Skip to main content
SpringerLink
Log in

Genomic characterization, molecular cloning and expression analysis of two terpene synthases from Thymus caespititius (Lamiaceae)

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

The identification, isolation and functional characterization of two genes encoding two monoterpene synthases—γ-terpinene synthase (Tctps2) and α-terpineol synthase (Tctps5)—from three chemically distinct Thymus caespititius (Lamiaceae) genotypes were performed. Genomic exon–intron structure was also determined for both terpene synthase genes, revealing an organization with seven exons and six introns. The cDNA of Tctps2 was 2,308 bp long and had an open reading frame of 1,794 bp encoding for a protein with 598 amino acids. Tctps5 was longer, mainly due to intron sequences, and presented high intraspecific variability on the plants analyzed. It encoded for a protein of 602 amino acids from an open reading frame of 1,806 bp comprising a total of 2,507 bp genomic sequence. The amino acid sequence of these two active Tctps genes shared 74 % pairwise identity, ranging between 42 and 94 % similarity with about 50 known terpene synthases of other Lamiaceae species. Gene expression revealed a multi-product Tctps2 and Tctps5 enzymes, producing γ-terpinene and α-terpineol as major components, respectively. These enzymatic results were consistent with the monoterpene profile present in T. caespititius field plants, suggesting a transcriptional regulation in leaves. Herewith reported for the first time for this species, these two newly characterized Tctps genes improve the understanding of the molecular mechanisms of reaction responsible for terpene biosynthesis and chemical diversity found in T. caespititius.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

DTT:

Dithiothreitol

GPP:

Geranyl diphosphate

FPP:

Farnesyl diphosphate

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

ISSR:

Inter-simple sequence repeat

PMSF:

Phenylmethylsulfonyl fluoride

RAPD:

Random amplification of polymorphic DNA

SPME:

Solid phase micro extraction

Tctps2 :

T. caespititius terpene synthase 2 gene (γ-terpinene synthase)

Tctps5 :

T. caespititius terpene synthase 5 gene (α-terpineol synthase)

TPS:

Terpene synthase

References

  • Aubourg S, Lecharny A, Bohlmann J (2002) Genomic analysis of the terpenoid synthase (AtTPS) gene family of Arabidopsis thaliana. Mol Genet Genomics 267:730–745

    Article  PubMed  CAS  Google Scholar 

  • Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95:4126–4133

    Article  PubMed  CAS  Google Scholar 

  • Chen F, Tholl D, Bohlmann J, Pichersky E (2011) The family of terpene synthases in plants: a mid-size family of genes specialized metabolism that is highly diversified throughout the kingdom. Plant J 66:212–229

    Article  PubMed  CAS  Google Scholar 

  • Crocoll C (2010) Biosynthesis of the phenolic monoterpenes, thymol and carvacrol, by terpene synthases and cytochrome P450s in oregano and thyme. PhD dissertation, Faculty of Biology and Pharmacy, University of Friedrich-Schiller, Germany

  • Crocoll C, Asbach J, Novak J, Gershenzon J, Degenhardt J (2010) Terpene synthases of oregano (Origanum vulgare L.) and their roles in the pathway and regulation of terpene biosynthesis. Plant Mol Biol 73:587–603

    Article  PubMed  CAS  Google Scholar 

  • Cseke L, Dudareva N, Pichersky E (1998) Structure and evolution of linalool synthase. Mol Biol Evol 15:1491–1498

    Article  PubMed  CAS  Google Scholar 

  • Daviet L, Schalk M (2010) Biotechnology in plant essential oil production: progress and perspectives in metabolic engineering of the terpene pathway. Flavour Fragr J 25:123–127

    Article  CAS  Google Scholar 

  • Degenhardt J, Crocoll C, Asbach J, Gershenzon J (2007) Terpene synthases. International Patent Application WO 2007/009958

  • Degenhardt J, Köllner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70:1621–1637

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Heled J, Kearse M, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A (2010) Geneious v5.3, Available from http://www.geneious.com

  • Emanuelsson O, Nielsen H, Von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984. Available online at http://www.cbs.dtu.dk/services/ChloroP/

    Google Scholar 

  • Fähnrich A, Krause K, Piechulla B (2011) Product variability of the ‘cineole cassette’ monoterpene synthases of related Nicotiana species. Mol Plant 4:965–984

    Article  PubMed  Google Scholar 

  • Figueiredo AC, Barroso JG, Pedro LG, Salgueiro L, Miguel MG, Faleiro ML (2008a) Portuguese Thymbra and Thymus species volatiles: chemical composition and biological activities. Curr Pharm Design 14:3120–3140

    Article  CAS  Google Scholar 

  • Figueiredo AC, Barroso JG, Pedro LG, Scheffer JC (2008b) Factors affecting secondary metabolite production in plants: volatile components and essential oils. Flavour Frag J 23:213–226

    Article  CAS  Google Scholar 

  • Figueiredo AC, Barroso JG, Pedro LG (2010) Volatiles from Thymbra and Thymus species of the Western Mediterranean Basin, Portugal and Macaronesia. Nat Prod Commun 5:1465–1476

    PubMed  CAS  Google Scholar 

  • Ito M, Honda G (2007) Geraniol synthases from perilla and their taxonomical significance. Phytochemistry 68:446–453

    Article  PubMed  CAS  Google Scholar 

  • 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 19:1994–2005

    Article  PubMed  CAS  Google Scholar 

  • Keszei A, Brubaker CL, Foley WJ (2008) A molecular perspective on terpene variation in Australian Myrtaceae. Aust J Bot 56:197–213

    Article  CAS  Google Scholar 

  • Keszei A, Brubaker CL, Carter R, Köllner T, Degenhardt J, Foley WJ (2010) Functional and evolutionary relationships between terpene synthases from Australian Myrtaceae. Phytochemistry 71:844–852

    Article  PubMed  CAS  Google Scholar 

  • Kollner 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 16:1115–1131

    Article  PubMed  Google Scholar 

  • Lane A, Boecklemann A, Woronuk GN, Sarker L, Mahmoud SS (2010) A genomic resource for investigating regulation of essential oil production in Lavandula angustifolia. Planta 231:835–845

    Article  PubMed  CAS  Google Scholar 

  • Li R, Fan Y (2010) Molecular cloning and expression analysis of a terpene synthase gene, HcTPS2, in Hedychium coronarium. Plant Mol Biol Rep 29:35–42

    Article  Google Scholar 

  • Likens SV, Nickerson GB (1964) Detection of certain hop oil constituents in brewing products. Am Soc Brew Chem Proc 5:13

    Google Scholar 

  • Lima AS, Trindade H, Figueiredo AC, Barroso JB, Pedro LG (2010) Molecular and volatiles characterization of Portuguese Thymus caespititius chemotypes. Acta Hort 860:81–86

    CAS  Google Scholar 

  • Lukas B, Samuel R, Novak J (2010) Oregano or marjoram? The enzyme γ-terpinene synthase affects chemotype formation in the genus Origanum. Isr J Plant Sci 58:211–220

    Article  Google Scholar 

  • Muñoz-Bertomeu J, Ros R, Arrillaga I, Segura J (2008) Expression of spearmint limonene synthase in transgenic spike lavender results in an altered monoterpene composition in developing leaves. Metab Eng 10:166–177

    Article  PubMed  Google Scholar 

  • Nagegowda DA (2010) Plant volatile terpenoid metabolism: biosynthetic genes, transcriptional regulation and subcellular compartmentation. FEBS Lett 584:2965–2973

    Article  PubMed  CAS  Google Scholar 

  • Poulose AJ, Croteau R (1978) γ-Terpinene synthetase: a key enzyme in the biosynthesis of aromatic monoterpenes. Arch Biochem Biophys 191:400–411

    Article  PubMed  CAS  Google Scholar 

  • Schmiderer C, Grausgruber-Gröger S, Grassi P, Steinborn R, Novak J (2010) Influence of gibberellins and daminozide on the expression of terpene synthases and on monoterpenes in common sage (Salvia officinalis). J Plant Physiol 167:779–786

    Article  PubMed  CAS  Google Scholar 

  • Tholl D (2006) Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol 9:297–304

    Article  PubMed  CAS  Google Scholar 

  • Trapp SC, Croteau RB (2001) Genomic organization of plant terpene synthases and molecular evolutionary implications. Genetics 158:811–832

    PubMed  CAS  Google Scholar 

  • Trindade H, Costa MM, Lima AS, Pedro LG, Figueiredo AC, Barroso JG (2008) Genetic diversity and chemical polymorphism of Thymus caespititius from Pico, São Jorge and Terceira islands (Azores). Biochem Sys Ecol 36:790–797

    Article  CAS  Google Scholar 

  • Trindade H, Costa MM, Lima AS, Pedro LG, Figueiredo AC, Barroso JG (2009) A combined approach using RAPD, ISSR and volatile analysis for the characterization of Thymus caespititius from Flores, Corvo and Graciosa islands (Azores, Portugal). Biochem Sys Ecol 37:670–677

    Article  CAS  Google Scholar 

  • Turner GW, Croteau R (2004) Organization of monoterpene biosynthesis in Mentha—immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase and pulegone reductase. Plant Physiol 136:4215–4227

    Article  PubMed  CAS  Google Scholar 

  • 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 120:879–886

    Article  PubMed  CAS  Google Scholar 

  • Weising K, Nybom H, Wolff K, Meyer W (1995) DNA fingerprinting in plants and fungi. CRC Press, Boca Raton, pp 43–152

    Google Scholar 

  • 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 37:12213–12220

    Article  PubMed  CAS  Google Scholar 

  • 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 273:14891–14899

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This study was partially funded by the Fundação para a Ciência e Tecnologia (FCT) under Pest/OE/EQB/LA0023/2011 and research contract PTDC/AGR-GPL/101334/2008. The funding from CRUP/DAAD, Ref. A11/12 supported the collaboration with AG Pharmaceutical Biotechnology group from Martin Luther University Halle-Wittenberg. The authors are grateful to Prof. Dr. K. Bahcevandziev (Escola Superior Agrária de Coimbra) for the maintenance of T. caespititius plants; Eng. V. Lopes (Banco Português de Germoplasma Vegetal) for T. caespititius samples from mainland Portugal; Prof. Dr. L. Domingues and to C. Oliveira (Universidade do Minho) for all the help, teaching and availability in the beginning of work lab with cloning and functional expression of Tctps; Prof. Dr. M. Oliveira and to M. Rodrigues (Instituto de Tecnologia Quimica e Biológica) for their availability and all the help with Gateway system.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Departmento de Biologia Vegetal, Faculdade de Ciências, Centro de Biotecnologia Vegetal, Instituto de Biotecnologia e Bioengenharia, Universidade de Lisboa, C2, Campo Grande, 1749-016, Lisbon, Portugal

    A. Sofia Lima, José G. Barroso, A. Cristina Figueiredo, Luis G. Pedro & Helena Trindade

  2. Institute for Pharmacy, AG Pharmaceutical Biotechnology, Martin Luther University Halle-Wittenberg, Hoher Weg 8, 06120, Halle (Saale), Germany

    Jette Schimmel & Jörg Degenhardt

  3. Institute for Applied Botany and Pharmacognosy, University of Veterinary Medicine, Veterinärplatz 1, 1210, Wien, Austria

    Brigitte Lukas & Johannes Novak

Authors
  1. A. Sofia Lima
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jette Schimmel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Brigitte Lukas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Johannes Novak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. José G. Barroso
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. Cristina Figueiredo
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Luis G. Pedro
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jörg Degenhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Helena Trindade
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Helena Trindade.

Additional information

Accession numbers

Tctps2-B1 (gDNA): KC181098; Tctps2-C2 (gDNA): KC691293; Tctps5-C2 (gDNA): KC181096; Tctps5-319 (gDNA): KC181095; Tctps2-B1-01 (cDNA): KC181099; Tctps2-C2-01 (cDNA): KC691294; Tctps5-C2-01 (cDNA): KC181101; Tctps5-319-01 (cDNA): KC181102; Tctps2-B1-02 (active enzyme): KC181103; Tctps2-C2-02 (active enzyme): KC691295; Tctps5-C2-02 (active enzyme): KC181104; Tctps5-319-02 (active enzyme): KC181105.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lima, A.S., Schimmel, J., Lukas, B. et al. Genomic characterization, molecular cloning and expression analysis of two terpene synthases from Thymus caespititius (Lamiaceae). Planta 238, 191–204 (2013). https://doi.org/10.1007/s00425-013-1884-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-013-1884-2

Keywords

Search

Navigation