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

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.

Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot

“…The L. nobilis 8-cineole synthase we characterized here produces a similar, but not identical, group of monoterpenes in addition to 1,8-cineole that previously characterized 8-cineole synthase from different plant species, and similar to them, it also belongs to the TPS-b clade (Wise et al, 1998;Chen et al, 2004;Demissie et al, 2012). For example, the Arabidopsis root-specific cineole synthase (AtTPC-Cin) produces, in addition to 1,8-cineole, (2)-(4S)-limonene and (E)-bocimene (Chen et al, 2004); the recombinant common sage (Salvia officinalis) cineole synthase produces 1,8-cineole as the major product along with (6)-limonene and (+)-campherene (Wise et al, 1998); and the cineole synthase from the glandular trichome of Lavandula 3 intermedia flowers produces, in addition to 1,8-cineole, (6)-limonene and linalool (Demissie et al, 2012). In contrast, the L. nobilis cineole synthase does not produce (6)-limonene, (E)-b-ocimene, linalool, or (+)-campherene (Fig.…”

The α-Terpineol to 1,8-Cineole Cyclization Reaction of Tobacco Terpene Synthases

“…C. unshiu and Lavandula spp.) is still unknown (Shimada et al, 2005;Demissie et al, 2012). Even though the open-chain cationic intermediates are achiral, Croteau et al (1994) pointed out that the stereochemical fate of the intermediate is already fixed upon binding and isomerization of the substrate GPP.…”

The Biosynthetic Origin of Irregular Monoterpenes in Lavandula

“…Grosso secretory cells of oil glands, tissues specialized for EO biosynthesis and secretion (31). All PDPS homologs in the database were retrieved by searching the EST database using the strings "diphosphate synthase" and "pyrophosphate synthase."…”

“…Grosso plants grown at the University of British Columbia, Okanagan campus lavender field following a previously described modified glass bead abrasion method (31). Total RNA was extracted from 100 mg of the tissue using an RNA extraction kit (OMEGA bio-tek) and reverse transcribed in a reaction containing the oligo(dT) primer (Fisher Scientific) and M-MuLV reverse transcriptase enzyme (New England Biolabs) following the manufacturer's directions.…”

“…Consequently, these oils are industrially utilized as ingredients of various cosmetic and antiseptic products (29). The biosynthetic pathway leading to these monoterpenes from IPP and DMAPP has been defined both experimentally (30 -32) and through in silico analysis of Lavandula EST databases (30,31,33). In this regard, four cDNAs encoding for mTPSs that transform GPP and NPP into linalool, 1,8-cineole, limonene, and ␤-phellandrene in vitro have been cloned from L. angustifolia and L. x intermedia species (30 -32).…”