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

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

Diane M Martin et al. BMC Plant Biol.

Abstract

Background: Terpenoids 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.

Results: We 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.

Conclusions: The 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.

Figures

Figure 1
Figure 1
Phylogeny and chromosome location of 69 putative intact VvTPS identified as gene models in the 12-fold coverage genome sequence assembly of Vitis vinifera (Pinot noir). Maximum likelihood analysis of the V. vinifera VvTPS gene family. Bootstrap values supported by ≥ 50% are designated * and those with values ≥ 80% are indicated with ^. Colors indicate chromosome location known at this time. Light blue = 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.
Figure 2
Figure 2
Genomic organization of a 690 kb multi-gene VvTPS cluster. A 690 kb long genomic region of chromosome 18 contains a 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 of other genes (orange arrows) and numerous vestiges of the Copia-like transposable element (yellow arrows).
Figure 3
Figure 3
Gene structure and classification of putative intact VvTPS. Exon-intron structures were predicted and manually curated for the putative intact VvTPS gene models. Green arrows and black lines represent at scale protein coding exons and introns, respectively. The conserved 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, by corresponding functional data. Subfamilies TPS-a, TPS-b and TPS-g are characterized by a highly conserved structure of seven exons. The three VvTPS genes of subfamilies TPS-c and TPS-e have distinct structures with 13 to 15 exons.
Figure 4
Figure 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 in this analysis. B: Alignment of the NSE/DTE motif within the 69 intact VvTPS annotated in this analysis. Dark grey shading indicates amino acids conserved with ≥ 80% and those shaded with light grey indicate conservation ≥ 60%.
Figure 5
Figure 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)-α-bergamotene syn, Vv(PN & Gw)GerD = germacrene D syn, VvCSaFar = (E,E)-α-farnesene syn, VvGwgCad = γ-cadinene syn, VvPNbCur = β-curcumene syn, VvPNSesq = sesquithujene syn, VvPNaZin = α-zingiberene syn, VvPNSeInt = Selina-4,11-diene/Intermedeol syn, VvPNCuCad = cubebol/δ-cadinene syn, VvPNaHum = α-humulene syn, and VvPNEb2epiCar = (E)-β-caryophyllene/2 epi-(E)-β-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, β-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)-β-farnesene syn (AAX39387), CjFarn = Citrus junos (E)-β-farnesene syn (AAK54279), LsGerA = Lactuca sativa germacrene A syn (AAM11627), LeVet = Lycopersicon esculentum vetispiradiene 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 x piperita (E)-β-farnesene syn (AAB95209).
Figure 6
Figure 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 = (+)-α-phellandrene syn, VvPNpPin = (+)-α-pinene syn, VvGwbOci &VvCSbOci = (E)-β-ocimene syn, VvCSEbOcM = (E)-β-ocimene/myrcene syn, VvGwEbOciF = (E)-β-ocimene/(E,E)-α-farnesene syn, and VvPNRLin = (3R)-linalool 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....). Abbreviations are as follows: CspaPS = Cannabis sativa (+)-α-pinene syn (ABI21838), LaRLin = Lavandula angustifolia (3R)-linalool syn (ABD77417), CtGerS = Cinnamomum tenuipile geraniol syn (CAD29734), VvaTer1 = V. vinifera (-)-α-terpineol syn (AAS79351), VvaTer2 = V. vinifera (-)-α-terpineol syn (AAS79352), QiMyr = Quercus ilex myrcene syn (Q93X23), ClgTer = C. limon γ-terpinene syn (AAM53943), LaLim = L. angustifolia (+)-limonene syn (ABB73044), SoSab = Salvia officinalis (+)-sabinene syn (O81193), MxDFS = Malus x domestica (E,E)-α-farnesene syn (AAX19772), AabPi = A. annua (-)-β-pinene syn (AAK58723), MsLim = Mentha spicata (-)-limonene syn (AAC37366), AtMyrOc = Arabidopsis thaliana myrcene/(E)-β-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)-β-ocimene syn (AAT86042), ObaZin = Ocimum basilicum α-zingiberene syn (AAV63788), LatBer = L. angustifolia (E)-α-bergamotene syn (ABB73046), ObFen = O. basilicum fenchol syn (AAV63790), SlLinNer = Solanum lycopersicum (3R)-linalool/(E)-nerolidol syn (AAX69063), SlPhe = S. lycopersicum β-phellandrene/myrcene/sabinene syn (AAX69064)
Figure 7
Figure 7
Phylogeny of the VvTPS-g subfamily. Maximum likelihood phylogenies of the V. vinifera TPS-g 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 VvPNLinNer1-5 = (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 are labeled with GenBank accession numbers (CAN....). Abbreviations for other tree members are as follows: AmMyr = Antirrhinum majus myrcene syn (AAO41727), AmMyr2 = A. majus myrcene syn (AAO41726), AmOci = A. majus (E)-β-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).
Figure 8
Figure 8
Phylogeny of TPS-c, e, and f subfamilies. Phylogenetic relationships as determined by maximum likelihood analysis of the V. vinifera TPS-c,e, and -f subfamilies. 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 VvCSENerGl and VvPNENerGl = (E)-nerolidol/(E,E)-geraniol linalool syn. VvTPS predicted from the 12-fold genome sequence assembly are in purple. TPS predicted by sequencing of the heterozygous Pinot noir are labeled with GenBank accession numbers (CAN....). TPS-c are all ent-copalyl diphosphate synthases - PtCPS = Populus trichocarpa (EEE81383), CmCPS1 = Cucurbita maxima (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 the analysis: 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 sativa Japonica group (AAQ72559). Abbreviations of the other included TPS are as follows: ZmFNF = Z. mays (E)-β-farnesene, (E)-nerolidol, (E,E)-farnesol syn (AAO18435), OsPim = O. sativa Indica group syn-pimara-7,15-diene syn (AAU05906), OS9Pim = O. sativa Japonica group 9b-pimara-7,15-diene syn (BAD54751), SHSanBer = Solanum habrochaites santalene/bergamotene syn (ACJ38409), SlPHS1 = S. lycopersicum β-phellandrene syn (ACO56896), NtTPS = Nicotiana tabacum terpene syn (AAS98912), AtGES = Arabidopsis thaliana geranyl linalool syn (NP_564772), CbSLin = Clarkia breweri (3S)-linalool syn (AAC49395), CbSLin2 = C. breweri (3S)-linalool syn 2 (AAD19840), AtTPS = A. thaliana tps (AAO85540).

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