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Cloning, functional characterization and genomic organization of 1,8-cineole synthases from Lavandula

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Abstract

Several members of the genus Lavandula produce valuable essential oils (EOs) that are primarily constituted of the low molecular weight isoprenoids, particularly monoterpenes. We isolated over 8,000 ESTs from the glandular trichomes of L. x intermedia flowers (where bulk of the EO is synthesized) to facilitate the discovery of genes that control the biosynthesis of EO constituents. The expression profile of these ESTs in L. x intermedia and its parents L. angustifolia and L. latifolia was established using microarrays. The resulting data highlighted a differentially expressed, previously uncharacterized cDNA with strong homology to known 1,8-cineole synthase (CINS) genes. The ORF, excluding the transit peptide, of this cDNA was expressed in E. coli, purified by Ni–NTA agarose affinity chromatography and functionally characterized in vitro. The ca. 63 kDa bacterially produced recombinant protein, designated L. x intermedia CINS (LiCINS), converted geranyl diphosphate (the linear monoterpene precursor) primarily to 1,8-cineole with K m and k cat values of 5.75 μM and 8.8 × 10−3 s−1, respectively. The genomic DNA of CINS in the studied Lavandula species had identical exon–intron architecture and coding sequences, except for a single polymorphic nucleotide in the L. angustifolia ortholog which did not alter protein function. Additional nucleotide variations restricted to L. angustifolia introns were also observed, suggesting that LiCINS was most likely inherited from L. latifolia. The LiCINS mRNA levels paralleled the 1,8-cineole content in mature flowers of the three lavender species, and in developmental stages of L. x intermedia inflorescence indicating that the production of 1,8 cineole in Lavandula is most likely controlled through transcriptional regulation of LiCINS.

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Abbreviations

CINS(s):

1,8-Cineole synthase(s)

dTPS(s):

Diterpene synthase(s)

EO(s):

Essential oil(s)

EST(s):

Expressed Sequence Tag(s)

GPP:

Geranyl diphosphate

mTPS(s):

Monoterpene synthase(s)

sTPS(s):

Sesquiterpene synthase(s)

TPS(s):

Terpene synthase(s)

LaCINS:

L. angustifolia 1,8-cineole synthase

LaLINS:

L. angustifolia linalool synthase

LaLIMS:

L. angustifolia limonene synthase

LaβPHLS:

L. angustifolia β-phellandrene synthase

LiCINS:

L. x intermedia 1,8-cineole synthase

LlCINS:

L. latifolia 1,8-cineole synthase

NPP:

Neryl diphosphate

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Acknowledgments

This work was supported through grants or in-kind contributions to SSM by UBC Okanagan campus, Investment Agriculture Foundation of British Columbia, NRC Plant Biotechnology Institute through the NAPGEN program, and Genome British Columbia, and to SSM and MRR by Natural Sciences and Engineering Research Council of Canada. ZAD would like to acknowledge the financial support through the Pacific Century Graduate Scholarships (PCGS) award from the province of British Columbia through the Ministry of Advanced Education. We would also like to thank Dr Tim Upson (Cambridge University, UK) for providing the L. latifolia leaf and flower tissues used in this study.

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Authors and Affiliations

  1. Department of Biology, University of British Columbia, 1177 Research Road, Kelowna, BC, V1V 1V7, Canada

    Zerihun A. Demissie, Monica A. Cella, Lukman S. Sarker, Travis J. Thompson, Mark R. Rheault & Soheil S. Mahmoud

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  1. Zerihun A. Demissie
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  2. Monica A. Cella
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Corresponding author

Correspondence to Soheil S. Mahmoud.

Additional information

Accession numbers: LiCINS: JN701459; LlCINS: JN701460; LaCINS: JN701461.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11103_2012_9920_MOESM1_ESM.pdf

Transcriptional activity of MEP-pathway genes in leaves and floral tissues of L. angustifolia, L. x intermedia and L. latifolia. FL: 30 % flower and LF: leaf. (PDF 199 kb)

11103_2012_9920_MOESM2_ESM.pdf

SDS-PAGE analysis of protein samples from bacterial cells expressing LiCINS, and those transformed with the empty expression vector. (a) protein marker, (b) total protein from cells expressing LiCINS, (c) soluble proteins from cells expressing LiCINS, (d) purified LiCINS and, (e) purified protein (GST) from cells transformed with the empty pET41(b+) vector. (PDF 64 kb)

11103_2012_9920_MOESM3_ESM.pdf

GC chromatograms of Ni-NTA affinity chromatography purified soluble fraction from induced cells transformed with empty pET41(b+) product from GPP. Peak (1) is linalool and asterisks represent peaks without hit in the National Institute of Standards and Technology (NIST) library. (PDF 36 kb)

11103_2012_9920_MOESM4_ESM.pdf

Multiple alignments of 1,8-cineole synthase cDNAs of L. latifolia, L. angustifolia and L. x intermedia. Asterisks indicate conserved nucleotides in the three cDNAs while the polymorphic nucleotides are in bold and bigger font size. (PDF 18 kb)

11103_2012_9920_MOESM5_ESM.pdf

GC chromatogram of the recombinant L. angustifolia 1,8-cineole synthase (LaCINS) catalyzed products from GPP. Peaks correspond to: 1) sabinene, 2) α-phellandrene, 3) limonene, 4) 1,8-cineole, 5) linalool and 6) α-terpineol. (PDF 35 kb)

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Demissie, Z.A., Cella, M.A., Sarker, L.S. et al. Cloning, functional characterization and genomic organization of 1,8-cineole synthases from Lavandula . Plant Mol Biol 79, 393–411 (2012). https://doi.org/10.1007/s11103-012-9920-3

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