03410nas a2200289   4500008004100000245014600041210006900187260000800256300001300264490000800277520240500285653002802690653002402718653002302742653002002765653001902785653002802804653002402832653001702856653002102873653001902894653001402913100002102927700002102948700002002969856013102989       2005                        eng d00aMonoterpene metabolism. Cloning, expression, and characterization of (-)-isopiperitenol/(-)-carveol dehydrogenase of peppermint and spearmint0 aMonoterpene metabolism Cloning expression and characterization o  cMar  a863–720 v1373 aThe essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicata) are distinguished by the oxygenation position on the p-menthane ring of the constitutive monoterpenes that is conferred by two regiospecific cytochrome P450 limonene-3- and limonene-6-hydroxylases. Following hydroxylation of limonene, an apparently similar dehydrogenase oxidizes (-)-trans-isopiperitenol to (-)-isopiperitenone in peppermint and (-)-trans-carveol to (-)-carvone in spearmint. Random sequencing of a peppermint oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes, including dehydrogenases. Full-length dehydrogenase clones were screened by functional expression in Escherichia coli using a recently developed in situ assay. A single full-length acquisition encoding (-)-trans-isopiperitenol dehydrogenase (ISPD) was isolated. The (-)-ISPD cDNA has an open reading frame of 795 bp that encodes a 265-residue enzyme with a calculated molecular mass of 27,191. Nondegenerate primers were designed based on the (-)-trans-ISPD cDNA sequence and employed to screen a spearmint oil gland secretory cell cDNA library from which a 5'-truncated cDNA encoding the spearmint homolog, (-)-trans-carveol-dehydrogenase, was isolated. Reverse transcription-PCR amplification and RACE were used to acquire the remaining 5'-sequence from RNA isolated from oil gland secretory cells of spearmint leaf. The full-length spearmint dehydrogenase shares >99% amino acid identity with its peppermint homolog and both dehydrogenases are capable of utilizing (-)-trans-isopiperitenol and (-)-trans-carveol. These isopiperitenol/carveol dehydrogenases are members of the short-chain dehydrogenase/reductase superfamily and are related to other plant short-chain dehydrogenases/reductases involved in secondary metabolism (lignan biosynthesis), stress responses, and phytosteroid biosynthesis, but they are quite dissimilar (approximately 13% identity) to the monoterpene reductases of mint involved in (-)-menthol biosynthesis. The isolation of the genes specifying redox enzymes of monoterpene biosynthesis in mint indicates that these genes arose from different ancestors and not by simple duplication and differentiation of a common progenitor, as might have been anticipated based on the common reaction chemistry and structural similarity of the substrate monoterpenes.10aAlcohol Oxidoreductases10aAmino Acid Sequence10aCloning: Molecular10aMentha piperita10aMentha spicata10aMolecular Sequence Data10aMolecular Structure10aMonoterpenes10aMultigene Family10aOils: Volatile10aPhylogeny1 aRinger, Kerry, L1 aDavis, Edward, M1 aCroteau, Rodney  uhttps://lamiaceae.myspecies.info/content/monoterpene-metabolism-cloning-expression-and-characterization-isopiperitenol-carveol02657nas a2200325   4500008004100000245016800041210006900209260000800278300001400286490000800300520151200308653002401820653003801844653002201882653002801904653002401932653002101956653001401977653003401991653001302025653001302038100001702051700003202068700001802100700001902118700001502137700002202152700002002174856013702194       2004                        eng d00aThe biochemical and molecular basis for the divergent patterns in the biosynthesis of terpenes and phenylpropenes in the peltate glands of three cultivars of basil0 abiochemical and molecular basis for the divergent patterns in th  cNov  a3724–360 v1363 aSurface glandular trichomes distributed throughout the aerial parts of sweet basil (Ocimum basilicum) produce and store monoterpene, sesquiterpene, and phenylpropene volatiles. Three distinct basil chemotypes were used to examine the molecular mechanisms underlying the divergence in their monoterpene and sesquiterpene content. The relative levels of specific terpenes in the glandular trichomes of each cultivar were correlated with the levels of transcripts for eight genes encoding distinct terpene synthases. In a cultivar that produces mostly (R)-linalool, transcripts of (R)-linalool synthase (LIS) were the most abundant of these eight. In a cultivar that synthesizes mostly geraniol, transcripts of geraniol synthase were the most abundant, but the glands of this cultivar also contained a transcript of an (R)-LIS gene with a 1-base insertion that caused a frameshift mutation. A geraniol synthase-LIS hybrid gene was constructed and expressed in Escherichia coli, and the protein catalyzed the formation of both geraniol and (R)-linalool from geranyl diphosphate. The total amounts of terpenes were correlated with total levels of terpene synthase activities, and negatively correlated with levels of phenylpropanoids and phenylalanine ammonia lyase activity. The relative levels of geranyl diphosphate synthase and farnesyl diphosphate synthase activities did not correlate with the total amount of terpenes produced, but showed some correlation with the ratio of monoterpenes to sesquiterpenes.10aAmino Acid Sequence10aGene Expression Regulation: Plant10aGenetic Variation10aMolecular Sequence Data10aMolecular Structure10aOcimum basilicum10aPhylogeny10aSequence Homology: Amino Acid10aStyrenes10aTerpenes1 aIijima, Yoko1 aDavidovich-Rikanati, Rachel1 aFridman, Eyal1 aGang, David, R1 aBar, Einat1 aLewinsohn, Efraim1 aPichersky, Eran  uhttps://lamiaceae.myspecies.info/content/biochemical-and-molecular-basis-divergent-patterns-biosynthesis-terpenes-and-phenylpropenes02853nas a2200349   4500008004100000245018100041210006900222260000800291300001400299490000800313520161000321653001001931653002401941653002301965653002101988653002302009653002702032653004402059653003802103653002802141653001402169653001702183653001902200653001402219653002702233653003402260653001202294100003202306700001902338700001802357856012802375       2004                        eng d00aCloning, functional expression, and characterization of the raffinose oligosaccharide chain elongation enzyme, galactan:galactan galactosyltransferase, from common bugle leaves0 aCloning functional expression and characterization of the raffin  cApr  a1377–870 v1343 aGalactan:galactan galactosyltransferase (GGT) is a unique enzyme of the raffinose family oligosaccharide (RFO) biosynthetic pathway. It catalyzes the chain elongation of RFOs without using galactinol (alpha-galactosyl-myoinositol) by simply transferring a terminal alpha-galactosyl residue from one RFO molecule to another one. Here, we report the cloning and functional expression of a cDNA encoding GGT from leaves of the common bugle (Ajuga reptans), a winter-hardy long-chain RFO-storing Lamiaceae. The cDNA comprises an open reading frame of 1215 bp. Expression in tobacco (Nicotiana plumbaginifolia) protoplasts resulted in a functional recombinant protein, which showed GGT activity like the previously described purified, native GGT enzyme. At the amino acid level, GGT shows high homologies (>60%) to acid plant alpha-galactosidases of the family 27 of glycosylhydrolases. It is clearly distinct from the family 36 of glycosylhydrolases, which harbor galactinol-dependent raffinose and stachyose synthases as well as alkaline alpha-galactosidases. Physiological studies on the role of GGT confirmed that GGT plays a key role in RFO chain elongation and carbon storage. When excised leaves were exposed to chilling temperatures, levels of GGT transcripts, enzyme activities, and long-chain RFO concentrations increased concomitantly. On a whole-plant level, chilling temperatures induced GGT expression mainly in the roots and fully developed leaves, both known RFO storage organs of the common bugle, indicating an adaptation of the metabolism from active growth to transient storage in the cold.10aAjuga10aAmino Acid Sequence10aCloning: Molecular10aCold Temperature10aDNA: Complementary10aGalactosyltransferases10aGene Expression Regulation: Enzymologic10aGene Expression Regulation: Plant10aMolecular Sequence Data10aPhylogeny10aPlant Leaves10aPlant Proteins10aRaffinose10aSequence Analysis: DNA10aSequence Homology: Amino Acid10aTobacco1 aTapernoux-Lüthi, Esther, M1 aBöhm, Andreas1 aKeller, Felix  uhttps://lamiaceae.myspecies.info/content/cloning-functional-expression-and-characterization-raffinose-oligosaccharide-chain02732nas a2200325   4500008004100000245008100041210006900122260000800191300001200199490000800211520172200219653003201941653002401973653002301997653001502020653002002035653001702055653002802072653002102100653001402121653002102135653003402156653001302190100001702203700001902220700001802239700002202257700002002279856010702299       2004                        eng d00aCharacterization of geraniol synthase from the peltate glands of sweet basil0 aCharacterization of geraniol synthase from the peltate glands of  cJan  a370–90 v1343 aThe monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn2+ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 microM were obtained for geranyl diphosphate and Mn2+, respectively. In the presence of 18O-labeled water, GES catalyzed the formation of 18O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral.10aAlkyl and Aryl Transferases10aAmino Acid Sequence10aDNA: Complementary10aDNA: Plant10aGene Expression10aGenes: Plant10aMolecular Sequence Data10aOcimum basilicum10aPhylogeny10aPlant Structures10aSequence Homology: Amino Acid10aTerpenes1 aIijima, Yoko1 aGang, David, R1 aFridman, Eyal1 aLewinsohn, Efraim1 aPichersky, Eran  uhttps://lamiaceae.myspecies.info/content/characterization-geraniol-synthase-peltate-glands-sweet-basil