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Planta

, 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

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.

Keywords

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

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

Notes

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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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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