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Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family… Keeling, Christopher I; Weisshaar, Sabrina; Ralph, Steven G; Jancsik, Sharon; Hamberger, Britta; Dullat, Harpreet K; Bohlmann, Jörg Mar 7, 2011

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RESEARCH ARTICLE Open AccessTranscriptome mining, functional characterization,and phylogeny of a large terpene synthase genefamily in spruce (Picea spp.)Christopher I Keeling1, Sabrina Weisshaar2, Steven G Ralph3, Sharon Jancsik1, Britta Hamberger4, Harpreet K Dullat1,Jörg Bohlmann1*AbstractBackground: In conifers, terpene synthases (TPSs) of the gymnosperm-specific TPS-d subfamily form a diversearray of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions andvolatile emissions. These compounds contribute to defence against herbivores and pathogens and perhaps alsoprotect against abiotic stress.Results: The availability of extensive transcriptome resources in the form of expressed sequence tags (ESTs) andfull-length cDNAs in several spruce (Picea) species allowed us to estimate that a conifer genome contains at least69 unique and transcriptionally active TPS genes. This number is comparable to the number of TPSs found in anyof the sequenced and well-annotated angiosperm genomes. We functionally characterized a total of 21 spruceTPSs: 12 from Sitka spruce (P. sitchensis), 5 from white spruce (P. glauca), and 4 from hybrid white spruce (P. glauca× P. engelmannii), which included 15 monoterpene synthases, 4 sesquiterpene synthases, and 2 diterpenesynthases.Conclusions: The functional diversity of these characterized TPSs parallels the diversity of terpenoids found in theoleoresin and volatile emissions of Sitka spruce and provides a context for understanding this chemical diversity atthe molecular and mechanistic levels. The comparative characterization of Sitka spruce and Norway sprucediterpene synthases revealed the natural occurrence of TPS sequence variants between closely related sprucespecies, confirming a previous prediction from site-directed mutagenesis and modelling.BackgroundConifer trees (order Coniferales; Gymnosperms) areextremely long-lived plants that must confront a multi-tude of biotic and abiotic stresses that vary with the sea-son and over their lifetime. Conifers have evolvedseveral resistance mechanisms that repel, kill, inhibit, orotherwise reduce the success of herbivores and patho-gens. These mechanisms include both mechanical andchemical defences that can be present constitutively orthat are induced upon challenge [1,2]. As a major partof their constitutive and inducible defensive repertoire,conifers produce an abundant and complex mixture ofterpenoids in the form of oleoresin secretions andvolatile emissions [2,3]. The diversity of the terpenoidsin conifers suggests that, like in other plants [4], anarms race has unfolded in the interactions of coniferswith other organisms through the production of specia-lized (i.e., secondary) metabolites. The diversity of coni-fer terpenoids includes predominantly monoterpenes,sesquiterpenes and diterpenes, which originate from theactivity of a family of terpene synthases (TPSs), andother enzymes, such as cytochromes P450, that mayfunctionalize some of the terpenes [2,5].Despite much work on individual conifer TPSs [2], thetotal number of TPSs present in any one conifer speciesis not yet known since no conifer genome has beensequenced to date. In contrast, the sequenced and anno-tated genomes of several angiosperm species provide anindication of the diversity of TPSs we might expect tosee in any one plant species. For example, the genes* Correspondence: bohlmann@msl.ubc.ca1Michael Smith Laboratories, University of British Columbia, 301-2185 EastMall, Vancouver BC, V6T 1Z4, CanadaFull list of author information is available at the end of the articleKeeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43© 2011 Keeling 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.encoding putatively active mono-, sesqui-, and di-TPSsnumber at least 32 in the Arabidopsis (Arabidopsisthaliana) genome [6], at least 31 in the rice (Oryzasativa) genome [7], at least 32 in the poplar (Populustrichocarpa) genome [8], and at least 69 in the genomeof a highly inbred grapevine (Vitis vinifera) Pinot Noirvariety [9,10]. All of these angiosperm genomes containclusters of duplicated TPS genes. The large genome sizeof conifers and the diversity of their terpenoid profilesmay suggest a similarly sized or potentially larger TPSgene family in conifer species. However, targeted BACsequencing of a few conifer TPSs from white spruce(Picea glauca) did not reveal any genomic clustering ofmultiple TPS genes in this conifer genome [11,12].Most of our current knowledge of the size, functionaldiversity and phylogeny of gymnosperm TPSs is based ontargeted cDNA cloning and characterization in two coni-fer species, grand fir (Abies grandis) and Norway spruce(P. abies), along with a few TPSs in other gymnosperms[2]. In grand fir, 11 different TPS genes have been func-tionally characterized [13]. Martin et al. [14] described aset of 9 different TPSs in Norway spruce (P. abies) andexamined the phylogeny of 29 gymnosperm TPSs, all ofwhich fell into the gymnosperm-specific TPS-d subfam-ily. A deeper understanding of the diversity and func-tional complexity of the conifer TPS-d subfamily requiresadditional gene discovery by transcriptome mining. Largecollections of expressed sequence tags (ESTs) and full-length cDNAs (FLcDNAs) exist for several conifer spe-cies [15-17] and provide a rich resource for identifyingand functionally characterizing new TPSs.Here, we have analyzed the ESTs and FLcDNAs fromSitka spruce (P. sitchensis), white spruce (P. glauca), andhybrid white spruce (P. glauca × P. engelmannii) toidentify a comprehensive set of expressed members ofthe spruce TPS gene family. We have functionally char-acterized several members from each species for a totalof 21 newly characterized spruce TPSs. This work com-plements previous work in Norway spruce [14] and pro-vides a molecular basis from which to explain much ofthe chemical complexity of the oleoresin and volatileterpenoids in spruce. Results of the functional genecharacterization are discussed in the context of pre-viously reported terpenoid metabolite profiles of oleore-sin and volatile emissions in Sitka spruce.Results and DiscussionIdentification of unique TPS sequences and isolation offull-length TPS cDNA clonesThe in silico analysis of 443,665 spruce ESTs identifieda total of 506 ESTs corresponding to putative TPSs(Table 1). Assembly of these ESTs into contigs and sing-lets allowed us to estimate the minimum number ofactively expressed TPS genes in each of the three sprucespecies of our analysis. We identified 69 unique TPSsequences in white spruce, 55 in Sitka spruce, and 20in hybrid white spruce. Although the rate of gene dis-covery was dependent on the depth of EST sequencing(Table 1), the substantially deeper EST sequence cover-age in white spruce (242,931 ESTs) did not result in aproportional increase of TPS discovery relative to Sitkaspruce (174,384 ESTs) and hybrid white spruce (26,350ESTs), suggesting that the majority of expressed TPSs inthe tissues sequenced were captured at the depth ofsequencing probed in white spruce and Sitka spruce.The estimate of at least 69 TPSs in white spruce is com-parable to the number of putatively active TPS genesfound in the sequenced genomes of angiosperms and isperhaps a good approximation of the total number oftranscriptionally active TPS genes in a conifer species.From the set of assembled TPS sequences, we examinedapproximately 170 of the corresponding cDNA clonesby restriction digest, colony PCR and/or sequencing toidentify those which contained full ORFs. EighteenFLcDNA clones were selected for subcloning and func-tional characterization. In addition, three full-lengthTPS cDNA clones were obtained by RACE cloning orhomology-based PCR cloning. As the Treenomix project[16], which generated the available cDNA clonesfocused its FLcDNA program on Sitka spruce, themajority of the full-length TPS cDNA clones were fromthis species (12 FLcDNAs). Five full-length TPS cDNAclones originated from white spruce, and four fromhybrid white spruce.Functional characterization of recombinant TPS enzymesMost previously described conifer TPSs are multi-product enzymes [14,18], and because the identity andrelative abundance of TPS products are very sensitive tosmall changes in amino acid sequence [19-24], it is notpossible to accurately predict function based solely uponTable 1 In silico identification of TPSs in the EST databases of Sitka spruce, white spruce, and hybrid white spruceSpruce Species Total ESTs Total Singlets Plus Contigs TPS ESTs* TPS Singlets TPS Contigs Total TPSsWhite 242,931 59,449 181 36 33 69Sitka 174,384 37,533 282 25 30 55Hybrid White 26,350 13,279 43 10 10 20*Conifer TPS protein sequences available from NCBI were used to query the three species-specific EST databases using the tBLASTn module of WU-BLAST 2.0 andan E-value cut off of 1 × 10-5. The resulting outputs were filtered to exclude duplicates, and then assembled separately by species using CAP3 [49]. The totalTPSs represents an estimated minimum number of unique TPSs found in each species.Keeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 2 of 14amino acid sequence similarity/phylogeny. While itmight be possible to infer a TPS gene function from thechemical phenotype of a corresponding plant mutant,the genetic resources for such an approach are availableonly for a very few model systems such as Arabidopsis[25]. Instead, in most systems, the functional annotationof each TPS requires expression and enzyme characteri-zation of recombinant protein.Recombinant spruce TPSs were expressed in E. coliand purified by Ni-affinity chromatography before assay-ing each individually with geranyl diphosphate (GPP),farnesyl diphosphate (E,E-FPP), and geranylgeranyldiphosphate (E,E,E-GGPP), the three respective trans-prenyl diphosphate substrates of conifer monoterpenesynthases, sesquiterpene synthases, and diterpenesynthases. Since two recent reports described the occur-rence and conversion of cis-prenyl diphosphate sub-strates in tomato [26-28], we also assessed if spruce islikely to produce these additional TPS substrates.Mining of all available spruce EST sequences did notreveal the presence of prenyltransferases for the forma-tion of cis-prenyl diphosphate substrates (D. Hall and J.Bohlmann; unpublished results).In the following sections we describe the specific func-tional characterization of the 21 spruce TPSs (Figure 1).With one exception, each of these TPSs only madesignificant use of one of the substrates. Based uponfunctional characterization, the 21 TPSs comprisedFigure 1 Phylogeny of functionally characterized gymnosperm TPSs. The ent-kaurene synthase from Physcomitrella patens was included asan outgroup. TPSs described in this paper are shown with white background. Protein alignments were prepared using MUSCLE [54] andphylogenetic trees were constructed using the neighbour-joining method with 100 bootstrap repetitions (asterisks are given at clades supportedby 80% and higher bootstrap values), within CLC Main Workbench (CLC bio, Århus, Denmark).Keeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 3 of 1415 monoterpene synthases, 4 sesquiterpene synthases,and 2 diterpene synthases. The product identities andabundance for each TPS, including quantitative compo-sition of multi-product profiles, is shown in Table 2,and representative GCMS traces are shown in Figures 2,3, and 4. A summary of the functional annotation alongwith NCBI GenBank accession numbers appears inTable 3. Results of the functional TPS characterizationare discussed in the context of previously reportedterpenoid metabolite profiles in Sitka spruce genotypeFB3-425 [see Supplemental Tables in [29]], from whichmany of the functionally characterized TPS FLcDNAswere isolated. Terpenoid profiles are also available froma collection of 111 Sitka spruce accessions [30].Functional characterization of monoterpene synthases:(-)-b-phellandrene synthasesWe identified four (-)-b-phellandrene synthases in Sitkaspruce (PsTPS-Phel-1, PsTPS-Phel-2, PsTPS-Phel-3, andPsTPS-Phel-4), which shared 99% amino acid identity witheach other, suggesting that these genes represent nearlyidentical allelic variants or very recently duplicated genesin the two genotypes that they originated from (Table 3).Interestingly, the Sitka spruce (-)-b-phellandrene synthaseswere only 70% identical to the (-)-b-phellandrene synthasefrom grand fir [13]. The phylogenetic distance betweenthe grand fir and Sitka spruce (-)-b-phellandrene synthases(Figure 1) suggests that this specific gene function evolvedindependently more than once. The identity and approxi-mate quantities of the major [(-)-b-phellandrene,(-)-b-pinene, and (-)-a-pinene] and minor products werenearly identical between the four Sitka spruce (-)-b-phellandrene synthases (Table 2), and the major productsand their approximate proportions were also the samebetween the (-)-b-phellandrene synthases of grand fir andspruce. To the best of our knowledge, a (-)-b-phellandrenesynthase has not previously been reported in any otherspecies of spruce [14,31]. In Sitka spruce, b-phellandreneis a major component of the constitutive monoterpenefraction in inner and outer stem tissue and in needles [seeSupplemental Tables in [29]]. In stems of Sitka spruce,accumulation of b-phellandrene increased in response totreatment of trees with methyl jasmonate (MeJA) or insectattack [29]. The Sitka spruce (-)-b-phellandrene synthasesidentified here are likely responsible for this major mono-terpenoid component of Sitka spruce oleoresin.Functional characterization of monoterpene synthases:(-)-a/b-pinene synthasesWe characterized one new (-)-a/b-pinene synthase inSitka spruce (PsTPS-Pin) and two in white spruce(PgTPS-Pin-1 and PgTPS-Pin-2; both originating fromthe same genotype) (Tables 1 and 2). These threeenzymes clustered closely with the two previouslyTable 2 Product profiles of recombinant TPS enzymesbased upon total ion current of GCMS analysis on aDB-WAX columnTPS Clone ID Products* PercenttotalMONOTERPENE SYNTHASESPg×eTPS-Car1WS0063_F08 (+)-3-Carene 53.7Terpinolene 17.2(+)-Sabinene 5.6Terpinen-4-ol 5.2(-)-a-Pinene 2.7a-Terpineol 2.6(-)-b-Phellandrene 2.3Myrcene 2.2g-Terpinene 0.9a-Terpinene 0.6a-Phellandrene 0.3a-Thujene 0.2Others 6.5PsTPS-Car1 WS02910_I02 (+)-3-Carene 66.4Terpinolene 16.3(+)-Sabinene 4.7(-)-a-Pinene 2.7Terpinen-4-ol 2.5(-)-b-Phellandrene 2.1Myrcene 2.1a-Terpineol 1.4g-Terpinene 0.8Others 1.1PgTPS-Cin WS02628_N22 1,8-Cineole 89.1(-)-a-Terpineol 4.7(+)-a-Pinene 1.9b-Pinene 1.9Unknown 1.4Myrcene 1.1Pg×eTPS-Cin WS00921_D15 1,8-Cineole 65.6(-)-a-Terpineol 18.3Myrcene 4.1(+)-a-Pinene 3.0b-Pinene 2.6g-Terpinene 1.8Others 4.6PsTPS-Cin WS0291_H24 1,8-Cineole 59.0(-)-a-Terpineol 12.2Myrcene 9.0b-Pinene 5.5(+)-a-Pinene 4.7Others 9.5PgTPS-Lin WS0054_P01 (-)-Linalool 100PsTPS-Lin-1 WS0285_L07 (-)-Linalool 100PsTPS-Lin-2 WS02915_K02 (-)-Linalool 100PsTPS-Phel-1 WS02729_A23 (-)-b-Phellandrene 61.9(-)-b-Pinene 18.6Keeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 4 of 14characterized (-)-a/b-pinene synthases from Sitkaspruce [32] and Norway spruce [14] in the TPS-d1clade (Figure 1). The topology of this group of five(-)-a/b-pinene synthases suggests that they representorthologs in the three spruce species of our compari-son. The two pairs of (-)-a/b-pinene synthase genes inwhite spruce and in Sitka spruce may representrecently duplicated genes or allelic variants in each ofthese two species. The two white spruce enzymes dif-fered in only four amino acids between each other,and the two Sitka spruce enzymes differed in only sixamino acids. The white spruce (-)-a/b-pinenesynthases were approximately 96% identical with the(-)-a/b-pinene synthase in Norway spruce, andapproximately 96% identical with the (-)-a/b-pinenesynthases in Sitka spruce. The Sitka spruce (-)-a/b-pinene synthases shared approximately 95% identitywith the Norway spruce enzyme. The (-)-a-pinenesynthase from loblolly pine (Pinus taeda) [33] and the(-)-a/b-pinene synthase from grand fir [34] clusteredoutside the group of the spruce (-)-a/b-pinenesynthases (Figure 1). These pine and grand fir(-)-pinene synthases may be the corresponding ortho-logs outside of the spruce genus.The two (-)-a/b-pinene synthases in white spruce(PgTPS-Pin-1 and PgTPS-Pin-2) contained only fouramino acid differences: Q/R94, R/G217, S/N221, andE/G599, but showed an opposing pattern in the rela-tive amounts of a- and b-pinene produced by therecombinant enzymes (67:33 and 29:71 a-pinene:b-pinene, respectively, Table 2). Based upon homologymodelling with the limonene synthase from Menthaspicata as a template [35], we examined whether anyof the four different residues were in or near the activesite. Only the residue at 599 (corresponding to M572of the template) was near the active site. Although thisresidue was not on the surface of the modelled activesite, it was directly behind the residues that contributeTable 2 Product profiles of recombinant TPS enzymesbased upon total ion current of GCMS analysis on aDB-WAX column (Continued)(-)-a-Pinene 12.3Myrcene 5.4a-Phellandrene 1.0a-Terpinolene 0.5PsTPS-Phel-2 WS0296_I22 (-)-b-Phellandrene 61.2(-)-b-Pinene 19.8(-)-a-Pinene 12.1Myrcene 5.5a-Phellandrene 0.9a-Terpinolene 0.5PsTPS-Phel-3 WS0276_M12 (-)-b-Phellandrene 60.9(-)-b-Pinene 20.9(-)-a-Pinene 12.5Myrcene 4.1a-Phellandrene 1.3a-Terpinolene 0.2PsTPS-Phel-4 WS01042_E12 (-)-b-Phellandrene 61.9(-)-b-Pinene 19.6(-)-a-Pinene 11.5Myrcene 5.2a-Phellandrene 1.2a-Terpinolene 0.6PgTPS-Pin-1 WS00725_G07c1 (-)-a-Pinene 66.7(-)-b-Pinene 33.3PgTPS-Pin-2 WS00725_G07c2 (-)-b-Pinene 70.5(-)-a-Pinene 29.5PsTPS-Pin WS0291_K15 (-)-a-Pinene 83.4(-)-b-Pinene 12.6Linalool 2.1b-Phellandrene 1.0Camphene 0.4Myrcene 0.4SESQUITERPENE SYNTHASESPg×eTPS-Far/OciWS00926_E08 (E,E)-a-Farnesene/(E)-b-ocimene100PgTPS-Hum WS0074_O16 a-Humulene 42.7(E)-b-Caryophyllene 37.9a-Longipinene 7.5Longifolene 3.1a-Muurolene 2.7g-Himachalene 2.6Others 3.4Pg×eTPS-LonfWS00927_M20 Longifolene 69.5a-Longipinene 30.5PsTPS-Lonp WS02712_A08 a-Longipinene 47.7Longifolene 19.9g-Himachalene 15.9(E)-b-Farnesene 7.0b-Longipinene 3.0Others 6.4Table 2 Product profiles of recombinant TPS enzymesbased upon total ion current of GCMS analysis on aDB-WAX column (Continued)DITERPENE SYNTHASESPsTPS-Iso pSW06061903 Isopimaradiene 98.3Sandaracopimaradiene 1.7PsTPS-LAS WS0299_C21 Abietadiene 49.4Levopimaradiene 23.8Neoabietadiene 23.3Palustradiene 3.5Compounds were identified by comparison of mass spectra and retentionindices with authentic standards if available, and retention indices, and/ormass spectra from Adams [52] and NIST, and combined mass spectra andretention index library searches in MassFinder [53] if standards were notavailable.Keeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 5 of 14to the active site surface near the tail of the substrateanalogue (approximately 8.5 Å away). One mighthypothesize that the E/G599 difference was the originof the observed product differences between the twowhite spruce PgTPS-Pin variants. However, this residueis glycine in a previously characterized (-)-a/b-pinenesynthase in Sitka spruce [32] and in a previously char-acterized (-)-a/b-pinene synthase in Norway spruce[14], which all produce different ratios of a/b-pinene.Therefore, the three other amino acid differencesfurther from the active site also contributed to productprofile differences.Figure 2 GCMS total ion chromatogram of products formed by the representative monoterpene synthases PsTPS-Car1, Pg×eTPS-Cin,PgTPS-Lin, PsTPS-Phel-1, and PsTPS-Pin when incubated with GPP. (A) PsTPS-Car1: 1. (-)-a-pinene, 2. (+)-sabinene, 3. myrcene, 4. (+)-3-carene, 5. b-phellandrene, 6. g-terpinene, 7. terpinolene, 8. terpinen-4-ol, 9. a-terpineol; (B) Pg×eTPS-Cin: 1. (+)-a-pinene, 2. b-pinene, 3. myrcene,4. 1,8-cineole, 5. g-terpinene, 6. unknown, 7. (-)-a-terpineol, 8. unknown; (C) PgTPS-Lin: 1. (-)-linalool; (D) PsTPS-Phel-1: 1. (-)-a-pinene, 2. (-)-b-pinene, 3. myrcene, 4. a-phellandrene, 5. b-phellandrene, 6. terpinolene; (E) PsTPS-Pin: 1. (-)-a-pinene, 2. camphene, 3. (-)-b-pinene, 4. myrcene,5. b-phellandrene, 6. linalool.Keeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 6 of 14In contrast to the white spruce enzymes PgTPS-Pin-1and PgTPS-Pin-2, the newly characterized Sitka sprucePsTPS-Pin enzyme produced a larger proportion of(-)-b-pinene (more than 80%) and lesser amounts of(-)-a-pinene (less than 13%), but also had four addi-tional minor products not observed with the PgTPS-Pin enzymes (Table 2, Figure 2). This product profilewas substantially different from that of the second, pre-viously characterized (-)-a/b-pinene synthase fromSitka spruce, which is dominated by (-)-a-pinene(more than 60% of total product) and lesser amountsof (-)-b-pinene (less than 20% of total product) [32]. Ofall five spruce (-)-a/b-pinene synthases, the knownNorway spruce enzyme shows the greatest productdiversity with (-)-b-pinene (57%), (-)-a-pinene (27%),and (-)-b-phellandrene (11%) as dominant productsalong with five other minor constituents [14]. Similarto the white spruce (-)-a/b-pinene synthase enzymes,the previously characterized and more distantly related(-)-a/b-pinene synthases from grand fir also producesonly (-)-a- and (-)-b-pinene (42% and 58%) [34]. Theknown product profile of loblolly pine (Pinus taeda)(-)-pinene synthase is substantially different, withmostly (-)-a-pinene (79%) with lesser amounts of(-)-b-pinene (4%) and additional minor products [33].These comparisons of product profiles and ratiosacross a set of orthologous, or likely orthologous, mul-tiproduct (-)-pinene synthases show that overallsequence relatedness is not a good indicator of the spe-cific product profiles and ratios even for closely relatedTPS enzymes.The monoterpenes (-)-a-pinene and (-)-b-pinene areprominent resin compounds in Sitka spruce [29,30] andin Norway spruce [36,37]. In Norway spruce, inducedaccumulation of these compounds in bark tissue ofMeJA-treated stems is the result of increased enzymeactivity, protein abundance, and transcript levels of(-)-a/b-pinene synthase [38]. Previous work inSitka spruce also showed strong accumulation of tran-scripts detected with a (-)-a/b-pinene synthase probe inFigure 3 GCMS total ion chromatogram of products formed by the sesquiterpene synthases Pg×eTPS-Far/Oci, Pg×eTPS-Lonf, PgTPS-Hum, and PsTPS-Lonp when incubated with FPP. (A) Pg×eTPS-Far/Oci: 1. (E,E)-a-farnesene; (B) Pg×eTPS-Lonf: 1. a-longipinene, 2. longifolene;(C) PgTPS-Hum: 1. a-longipinene, 2. longifolene, 3. (E)-b-caryophyllene, 4. a-humulene, 5. g-himachalene, 6. a-muurolene; (D) PsTPS-Lonp: 1. a-longipinene, 2. b-longipinene, 3. longifolene, 4. (E)-b-farnesene, 5. g-himachalene.Keeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 7 of 14MeJA- and insect-treated stems, both at the site ofinsect feeding and some distance away [29].Functional characterization of monoterpene synthases:(-)-Linalool synthasesWe characterized two new (-)-linalool synthases in Sitkaspruce (PsTPS-Lin-1 and PsTPS-Lin-2) and one inwhite spruce (PgTPS-Lin) (Tables 2 and 3, Figure 2).Within the TPS-d1 clade, the Sitka spruce and whitespruce (-)-linalool synthases formed a group of ortholo-gous genes with the previously cloned Norway spruce(-)-linalool synthase (PaTPS-Lin) [14] (Figure 1). All ofthese monoterpene synthases were single-productenzymes producing exclusively an acyclic monoterpenealcohol. They shared 86 to 98% amino acid sequenceidentity, with Sitka spruce PsTPS-Lin1 and white sprucePgTPS-Lin being the most closely related. Since the two(-)-linalool synthases from Sitka spruce (91% identitybetween them) originated from the same genotype (FB3-425; Table 3), they are likely recently duplicated genes.(-)-Linalool was previously detected as the major vola-tile emission of MeJA-treated and weevil-attacked Sitkaspruce saplings in the genotype FB3-425 [29], similar tothe MeJA-induced emission of linalool from Norwayspruce [37]. Transcripts detected with a PaTPS-Linprobe were strongly induced in needles of MeJA-treatedSitka spruce [29]. Linalool volatiles are thought to func-tion in indirect defence against herbivores. Apparently,the (-)-linalool emissions in spruce do not originatefrom the oleoresin reservoirs of severed resin ducts, butfrom the induced de novo biosynthesis in other tissues.The cloning of (-)-linalool synthase genes from Sitkaspruce and white spruce makes it possible to investigate,in future work, the localization of these enzymes andthe corresponding transcripts in the needles using themethods of laser-assisted tissue microdissection techni-ques [39] or immunofluorescence localization [40].Functional characterization of monoterpene synthases:(+)-3-Carene synthasesWe recently identified a small clade of (+)-3-carenesynthases and sabinene synthases in two genotypes ofSitka spruce that are resistant [genotype H898; PsTPS-car1(R), PsTPS-car2(R), and PsTPS-sab(R)] or susceptible[genotype Q903; PsTPS-car1(S), PsTPS-car3(S), andPsTPS-sab(S)] to white pine weevil, Pissodes strobi [41].Here, we identified two additional (+)-3-carene synthases,one in a different genotype of Sitka spruce (genotypeFB3-425; PsTPS-Car1), and one in hybrid white spruce(Pg×eTPS-Car1) (Tables 2 and 3, Figure 2). These two(+)-3-carene synthases shared approximately 99% aminoacid identity to each other, and were likely the ortholo-gues of the (+)-3-carene synthases PsTPS-car1(R) andPsTPS-car1(S) recently described (Figure 1). Their pro-duct profiles were also highly similar for all of the majorand most of the minor products. These Sitka and hybridwhite spruce (+)-3-carene synthase genes were less simi-lar to the previously characterized Norway spruce(+)-3-carene synthase [42]. A (+)-3-carene synthase genehas not yet been characterized for any conifer outside ofthe genus Picea. The (+)-3-carene synthases were multi-product enzymes, producing predominantly (+)-3-carenesynthase (approximately 53 to 66%) and terpinolene(approximately 16%), with lesser amounts of (+)-sabineneand several other minor products (Table 2). Despite thesimilarity of product profiles, the Sitka spruce and hybridwhite spruce (+)-3-carene synthase both shared only 84%percent amino acid sequence identity with the Norwayspruce TPS. This highlights how even enzymes with fairlydivergent primary sequence can share a similar, complexproduct profile. The two most abundant products of theSitka spruce (+)-3-carene synthases, the monoterpenes(+)-3-carene and terpinolene, have recently been identi-fied as indicators for resistance against weevils in aFigure 4 GCMS total ion chromatogram of products formed byditerpene synthases PsTPS-LAS and PsTPS-Iso when incubatedwith GGPP. (A) PsTPS-LAS: 1. palustradiene, 2. levopimaradiene, 3.abietadiene, 4. neoabietadiene; (B) PsTPS-Iso: 1.sandaracopimaradiene, 2. isopimaradiene.Keeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 8 of 14particular geographic region of Sitka spruce origin [30].Substantial variation exists in the levels of (+)-3-careneacross the range of Sitka spruce [30]. The cloning of(+)-3-carene synthases from resistant and susceptibleSitka spruce enabled a detailed characterization of thegenetic variability and the molecular underpinnings of(+)-3-carene formation in resistant and susceptible geno-types [41]. Previous work in Sitka spruce showed MeJA-and weevil-induced accumulation of transcripts hybridiz-ing to the Norway spruce (+)-3-carene synthase probe[29]. Similarly, (+)-3-carene synthase was very stronglyinduced at the transcript, protein, and enzyme activitylevels in Norway spruce treated with MeJA [38].Functional characterization of monoterpene synthases:1,8-Cineole synthasesIn each of the three spruce species studied we identifiedand characterized a single 1,8-cineole synthase, PgTPS-Cin, Pg×eTPS-Cin, and PsTPS-Cin (Tables 2 and 3, Fig-ure 2). The three enzymes shared approximately 99%sequence identity to each other and form a distinctgroup in the TPS-d1 clade most closely related to thelinalool synthases. The 1,8-cineole synthases and thelinalool synthases are among only a few known conifermonoterpene synthases that produce mainly oxygenatedmonoterpenes instead of olefins. All three 1,8-cineolesynthases were multi-product enzymes with the amountof the major 1,8-cineole product varying from approxi-mately 60% of total product for PsTPS-Cin to approxi-mately 90% for PgTPS-Cin. These three spruce enzymesalso had similar profiles of minor products (-)-a-terpineol, (+)-a-pinene, b-pinene, myrcene and others(Table 2 and Figure 2). Although 1,8-cineole has beenidentified as a monoterpenoid component in needlesand MeJA-induced volatile emissions of Norway spruce[37], and has recently been shown to inhibit attractionin the field and response of an olfactory receptor neuronto pheromone of a spruce beetle [43], this is the firstcharacterization of gymnosperm TPSs that produce thiscompound.Functional characterization of sesquiterpene synthasesA complex blend of sesquiterpenes is found in minorquantities in the oleoresin of conifers, including Sitkaspruce [29] and Norway spruce [37]. Sesquiterpenes arealso present in the MeJA-induced volatile emissions ofNorway spruce [37] and in the MeJA- and weevil-induced volatile emissions in Sitka spruce [29]. For theTable 3 Gene name, origin, accession numbers, and functional annotation of spruce TPSGene Clone ID (genotype) Functional Annotation* NCBI AccessionMONOTERPENE SYNTHASESPg×eTPS-Car1 WS0063_F08 (Fa1-1028) (+)-3-Carene synthase HQ426152PsTPS-Car1 WS02910_I02 (FB3-425) (+)-3-Carene synthase HQ426167PgTPS-Cin WS02628_N22 (PG29) 1,8-Cineole synthase HQ426160PgxeTPS-Cin WS00921_D15 (Fa1-1028) 1,8-Cineole synthase HQ426156PsTPS-Cin WS0291_H24 (FB3-425) 1,8-Cineole synthase HQ426165PgTPS-Lin WS0054_P01 (PG29) (-)-Linalool synthase HQ426151PsTPS-Lin-1 WS0285_L07 (FB3-425) (-)-Linalool synthase HQ426164PsTPS-Lin-2 WS02915_K02 (FB3-425) (-)-Linalool synthase HQ426168PsTPS-Phel-1 WS02729_A23 (FB3-425) (-)-b-Phellandrene synthase HQ426162PsTPS-Phel-2 WS0296_I22 (FB3-425) (-)-b-Phellandrene synthase HQ426169PsTPS-Phel-3 WS0276_M12 (FB3-425) (-)-b-Phellandrene synthase HQ426163PsTPS-Phel-4 WS01042_E12 (Gb2-229) (-)-b-Phellandrene synthase HQ426159PgTPS-Pin-1 WS00725_G07c1 (PG29) (-)-a/b-Pinene synthase HQ426153PgTPS-Pin-2 WS00725_G07c2 (PG29) (-)-a/b-Pinene synthase HQ426154PsTPS-Pin WS0291_K15 (FB3-425) (-)-a/b-Pinene synthase HQ426166SESQUITERPENE SYNTHASESPg×eTPS-Far/Oci WS00926_E08 (Fa1-1028) (E,E)-a-Farnesene/(E)-b-ocimene synthase HQ426157PgTPS-Hum WS0074_O16 (PG29) a-Humulene synthase HQ426155Pg×eTPS-Lonf WS00927_M20 (Fa1-1028) Longifolene synthase HQ426158PsTPS-Lonp WS02712_A08 (FB3-425) a-Longipinene synthase HQ426161DITERPENE SYNTHASESPsTPS-Iso pSW06061903 (Haney 898) Isopimaradiene synthase HQ426150PsTPS-LAS WS0299_C21 (FB3-425) Levopimaradiene/abieta-diene synthase HQ426170*Functional annotation is based on the main terpenoid product(s) of recombinant enzymes expressed in E. coli. Most TPSs produced multiple products, as shownin Table 2.Keeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 9 of 14three spruce species of our EST analysis, we cloned andfunctionally characterized four FLcDNAs, PgTPS-Hum,Pg×eTPS-Lonf, PsTPS-Lonp, and Pg×eTPS-Far/Oci, asbona fide sesquiterpene synthases (Tables 2 and 3; Fig-ure 3). PgTPS-Hum, Pg×eTPS-Lonf, PsTPS-Lonp onlyused FPP as substrate and were typical multi-productconifer sesquiterpene synthases such as those first iden-tified in grand fir [18]. In contrast, Pg×eTPS-Far/Ociwas active both with GPP and FPP. This enzyme pro-duced only (E,E)-a-farnesene, when assayed with FPP,and (E)-b-ocimene and a small amount of myrcene,when assayed with GPP. The previously characterized(E,E)-a-farnesene synthases cloned from Norway spruce[14] and loblolly pine [33] did not show this dual sub-strate utilization [14,33], although it has been observedwith apple (Malus × domestica) (E,E)-a-farnesenesynthase [44]. (E,E)-a-farnesene is major sesquiterpenecomponent of the MeJA- and weevil-induced volatileemissions of Sitka spruce [29].PgTPS-Hum produced predominantly a-humulene(approximately 43%) and (E)-b-caryophyllene (approxi-mately 38%), along with several minor products, similarto the a-humulene synthase previously characterized inScots pine (Pinus sylvestris) [45]. Pg×eTPS-Lonf pro-duced longifolene (approximately 70%) and a-longipi-nene (approximately 30%). Unlike the longifolenesynthase from Norway spruce [14], this TPS did notproduce other minor products. Sitka spruce PsTPS-Lonp produced predominantly a-longipinene (approxi-mately 48%) but also substantial amounts of longifolene,g-himachalene, and other minor products. Longifoleneand a-longipinene were previously found in the resin ofuntreated and induced Sitka spruce stems [29] and wee-vil attack caused an increase of these compounds.PgTPS-Hum, Pg×eTPS-Lonf, PsTPS-Lonp belong tothe TPS-d2 clade of the gymnosperm TPS-d subfamily,together with other conifer multi-product sesquiterpenesynthases (Figure 1). The hybrid white spruce Pg×eTPS-Far/Oci appeared to be orthologous with farnesenesynthases from loblolly pine and Norway spruce in theTPS-d1 clade.Functional characterization of diterpene synthasesTwo paralogous diterpene synthases, PsTPS-LAS andPsTPS-Iso, were characterized in Sitka spruce (Tables 2and 3, Figure 4). These TPSs shared 90% identity andthey are the orthologues of levopimaradiene/abietadienesynthase (PaTPS-LAS) and isopimaradiene synthase(PaTPS-Iso) from Norway spruce [14] (Figure 1). Theybelong to the TPS-d3 clade of the gymnosperm TPS-dfamily. PsTPS-LAS produced a similar multi-productprofile as its ortholog in Norway spruce, composed ofabietadiene (49%), levopimaradiene (24%), neoabieta-diene (23%), and palustradiene (4%). In contrast to thesingle-product isopimaradiene synthase from Norwayspruce [14], Sitka spruce PsTPS-Iso produced minoramounts of sandaracopimaradiene (2%) in addition toisopimaradiene (98%) (Table 2, Figure 4). PsTPS-Iso isthe first gymnosperm TPS identified to naturally pro-duce sandaracopimaradiene, albeit in minor amounts.An ent-sandaracopimaradiene synthase has been charac-terized in rice [46].PsTPS-LAS and PsTPS-Iso play an important role inthe overall diterpene resin acid defence systems of Sitkaspruce. The six products of the two Sitka spruce diter-pene synthases are present as the corresponding diter-pene resin acids in the oleoresin of Sitka spruce stemtissues [29]. Accumulation of all of these diterpene resinacids was induced by MeJA treatment or insect attack,along with increased transcript levels detected with theorthologous PaTPS-LAS and PaTPS-Iso probes [29].The sequences of PsTPS-LAS and PaTPS-LAS differedby only 12 amino acids, and PsTPS-Iso and PaTPS-Isodiffered by only 35 amino acids. In a detailed investiga-tion of the PaTPS-LAS and PaTPS-Iso enzymes, usingreciprocal site-directed mutagenesis and domain-swapping, we have recently shown that four amino acidresidues determine the different product profiles ofthese Norway spruce diterpene synthases [24]. Theseproduct-determining residues are identical between thelevopimaradiene/abietadiene synthases (PsTPS-LAS andPaTPS-LAS) in Sitka and Norway spruce, consistentwith their similar product profiles. However, only threeof these residues are identical between the isopimara-diene synthases (PsTPS-Iso and PaTPS-Iso) in Sitka andNorway spruce; the fourth residue (V732) is the sameas that found in the Norway spruce levopimaradiene/abietadiene synthase. In our previous study [24], thecorresponding reciprocal L725V mutation obtained bysite-directed mutagenesis of PaTPS-Iso resulted in theformation of sandaracopimaradiene as a minor product.This product profile change is consistent with the newobservation that the isopimaradiene synthase from Sitkaspruce (PsTPS-Iso) naturally produced sandaracopimar-adiene as a minor compound (Table 2, Figure 4).Overall, these results highlight how mutations producedin the laboratory that determine product profile differ-ences also exist in nature and do result in the evolutionof altered TPS product profiles between species orgenotypes.Phylogeny of gymnosperm TPSsAll known conifer TPSs of specialized (i.e., secondary)metabolism are members of the gymnosperm-specificTPS-d subfamily, which is a distinct clade of the largerplant TPS gene family [47]. The TPS-d subfamily hasbeen subdivided into three clades TPS-d1 through TPS-d3 based on a previous phylogeny of 29 gymnospermKeeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 10 of 14TPSs [14]. Here, we have substantially expanded thephylogeny of functionally characterized gymnospermTPSs to a total of 72 members (Figure 1), of which 41are from spruce species with 20 different TPSs fromSitka spruce. The number of TPSs functionally charac-terized in Sitka spruce is one of the largest for any spe-cies, but is not yet approaching our in silico minimumestimate for the number of TPSs in a spruce genome (atleast 69 transcriptionally active TPS genes). The diverseset of newly characterized spruce TPSs broadly repre-sent the major TPS-d1, TPS-d2 and TPS-d3 clades, andallowed us to identify groups of likely orthologous TPSgenes across the spruce species. Examples for suchgroups of orthologous TPSs in the TPS-d1 clade are the(-)-a/b-pinene synthases, the (-)-linalool synthases, (E,E)-a-farnesene synthases; in the TPS-d2 clade are thelongifolene synthases; and in the TPS-d3 clade are thelevopimaradiene/abietadiene synthases and isopimara-diene synthases. These groups represent genes whosefunctions had apparently evolved prior to speciation ofthe spruce genus. In the TPS-d3 group of conifer diter-pene synthases, the basal function of a multi-productlevopimaradiene/abietadiene synthase had apparentlyevolved prior to conifer speciation, as this functionexists in a group of closely related genes from the gen-era Abies, Pinus and Picea.Overall, the large diversity of gene functions amongthe many closely related genes of the conifer TPS-d1group illustrates the many events of gene duplicationsand sub- or neo-functionalizations that have occurred inthe evolution of this amazing family of conifer genes ofspecialized metabolism. The functionally identifiedspruce TPS genes account for many of the major andminor terpenoid compounds of the defensive oleoresinand volatile emissions. However, there are several dis-tinct types of TPSs still to be found in spruce basedupon the terpenoid components identified in oleoresin.Based on the current phylogeny of functionally charac-terized spruce TPSs, we predict that most of theremaining TPSs to be identified will be highly similar insequence to previously identified TPS, but with the pos-sibility of diverse function due to relatively minorsequence divergence.In contrast to the many duplicated TPS-d genes ofterpenoid specialized metabolism, the related spruceTPS genes of general gibberellin phytohormone bio-synthesis, specifically ent-copalyl diphosphate synthase(TPS-c) and ent-kaurene synthase (TPS-e), appear to beexpressed as single copy genes [12]. These primarymetabolism TPS genes are basal to the specialized meta-bolism genes and are the descendants of an ancestralplant diterpene synthase similar to the one found in thenon-vascular plant Physcomitrella patens [12,48]. Themechanisms that suppress manifestation or retention ofTPS gene duplication in diterpenoid primary metabo-lism and those that enhance TPS gene duplication andfunctional diversification in specialized metabolism in aconifer genome are not known but are worthy of futureinvestigation. The high functional plasticity of the TPS-dfamily and the great diversity of terpenoids producedmay impart fitness advantages against a multitude ofpests and pathogens. We speculate that the TPS-d genesof specialized metabolism originating from gene duplica-tion are slower, or less likely, to become inactive pseu-dogenes compared to those genes with less functionalplasticity in primary metabolism.ConclusionsBased upon estimates from EST and FLcDNA sequen-cing in three species of spruce, the TPS gene family inconifers appears to be at least of comparable size tothose found in angiosperms with sequenced genomes.This study highlights the great diversity of TPSs of spe-cialized metabolism in conifers, which resulted fromgene duplication and functional diversification.Functional differences can occur naturally due to smalldifferences in amino acid sequence.MethodsIn silico identification of spruce terpene synthases in theEST and FLcDNA databasesQuality trimmed and filtered nucleotide sequences wereobtained from spruce genomic resources developed inthe Genome Canada-funded Treenomix (http://www.treenomix.ca) and Arborea (http://www.arborea.ulaval.ca) projects as follows: white spruce (242,931 ESTs),Sitka spruce (174,384 ESTs), and hybrid white spruce(also referred to as interior spruce; 26,350 ESTs) [15,16].Conifer TPS protein sequences available from NCBIwere used to query the three species-specific databasesusing the tBLASTn module of WU-BLAST 2.0 and anE-value cut off of 1 × 10-5. The resulting outputs werefiltered to exclude duplicates, and then assembled sepa-rately by species using CAP3 [49] using an overlap of 40bp and a percent identity of 95%. The assembled TPScandidate sequences were then tentatively annotatedusing NCBI BLASTx using the nr database (downloadedOct. 2008).Selection of FLcDNA clones for functional characterizationAuthentic cDNA clones corresponding to the above-identified TPS candidate sequences were examinedfurther by restriction digest, colony PCR, and/or sequen-cing. Those clones that potentially contained a full-lengthTPS cDNA (i.e. complete ORF) were fully sequenced andif a unique full-ORF TPS was found, the insert was sub-cloned for expression as described below. In one case,two full ORFs (WS00725_G07c1, WS00725_G07c2) wereKeeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 11 of 14obtained by 5’-RACE and the full-length genes were sub-sequently cloned into pCR Blunt II TOPO (Invitrogen).Cloning of PsTPS-IsoBecause of our particular interest in conifer diTPSs[12,24] and the low abundance of putative diTPSs in theESTs, we chose to isolate an isopimaradiene synthase(PsTPS-Iso) cDNA from Sitka spruce using homology-based cloning to allow functional comparison with itsputative levopimaradiene/abietadiene synthase paralog(PsTPS-LAS, WS0299_C21, described here). Examinationof the spruce EST resources [15,16] identified a 3’-readfor clone WS00752_D05 from white spruce with highsimilarity to the isopimaradiene synthase from Norwayspruce (PaTPS-Iso; [14]). Full sequencing of this cDNAclone indicated that it was an incomplete transcript.Using PCR with primers designed for the 3’-UTR of thissequence and the 5’-UTR of WS0299_C21, we amplifieda 2,700 bp cDNA from the bark of methyl jasmonate-treated Sitka spruce (genotype Haney 898). The ampli-con was cloned into pCR Blunt II TOPO and fullysequenced (PsTPS-Iso, pSW06061903).Expression and purification of recombinant TPS enzymesTPS cDNAs were amplified using proof-reading poly-merase (Phusion, Finnzymes, Espoo, Finland) and sub-cloned into NdeI/HindIII-digested pET28b(+) (Novagen)using a sticky-end PCR approach [50], or via topoisome-rase-mediated insertion into pET100 TOPO/D orpET200 TOPO/D (Invitrogen). All resulting recombi-nant proteins were full-length and N-terminally His-tagged. Expression constructs were fully sequenceverified.Plasmids were transformed into chemically competentC41 E. coli cells (http://www.overexpress.com) contain-ing the pRARE 2 plasmid (coding for rare tRNAs) pre-pared from Novagen Rosetta 2 cells (EMD Biosciences,Inc., Madison, WI, USA). Luria-Bertani medium (5 mL)containing appropriate antibiotics was inoculated withthree individual colonies and cultured overnight at 37°C,220 rpm. Terrific Broth medium (50 mL) containingappropriate antibiotics was then inoculated with 0.5 mLof the overnight culture and grown in a 250 mL baffleflask at 37°C and 300 rpm until an optical density at600 nm of at least 0.8 was reached. Cultures were thencooled to 16°C, induced with 0.2 mM IPTG, and thencultured for approximately 16-20 h at 16°C and220 rpm before pelleting and freezing.Cell pellets were resuspended, lysed, and sonicated in(1.5 mL g-1 pellet) ice cold 20 mM NaPO4, 500 mM NaCl,30 mM imidazole, 0.04 mg mL-1 DNase, 1 mM MgCl2,5 mM PMSF, and 0.5 mg mL-1 lysozyme, pH 7.4 and thenclarified by centrifugation (30 min, 12,000 × g, 4°C). Thecleared lysates were applied to His SpinTrap Ni-affinitycolumns (GE Healthcare, Piscataway, NJ, USA) and elutedwith 20 mM NaPO4, 500 mM NaCl, and 500 mM imida-zole, pH 7.4 at 4°C following the manufacturer’s protocol.Purified enzymes were desalted at 4°C into 25 mM HEPESpH 7.2, 100 mM KCl, and 10% glycerol using PD Mini-Trap G-25 desalting columns (GE Healthcare) and thenused immediately for enzyme assays.Enzyme assays and gas chromatography-massspectrometry (GCMS) analysesSingle-vial enzyme assays were completed in triplicate in2 mL amber glass GC sample vials as previouslydescribed [51] in three different buffer/substrate combi-nations with approximately 60 μg of purified protein per500 μL assay. Buffers consisted of: monoTPS assays;25 mM HEPES, pH 7.2, 100 mM KCl, 10 mM MnCl2,5 mM fresh DTT, 10% glycerol, and 51 μM GPP (gera-nyl diphosphate, Sigma-Aldrich, Oakville, ON); ses-quiTPS assays; 25 mM HEPES, pH 7.2, 10 mM MgCl2,5 mM fresh DTT, 10% glycerol, and 43 μM FPP ((E,E)-farnesyl diphosphate, Sigma-Aldrich); diTPS assays;50 mM HEPES, pH 7.2, 100 mM KCl, 7.5 mM MgCl2,20 μM MnCl2, 5 mM fresh DTT, 5% glycerol, and37 μM GGPP ((E,E,E)-geranylgeranyl diphosphate,Sigma-Aldrich). Assays were overlaid with 500 μL ofpentane and incubated at 30°C for 90 min after whichthey were vortexed for 30 s to denature the proteinsand extract the products into the pentane layer. Tocompletely separate the phases prior to GCMS analysis,samples were frozen at -80°C and then the vials werecentrifuged for 30 min at 1,000 × g at 4°C.Assay products were analyzed on an Agilent HP-5mscolumn (5% phenyl methyl siloxane, 30 m × 250 μm ID,0.25 μm film) at 1 mL min-1 He on an Agilent 6890N gaschromatograph, 7683B series autosampler (vertical syr-inge position of 8 to sample the pentane layer), and 5975Inert XL MS Detector. GC temperature program as fol-lows: 40°C, hold 1 min, 7.5°C min-1 to 250°C, hold 2 min,pulsed splitless injector held at 250°C. Samples were alsoanalyzed on an Agilent DB-WAX column (polyethyleneglycol, 30 m × 250 μm ID, 0.25 μm film) with the follow-ing temperature program: 40°C, hold 3 min, 10°C min-1to 240°C, hold 15 min, pulsed splitless injector held at240°C. Compounds were identified by comparison ofmass spectra and retention indices with authentic stan-dards if available, and retention indices, and/or massspectra from Adams [52] and NIST, and combined massspectra and retention index library searches in MassFin-der [53] if standards were not available.When possible, stereochemistry of enzyme productswere compared to authentic chiral standards on an Agi-lent Cyclodex B column (permethylated b-cyclodextrin inDB 1701 ((14%-cyanopropyl-phenyl)-methylpolysilox-ane), 30 m × 250 μm ID, 0.25 μm film) with the followingKeeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 12 of 14temperature program: 55°C, hold 1 min, 1°C min-1 to100°C, 10°C min-1 to 240°C, hold 10 min, pulsed splitlessinjector held at 230°C.Phylogenetic analysisProtein alignments were prepared using MUSCLE [54]and phylogenetic trees were constructed using theneighbour-joining method with 100 bootstrap repeti-tions, both within CLC Main Workbench 5.6.1 (CLCbio, Århus, Denmark).Molecular modellingWe used Deep View/Swiss-PDBViewer (Mac version3.9.1b) and SWISS-MODEL [55-57] to develop a 3Dhomology model of WS00725_G07c1 truncated at Q63based on the structure of limonene synthase from Menthaspicata containing the substrate analogue 2-fluorogeranyldiphosphate (Protein Data Bank 2ONGB) [35].List of abbreviationsTPS: terpene synthase; EST: expressed sequence tag; FLcDNA: full-lengthcDNA; GPP: geranyl diphosphate; FPP: farnesyl diphosphate; GGPP:geranylgeranyl diphosphate; GC: gas chromatography; MS: massspectrometry; ORF: open reading frame; MeJA: methyl jasmonate; gene andenzyme names abbreviations are shown in Table 3.Acknowledgements and FundingWe thank Ms. Lina Madilao for GCMS support, and Ms. Karen Reid forexcellent laboratory management support and for generating much of thesequence information used for the analysis shown in Table 1. This researchwas supported by a discovery grant to JB from the Natural Sciences andEngineering Research Council (NSERC), and with funds from Genome BritishColumbia and Genome Canada to JB in support of the Treenomix ConiferForest Health Project (http://www.treenomix.ca). JB was supported in part bythe UBC Distinguished University Scholars program and an NSERC SteacieMemorial Fellowship.Author details1Michael Smith Laboratories, University of British Columbia, 301-2185 EastMall, Vancouver BC, V6T 1Z4, Canada. 2Roche Diagnostics Ltd., Forrenstrasse,CH-6343 Rotkreuz, Switzerland. 3Department of Biology, University of NorthDakota, Grand Forks, ND, 58202-9019, USA. 4Department of Plant Biologyand Biotechnology, University of Copenhagen, Thorvaldsensvej 40, opg. 10,1.-1871 Frederiksberg, Denmark.Authors’ contributionsCIK, SGR, and JB conceived the research. SGR and SJ selected andsequenced the clones for functional characterization and completed RACE.CIK, SW, BH, and HKD cloned, expressed and/or functionally characterizedthe clones. CIK and JB wrote the manuscript. All authors read and approvedthe final manuscript.Received: 21 December 2010 Accepted: 7 March 2011Published: 7 March 2011References1. Franceschi VR, Krokene P, Christiansen E, Krekling T: Anatomical andchemical defenses of conifer bark against bark beetles and other pests.New Phytol 2005, 167:353-376.2. Keeling CI, Bohlmann J: Genes, enzymes and chemicals of terpenoiddiversity in the constitutive and induced defence of conifers againstinsects and pathogens. New Phytol 2006, 170:657-675.3. Keeling CI, Bohlmann J: Diterpene resin acids in conifers. Phytochemistry2006, 67:2415-2423.4. Becerra JX, Noge K, Venable DL: Macroevolutionary chemical escalation inan ancient plant-herbivore arms race. Proc Natl Acad Sci USA 2009,106:18062-18066.5. 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Schwede T, Kopp J, Guex N, Peitsch MC: SWISS-MODEL: An automatedprotein homology-modeling server. Nucleic Acids Res 2003, 31:3381-3385.doi:10.1186/1471-2229-11-43Cite this article as: Keeling et al.: Transcriptome mining, functionalcharacterization, and phylogeny of a large terpene synthase genefamily in spruce (Picea spp.). BMC Plant Biology 2011 11:43.Keeling et al. BMC Plant Biology 2011, 11:43http://www.biomedcentral.com/1471-2229/11/43Page 14 of 14

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