geranyl-pyrophosphate and methyl-jasmonate

Plant terpenoids are a large and highly diverse class of metabolites with an important role in the immune defense. They find wide industrial application as active pharmaceutical ingredients, aroma and fragrance compounds. Several Eremophila sp. derived terpenoids have been documented. To elucidate the terpenoid metabolism, the transcriptome of juvenile and mature Eremophila serrulata (A.DC.) Druce (Scrophulariaceae) leaves was sequenced and a transcript library was generated. We report on the first transcriptomic dataset of an Eremophila plant. IlluminaMiSeq sequencing (2 × 300 bp) revealed 7,093,266 paired reads, which could be assembled to 34,505 isogroups. To enable detection of terpene biosynthetic genes, leaves were separately treated with methyl jasmonate, a well-documented inducer of plant secondary metabolites. In total, 21 putative terpene synthase genes were detected in the transcriptome data. Two terpene synthase isoenzymatic genes, termed ES01 and ES02, were successfully expressed in E. coli. The resulting proteins catalyzed the conversion of geranyl pyrophosphate, the universal substrate of monoterpene synthases to myrcene and Z-(b)-ocimene, respectively. The transcriptomic data and the discovery of the first terpene synthases from Eremophila serrulata are the initial step for the understanding of the terpene metabolism in this medicinally important plant genus.

Topics: Acetates; Acyclic Monoterpenes; Alkenes; Alkyl and Aryl Transferases; Australia; Cyclopentanes; Eremophila Plant; Escherichia coli; Gene Expression Profiling; Intramolecular Lyases; Monoterpenes; Oxylipins; Plant Proteins; Polyisoprenyl Phosphates; Scrophulariaceae; Terpenes

Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.

Topics: Acetates; Alkyl and Aryl Transferases; Bicyclic Monoterpenes; Bridged Bicyclo Compounds; Cyclohexane Monoterpenes; Cyclohexenes; Cyclopentanes; Gas Chromatography-Mass Spectrometry; Gossypium; Intramolecular Lyases; Monocyclic Sesquiterpenes; Monoterpenes; Oxylipins; Phytoalexins; Polycyclic Sesquiterpenes; Polyisoprenyl Phosphates; Sesquiterpenes; Sesquiterpenes, Guaiane; Terpenes; Volatile Organic Compounds

Cotton (Gossypium hirsutum L.) plants damaged by insects emit a blend of volatiles, including monoterpenes and sesquiterpenes, which can directly repel herbivores and/or indirectly protect the plant by attracting natural enemies of the herbivores. To understand the molecular basis of terpene biosynthesis and regulation in cotton, two terpene synthase genes, GhTPS1 and GhTPS2, were heterologously expressed and characterized. Recombinant GhTPS1 accepted farnesyl pyrophosphate as substrate and produced (E)-β-caryophyllene and α-humulene. GhTPS2 was characterized as a monoterpene synthase which formed α-pinene and β-pinene using geranyl pyrophosphate as substrate. Quantitative real-time PCR analysis revealed that GhTPS1 and GhTPS2 gene expression was elevated after methyl jasmonate (MeJA) treatment in cotton leaves. Moreover, feeding of the green plant bug Apolygus lucorum, a major cotton pest in northern China, resulted in increased GhTPS2 expression in young leaves, suggesting that GhTPS2 might be involved in plant defense in cotton.

Topics: Acetates; Adaptation, Physiological; Alkyl and Aryl Transferases; Animals; Bicyclic Monoterpenes; Bridged Bicyclo Compounds; Carbon-Oxygen Lyases; China; Cyclopentanes; Gene Expression; Genes, Plant; Gossypium; Herbivory; Insecta; Monocyclic Sesquiterpenes; Monoterpenes; Oxylipins; Plant Diseases; Plant Leaves; Plant Proteins; Polycyclic Sesquiterpenes; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes

Norway spruce (Picea abies) defends itself against herbivores and pathogens by formation of traumatic resin ducts filled with terpenoid-based oleoresin. An important group of enzymes in terpenoid biosynthesis are the short-chain isoprenyl diphosphate synthases which produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of monoterpenes, sesquiterpenes, and diterpene resin acids, respectively. After treatment with methyl jasmonate (MJ) we investigated the expression of all isoprenyl diphosphate synthase genes characterized to date from Norway spruce and correlated this with formation of traumatic resin ducts and terpene accumulation. Formation of traumatic resin ducts correlated with higher amounts of monoterpenes, sesquiterpenes and diterpene resin acids and an upregulation of isoprenyl diphosphate synthase genes producing geranyl diphosphate or geranylgeranyl diphosphate. Among defense hormones, jasmonate and jasmonate-isoleucine conjugate accumulated to higher levels in trees with extensive traumatic resin duct formation, whereas salicylate did not. Jasmonate and ethylene are likely to both be involved in formation of traumatic resin ducts based on elevated transcripts of genes encoding lipoxygenase and 1-aminocyclopropane-1-carboxylic acid oxidase associated with resin duct formation. Other genes involved in defense signalling in other systems, mitogen-activated protein kinase3 and nonexpressor of pathogenesis-related gene1, were also associated with traumatic resin duct formation. These responses were detected not only at the site of MJ treatment, but also systemically up to 60 cm above the site of treatment on the trunk.

Topics: Acetates; Alkyl and Aryl Transferases; Cyclopentanes; Diphosphates; Diterpenes; Oxylipins; Picea; Plant Growth Regulators; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes

Geranyl diphosphate (GPP), the universal precursor of monoterpenes, is formed from isopentenyl diphosphate and dimethylallyl diphosphate by the action of geranyl diphosphate synthase, one of the key branchpoint enzymes of terpene biosynthesis. Three types of GPP synthase can be distinguished in plants based on sequence similarity and subunit architecture, but until now individual species have been reported to contain only one of these types. Here we show that the conifer, Norway spruce (Picea abies), contains two different types of GPP synthase belonging to two separate groups of homodimeric proteins. One enzyme, designated PaIDS2 (P. abies isoprenyl diphosphate synthase 2), has high sequence similarity to other gymnosperm GPP synthases. It produces solely GPP in in vitro assays after expression in Escherichia coli and likely participates in monoterpene biosynthesis accompanying induced oleoresin formation, based on dramatic increases in transcript level after methyl jasmonate application. The other enzyme, designated PaIDS3, has highest similarity to the previously reported Arabidopsis thaliana GPP synthase and several other angiosperm sequences, and is not associated with induced oleoresin formation in Norway spruce. In vitro assay of this protein and one encoded by a similar gene sequence from Quercus robur gave substantial amounts of the larger prenyl diphosphates, FPP and GGPP, in addition to GPP. Hence these proteins may not be involved in monoterpene formation and could conceivably form products in addition to GPP in planta.

Topics: Acetates; Alkyl and Aryl Transferases; Amino Acid Sequence; Cloning, Molecular; Cyclopentanes; Diphosphates; Diterpenes; DNA, Complementary; Gene Expression Regulation, Enzymologic; Molecular Sequence Data; Oxylipins; Phylogeny; Picea; Quercus; Terpenes