Skip to main content
SpringerLink
Log in

Molecular cloning and characterization of monoterpene synthases from Litsea cubeba (Lour.) Persoon

  • Original Paper
  • Published:
Tree Genetics & Genomes Aims and scope Submit manuscript

Abstract

Mountain pepper (Litsea cubeba (Lour.) Persoon) is an evergreen tree in the Lauraceae family and has attracted attention for the antioxidant and antibacterial effects of its essential oils. The dominant components of the essential oil of L. cubeba are monoterpenes. To investigate the biosynthesis of monoterpenes that have bioactivity in L. cubeba, three cDNAs corresponding to monoterpene synthase genes were isolated, and the corresponding enzymes were functionally characterized in vitro. The genes obtained were named LcTPS1, LcTPS2, and LcTPS3. Transcript levels for LcTPS1 were most prominent in the leaf tissues, and levels of LcTPS2 and LcTPS3 were more evident in fruit tissues than in leaf tissues. Phylogenetic analyses indicated that LcTPS1, LcTPS2, and LcTPS3 all belong to TPS-b subfamily of angiosperm monoterpene synthases. For each genes, the corresponding enzymes were expressed and functionally characterized. LcTPS1 converted geranyl diphosphate to trans-ocimene, and LcTPS2 converted geranyl diphosphate to α-thujene, while LcTPS3 is a mutifunctional enzyme that converted geranyl diphosphate to α-thujene and (+)-sabinene, respectively.

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

mono-TPS:

Monoterpene synthase

TPS:

Terpene synthase

References

  • Arimura G, Ozawa R, Shimoda T, Nishioka T, Boland W, Takabayashi J (2000) Herbivory-induced volatiles elicit defence genes in lima bean leaves. Nature 406:512–515

    Article  PubMed  CAS  Google Scholar 

  • Bergstrom G, Dobson HEM, Groth I (1995) Spatial fragrance patterns within the flowers of Ranunculus acris (Ranunculaceae). Plant Syst Evol 195:221–242

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Bohlmann J, Martin D, Oldham NJ, Gershenzon J (2000) Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-β-ocimene synthase. Arch Biochem Biophys 375:261–269

    Article  PubMed  CAS  Google Scholar 

  • Borg-Karlson AK, Englund FO, Unelius CR (1994) Dimethyl oligosulphides, major volatiles released from Sauromatum gottatum and Phallus impudicus. Phytochemistry 35:321–323

    Article  CAS  Google Scholar 

  • Buckingham J (1998) Dictionary of natural products on CD-ROM, version 6.1. Chapman and Hall, London

    Google Scholar 

  • Chen F, Tholl D, D’Auria JC, Farooq A, Pichersky E, Gershenzon J (2003) Biosynthesis and emission of terpenoid volatiles from Arabidopsis flowers. Plant Cell 15:481–494

    Article  PubMed  CAS  Google Scholar 

  • Chen F, Ro DK, Petri J, Gershenzon J, Bohlmann J, Pichersky E, Tholl D (2004) Characterization of a root-specific Arabidopsis terpene synthase responsible for the formation of the volatile monoterpene 1,8-cineole1. Plant Physiol 135:1956–1966

    Article  PubMed  CAS  Google Scholar 

  • Christianson DW (2006) Structural biology and chemistry of the terpenoid cyclases. Chem Rev 106:3412–3442

    Article  PubMed  CAS  Google Scholar 

  • Coudhunry S, Ahmed R, Barthel A, Leclercq PA (1998) Composition of the stem, flower and fruit oils of Litsea cubeba Pers. From two locations of Assam, India. J Essent Oil Res 10:381–386

    Google Scholar 

  • Cox B, Kislinger T, Emili A (2004) Integrating gene and protein expression data: pattern analysis and profile mining. Methods 35:303–314

    Article  Google Scholar 

  • Croteau R (1987) Biosynthesis and catabolism of monoterpenoids. Chem Rev 87:929–954

    Article  CAS  Google Scholar 

  • Davis EM, Croteau R (2000) Cyclization enzymes in the biosynthesis of monoterpenes, sesquiterpenes, and diterpenes. Top Curr Chem 209:53–95

    Article  CAS  Google Scholar 

  • 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 

  • Duke JA, Ayensu ES (1985) Medicinal plants of China. Reference Publications, Michigan, pp 390–391

    Google Scholar 

  • Elzen GW, Williams HJ, Bell AA, Stipanovic RD, Vinson SB (1985) Quantification of volatile terpenes of glanded and glandless Gossypium hirsutum L. cultivars and lines by gas chromatography. J Agric Food Chem 33:1079–1082

    Article  CAS  Google Scholar 

  • Fabbro SD, Nazzi F (2008) Repellent effect of sweet basil compounds on Ixodes ricinus ticks. Exp Appl Acarol 45:219–228

    Article  PubMed  Google Scholar 

  • Fäldt J, Martin D, Miller B, Rawat S, Bohlmann J (2003) Traumatic resin defense in Norway spruce (Picea abies): methyl jasmonate-induced terpene synthase gene expression, and cDNA cloning and functional characterization of (+)-3-carene synthase. Plant Mol Biol 51:119–133

    Article  PubMed  Google Scholar 

  • Ho CL, Jie-Pinge O, Liu YC, Hung CP, Tsai MC, Liao PC, Wang EI, Chen YL, Su YC (2010) Compositions and in vitro anticancer activities of the leaf and fruit oils of Litsea cubeba from Taiwan. Nat Prod Commun 5:617–620

    PubMed  CAS  Google Scholar 

  • Hwang JK, Choi EM, Lee JH (2005) Antioxidant activity of Litsea cubeba. Fitoterapia 76:684–686

    Article  PubMed  Google Scholar 

  • Hyatt DC, Youn B, Zhao Y, Santhamma B, Coates RM, Croteau RB, Kang CH (2007) Structure of limonene synthase, a simple model for terpenoid cyclase catalysis. Proc Natl Acad Sci USA 104:5360–5365

    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 

  • Kessler A, Baldwin IT (2010) Defensive function of herbivore-induced plant volatile emissions in nature. Sci 291:2141–2144

    Article  Google Scholar 

  • Kohzaki K, Gomi K, Kokudo YY, Ozawa R, Takabayashi J, Akimitsu K (2009) Characterization of a sabinene synthase gene from rough lemon (Citrus jambhiri). J Plant Physiol 166:1700–1704

    Article  PubMed  CAS  Google Scholar 

  • Lagalante AF, Montgomery ME (2003) Analysis of terpenoids from Hemlock (Tsuga) species by solid-phase microextraction/gas chromatography/ion–trap mass spectrometry. J Agric Food Chem 51:2115–2120

    Article  PubMed  CAS  Google Scholar 

  • Liu ZL, Goh SH, Ho SH (2007) Screening of Chinese medicinal herbs for bioactivity against Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst). J Stored Prod Res 43:290–296

    Article  Google Scholar 

  • Lücker J, Bowen P, Bohlmann J (2004) Vitis vinifera terpenoid cyclases: functional identification of two sesquiterpene synthase cDNAs encoding (+)-valencene synthase and (+)-germacrene D synthase and expression of mono- and sesquiterpene synthases in grapevine flowers and berries. Phytochemistry 65:2649–2659

    Article  Google Scholar 

  • Marriacci L, Dicke M, Posthumus MA (1994) Induction of parasitoid attracting synomone in brussels sprouts plants by feeding of Pieris brassicae larvae: role of mechanical damage and herbivore elicitor. J Chem Ecol 20:2229–2247

    Article  Google Scholar 

  • Martin DM, Faldt J, Bohlmann J (2004) Functional characterization of nine Norway spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily. Plant Physiol 135:1908–1927

    Article  PubMed  CAS  Google Scholar 

  • Page RDM (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358

    PubMed  CAS  Google Scholar 

  • Schnee C, Kollner TG, Gershenzon J, Degenhardt J (2002) The maize gene terpene synthase 1 encodes a sesquiterpene synthase catalyzing the formation of (E)-beta-farnesene, (E)-nerolidol, and (E, E)-farnesol after herbivore damage. Plant Physiol 130:2049–2052

    Article  PubMed  CAS  Google Scholar 

  • Seemann M, Zhai GZ, de Kraker JW, Paschall CM, Christianson DW, Cane DE (2002) Pentalenene synthase. Analysis of active site residues by site-directed mutagenesis. J Am Chem Soc 124:7681–7689

    Article  PubMed  CAS  Google Scholar 

  • Starks CM, Back K, Chappell J, Noel JP (1997) Structural basis for cyclic terpene biosynthesis by tobacco 5-epi-aristolochene synthase. Sci 277:1815–1820

    Article  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 

  • Wang H, Liu Y (2010) Chemical composition and antibacterial activity of essential oils from different parts of Litsea cubeba. Chem Biodiv 7:229–235

    Article  CAS  Google Scholar 

  • Whittington DA, Wise ML, Urbansky M, Coates RM, Croteau RB, Christianson DW (2002) Bornyl diphosphate synthase: structure and strategy for carbocation manipulation by a terpenoid cyclase. Natl Acad Sci USA 99:15375–15380

    Article  CAS  Google Scholar 

  • Wise ML, Croteau R (1999) Comprehensive natural products chemistry: isoprenoids including caroteinoids and steroids. Sci 2:97–135

    CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful for expert technical assistance from Yi-Ru Lee and the Huisun Experimental Forest of the National Chung Hsing University for providing various organ samples of L. cubeba. Financial assistance from the Council of Agriculture Executive Yuan is gratefully acknowledged.

Author information

Authors and Affiliations

  1. School of Forestry and Resource Conservation, National Taiwan University, Taipei, 106, Taiwan

    Yueh-Te Chang & Fang-Hua Chu

Authors
  1. Yueh-Te Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fang-Hua Chu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fang-Hua Chu.

Additional information

Communicated by R. Sederoff

Electronic supplementary materials

Below is the link to the electronic supplementary material.

Figure 1

Homology models for LcTPS1. The models were constructed using SWISS protein structure prediction server by threading LcTPS1 sequences into the coordinate of (4S)-limonene synthase (PDB code: 2ongB) three-dimensional structure. a Homology model for LcTPS1 that colored by secondary structure succession from N-terminal to C-terminal domain. b Homology model for LcTPS1 which the DDxxD motif (green), the NSE/DTE motif (blue), and the H-α1 loop region (purple) with their associated bound magnesium atoms (white tri-atom cluster) and the diphosphate moiety of 2-fluoro GPP (analogous of GPP) which is shown in sticks are also indicated. (DOC 513 kb)

Figure 2

Amino acids alignment showing the topological positioning of the H-α1 loop in nine TPS-d enzymes and the three TPS-b enzymes from L. cubeba. The nine TPS-d sequences were selected on the basis that they account for all possible variations of amino acids in the H-α1 loop of 24 TPS-d protein sequences. Aa linalool, Artemisia annua linalool synthase (AAF13356); Aa pinene, Artemisia annua ß -pinene synthase (AAK58723); At myrcene, Arabidopsis thaliana putative limonene cyclase (AAD03382); At limonene, Arabidopsis thalian limonene cyclase (BAB01181); Cu pinene, Citrus unshiu ß-pinene synthase (BAD27260); La bergamotene, Lavandula angustifolia trans-α-bergamotene synthase (ABB73046); La limonene, Lavandula angustifolia limonene synthase (ABB73044); Ma linalool Mentha aquati linalool synthase (AAL99381); Ob myrcene, Ocimum basilicum ß-myrcene synthase (AAV63791); Pml isoprene, Pueraria montana var. lobata isoprene synthase (AAQ84170); Pn isoprene, Populus nigra isoprene synthase (CAL69918); Qi myrcene, Quercus ilex putative chloroplast terpene synthase (CAC41012); Sf cineole Salvia fruticosa cineole synthase (ABH07677); So bornyl, Salvia officinalis (+)-bornyl diphosphate synthase (AAC26017). (DOC 320 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chang, YT., Chu, FH. Molecular cloning and characterization of monoterpene synthases from Litsea cubeba (Lour.) Persoon. Tree Genetics & Genomes 7, 835–844 (2011). https://doi.org/10.1007/s11295-011-0377-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11295-011-0377-3

Keywords

Search

Navigation