene(56%), (E)- b-caryophyllene (6.5%) and a sesquiterpenealcohol (37.5%) that we initially failed to identify by GCMS;the latter compound was then produced in milligramquantities, purified by liquid chromatography and identi-fied by 1H- and 13C-NMR spectroscopy as hyemalol(3,7,10,10-tetramethylcycloundeca-3,7-dien-1-ol) a humu-lane-type sesquiterpenoid recently discovered in Zanthoxy-lum hyemale [60]. VvPNEb/2epiCar (CAO39418), wasidentified as a third type of caryophyllene synthase, whichin contrast to VvPNECar and VvGwECar also catalyzedthe formation of 2-epi-(E)-b-caryophyllene as a substantial(25%) product.Collectively, these characterized VvTPS-a enzymesproduce some of the major sesquiterpenes identifiedfrom grapevine. Furthermore, the prevalence of VvTPSproducing (E)-b-caryophyllene, a-humulene and germa-crene D may in part explain the reported prominence ofthese compounds in grapevine berries and vegetative tis-sues [12-15].Functions of VvTPS FLcDNAs of the TPS-b subfamilyWe functionally characterized seven unique VvTPS (ninedifferent FLcDNA clones) from the TPS-b subfamily(Table 2). All of the characterized VvTPS genes of thisgroup produce monoterpenes, and most are multi-pro-duct enzymes. The products of VvGwaPhe included(+)-a-phellandrene (40%), myrcene (15%), terpinolene(15%), and seven other minor products. VvPNaPIN pro-duced (+)-a-pinene (47%), (+)-limonene (25%), (+)-cam-phene (9%), and eight other minor products. Five of thenine VvTPS of the TPS-b subfamily (VvGwbOci,VvCSbOci, VvCSbOciM, VvGwbOciF, VvCSbOciF) pro-duced (E)-b-ocimene as a major product with individualvariations of additional products. VvGwbOci andVvCSbOci produced additional minor amounts of (Z)-b-ocimene; VvCSbOciM produced additional majoramounts of myrcene (42%) along with minor amountsof b-pinene and limonene; VvGwbOciF and VvCSbOciFalso converted FPP into (E,E)-a-farnesene. Lastly,VvPNRLin produced a single oxygenated product, (3R)-linalool. Together the VvTPS-b subfamily membersaccount for many of the acyclic and cyclic monoterpenehydrocarbons and a few of the monoterpene alcoholsfound in Vitis vinifera.Functions of VvTPS FLcDNAs of the TPS-g subfamilyThe TPS-g subfamily is greatly expanded in V. vinifera(Figure 2, Addiitonal File 1). Functional characterizationof ten different FLcDNA clones of this subfamilyTable 3 Experimental Functional Annotation of VvTPS Genes of the VvTPS-g SubfamilyFunctional GeneIDFunction Access. # FLcDNA ID Nearest VvTPS genemodelRepresentativeProducts%VvPNLinNer1 (3S)- Linalool/(E)- Nerolidol syn HM807391 PN25M6 VvTPS54 (3S)-Linalool 100*VvPNLinNer2 HM807392 PN55M1 VvTPS56VvCSLinNer HM807393 CS2251F VvTPS56 (E)-Nerolidol 100**VvPNLNGl1 Linalool/(E)- Nerolidol/(E,E)-Geranyllinalool synHM807394 PNTPS271M2 VvTPS57 (3S)-Linalool 100*VvPNLNGl2 HM807395 PNTPS271M5 VvTPS63 (E)-Nerolidol 100**VvPNLNGl3 HM807396 PNTPS271M4 VvTPS58 (E,E)-Geranyl linalool 100***VvPNLNGl4 HM807397 PN3M2 VvTPS61VvGwGer Geraniol syn HM807398 Gw63YB3 VvTPS52 Geraniol 100VvCSGer HQ326231 CS5M2 VvTPS52VvPNGer HM807399 PN5L1 VvTPS52Terpenoids produced by individual VvTPS clones when incubated with GPP (*) or FPP (**) are listed. Where noted are trifunctional TPS capable of producingproducts also with GGPP (***). The tissue specific cDNA used to clone a particular gene is indicated in FLcDNA ID with either YB (young berry), F (flower), L(leaves) or M (mixed template consisting of stems, berries, flowers and leaves). FLcDNA ID in bold indicates clone came from cDNA of methyl jasmonate treatedtissue. Clones with redundant functions are listed. Bolded VvTPS gene models indicate proof of transcript not previously known from the available ESTs.Table 4 Experimental Functional Annotation of VvTPS Genes of the VvTPS-f SubfamilyFunctional GeneIDFunction Access. # FLcDNAIDNearest VvTPS genemodelRepresentative ProductProfile%VvCSENerGl (E)-Nerolidol/(E,E)-Geranyl linaloolsynHM807400 CS34137F NA (E)-Nerolidol 100*VvPNENerGl HM807401 PNTPS51F2 NA (E,E)-Geranyl linalool 100**Terpenoids produced by individual VvTPS clones when incubated with FPP (*) or GGPP (**) are listed. The tissue specific cDNA used to clone a particular gene isindicated in FLcDNA ID with F (flower). Clones with redundant functions are listed also.Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 11 of 22identified three unique gene functions (Table 3). AllVvTPS of this group produce exclusively acyclic terpenealcohols, but the three types differ by their range of sub-strates. The first type of VvTPS gene function within theTPS-g subfamily is represented by three genes,VvPNLinNer1, VvPNLinNer2, and VvCSLinNer. Theseenzymes accept two substrates, C10 GPP and C15 FPP,and produce (3S)-linalool and (E)-nerolidol, respectively(Table 3). The second group is represented by fourVvPNLNGl enzymes which also accept the additionalC20 substrate GGPP to produce (E,E)-geranyl linalool.The third unique function in this subfamily is repre-sented by three genes, VvGwGer, VvCSGer andVvPNGer, which had activity only with GPP to producegeraniol. While seven of the VvTPS of the TPS-g sub-family accept more than one substrate in vitro and con-tribute potentially to the formation of terpene alcoholsof different chain lengths, it is not known whether theseenzymes indeed encounter more than one type of sub-strate in vivo.Functions of VvTPS FLcDNAs of the TPS-f subfamilyAlthough the analysis of the 12-fold genome sequencecoverage did not identify any intact VvTPS genes of theTPS-f subfamily, a unique VvTPS function of the TPS-fsubfamily was characterized with the two FLcDNAsVvCSENerGl and VvPNENerGl (Table 4). Theseenzymes accepted either FPP or GGPP to produce (E)-nerolidol or (E,E)-geranyl linalool, respectively. Unlikethe VvTPS of the TPS-g these enzymes had no activitywith GPP. VvCSENerGl and VvPNENerGl are only 62%identical and 76% similar on an amino acid level.Phylogeny of functionally characterized VvTPS and VvTPSgene modelsThe phylogenetic analyses presented here include V.vinifera TPS from the 12-fold sequence assembly of thenearly homozygous Pinot noir genotype [1] and thefunctionally characterized VvTPS described here. Theanalyses also included full-length TPS sequences thatcontained the known TPS motifs predicted from thegenome assembly of the heterozygous Pinot noir geno-type [36] for a more complete annotation of the VvTPSfamily. In this way, we have integrated the predictionsof VvTPS gene models from the two grapevine genomesequences [1,36] in a compatible fashion and we areproposing a unified VvTPS classification andnomenclature.Within the TPS-a subfamily of sesquiterpenesynthases the functionally characterized VvTPS are closeto most of the VvTPS predicted in the 12-fold genomesequence assembly (Figure 5). This topology suggeststhat the diversity of functions for grapevine sesquiter-pene synthases is well represented with the functionallycharacterized VvTPS described in this work. Relative toTPS-a enzymes of other plant species, the VvTPS exhibita large paralogous cluster with VvTPS-a members moreclosely related to one another than they are to TPSfrom other species, regardless of function. ParalogousTPS gene clusters were found previously for other spe-cies examined in depth such as A. thaliana [16] andNorway spruce [56] and indicate post-speciation geneduplication events. The large number of VvTPS-a sug-gests that this subfamily plays an important role ingrapevine biology.VvTPS of the TPS-b subfamily fall into two clades,VvTPS-b clade I and VvTPS-b clade II, bifurcated byrepresentative TPS from other plants (Figure 6). Themajority of the VvTPS of clade I make cyclic productswhile those of clade II produce only acyclic terpenoids.It is possible that clade-specific conserved sequence fea-tures determine whether a TPS is able to produce cyclicor acyclic products [53]; thus, the two clades may repre-sent an evolutionary pattern of sub-functionalizationfrom cyclic-product TPS in clade I to those TPS produ-cing acyclic products in clade II. In contrast to otherTPS subfamilies, the VvTPS clades of the TPS-b subfam-ily include members that have functional equivalents indistantly related species. For example, Lotus japonica(E)-b-ocimene synthase clusters closely with grapevineocimene synthases. Malus x domestica (E,E)-a-farnesenesynthases also clusters closest to VvTPS of the samefunction. This pattern suggests that these functionsarose prior to speciation events.The TPS-g subfamily of plant TPS was defined by pre-vious work on TPS of floral scent biosynthesis in snap-dragon (Anthirrrhinum majus) [37]. Phylogeneticanalyses that include the large number of VvTPS gene(Figure 1) conclusively resolved a bifurcation of theTPS-b and TPS-g subfamilies at a juncture that was pre-viously ambiguous and had misclassified some TPS-ggenes as TPS-b members. Specifically, the newly charac-terized grapevine geraniol synthase VvPNGer whichmatches gene model VvTPS52 (Figure 1) as well as thegeraniol and linalool synthases from basil (Ocimumbasilicum) (Figure 7), clustered closely with the TPS-gsubfamily. The phylogenetic proximity between the basiland grapevine geraniol synthases indicates that theseTPS functions already existed in a common ancestor. Incontrast, the remaining VvTPS of the TPS-g subfamily,which are all linalool/nerolidol synthases, cluster closestto other VvTPS. As mentioned above, the entire pre-dicted VvTPS-g subfamily has a conserved NSF/DTEmotif (Figure 4). This same motif is present in thecloned VvTPS cDNAs as well as other members of theTPS-g subfamily from different species. Prominent inthis motif of the VvTPS-g members is a W in the secondposition; this residue may affect the magnesium bindingMartin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 12 of 22and/or substrate orientation. Also noteworthy is thehighly modified or absent RRX8W motif from thisgroup of TPS (Figure 4) and which may imply thatthese acyclic products are formed via the geranyl cationrather than the linalyl cation [37,53].The TPS-e and TPS-c subfamilies in V. vinifera con-tain one and two members, respectively (Figure 8).Although these were not functionally characterized inthis paper, they are almost certainly involved as diter-pene synthases in ent-kaurene biosynthesis [61]. Surpris-ingly, the 12-fold sequence coverage of the grapevinegenome did not reveal any members of the TPS-f sub-family; however, our FLcDNA cloning identified twomembers of this subfamily and both were characterizedas nerolidol/geranyl linalool synthases (Figure 8). Theseare related to the Clarkia brewerii linalool synthases[62] and the recently characterized A. thaliana geranyllinalool synthase [20], each of which produces acyclicterpene alcohols as a functional signature of thissubfamily.ConclusionsThe present study provides the first comprehensiveannotation of the very large VvTPS gene family withregard to chromosomal localization, enzyme functions,and phylogeny relative to the overall plant TPS genefamily. The VvTPS gene family is one of the largestgene families of specialized (i.e., secondary) metabolismin grapevine where TPS enzymes contribute to berryand wine flavour, floral scent and potentially a diversityof other biological functions such as defense and resis-tance. The emerging profile of the VvTPS familydescribed here illustrates how this large gene family hasexpanded across the genome through gene duplicationevents and functional diversification. Notably, the largenumber of functionally diverse sesquiterpene synthasesidentified in our biochemical characterization of theVvTPS-a genes suggests that these enzymes and theirproducts may contribute substantially to grapevine biol-ogy and wine quality. The recent reports of sesquiter-penes in Shiraz grapes and wine [10,11] or theidentification of sesquiterpene volatiles in anthers andpollen of grapevine flowers [32] are early insights to theemerging roles for sesquiterpene metabolism in V.vinifera.Phylogenetic analyses of the VvTPS show a resultsimilar to many plant species studied thus far in thatmost of the VvTPS form clusters of paralogous geneswithin the plant TPS family. This finding indicates adominant process of post-speciation gene duplications,although there are also examples of conserved TPSfunctions of a more ancient order. Furthermore, ouranalyses substantiate separation of the TPS-b and theTPS-g subfamilies. The separation based on sequencerelatedness is matched by separation of gene functions,since all known members of the TPS-g subfamily pro-duce acyclic products.Of the monoterpene synthases presented here, thosethat produce (3S)-linalool (VvTPS-g), geraniol (VvTPS-g), and the previously identified a-terpineol synthase(VvTPS-b) will be of much interest to viticulturalists andwine makers as these are some of the most prevalentcompounds responsible for the floral characteristics ofaromatic varieties. Furthermore, compounds such asgeraniol and linalool can be further modified in grapemusts and wine to produce citronellol, rose oxide, andwine lactone [63,64]. Linalool and a-terpineol have alsobeen found to contribute to the character of non-aromatic red grapevine varieties [65]. It is conceivablethat different viticultural regimes may modify theexpression of these TPS in grape berries and can therebyimpact the quality of the resultant wines.While many of the terpenoid products of the VvTPSenzymes characterized here have been described in theviticulture and oenology literature [8,66], still severalhave yet to be associated with traits in grapes or wine.Taken together, the present VvTPS genomic annotationsand the VvTPS functional characterizations provide areference framework for future studies, including tran-script and protein expression profiling, as well as terpe-noid molecular marker development through, forexample association mapping.MethodsVvTPS gene discovery and manual annotationThe predicted proteome of the 12-fold coverage grape-vine genome sequence assembly (GenBank, NCBI pro-ject ID 18785; Genoscope website: http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/) wasscreened with two HMM profiles of the PFAM motifs[45] PF01397 (N-terminal TPS domain) and PF03936(TPS, metal binding domain). In addition, the 12-foldgenome sequence assembly was screened (TBLASTN)with known TPS sequences from Swiss-Prot in order tobe not dependent of the automatic annotation. The 152loci exhibiting significant similarities with known TPS(all BLAST hits with an e-value lower than 1.e-4 wereindividually evaluated) were manually annotated to cor-rect erroneous automatic annotation and to discriminatebetween complete, partial and pseudo-TPS. Genomicregions with similarities spanning on less than 50 aminoacids with TPS have not been considered. The manualannotation is based on the results of the EuGène predic-tor-combiner software [67] that was specifically trainedfor Vitis vinifera, sequence alignments with previouslycharacterized TPS proteins and related PFAM motifs,spliced alignments [68] of cognate EST and cDNAsequences and knowledge of TPS gene structure andMartin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 13 of 22Figure 5 Phylogeny of the VvTPS-a subfamily. Maximum likelihood analysis of the V. vinifera TPS-a subfamily. Bootstrap values supported by≥ 50% are designated * and those with values ≥ 80%are indicated with ^. TPS characterized in this paper are in teal and include Vv(PN & Gw)ECar# = (E)-caryophyllene syn, VvPNGerA = germacrene A syn, VvGwaBer = (E)-a-bergamotene syn, Vv(PN & Gw)GerD = germacrene D syn,VvCSaFar = (E,E)-a-farnesene syn, VvGwgCad = g-cadinene syn, VvPNbCur = b-curcumene syn, VvPNSesq = sesquithujene syn, VvPNaZin = a-zingiberene syn, VvPNSeInt = Selina-4,11-diene/Intermedeol syn, VvPNCuCad = cubebol/δ-cadinene syn, VvPNaHum = a-humulene syn, andVvPNEb2epiCar = (E)-b-caryophyllene/2 epi-(E)-b-caryophyllene syn. VvTPS predicted from the 12-fold genome sequence assembly are in purple.Previously cloned VvTPS are in brown. TPS predicted by sequencing of the heterozygous Pinot noir are labeled with GenBank accession numbers(CAN....) or marked with ~ if they were also cloned and characterized. Abbreviations are as follows: AabCS = Artemisia annua, b-caryophyllene syn(AAL79181), VvGwGerD = Vitis vinifera (-)-germacrene D syn (AAS66357), VvCsVal = V. vinifera (+)-valencene syn (ACO36239), Aaced = A. annua 8-epi-cedrol syn (AAF80333), ScGerD = Solidago canadensis (-)-germacrene D syn (AAR31145), AaEbFar = A. annua (E)-b-farnesene syn (AAX39387),CjFarn = Citrus junos (E)-b-farnesene syn (AAK54279), LsGerA = Lactuca sativa germacrene A syn (AAM11627), LeVet = Lycopersicon esculentumvetispiradiene syn (AAG09949), GadCad = Gossypium arboreum (+)-δ-cadinene syn (O49853), PfVal = Perilla frutescens var. frutescens valencene syn(AAX16077), CsVal = C. sinensis valencene syn (AAQ04608), Nt5eAri = Nicotiana attenuata 5-epi-aristolochene syn (AAP05761), MpFar = Mentha xpiperita (E)-b-farnesene syn (AAB95209).Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 14 of 22Figure 6 Phylogeny of the TPS-b subfamily. Maximum likelihood analysis of the V. vinifera TPS-b subfamily. Bootstrap values supported by ≥50% are designated * and those with values ≥ 80%are indicated with ^. TPS characterized in this paper are in teal and include VvGwPhe =(+)-a-phellandrene syn, VvPNpPin = (+)-a-pinene syn, VvGwbOci &VvCSbOci = (E)-b-ocimene syn, VvCSEbOcM = (E)-b-ocimene/myrcene syn,VvGwEbOciF = (E)-b-ocimene/(E,E)-a-farnesene syn, and VvPNRLin = (3R)-linalool syn. VvTPS predicted from the 12-fold genome sequenceassembly are in purple. Previously cloned VvTPS are in brown. TPS predicted by sequencing of the heterozygous Pinot noir are labeled withGenBank accession numbers (CAN....). Abbreviations are as follows: CspaPS = Cannabis sativa (+)-a-pinene syn (ABI21838), LaRLin = Lavandulaangustifolia (3R)-linalool syn (ABD77417), CtGerS = Cinnamomum tenuipile geraniol syn (CAD29734), VvaTer1 = V. vinifera (-)-a-terpineol syn(AAS79351), VvaTer2 = V. vinifera (-)-a-terpineol syn (AAS79352), QiMyr = Quercus ilex myrcene syn (Q93X23), ClgTer = C. limon g-terpinene syn(AAM53943), LaLim = L. angustifolia (+)-limonene syn (ABB73044), SoSab = Salvia officinalis (+)-sabinene syn (O81193), MxDFS = Malus xdomestica (E,E)-a-farnesene syn (AAX19772), AabPi = A. annua (-)-b-pinene syn (AAK58723), MsLim = Mentha spicata (-)-limonene syn (AAC37366),AtMyrOc = Arabidopsis thaliana myrcene/(E)-b-ocimene syn (NP_179998), MaLin = M. aquatica (3R)-linalool syn (AAL99381), SoBDS = S. officinalis(+)-bornyl diphosphate syn (O81192), So18Cs = S. officinalis 1,8-cineole syn (O81191), LcjOs = Lotus corniculatus var. japonicus (E)-b-ocimene syn(AAT86042), ObaZin = Ocimum basilicum a-zingiberene syn (AAV63788), LatBer = L. angustifolia (E)-a-bergamotene syn (ABB73046), ObFen = O.basilicum fenchol syn (AAV63790), SlLinNer = Solanum lycopersicum (3R)-linalool/(E)-nerolidol syn (AAX69063), SlPhe = S. lycopersicum b-phellandrene/myrcene/sabinene syn (AAX69064)Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 15 of 22Figure 7 Phylogeny of the VvTPS-g subfamily. Maximum likelihood phylogenies of the V. vinifera TPS-g subfamily. Bootstrap values supportedby ≥ 50% are designated * and those with values ≥ 80%are indicated with ^. TPS characterized in this paper are in teal and includeVvPNLinNer1-5 = (3S)-linalool/(E)-nerolidol syn, VvPNLNGl = (3S)-linalool/(E)-nerolidol/(E,E)-geraniol linalool syn, and VvGwGer = geraniol syn.VvTPS predicted from the 12-fold genome sequence assembly are in purple. TPS predicted by sequencing of the heterozygous Pinot noir arelabeled with GenBank accession numbers (CAN....). Abbreviations for other tree members are as follows: AmMyr = Antirrhinum majus myrcenesyn (AAO41727), AmMyr2 = A. majus myrcene syn (AAO41726), AmOci = A. majus (E)-b-ocimene syn (AAO42614.1), FaNLS = Fragaria x ananassa(3S)-linalool/(E)-nerolidol syn (CAD57106), AmLinNer1 = A. majus nerolidol/(3S)-linalool syn 1 (ABR24417), AmLinNer2 = A. majus nerolidol/(3S)-linalool syn 2 (ABR24418), AtSLin = A. thaliana (3S)-linalool syn (NP_176361), ObGES = O. basilicum geraniol syn (AAR11765), OCLiS = O. basilicum(3R)-linalool syn (AAV63789).Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 16 of 22Figure 8 Phylogeny of TPS-c, e, and f subfamilies. Phylogenetic relationships as determined by maximum likelihood analysis of the V. viniferaTPS-c,e, and -f subfamilies. Bootstrap values supported by ≥ 50% are designated * and those with values ≥ 80% are indicated with ^. TPScharacterized in this paper are in teal and include VvCSENerGl and VvPNENerGl = (E)-nerolidol/(E,E)-geraniol linalool syn. VvTPS predicted fromthe 12-fold genome sequence assembly are in purple. TPS predicted by sequencing of the heterozygous Pinot noir are labeled with GenBankaccession numbers (CAN....). TPS-c are all ent-copalyl diphosphate synthases - PtCPS = Populus trichocarpa (EEE81383), CmCPS1 = Cucurbitamaxima (AAD04292), LsCPS1 = Lactuca sativa (BAB12440), SrCPS = Stevia rebaudiana (AAB87091), CmCPS2 = C. maxima (AAD04293), TaCPS =Triticum aestivum (BAH56560), OsCPS = Oryza sativa Japonica group (BAD42452). The following ent-kaurene synthases (Tps-e) are included in theanalysis: PteKS = Populus trichocarpa (EEE88653), RceKS = Ricinus communis (EEF28689), HveKS = Hordeum vulgare subsp. Vulgare (AAT49066),LseKS1 = Lactuca sativa (BAB12441), SreKS = Stevia rebaudiana (AAD34295), ZmeKS = Zea mays (NP_001148059), OseKS1a = Oryza sativaJaponica group (AAQ72559). Abbreviations of the other included TPS are as follows: ZmFNF = Z. mays (E)-b-farnesene, (E)-nerolidol, (E,E)-farnesolsyn (AAO18435), OsPim = O. sativa Indica group syn-pimara-7,15-diene syn (AAU05906), OS9Pim = O. sativa Japonica group 9b-pimara-7,15-dienesyn (BAD54751), SHSanBer = Solanum habrochaites santalene/bergamotene syn (ACJ38409), SlPHS1 = S. lycopersicum b-phellandrene syn(ACO56896), NtTPS = Nicotiana tabacum terpene syn (AAS98912), AtGES = Arabidopsis thaliana geranyl linalool syn (NP_564772), CbSLin = Clarkiabreweri (3S)-linalool syn (AAC49395), CbSLin2 = C. breweri (3S)-linalool syn 2 (AAD19840), AtTPS = A. thaliana tps (AAO85540).Martin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 17 of 22protein sequences. Data and other related informationwere imported and merged in the ARTEMIS tool [69]to evaluate each resource and produce the final annota-tion. The EuGène predictions, the manual structuralannotation of the 152 loci and the correspondingsequences are available in the FLAGdb++ databasehttp://urgv.evry.inra.fr/FLAGdb[70]. Protein sequencesdeduced from the 69 full VvTPS genes were analyzedwith ChloroP for prediction of N-terminal plastidial tar-geting peptides [71]Phylogenetic AnalysesAmino acid alignments were made using Dialign (dia-lign.gobics.de/anchor/submission.php) with a thresholdvalue of 10. Manual adjustments such as aligning con-served motifs and manual trimming were performedusing GeneDoc http://www.nrbsc.org/gfx/genedoc. Forall analyses, sequence information upstream of the par-tially conserved RR(X)8W motif was trimmed. Maxi-mum likelihood analyses were completed using Phyml[72] available at http://www.atgc-montpellier.fr/phyml/.For each analysis, the LG amino acid substitution modeland four substitution rate categories were used, the pro-portion of invariable sites and the gamma distributionparameter were estimated, and the branch lengths andtree topology were optimized from the data set. Theestimated values for the proportion of invariable sitesand the gamma shape parameter were then used whenperforming 100 bootstrap replicas. Phylogenetic treeswere visualized using TreeView http://taxonomy.zool-ogy.gla.ac.uk/rod/treeview.html.RNA isolation and VvTPS cDNA cloningRNA was isolated from Gewürztraminer, Pinot noir, andCabernet Sauvignon grapevine shoot cuttings grown inthe greenhouse as previously described [32]. RNA wasisolated from stem (S), leaf (L), berry (B), root (R) andflower (F) tissues (see FLcDNA ID in Table 1, Table 2,Table 3, and Table 4) as detailed in Reid et al. [73]. Toup-regulate expression of TPS genes, a subset of grape-vine cuttings were treated with methyl jasmonate (0.01%v/v in water and 0.1% Tween) two days prior to RNAisolation. The Superscript Vilo cDNA synthesis kit(http://www.invitrogen.com was used according to themanufacturer’s instructions. Primers for VvTPS cDNAcloning were designed based on TPS sequences obtainedthrough iterative BLAST searches in NCBI GenBankusing members of each of the TPS subfamilies. Addi-tional information for the design of PCR primers camefrom the predicted VvTPS gene models identified in the12-fold genome sequence assembly. To increase thelikelihood of a successful PCR amplification of VvTPScDNAs, cDNA templates from various tissues from thesame cultivar were often combined. These clones aredesignated “M” for mixed template while those TPScloned from individual tissues are labeled with the singleletter abbreviations described above (Table 1, Table 2,Table 3, and Table 4). PCR reactions were done usingtouchdown PCR with proofreading polymerases, as perthe product manufacturer’s instructions. PCR productsof the expected sizes were cloned into the pJet1.2 clon-ing vector http://www.fermentas.com and transformedinto E. coli a-select cells http://www.bioline.com. Plas-mids containing a correctly sized insert were sequencedfollowed by insert amplification and ligation into thepET28b expression vector (Novagen, http://www.emd-chemicals.com) using sticky end PCR [74]. When clon-ing difficulties were encountered, as was the case withseveral sequences, pASK-IBA3plus (IBA, http://www.iba-go.com) vector was used [20].Expression of recombinant VvTPS proteins, in vitroenzyme assays and product identifications by GCMSFor TPS protein expression, Cip41 Rare E. coli cells [75]containing recombinant VvTPS plasmids were grownuntil 0.8 OD, induced with 0.5 mM IPTG and thengrown for an additional 16 h at 16 °C. RecombinantVvTPS were partially purified using His SpinTrapcolumns (GE Biosciences, http://www.apbiotech.com).Protein expression was verified using silver stained SDS-PAGE gels and western blot analysis using Murine anti-polyHistidine Monoclonal antibody (1/4000 dilution,Sigma-Aldrich, http://www.sigmaaldrich.com) and5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazo-lium (CalBiochem, http://www.emdchemicals.com). ForStrep-tag II clones, the pASK-IBA3plus vector combinedwith Cip41 Rare cells were used for expression and proteinwas isolated as per the manufacturer’s instructions using aStrep-Tactin affinity purification column (IBA).For functional enzyme characterization, approximately100 μg total protein of semi-purified recombinantVvTPS were added to a single vial assay in 50 μL totalvolume as described [32,75,76]. Each recombinantVvTPS was incubated with GPP (109 μM), (E,E)-FPP(92 μM) or (E,E,E)-GGPP (20 μM) for 2 h at 30 °C. Buf-fers used for sesquiterpene and diterpene synthaseassays contained 50 mM HEPES pH 7.0, 10 mM MgCl2,20 μM MnCl2, 10% glycerol (v/v), and 1 mM DTT. Formonoterpene synthase assays, the buffer consisted of 50mM HEPES pH 7.0, 10 mM MgCl2, 20 μM MnCl2 100mM KCl, 10% glycerol (v/v), and 1 mM DTT.Gas chromatography coupled with mass spectrometry(GCMS) analysis was employed to determine the pro-duct profiles of each TPS as previously described[32,56]. Columns used to separate product mixturesincluded HP-5MS (Agilent), DB-Wax (Agilent), SolGel-Wax (SGE), and Cyclodex-B (Aglient) for chiral ana-lyses. For sesquiterpene analysis, a cool-on column inletMartin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 18 of 22(starting temperature of 35 °C and track oven programafter injection) was used to prevent thermo-rearrange-ments of terpenes in the injector. Identities of individualterpenes were made using a combination of authenticstandards and/or retention indices along with MS librarymatches (including WileyNist 2005, [77], and/or Mass-Finder4 [massfinder.com/wiki/MassFinder_4]).In vivo enzyme assays using metabolically engineeredE. coliA subset of VvTPS encoding sesquiterpene synthaseswere codon-optimized for expression in E. coli, usinggene synthesis by DNA2.0 https://www.dna20.com/ andsubcloned into the pET-3a or pET-Duet1 expressionplasmids (Novagen, http://www.emdchemicals.com).Enzymatic activity was assessed in E. coli cells co-expressing the TPS together with the enzymes of a five-step biosynthetic pathway converting mevalonic acid toFPP. The FPP synthase gene was amplified from S. cere-visiae genomic DNA and ligated into the first multiplecloning site (MCS) of the pACYCDuet-1 expressionplasmid (Novagen, http://www.emdchemicals.com) pro-viding the plasmid FPPs-pACYCDuet. An operonencoding the genes for a mevalonate kinase (MvaK1), aphosphomevalonate kinase (MvaK2), a mevalonatediphosphate decarboxylase (MvaD) and an isopentenyldiphospahte isomerase (idi) was amplified from genomicDNA of Streptococcus pneumoniae (ATCC BAA-334)and ligated into the second MCS of the FPPs-pACYC-Duet plasmid providing the plasmid pACYCDuet-4506.BL21 Star™(DE3) E. coli cells http://www.invitrogen.comwere co-transformed with the plasmids, pACYCDuet-4506 and either of the pET series plasmids harboringcandidate VvTPS coding sequences. Single colonies wereused to inoculate 5 mL of LB medium supplementedwith carbenicillin (50 mg/mL) and chloramphenicol (34mg/mL). Cultures were incubated overnight at 37°C.The next day 250 mL to 1 L of Terrific Broth mediumsupplemented with the appropriate antibiotics wereinoculated with 1/100 volume of the overnight culture.After 6 h incubation at 37°C, cultures were cooled downto 28°C and then 1 mM IPTG, 2 mg/mL mevalonateprepared by dissolving mevalonolactone http://www.sig-maaldrich.com in 0.5N NaOH at a concentration of 1 g/mL and incubating the solution for 30 min at 37°C) and0.1 volume of decane were added to the cultures. After48 h incubation, the decane fraction was directly ana-lysed by GCMS on a Hewlett Packard 6890 series GCsystem equipped with a DB1 column 30 m × 0.25 mm ×0.25 mm film thickness http://www.agilent.com andcoupled with a 5975 series mass spectrometer. The car-rier gas was helium at a constant flow of 1 ml.min-1.Injection was in splitless mode with the injector tem-perature set at 120°C and the oven temperature wasprogrammed from 60°C to 265°C at 5°C.min-1. Identifi-cation of VvTPS products was based on retention timeindex, mass spectra of authentic standards, and on pub-lished [78] or Firmenich MS database. Co-injectionswere also used for some of the compounds (caryophyl-lene, humulene, cubebol, cubebene, intermedeol). Forcompounds that could not be identified by GCMS, theproducts were purified and their structures determinedby NMR spectroscopy. Sesquithujene was isolated usingmanual, preparative-GC. The crude sample (bi-phasicbacteria culture) was first distilled using a Fisher columnto remove the decane (temperature: 95 °C; pressure: 25mbar). The compounds were then separated using a GCequipped with a.1.83 m × 2.1 mm i.d., 10% OV -1packed column at a flow rate of 10 mL/min. The oventemperature was programmed from 160 °C (held 10minutes) to 230 °C at 10 °C/min. Helium was used asthe carrier gas. Hyemalol was purified by silica gel flashcolumn chromatography (Silicagel 60, 12*150 mm, 40-63 μM, Merck) using a 98:2 mixture of toluene anddiethyl ether as solvent system. NMR data wereacquired at 298 K using a Bruker Avance 500 MHzspectrometer. The structure was established by 1 D 1H-and 13C- NMR, as well as 2 D HSQC, COSY andHMBC experiments.Additional materialAdditional file 1: The Excel file contains all the information relativeto the 152 TPS loci detected and curated in the 12-fold PN40024grapevine genome. The content of each column is: - Name: VvTPSnomenclature for the 69 complete terpene synthases. - Gene ID (12x):The official ID of the gene automatically annotated by IGGP(International Grape Genome Program) and used by GenBank/EMBL. Thegenes called ‘newX’ correspond to TPS loci completely missed by theautomatic annotation pipeline. The curated gene structures andsequences are available in the FLAGdb++ database http://urgv.evry.inra.fr/FLAGdb. - merged ID: If the re-annotated TPS genes fit or overlap withseveral IGGP consecutive genes (erroneous splitting of the automaticannotation), their IDs are mentioned here. - Prot size: The size (in aminoacids) of the re-annotated TPS proteins. For not complete or coherentCDS (partial or pseudo-TPS), the size fits with the longest rebuilt proteinsequence (in italic). - Chr: The chromosome number. ‘10R’ means thatthe gene is on the chromosome 10 but on a not mapped scaffold. ‘R’means that the gene is inside an unmapped scaffold. - Exons: Number ofannotated exons in the CDS (after curation). - Subfamily: Classification ofTPS (TPS-a to TPS-g) according to the phylogeny and functional studies.The table is colored according to this column. - DDxxD: ‘yes’ means thatthe exact motif is present at the expected position (end of the exon 4).‘?’ means that the corresponding part of the gene is absent (partial geneor pseudogene). - RRx(8)W: ‘yes’ means that the exact motif is present atthe expected position (end of the exon 1). ‘?’ means that thecorresponding part of the gene is absent (partial gene or pseudogene). -NSE/DTE: sequence of the NSE/DTE motif present in the C-terminalregion of the proteins from TPS-a, -b and -g subfamilies. - Start: firstposition of the curated CDS in the 12-fold pseudo-chromosomes. - Stop:last position of the curated CDS in the 12-fold pseudo-chromosomes. -Strand: ‘m’ means minus strand and ‘p’ means plus strand relatively tothe pseudo-chromosomes. - Manual re-annotation result: informationabout the evaluation and curation process. Protein IDs are listed for theVvTPS genes where automatic annotation predicted a correct structure. -Type: ‘full’ means that the TPS gene (CDS) is complete, without sequenceMartin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 19 of 22problem. ‘full ?’ means that CDS is complete excepted one punctualsequence problem. A sequencing error is therefore possible and thegene could be functional. ‘partial’ means that the gene is disrupted byan un-sequenced region (gap of N) and additional sequencing isnecessary to have a full CDS. ‘pseudo’ means that the gene structure isdisturbed by stop(s) in frame, frameshift(s) and/or deletion(s). As it is, thegene cannot be functional and is qualified as pseudogene. - EST: Thenumber of available cognate transcript sequences. (+) means that theCDS is fully covered by the EST contig. - Note: Additional informationabout the partial genes and pseudogenes (nature of the problem, gappositions...). - Cluster: TPS with the same letter are organized in tandemin the same physical cluster. - Subcell. loc.: The result of the ChloroPprediction tool. A score greater than 0.5 means that the TPS protein isprobably targeted to the plastids. ‘x’ means that the prediction has notbeen done because the N-terminal region of the protein is lacking. - Besthit in SwissProt: Biochemical function of the first hit obtained in theSwissProt database with BLASTP.AbbreviationsVvTPS: Vitis vinifera terpene synthase; AtTPS: Arabidopsis thaliana terpenesynthase; FLcDNA: full length cDNA; PN: Pinot noir; CS: Cabernet Sauvignon;Gw: Gewürztraminer; FPP: farnesyl diphosphate; GPP: geranyl diphosphate;GGPP: geranylgeranyl diphosphate.AcknowledgementsWe are grateful to Eric J. Abbott for bioinformatics and technical work onthe cloning of TPS. We acknowledge Vincor International for allowing us tocollect grape shoot material from their vineyards in Okanagan Falls, BC, andOsoyoos, BC. DMM was supported by a postdoctoral fellowship from theNatural Sciences and Engineering Research Council of Canada (NSERC).Salary support for JB was provided, in part, by the UBC DistinguishedUniversity Scholar program. Funding for this project was provided by theNatural Sciences and Engineering Research Council of Canada (to JB),Genome British Columbia, and Genome Canada (to JB and STL). FabienneDeguerry, Laurence Pastore and Eggen Xian-Wen Gan (Firmenich SA) arethanked for excellent technical assistance.Author details1Michael Smith Laboratories, University of British Columbia, 2185 East Mall,Vancouver, B.C, V6T 1Z4, Canada. 2Wine Research Centre, University of BritishColumbia, 2205 East Mall, Vancouver, B.C., V6T 1Z4, Canada. 3Unité deRecherche en Génomique Végétale (URGV) UMR INRA 1165 - Universitéd’Evry Val d’Essonne - ERL CNRS 8196, 2 Rue Gaston Crémieux, 91057 EvryCedex, France. 4Firmenich SA, Corporate R&D Division, Geneva, CH-1211,Switzerland.Authors’ contributionsDMM, OT, MBS, LD, and SA, performed experiments and analyzed data.DMM, JB, LD, MS and SA conceived of the study, interpreted results andwrote the paper. OT, LD, MS and STL reviewed the paper prior tosubmission and provided valuable comments on the interpretation andpresentation of results. JB and STL secured funding. 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BMC Plant Biology 2010 10:226.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitMartin et al. BMC Plant Biology 2010, 10:226http://www.biomedcentral.com/1471-2229/10/226Page 22 of 22

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