, Volume 233, Issue 4, pp 685–696 | Cite as

Cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia

  • Zerihun A. Demissie
  • Lukman S. Sarker
  • Soheil S. MahmoudEmail author
Original Article


En route to building genomics resources for Lavandula, we have obtained over 14,000 ESTs for leaves and flowers of L. angustifolia, a major essential oil crop, and identified a number of previously uncharacterized terpene synthase (TPS) genes. Here we report the cloning, expression in E. coli, and functional characterization of β-phellandrene synthase, LaβPHLS. The ORF—excluding the transit peptide—for this gene encoded a 62.3 kDa protein that contained all conserved motifs present in plant TPSs. Expression in bacteria resulted in the production of a soluble protein that was purified by Ni–NTA agarose affinity chromatography. While the recombinant LaβPHLS did not utilize FPP as a substrate, it converted GPP (the preferred substrate) and NPP into β-phellandrene as the major product, with Km and kcat of 6.55 μM and 1.75 × 10−2 s−1, respectively, for GPP. The LaβPHLS transcripts were highly abundant in young leaves where β-phellandrene is produced, but were barely detectable in flowers and older leaves, where β-phellandrene is not synthesized in significant quantities. This data indicate that β-phellandrene biosynthesis is transcriptionally and developmentally regulated. We also cloned and expressed in E. coli a second TPS-like protein, LaTPS-I, that lacks an internal stretch of 73 amino acids, including the signature DDxxD divalent metal binding motif, compared to other plant TPSs. The recombinant LaTPS-I did not produce detectable products in vitro when assayed with GPP, NPP or FPP as substrates. The lack of activity is most likely due to the absence of catalytically important amino acid residues within the missing region.


L. angustifolia Essential oil Terpene biosynthesis Monoterpene synthases β-Phellandrene 



Essential oil(s)


Terpene synthase(s)


Monoterpene synthase(s)


Sesquiterpene synthase(s)


Geranyl diphosphate


Nerolidyl diphosphate


L. angustifolia β-phellandrene synthase


L. angustifolia terpene synthase-like


L. angustifolia linalool synthase


L. angustifolia limonene synthase



This work was supported through grants or in-kind contributions to Soheil Mahmoud by UBC Okanagan, Natural Sciences and Engineering Research Council of Canada, Investment Agriculture Foundation of British Columbia, and NRC Plant Biotechnology Institute through the NAPGEN program. We are grateful for Dr Mark Rheault and Dr Kirsten Wolthers of UBC Okanagan for their advice on the enzyme kinetic analysis.


  1. Boeckelmann A (2008) Monoterpene production and regulation in lavenders (Lavandula angustifolia and Lavandula × intermedia). MSc Thesis, University of British Columbia Okanagan, pp 70–71Google Scholar
  2. Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95(8):4126–4133PubMedCrossRefGoogle Scholar
  3. Bohlmann J, Phillips M, Ramachandiran V, Katoh S, Croteau R (1999) cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis). Arch Biochem Biophys 368(2):232–243PubMedCrossRefGoogle Scholar
  4. Boira H, Blanquer A (1998) Environmental factors affecting chemical variability of essential oils in Thymus piperella L. Biochem Syst Ecol 26(8):811–822CrossRefGoogle Scholar
  5. Cai Y, He J, Li X, Feng K, Lu L, Feng K, Kong X, Lu W (2010) Prediction of protein subcellular locations with feature selection and analysis. Protein Pept Lett 17(4):464–472PubMedCrossRefGoogle Scholar
  6. Cavanagh HMA, Wilkinson JM (2002) Biological activities of lavender essential oil. Phytother Res 16(4):301–308PubMedCrossRefGoogle Scholar
  7. Crowell AL, Williams DC, Davis EM, Wildung MR, Croteau R (2002) Molecular cloning and characterization of a new linalool synthase. Arch Biochem Biophys 405(1):112–121PubMedCrossRefGoogle Scholar
  8. Degenhardt J, Köllner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70(15–16):1621–1637PubMedCrossRefGoogle Scholar
  9. Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard J, Guindon S, Lefort V, Lescot M, Claverie J, Gascuel O (2008) robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 1(36):W465–W469CrossRefGoogle Scholar
  10. Falk L, Biswas K, Boeckelmann A, Lane A, Mahmoud SS (2009) An efficient method for the micropropagation of lavenders: regeneration of a unique mutant. J Essent Oil Res 21(3):225–228Google Scholar
  11. Felicetti B, Cane DE (2004) Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis. J Am Chem Soc 126(23):7212–7221PubMedCrossRefGoogle Scholar
  12. Figueiredo AC, Barroso JG, Pedro LG, Sevinate-Pinto I, Antunes T, Fontinha SS, Looman A, Scheffer JS (1995) Composition of the essential oil of Lavandula pinnata L. fil. var. pinnata grown on madeira. Flavour Fragr J 10(2):93–96CrossRefGoogle Scholar
  13. Flores G, Blanch GP, Castillo M, del Ruiz L, Herraiz M (2005) Enantiomeric composition studies in Lavandula species using supercritical fluids. J Sep Sci 28(17):2333–2338PubMedCrossRefGoogle Scholar
  14. Harborne JB, Williams CA (2002) Phytochemistry of the genus Lavandula. In: Lis-Balchin M (ed) Lavender: the genus lavandula. Tayler & Francis, London, pp 86–100Google Scholar
  15. Jia JW, Crock J, Lu S, Croteau R, Chen XY (1999) (3R)-Linalool synthase from Artemisia annua L.: cDNA isolation, characterization, and wound Induction. Arch Biochem Biophys 372:143–149PubMedCrossRefGoogle Scholar
  16. Keegstra K, Olsen LJ, Theg SM (1989) Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Plant Phys 40(1):471–501CrossRefGoogle Scholar
  17. Kreis P, Mosandl A (1992) Chiral compounds of essential oils. Part XI. Simultaneous stereoanalysis of Lavandula oil constituents. Flavour Fragr J 7(4):187–193CrossRefGoogle Scholar
  18. Landmann C, Fink B, Festner M, Dregus M, Engel K, Schwab W (2007) Cloning and functional characterization of three terpene synthases from lavender (Lavandula angustifolia). Arch Biochem Biophys 465(2):417–429PubMedCrossRefGoogle Scholar
  19. Lane A, Boecklemann A, Woronuk G, Sarker L, Mahmoud S (2010) A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia. Planta 231(4):835–845PubMedCrossRefGoogle Scholar
  20. Lis-Balchin M (2002) Chemical composition of essential oils from different species, hybrids and cultivars of Lavandula. In: Lis-Balchin M (ed) Lavender: the genus Lavandula. Taylor & Francis, London, pp 251–262Google Scholar
  21. Mahmoud SS, Croteau RB (2003) Menthofuran regulates essential oil biosynthesis in peppermint by controlling a downstream monoterpene reductase. Proc Natl Acad Sci USA 100(24):14481–14486PubMedCrossRefGoogle Scholar
  22. Mahmoud SS, Williams M, Croteau R (2004) Cosuppression of limonene-3-hydroxylase in peppermint promotes accumulation of limonene in the essential oil. Phytochemistry 65(5):547–554PubMedCrossRefGoogle Scholar
  23. McConkey ME, Gershenzon J, Croteau RB (2000) Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint. Plant Physiol 122(1):215–224PubMedCrossRefGoogle Scholar
  24. Muñoz-Bertome J, Arrillaga I, Segura J (2007) Essential oil variation within and among natural populations of Lavandula latifolia and its relation to their ecological areas. Biochem Syst Ecol 35(8):479–488CrossRefGoogle Scholar
  25. Nagegowda DA (2010) Plant volatile terpenoid metabolism: biosynthetic genes, transcriptional regulation and subcellular compartmentation. FEBS Lett 584(14):2965–2973PubMedCrossRefGoogle Scholar
  26. Nagegowda DA, Gutensohn M, Wilkerson CG, Dudareva N (2008) Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers. Plant J. 55(2):224–239PubMedCrossRefGoogle Scholar
  27. Nieuwenhuizen NJ, Wang MY, Matich AJ, Green SA, Chen X, Yauk Y, Beuning LL, Nagegowda DA, Dudareva N, Atkinson RG (2009) Two terpene synthases are responsible for the major sesquiterpenes emitted from the flowers of kiwifruit (Actinidia deliciosa). J Exp Bot 60(11):3203–3219PubMedCrossRefGoogle Scholar
  28. Pichersky E, Lewinsohn E, Croteau R (1995) Purification and characterization of S-linalool synthase, an enzyme involved in the production of floral scent in Clarkia Breweri. Arch Biochem Biophys 316(2):803–807PubMedCrossRefGoogle Scholar
  29. Roeder S, Hartmann A, Effmert U, Piechulla B (2007) Regulation of simultaneous synthesis of floral scent terpenoids by the 1, 8-cineole synthase of Nicotiana suaveolens. Plant Mol Biol 65(1):107–124PubMedCrossRefGoogle Scholar
  30. Schilmiller AL, Schauvinhold I, Larson M, Xu R, Charbonneau AL, Schmidt A, Wilkerson C, Last RL, 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 USA 106(26):10865–10870PubMedCrossRefGoogle Scholar
  31. Seemann M, Zhai G, de Kraker J, Paschall CM, Christianson DW, Cane DE (2002) Pentalenene synthase. analysis of active site residues by site-directed mutagenesis. J Am Chem Soc 124(26):7681–7689Google Scholar
  32. Shimada T, Endo T, Fujii H, Hara M, Omura M (2005) Isolation and characterization of (E)-beta-ocimene and 1, 8 cineole synthases in Citrus unshiu Marc. Plant Sci 168(4):987–995CrossRefGoogle Scholar
  33. Tholl D, Croteau R, Gershenzon J (2001) Partial purification and characterization of the short-chain prenyltransferases, geranyl diphosphate synthase and farnesyl diphosphate synthase, from Abies grandis (grand fir). Arch Biochem Biophys 386(2):233–242PubMedCrossRefGoogle Scholar
  34. Von Heijne G, Steppuhn J, Herrmann RG (1989) Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180(3):535–545CrossRefGoogle Scholar
  35. Williams DC, McGarvey DJ, Katahira EJ, Croteau R (1998) Truncation of limonene synthase pre-protein provides a fully active ‘pseudomature’ form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair. Biochemistry 37(35):12213–12220PubMedCrossRefGoogle Scholar
  36. Wise ML, Croteau R (1999) Monoterpene biosynthesis. In: Cane DD (ed) Comprehensive natural products chemistry: isoprenoids including carotenoids and steroids, vol 2. Elsevier Science, Amsterdam, pp 97–153Google Scholar
  37. Wise ML, Savage TJ, Katahira E, Croteau R (1998) Monoterpene synthases from common sage (Salvia officinalis). J Biol Chem 273:14891–14899PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Zerihun A. Demissie
    • 1
  • Lukman S. Sarker
    • 1
  • Soheil S. Mahmoud
    • 1
    Email author
  1. 1.Department of BiologyUniversity of British Columbia-OkanaganKelownaCanada

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