Plant Molecular Biology

, Volume 93, Issue 6, pp 641–657 | Cite as

Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia

  • Ayelign M. Adal
  • Lukman S. Sarker
  • Ashley D. Lemke
  • Soheil S. MahmoudEmail author


Key message

A methyl jasmonate responsive 3-carene synthase (Li3CARS) gene was isolated from Lavandula x intermedia and functionally characterized in vitro.


Lavenders produce essential oils consisting mainly of monoterpenes, including the potent antimicrobial and insecticidal monoterpene 3-carene. In this study we isolated and functionally characterized a leaf-specific, methyl jasmonate (MeJA)-responsive monoterpene synthase (Li3CARS) from Lavandula x intermedia. The ORF excluding transit peptides encoded a 64.9 kDa protein that was expressed in E. coli, and purified with Ni–NTA agarose affinity chromatography. The recombinant Li3CARS converted GPP into 3-carene as the major product, with K m and k cat of 3.69 ± 1.17 µM and 2.01 s−1 respectively. Li3CARS also accepted NPP as a substrate to produce multiple products including a small amount of 3-carene. The catalytic efficiency of Li3CARS to produce 3-carene was over ten fold higher for GPP (k cat /K m = 0.56 µM−1s−1) than NPP (k cat /K m = 0.044 µM−1s−1). Production of distinct end product profiles from different substrates (GPP versus NPP) by Li3CARS indicates that monoterpene metabolism may be controlled in part through substrate availability. Li3CARS transcripts were found to be highly abundant in leaves (16-fold) as compared to flower tissues. The transcriptional activity of Li3CARS correlated with 3-carene production, and was up-regulated (1.18- to 3.8-fold) with MeJA 8–72 h post-treatment. The results suggest that Li3CARS may have a defensive role in Lavandula.


3-Carene synthase Lavandula Lavandula x intermedia Methyl jasmonate Monoterpene synthase Transcriptional regulation 



Bornyl pyrophosphate synthase


Essential oil


Geranyl pyrophosphate


Methyl jasmonate


Monoterpene synthase


L. x intermedia 3-carene synthase


Neryl pyrophosphate


Quantitative real-time PCR


Terpene synthase



This work was supported through grants and/or in-kind contributions to SSM by UBC, Genome British Columbia, Natural Sciences and Engineering Research Council of Canada, Agriculture and Agri-Food Canada and the BC Ministry of Agriculture (through programs delivered by the Investment Agriculture Foundation of BC).

Supplementary material

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Supplementary material 1 (DOCX 11 KB)
11103_2017_588_MOESM2_ESM.docx (59 kb)
Supplementary material 2 (DOCX 59 KB)
11103_2017_588_MOESM3_ESM.docx (55 kb)
Supplementary material 3 (DOCX 54 KB)


  1. Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci U S A 95:4126–4133. doi: 10.1073/pnas.95.8.4126 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Boone CK, Aukema BH, Bohlmann J, Carroll AL, Raffa KF (2011) Efficacy of tree defense physiology varies with bark beetle population density: a basis for positive feedback in eruptive species. Can J For Res 41:1174–1188. doi: 10.1139/x11-041 CrossRefGoogle Scholar
  3. Bordoli L, Kiefer F, Arnold K, Benkert P, Battey J, Schwede T (2009) Protein structure homology modeling using SWISS-MODEL workspace. Nat Protoc 4:1–13. doi: 10.1038/nprot.2008.197 CrossRefPubMedGoogle Scholar
  4. Burke CC, Wildung MR, Croteau R (1999) Geranyl diphosphate synthase: cloning, expression, and characterization of this prenyltransferase as a heterodimer. Proc Natl Acad Sci U S A 96:13062–13067. doi: 10.1073/pnas.96.23.13062 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Byun-McKay A, Godard K-A, Toudefallah M, Martin DM, Alfaro R, King J, Bohlmann J, Plant AL (2012) Wound-induced terpene synthase gene expression in Sitka spruce that exhibit resistance or susceptibility to attack by the white pine weevil. New Phytol 3:3043–3052. doi: 10.1104/pp.105.071803 Google Scholar
  6. Chen F, Tholl D, Bohlmann J, Pichersky E (2011) The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J 66:212–229. doi: 10.1111/j.1365-313X.2011.04520.x CrossRefPubMedGoogle Scholar
  7. De Rapper S, Kamatou G, Viljoen A, Van Vuuren S (2013) The in vitro antimicrobial activity of Lavandula angustifolia essential oil in combination with other aroma-therapeutic oils. Evid Based Complement Altern Med. doi: 10.1155/2013/852049 Google Scholar
  8. De Vega C, Herrera CM, Dotterl S (2014) Floral volatiles play a key role in specialized ant pollination. Perspect Plant Ecol Evol Syst 16:32–42. doi: 10.1016/j.ppees.2013.11.002 CrossRefGoogle Scholar
  9. Degenhardt J, Köllner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70:1621–1637. doi: 10.1016/j.phytochem.2009.07.030 CrossRefPubMedGoogle Scholar
  10. Demissie ZA, Sarker LS, Mahmoud SS (2011) Cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia. Planta 233:685–696. doi: 10.1007/s00425-010-1332-5 CrossRefPubMedGoogle Scholar
  11. Demissie ZA, Cella MA, Sarker LS, Thompson TJ, Rheault MR, Mahmoud SS (2012) Cloning, functional characterization and genomic organization of 1,8-cineole synthases from Lavandula. Plant Mol Biol 79:393–411. doi: 10.1007/s11103-012-9920-3 CrossRefPubMedGoogle Scholar
  12. Demissie ZA, Erland LAE, Rheault MR, Mahmoud SS (2013) The biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase. J Biol Chem 288:6333–6341. doi: 10.1074/jbc.M112.431171 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Dudareva N, Klempien A, Muhlemann JK, Kaplan I (2013) Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol 198:16–32. doi: 10.1111/nph.12145 CrossRefPubMedGoogle Scholar
  14. Erland LAE (2015) Enhancement of specialized metabolism, regeneration efficiency and biological activity in lavandin (Lavandula x intermedia cv ‘Grosso’). MSc Thesis, University of british Columbia, pp:40–41Google Scholar
  15. Erland LAE, Rheault MR, Mahmoud SS (2015) Insecticidal and oviposition deterrent effects of essential oils and their constituents against the invasive pest Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). Crop Prot 78:20–26. doi: 10.1016/j.cropro.2015.08.013 CrossRefGoogle Scholar
  16. Fäldt J, Martin D, Miller B, Rawat S, Bohlmann J (2003) Traumatic resin defense in Norway spruce (Picea abies): Methyljasmonate-induced terpene synthase gene expression, and cDNA cloningand functional characterization of (+)-3-carene synthase. Plant Mol Biol 51:119–133CrossRefPubMedGoogle Scholar
  17. Fontana A, Held M, Fantaye CA, Turlings TC, Degenhardt J, Gershenzon J (2011) Attractiveness of constitutive and Herbivore-induced Sesquiterpene blends of Maize to the Parasitic Wasp Cotesia marginiventris (Cresson). J Chem Ecol 37:582–591. doi: 10.1007/s10886-011-9967-7 CrossRefPubMedGoogle Scholar
  18. Frost CJ, Appel HM, Carlson JE, De Moraes CM, Mescher MC, Schulz JC (2007) Within-plant signalling via volatiles overcomes vascular constraints on systemic signalling and primes responses against herbivores. Ecol Lett 10:490–498. doi: 10.1111/j.1461-0248.2007.01043.x CrossRefPubMedGoogle Scholar
  19. Fujimoto T, Tomitaka Y, Abe H, Tsuda S, Futai K, Mizukubo T (2011) Expression profile of jasmonic acid-induced genes and the induced resistance against the root-knot nematode (Meloidogyne incognita) in tomato plants (Solanum lycopersicum) after foliar treatment with methyl jasmonate. J Plant Physiol 168:1084–1097. doi: 10.1016/j.jplph.2010.12.002 CrossRefPubMedGoogle Scholar
  20. Guex N, Peitsch MC, Schwede T (2009) Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis 30:162–173. doi: 10.1002/elps.200900140 CrossRefGoogle Scholar
  21. Hall DE, Robert JA, Keeling CI, Domanski D, Quesada AI, Jancsik S, Kuzyk MA, Hamberger B, Borchers CH, Bohlmann J (2011) An integrated genomic, proteomic and biochemical analysis of (+)-3-carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil. Plant J 65:936–948. doi: 10.1111/j.1365-313X.2010.04478.x CrossRefPubMedGoogle Scholar
  22. Hasegawa M, Mitsuhara I, Seo S, Imai T, Koga J, Okada K, Yamane H, Ohashi Y (2010) Phytoalexin accumulation in the interaction between rice and the blast fungus. Mol Plant Microbe Interact 23:1000–1011. doi: 10.1094/MPMI-23-8-1000 CrossRefPubMedGoogle Scholar
  23. Heiling S, Schuman MC, Schoettner M, Mukerjee P, Berger B, Schneider B, Jassbi AR, Baldwin IT (2010) Jasmonate and ppHsystemin regulate key Malonylation steps in the biosynthesis of 17-Hydroxygeranyllinalool Diterpene Glycosides, an abundant and effective direct defense against herbivores in Nicotiana attenuata. Plant Cell 22:273–292. doi: 10.1105/tpc.109.071449 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hoelscher DJ, Williams DC, Wildung MR, Croteau R (2003) A cDNA clone for 3-carene synthase from Salvia stenophylla. Phytochemistry 62:1081–1086. doi: 10.1016/S0031-9422(02)00674-X CrossRefPubMedGoogle Scholar
  25. Huang M, Abel C, Sohrabi R, Petri J, Haupt I, Cosimano J, Gershenzon J, Tholl D (2010) Variation of herbivore-induced volatile terpenes among Arabidopsis ecotypes depends on allelic differences and subcellular targeting of two terpene synthases, TPS02 and TPS03. Plant Physiol 153:1293–1310. doi: 10.1104/pp.110.154864 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Hyatt DC, Youn B, Zhao Y, Santhamma B, Coates RM, Croteau RB, Kang C (2007) Structure of limonene synthase, a simple model for terpenoid cyclase catalysis. Proc Natl Acad Sci U S A 104:5360–5365. doi: 10.1073/pnas.0700915104 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Jullien F, Moja S, Bony A, Legrand S, Petit C, Benabdelkader T, Poirot K, Fiorucci S, Guitton Y, Nicole F, Baudino S, Magnard JL (2014) Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia. Plant Mol Biol 84:227–241. doi: 10.1007/s11103-013-0131-3 CrossRefPubMedGoogle Scholar
  28. Krieger E, Joo K, Lee J, Lee J, Raman S, Thompson J, Tyka M, Baker D, Karplus K (2009) Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8. Proteins Struct Funct Bioinforma 77:114–122. doi:  10.1002/prot.22570 CrossRefGoogle Scholar
  29. Landmann C, Fink B, Festner M, Dregus M, Engel KH, Schwab W (2007) Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia). Arch Biochem Biophys 465:417–429. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  30. Lane A, Boecklemann A, Woronuk GN, Lukman S, Mahmoud SS (2010) A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia. Planta 231:835–845. doi: 10.1007/s00425-009-1090-4 CrossRefPubMedGoogle Scholar
  31. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2^(-∆∆CT) method. Methods 25:402–408. doi: 10.1006/meth.2001.1262 CrossRefPubMedGoogle Scholar
  32. Martin D, Tholl D, Gershenzon J (2002) Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of norway spruce stems 1. Society 129:1003–1018. doi: 10.1104/pp.011001 McGarvey Google Scholar
  33. Menzel TR, Weldegergis BT, David A, Boland W, Gols R, Van Loon JJA, Dicke M (2014) Synergism in the effect of prior jasmonic acid application on herbivore-induced volatile emission by Lima bean plants: transcription of a monoterpene synthase gene and volatile emission. J Exp Bot 65:4821–4831. doi: 10.1093/jxb/eru242 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Miller B, Madilao LL, Ralph S, Bohlmann J (2005) Insect-induced conifer defense. White pine weevil and methyl jasmonate induce traumatic resinosis, de novo formed volatile emissions, and accumulation of terpenoid synthase and putative octadecanoid pathway transcripts in Sitka spruce. Plant Physiol 137:369–382. doi: 10.1104/pp.104.050187 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Moon T, Wilkinson JM, Cavanagh HMA (2006) Antibacterial activity of essential oils, hydrosols and plant extracts from Australian grown Lavandula spp. Int J Aromather 16:9–14. doi: 10.1016/j.ijat.2006.01.007 CrossRefGoogle Scholar
  36. Moreira X, Sampedro L, Zas R (2009) Defensive responses of Pinus pinaster seedlings to exogenous application of methyl jasmonate: Concentration effect and systemic response. Environ Exp Bot 67:94–100. doi: 10.1016/j.envexpbot.2009.05.015 CrossRefGoogle Scholar
  37. Navia-Giné WG, Gomez SK, Yuan J, Chen F, Korth KL (2009) Insect-induced gene expression at the core of volatile terpene release in Medicago truncatula. Plant Signal Behav 4:639–641. doi: 10.1016/j.plaphy.2009.01.008 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Ott DS, Yanchuk AD, Huber DPW, Wallin KF (2011) Genetic variation of lodgepole pine, Pinus contorta var. latifolia, chemical and physical defenses that affect mountain pine beetle, Dendroctonus ponderosae, attack and tree mortality. J Chem Ecol 37:1002–1012. doi: 10.1007/s10886-011-0003-8 CrossRefPubMedGoogle Scholar
  39. Rivera SB, Swedlund BD, King GJ, Bell RN, Hussey CE, Shattuck-Eidens DM, Wrobel WM, Peiser GD, Poulter CD (2001) Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium. Proc Natl Acad Sci USA 98:4373–4378. doi: 10.1073/pnas.071543598 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Roach CR, Hall DE, Zerbe P, Bohlmann J (2014) Plasticity and evolution of (+)-3-carene synthase and (-)-sabinene synthase functions of a Sitka spruce monoterpene synthase gene family associated with weevil resistance. J Biol Chem 289:23859–23869. doi: 10.1074/jbc.M114.571703 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Robert JA, Madilao LL, White R, Yanchuk A, King JR, Bohlmann J (2010) Terpenoid metabolite profiling in Sitka spruce identifies association of dehydroabietic acid, (+)-3-carene, and terpinolene with resistance against white pine weevil. Botany-Botanique 88:810–820. doi: 10.1139/B10-068 CrossRefGoogle Scholar
  42. Ruijter JM, Ramakers C, Hoogaars WMH, Karlen Y, Bakker O, Van Den Hoff MJB, Moorman AFM (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucl Acids Res. doi: 10.1093/nar/gkp045 Google Scholar
  43. Rynkiewicz MJ, Cane DE, Christianson DW (2001) Structure of trichodiene synthase from Fusarium sporotrichioides provides mechanistic inferences on the terpene cyclization cascade. Proc Natl Acad Sci U S A 98:13543–13548. doi: 10.1073/pnas.231313098 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Sarker LS, Galata M, Demissie ZA, Mahmoud SS (2012) Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula x intermedia. Arch Biochem Biophys 528:163–170. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  45. Savage TJ, Croteau R (1993) Biosynthesis of monoterpenes—regiochemistry and stereochemistry of (+)-3-carene biosynthesis. Arch Biochem Biophys 305:581–587CrossRefPubMedGoogle Scholar
  46. Schilmiller AL, Schauvinhold I, Larson M, Xu R, Charbonneau AL, Schmidt A, Wilkerson C, Last RT, Pichersky E (2009) Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate. Proc Natl Acad Sci U S A 106:10865–10870. doi: 10.1073/pnas.0904113106\r0904113106 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  47. Schüttelkopf AW, Van Aalten DMF (2004) PRODRG: A tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr Sect D Biol Crystallogr 60:1355–1363. doi:  10.1107/S0907444904011679 CrossRefGoogle Scholar
  48. Shi J, Ma C, Qi D, Lv H, Yang T, Peng Q, Chen Z, Lin Z (2015) Transcriptional responses and flavor volatiles biosynthesis in methyl jasmonate-treated tea leaves. BMC Plant Biol 15:233. doi: 10.1186/s12870-015-0609-z CrossRefPubMedPubMedCentralGoogle Scholar
  49. Taniguchi S, Hosokawa-Shinonaga Y, Tamaoki D, Yamada S, Akimitsu K, Gomi K (2014) Jasmonate induction of the monoterpene linalool confers resistance to rice bacterial blight and its biosynthesis is regulated by JAZ protein in rice. Plant Cell Environ 37:451–461. doi: 10.1111/pce.12169 CrossRefPubMedGoogle Scholar
  50. Tholl D, Lee S (2011) Terpene Specialized Metabolism in Arabidopsis thaliana. Arabidopsis Book 9:e0143. doi: 10.1199/tab.0143 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Van Schie CCN, Haring MA, Schuurink RC (2007) Tomato linalool synthase is induced in trichomes by jasmonic acid. Plant Mol Biol 64:251–263. doi: 10.1007/s11103-007-9149-8 CrossRefPubMedPubMedCentralGoogle Scholar
  52. Vickers CE, Gershenzon J, Lerdau MT, Loreto F (2009) A unified mechanism of action for volatile isoprenoids in plant abiotic stress. Nat Chem Biol 5:283–291. doi: 10.1038/nchembio.158 CrossRefPubMedGoogle Scholar
  53. War A, Sharma HC, Paulraj MG, War MY, Ignacimuthu S (2011) Herbivore induced plant volatiles their role in plant defense for pest management. Plant Signal Behav 6:1973–1978. doi: 10.4161/psb/6.12.18053 CrossRefPubMedPubMedCentralGoogle Scholar
  54. Whittington DA, Wise ML, Urbansky M, Urbansky M, Coates RM, Croteau RB, Christianson DW (2002) Bornyl diphosphate synthase: structure and strategy for carbocation manipulation by a terpenoid cyclase. Proc Natl Acad Sci USA 99:15375–15380. doi: 10.1073/pnas.232591099 CrossRefPubMedPubMedCentralGoogle Scholar
  55. Williams DC, Mcgarvey DJ, Katahira EJ, Croteau R (1998) Truncation of limonene synthase preprotein provides a fully active “pseudomature” form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair. BioChemistry 37:12213–12220CrossRefPubMedGoogle Scholar
  56. Wise M, Savage T, Katahira E (1998) Monoterpene synthases from common sage (Salvia officinalis). J Biol 273:14891–14899. doi: 10.1074/jbc.273.24.14891 Google Scholar
  57. Wittstock U, Gershenzon J (2002) Constitutive plant toxins and their role in defense against herbivores and pathogens. Curr Opin Plant Biol 5:300–307. doi: 10.1016/S1369-5266(02)00264-9 CrossRefPubMedGoogle Scholar
  58. Xu Y-H, Liao Y-C, Zhang Z, Liu J, Sun P-W, Gao Z-H, Sui C, Wei J-H (2016) Jasmonic acid is a crucial signal transducer in heat shock induced sesquiterpene formation in Aquilaria sinensis. Sci Rep 6:21843. doi: 10.1038/srep21843 CrossRefPubMedPubMedCentralGoogle Scholar
  59. Zeng G (1998) Sticky-end PCR: new method for subcloning. Biotechniques 25:206–208PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Ayelign M. Adal
    • 1
  • Lukman S. Sarker
    • 1
  • Ashley D. Lemke
    • 1
  • Soheil S. Mahmoud
    • 1
    Email author
  1. 1.Department of BiologyUniversity of British ColumbiaKelownaCanada

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