Plant Molecular Biology

, Volume 73, Issue 6, pp 587–603

Terpene synthases of oregano (Origanum vulgare L.) and their roles in the pathway and regulation of terpene biosynthesis

  • Christoph Crocoll
  • Julia Asbach
  • Johannes Novak
  • Jonathan Gershenzon
  • Jörg Degenhardt


The aroma, flavor and pharmaceutical value of cultivated oregano (Origanum vulgare L.) is a consequence of its essential oil which consists mostly of monoterpenes and sesquiterpenes. To investigate the biosynthetic pathway to oregano terpenes and its regulation, we identified and characterized seven terpene synthases, key enzymes of terpene biosynthesis, from two cultivars of O. vulgare. Heterologous expression of these enzymes showed that each forms multiple mono- or sesquiterpene products and together they are responsible for the direct production of almost all terpenes found in O. vulgare essential oil. The correlation of essential oil composition with relative and absolute terpene synthase transcript concentrations in different lines of O. vulgare demonstrated that monoterpene synthase activity is predominantly regulated on the level of transcription and that the phenolic monoterpene alcohol thymol is derived from γ-terpinene, a product of a single monoterpene synthase. The combination of heterologously-expressed terpene synthases for in vitro assays resulted in blends of mono- and sesquiterpene products that strongly resemble those found in vivo, indicating that terpene synthase expression levels directly control the composition of the essential oil. These results will facilitate metabolic engineering and directed breeding of O. vulgare cultivars with higher quantity of essential oil and improved oil composition.


Origanum vulgare L. Terpene synthase γ-Terpinene Thymol Essential oil Transcript regulation 



Flame ionization detector


Farnesyl diphosphate


Gas chromatography


Geranyl diphosphate


Mass spectrometry


Terpene synthase


Quantitative realtime polymerase chain reaction

Supplementary material

11103_2010_9636_MOESM1_ESM.doc (2.2 mb)
Supplementary material 1 (DOC 2231 kb)


  1. Aharoni A, Giri AP, Verstappen FWA, Bertea CM, Sevenier R, Sun ZK, Jongsma MA, Schwab W, Bouwmeester HJ (2004) Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species. Plant Cell 16:3110–3131CrossRefPubMedGoogle Scholar
  2. Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95:4126–4133CrossRefPubMedGoogle Scholar
  3. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  4. Braga PC, Culici M, Alfieri M, Dal Sasso M (2008) Thymol inhibits Candida albicans biofilm formation and mature biofilm. Intl J Antimicrob Agents 31:472–477CrossRefGoogle Scholar
  5. Colby SM, Alonso WR, Katahira EJ, Mcgarvey DJ, Croteau R (1993) 4S-Limonene synthase from the oil glands of spearmint (Mentha spicata)—cDNA isolation, characterization, and bacterial expression of the catalytically active monoterpene cyclase. J Biol Chem 268:23016–23024PubMedGoogle Scholar
  6. Dudareva N, Martin D, Kish CM, Kolosova N, Gorenstein N, Faldt J, Miller B, Bohlmann J (2003) (E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily. Plant Cell 15:1227–1241CrossRefPubMedGoogle Scholar
  7. Emanuelsson O, Nielsen H, Brunak S, von Heijne G (2000) Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300:1005–1016CrossRefPubMedGoogle Scholar
  8. Gang DR (2005) Evolution of flavors and scents. Ann Rev Plant Biol 56:301–325CrossRefGoogle Scholar
  9. Gershenzon J, Dudareva N (2007) The function of terpene natural products in the natural world. Nat Chem Biol 3:408–414CrossRefPubMedGoogle Scholar
  10. Gershenzon J, Kreis W (1999) Biochemistry of terpenoids: monoterpenes, sesquiterpenes, diterpenes, sterols, cardiac glycosides and steroid saponins. In: Wink M (ed.) Biochemistry of plant secondary metabolism, annual plant reviews, vol 2. Sheffield Academic Press, Sheffield, pp 222–299Google Scholar
  11. Gershenzon J, Maffei M, Croteau R (1989) Biochemical and histochemical-localization of monoterpene biosynthesis in the glandular trichomes of spearmint (Mentha spicata). Plant Physiol 89:1351–1357CrossRefPubMedGoogle Scholar
  12. Gershenzon J, Mccaskill D, Rajaonarivony JIM, Mihaliak C, Karp F, Croteau R (1992) Isolation of secretory-cells from plant glandular trichomes and their use in biosynthetic-studies of monoterpenes and other gland products. Anal Biochem 200:130–138CrossRefPubMedGoogle Scholar
  13. Granger R, Passet J, Verdier R (1964) Le gamma-terpinene precurseur du p-cymene dans Thymus vulgaris L. C R Hebd Seances Acad Sci 258:5539–5541PubMedGoogle Scholar
  14. Hallahan TW, Croteau R (1988) Monoterpene biosynthesis—demonstration of a geranyl pyrophosphate—sabinene hydrate cyclase in soluble enzyme preparations from sweet marjoram (Majorana hortensis). Arch Biochem Biophys 264:618–631CrossRefPubMedGoogle Scholar
  15. Hallahan TW, Croteau R (1989) Monoterpene biosynthesis—mechanism and stereochemistry of the enzymatic cyclization of geranyl pyrophosphate to (+)-cis-sabinene and (+)-trans-sabinene hydrate. Arch Biochem Biophys 269:313–326CrossRefPubMedGoogle Scholar
  16. Haudenschild C, Schalk M, Karp F, Croteau R (2000) Functional expression of regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha spp.) in Escherichia coli and Saccharomyces cerevisiae. Arch Biochem Biophys 379:127–136CrossRefPubMedGoogle Scholar
  17. Hummelbrunner LA, Isman MB (2001) Acute, sublethal, antifeedant, and synergistic effects of monoterpenoid essential oil compounds on the tobacco cutworm, Spodoptera litura (Lep., Noctuidae). J Agric Food Chem 49:715–720CrossRefPubMedGoogle Scholar
  18. Iijima Y, Davidovich-Rikanati R, Fridman E, Gang DR, Bar E, Lewinsohn E, Pichersky E (2004) The biochemical and molecular basis for the divergent patterns in the biosynthesis of terpenes and phenylpropenes in the peltate glands of three cultivars of basil. Plant Physiol 136:3724–3736CrossRefPubMedGoogle Scholar
  19. Isman MB (2000) Plant essential oils for pest and disease management. Crop Prot 19:603–608CrossRefGoogle Scholar
  20. Jez JM, Ferrer JL, Bowman ME, Dixon RA, Noel JP (2000) Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry 39:890–902CrossRefPubMedGoogle Scholar
  21. Kampranis SC, Ioannidis D, Purvis A, Mahrez W, Ninga E, Katerelos NA, Anssour S, Dunwell JM, Degenhardt J, Makris AM, Goodenough PW, Johnson CB (2007) Rational conversion of substrate and product specificity in a Salvia monoterpene synthase: structural insights into the evolution of terpene synthase function. Plant Cell 19:1994–2005CrossRefPubMedGoogle Scholar
  22. Kintzios SE (2002) Profile of the multifaced prince of the herbs. In: Kintzios SE (ed) Oregano: the genera Origanum and Lippia. Taylor & Francis, London, pp 3–8Google Scholar
  23. Kokkini S, Vokou D (1989) Carvacrol-rich plants in Greece. Flavour Frag J 4:1–7CrossRefGoogle Scholar
  24. Köllner TG, Schnee C, Gershenzon J, Degenhardt J (2004) The variability of sesquiterpenes cultivars is controlled by allelic emitted from two Zea mays variation of two terpene synthase genes encoding stereoselective multiple product enzymes. Plant Cell 16:1115–1131CrossRefPubMedGoogle Scholar
  25. Köllner TG, Held M, Lenk C, Hiltpold I, Turlings TCJ, Gershenzon J, Degenhardt J (2008) A maize (E)-β-caryophyllene synthase implicated in indirect defense responses against herbivores is not expressed in most American maize varieties. Plant Cell 20:482–494CrossRefPubMedGoogle Scholar
  26. Lupien S, Karp F, Wildung M, Croteau R (1999) Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species: cDNA isolation, characterization, and functional expression of (−)-4S-limonene-3-hydroxylase and (−)-4S-limonene-6-hydroxylase. Arch Biochem Biophys 368:181–192CrossRefPubMedGoogle Scholar
  27. Mahmoud SS, Croteau RB (2001) Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. Proc Natl Acad Sci USA 98:8915–8920CrossRefPubMedGoogle Scholar
  28. Novak J, Lukas B, Bolzer K, Grausgruber-Groger S, Degenhardt J (2008) Identification and characterization of simple sequence repeat markers from a glandular Origanum vulgare expressed sequence tag. Mol Ecol Resour 8:599–601CrossRefGoogle Scholar
  29. Palovaara J, Hakman I (2008) Conifer WOX-related homeodomain transcription factors, developmental consideration and expression dynamic of WOX2 during Picea abies somatic embryogenesis. Plant Mol Biol 66:533–549CrossRefPubMedGoogle Scholar
  30. Pechous SW, Whitaker BD (2004) Cloning and functional expression of an (E, E)-α-farnesene synthase cDNA from peel tissue of apple fruit. Planta 219:84–94CrossRefPubMedGoogle Scholar
  31. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefPubMedGoogle Scholar
  32. Poulose AJ, Croteau R (1978a) Biosynthesis of aromatic monoterpenes: conversion of γ-terpinene to p-cymene and thymol in Thymus vulgaris L. Arch Biochem Biophys 187:307–314CrossRefPubMedGoogle Scholar
  33. Poulose AJ, Croteau R (1978b) γ-Terpinene synthetase—key enzyme in biosynthesis of aromatic monoterpenes. Arch Biochem Biophys 191:400–411CrossRefPubMedGoogle Scholar
  34. Ren MZ, Chen QJ, Li L, Zhang R, Guo SD (2005) Successive chromosome walking by compatible ends ligation inverse PCR. Mol Biotechnol 30:95–101CrossRefPubMedGoogle Scholar
  35. Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  36. Sedy KA, Koschier EH (2003) Bioactivity of carvacrol and thymol against Frankliniella occidentalis and Thrips tabaci. J Appl Entomol 127:313–316CrossRefGoogle Scholar
  37. Skoula M, Harborne JB (2002) The taxonomy and chemistry of Origanum. In: Kintzios SE (ed) Oregano: the genera Origanum and Lippia. Taylor & Francis, London, pp 67–108Google Scholar
  38. Skoula M, Gotsiou P, Naxakis G, Johnson CB (1999) A chemosystematic investigation on the mono- and sesquiterpenoids in the genus Origanum (Labiatae). Phytochemistry 52:649–657CrossRefGoogle Scholar
  39. Starks CM, Back KW, Chappell J, Noel JP (1997) Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase. Science 277:1815–1820CrossRefPubMedGoogle Scholar
  40. Tholl D (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol 9:297–304CrossRefPubMedGoogle Scholar
  41. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefPubMedGoogle Scholar
  42. Turner G, Gershenzon J, Nielson EE, Froehlich JE, Croteau R (1999) Limonene synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localized to leucoplasts of oil gland secretory cells. Plant Physiol 120:879–886CrossRefPubMedGoogle Scholar
  43. Van de Peer Y, De Wachter R (1994) Treecon for Windows—a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10:569–570PubMedGoogle Scholar
  44. Vokou D, Kokkini S, Bessiere JM (1993) Geographic-variation of Greek oregano (Origanum vulgare ssp hirtum) essential oils. Biochem Syst Ecol 21:287–295CrossRefGoogle Scholar
  45. 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
  46. Wise ML, Croteau R (1999) Monoterpene biosynthesis. In: Cane DE (ed) Comprehensive natural products chemistry. Elsevier, Amsterdam, pp 97–153CrossRefGoogle Scholar
  47. Wise ML, Savage TJ, Katahira E, Croteau R (1998) Monoterpene synthases from common sage (Salvia officinalis)—cDNA isolation, characterization, and functional expression of (+)-sabinene synthase, 1, 8-cineole synthase, and (+)-bornyl diphosphate synthase. J Biol Chem 273:14891–14899CrossRefPubMedGoogle Scholar
  48. Yu XH, Liu CJ (2006) Development of an analytical method for genome-wide functional identification of plant acyl-coenzyme A-dependent acyltransferases. Anal Biochem 358:146–148CrossRefPubMedGoogle Scholar
  49. Zavala-Paramo G, Chavez-Moctezuma MP, Garcia-Pineda E, Yin S, Chappell J, Lozoya-Gloria E (2000) Isolation of an elicitor-stimulated 5-epi-aristolochene synthase gene (gPEAS1) from chili pepper (Capsicum annuum). Physiol Plantarum 110:410–418CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Christoph Crocoll
    • 1
  • Julia Asbach
    • 1
  • Johannes Novak
    • 2
  • Jonathan Gershenzon
    • 1
  • Jörg Degenhardt
    • 3
  1. 1.Department of BiochemistryMax Planck Institute for Chemical EcologyJenaGermany
  2. 2.Institute for Applied Botany and PharmacognosyUniversity of Veterinary MedicineWienAustria
  3. 3.Institute of PharmacyMartin Luther University HalleHalleGermany

Personalised recommendations