title: 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.
authors.affiliation: Department of Biology, University of British Columbia, 1177 Research Road, Kelowna, BC, V1V 1V7, Canada.
authors.forename: Zerihun A
authors.initials: ZA
authors.lastname: Demissie
authors.forename: Monica A
authors.initials: MA
authors.lastname: Cella
authors.forename: Lukman S
authors.initials: LS
authors.lastname: Sarker
authors.forename: Travis J
authors.initials: TJ
authors.lastname: Thompson
authors.forename: Mark R
authors.initials: MR
authors.lastname: Rheault
authors.forename: Soheil S
authors.initials: SS
authors.lastname: Mahmoud
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019755:Carbon-Carbon Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003511:Cyclohexanols / Q000378:metabolism; D018744:DNA, Plant / Q000235:genetics; D000077591:Eucalyptol; D020224:Expressed Sequence Tags; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D018745:Genome, Plant; D027523:Lavandula / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000378:metabolism; D009822:Oils, Volatile / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D003511:Cyclohexanols; D018744:DNA, Plant; D039821:Monoterpenes; D009822:Oils, Volatile; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; C019899:1,8-cineole synthetase; D019755:Carbon-Carbon Lyases; D000077591:Eucalyptol