Publications

• Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Picea spp.).

  Keeling C.I., Weisshaar S., Ralph S.G., Jancsik S., Hamberger B., Dullat H.K., Bohlmann J.

  <h4>Background</h4>In conifers, terpene synthases (TPSs) of the gymnosperm-specific TPS-d subfamily form a diverse array of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions and volatile emissions. These compounds contribute to defence against herbivores a ... >> More

  <h4>Background</h4>In conifers, terpene synthases (TPSs) of the gymnosperm-specific TPS-d subfamily form a diverse array of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions and volatile emissions. These compounds contribute to defence against herbivores and pathogens and perhaps also protect against abiotic stress.<h4>Results</h4>The availability of extensive transcriptome resources in the form of expressed sequence tags (ESTs) and full-length cDNAs in several spruce (Picea) species allowed us to estimate that a conifer genome contains at least 69 unique and transcriptionally active TPS genes. This number is comparable to the number of TPSs found in any of the sequenced and well-annotated angiosperm genomes. We functionally characterized a total of 21 spruce TPSs: 12 from Sitka spruce (P. sitchensis), 5 from white spruce (P. glauca), and 4 from hybrid white spruce (P. glauca × P. engelmannii), which included 15 monoterpene synthases, 4 sesquiterpene synthases, and 2 diterpene synthases.<h4>Conclusions</h4>The functional diversity of these characterized TPSs parallels the diversity of terpenoids found in the oleoresin and volatile emissions of Sitka spruce and provides a context for understanding this chemical diversity at the molecular and mechanistic levels. The comparative characterization of Sitka spruce and Norway spruce diterpene synthases revealed the natural occurrence of TPS sequence variants between closely related spruce species, confirming a previous prediction from site-directed mutagenesis and modelling. << Less

  BMC Plant Biol. 11:43-43(2011) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis).

  Bohlmann J., Phillips M., Ramachandiran V., Katoh S., Croteau R.B.

  Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesi ... >> More

  Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)) and geranylgeranyl diphosphate (C(20)), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50-60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C(15)) synthases and diterpene (C(20)) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure-function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization-cyclization reaction. << Less

  Arch. Biochem. Biophys. 368:232-243(1999) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Monoterpene synthases of Pinus contorta and related conifers. A new class of terpenoid cyclase.

  Savage T.J., Hatch M.W., Croteau R.

  A cell-free extract from the xylem of lodgepole pine (Pinus contorta) catalyzes the conversion of [1-3H1]geranyl pyrophosphate to a variety of monoterpene olefins found in lodgepole pine oleoresin. This monoterpene synthase activity is similar to previously described terpenoid cyclases from grand ... >> More

  A cell-free extract from the xylem of lodgepole pine (Pinus contorta) catalyzes the conversion of [1-3H1]geranyl pyrophosphate to a variety of monoterpene olefins found in lodgepole pine oleoresin. This monoterpene synthase activity is similar to previously described terpenoid cyclases from grand fir (Abies grandis) and other higher plants in molecular mass (67 +/-2 kDa as estimated by size-exclusion chromatography), Km for geranyl pyrophosphate (7.8 +/- 1.9 microM), and isoelectric point (4.75 +/-0.2 as determined by isoelectric focusing), but the cyclases from both lodgepole pine and grand fir are unlike previously characterized terpenoid cyclases from angiosperms and fungi, in that they have an alkaline pH optimum (pH 7.8), are activated by K+, Rb+, Cs+, or NH+4 (Li+ and Na+ are not effective), require either Mn2+ or Fe2+ as divalent metal ion cofactors (Mg2+ is not effective), and are not protected by the substrate-metal ion complex against inhibition by the histidine-directed reagent diethyl pyrocarbonate. Chromatography of the pine xylem extracts on a quaternary amino anion-exchange resin results in the separation of four similar, but distinct, multiple product monoterpene synthases that produce sabinene, beta-phellandrene, 3-carene, and beta-pinene as the principal components, respectively. The major cyclase (phellandrene synthase) was subsequently purified by hydroxyapatite chromatography and electrophoresis. V8 proteolysis provided a peptide map significantly different from that obtained with limonene synthase from spearmint (Mentha spicata), and limited NH2-terminal sequencing of the phellandrene synthase fragments revealed no significant similarity to the deduced amino acid sequence of the angiosperm limonene synthase, the only monoterpene cyclase to be cloned and sequenced thus far. Furthermore, polyclonal antibodies raised against the angiosperm limonene synthase did not detectably cross-react with any proteins in extracts from either lodgepole pine or grand fir by immunoblotting analysis. In addition to these structural differences between cyclases from conifers and herbaceous angiosperms, the unusual pH optimum, mono- and divalent metal ion requirement, and reactivity toward histidine carbethoxylation indicate that monoterpene cyclases isolated from conifers may also have a different complement of active-site amino acid residues involved in substrate binding and catalysis than those of terpenoid cyclases previously isolated from angiosperms. << Less

  J Biol Chem 269:4012-4020(1994) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Hydride shifts in the biosynthesis of the p-menthane monoterpenes alpha-terpinene, gamma-terpinene, and beta-phellandrene.

  LaFever R.E., Croteau R.

  The biosynthesis of several monoterpenes from the acyclic precursor geranyl pyrophosphate requires the migration of positive charge from the isopropyl side chain into the cyclohexenoid ring of the universal alpha-terpinyl cation intermediate of the reaction. The hydride shifts responsible for char ... >> More

  The biosynthesis of several monoterpenes from the acyclic precursor geranyl pyrophosphate requires the migration of positive charge from the isopropyl side chain into the cyclohexenoid ring of the universal alpha-terpinyl cation intermediate of the reaction. The hydride shifts responsible for charge migration in the formation of three p-menthane olefin isomers were examined in a range of plant species: alpha-terpinene in American wormseed, gamma-terpinene in thyme, and (-)-beta-phellandrene in lodgepole pine. The experimental approach utilized soluble cell-free enzyme systems and specifically labeled geranyl substrates, with the determination of the labeling patterns in the resulting cyclic products. The results were consistent with stereoelectronic features of the cyclization and support the general model for monoterpene formation. << Less

  Arch Biochem Biophys 301:361-366(1993) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.