Abstract
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.
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Abbreviations
- EO(s):
-
Essential oil(s)
- TPS(s):
-
Terpene synthase(s)
- mTPS(s):
-
Monoterpene synthase(s)
- sTPS(s):
-
Sesquiterpene synthase(s)
- GPP:
-
Geranyl diphosphate
- NPP:
-
Nerolidyl diphosphate
- LaβPHLS :
-
L. angustifolia β-phellandrene synthase
- LaTPS-I:
-
L. angustifolia terpene synthase-like
- LaLINS :
-
L. angustifolia linalool synthase
- LaLIMS :
-
L. angustifolia limonene synthase
References
Boeckelmann A (2008) Monoterpene production and regulation in lavenders (Lavandula angustifolia and Lavandula × intermedia). MSc Thesis, University of British Columbia Okanagan, pp 70–71
Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95(8):4126–4133
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):232–243
Boira H, Blanquer A (1998) Environmental factors affecting chemical variability of essential oils in Thymus piperella L. Biochem Syst Ecol 26(8):811–822
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):464–472
Cavanagh HMA, Wilkinson JM (2002) Biological activities of lavender essential oil. Phytother Res 16(4):301–308
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):112–121
Degenhardt J, Köllner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70(15–16):1621–1637
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) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 1(36):W465–W469
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):225–228
Felicetti B, Cane DE (2004) Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis. J Am Chem Soc 126(23):7212–7221
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):93–96
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):2333–2338
Harborne JB, Williams CA (2002) Phytochemistry of the genus Lavandula. In: Lis-Balchin M (ed) Lavender: the genus lavandula. Tayler & Francis, London, pp 86–100
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 372:143–149
Keegstra K, Olsen LJ, Theg SM (1989) Chloroplastic precursors and their transport across the envelope membranes. Annu Rev Plant Phys 40(1):471–501
Kreis P, Mosandl A (1992) Chiral compounds of essential oils. Part XI. Simultaneous stereoanalysis of Lavandula oil constituents. Flavour Fragr J 7(4):187–193
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):417–429
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):835–845
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 251–262
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):14481–14486
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):547–554
McConkey ME, Gershenzon J, Croteau RB (2000) Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint. Plant Physiol 122(1):215–224
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):479–488
Nagegowda DA (2010) Plant volatile terpenoid metabolism: biosynthetic genes, transcriptional regulation and subcellular compartmentation. FEBS Lett 584(14):2965–2973
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):224–239
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):3203–3219
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):803–807
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):107–124
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):10865–10870
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):7681–7689
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):987–995
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):233–242
Von Heijne G, Steppuhn J, Herrmann RG (1989) Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180(3):535–545
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):12213–12220
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 97–153
Wise ML, Savage TJ, Katahira E, Croteau R (1998) Monoterpene synthases from common sage (Salvia officinalis). J Biol Chem 273:14891–14899
Acknowledgments
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.
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Demissie, Z.A., Sarker, L.S. & Mahmoud, S.S. Cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia . Planta 233, 685–696 (2011). https://doi.org/10.1007/s00425-010-1332-5
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DOI: https://doi.org/10.1007/s00425-010-1332-5