Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus.     

Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants

The multitude of terpene carbon skeletons in plants is formed by enzymes known as terpene synthases. This review covers the monoterpene and sesquiterpene synthases presenting an up-to-date list of enzymes reported and evidence for their ability to form multiple products. The reaction mechanisms of these enzyme classes are described, and information on how terpene synthase proteins mediate catalysis is summarized. Correlations between specific amino acid motifs and terpene synthase function are described, including an analysis of the relationships between active site sequence and cyclization type and a discussion of whether specific protein features might facilitate multiple product formation.     

Protonation of a neutral (S)-beta-bisabolene intermediate is involved in (S)-beta-macrocarpene formation by the maize sesquiterpene synthases TPS6 and TPS11

Terpene synthases are responsible for the large diversity of terpene carbon skeletons found in plants. The unique, carbocationic reaction mechanism of these enzymes can form multiple products from a single prenyl diphosphate substrate. Two maize genes were isolated that encode very similar sesquiterpene synthases, TPS6 and TPS11, which both produce beta-bisabolene, a common monocyclic sesquiterpene, and beta-macrocarpene, an uncommon bicyclic olefin. Investigation of the reaction mechanism showed that the formation of beta-macrocarpene proceeds via a neutral beta-bisabolene intermediate and requires reprotonation by a proton that may ultimately be abstracted from water. This reprotonation is dependent on the pH and the presence of a Mg(2+) cofactor. Mutational analysis of the enzyme demonstrated that a highly conserved tyrosine residue in the active center of the enzymes is important for the protonation process. TPS6 and TPS11 are transcribed both in leaves and roots of maize, but the respective terpene products were only detected in roots. The expression in roots was up-regulated by herbivore damage to the leaves, suggesting a long distance signal transduction cascade between leaves and roots.     

桂花萜烯合酶(TPS)的生物信息学与原核表达分析

挥发性萜烯类化合物是植物花和果实中重要的香气活性物质,萜烯合酶(TPS)的种类和功能决定物种中萜烯类物质的多样性。桂花作为重要的香花植物,萜烯类物质是其花香的重要成分,但有关桂花萜烯合酶的研究却并不多。为揭示桂花萜烯类物质的生物合成机理,该研究利用生物信息学分析了4个TPS蛋白的理化性质、亚细胞定位及其结构,在原核表达系统中进行4个TPS蛋白的体外表达,并对可溶性的TPS4重组蛋白进行了体外酶反应功能分析。结果表明:(1) 4个TPS蛋白的理化性质差异较小,但仅有TPS4蛋白定位于其他靶向,不含信号肽,延伸链的比例较低,在靠近氨基端的区域内不含延伸链。(2) 4个TPS蛋白在原核系统中均可成功表达,但仅有TPS4获得了可溶性重组蛋白。(3)将纯化的TPS4重组蛋白分别与GPP、NDP和FPP进行反应,均只检测到一种产物,分别为反式-β-罗勒烯、β-水芹烯和α-法呢烯。这为揭示植物萜烯类物质生物合成的分子机理提供了参考,对从蛋白水平研究桂花花香基因功能奠定了基础。     

Evaluating type III polyketide synthase (PKS) and terpene synthase (TPS) expression in incompatible interactions of Zingiber zerumbet to Pythium myriotylum Drechsler

Abstract

Pythium species are aggressive soil-borne necrotrophic oomycetes causing soft-rot disease of ginger. Disease severity indices determined following infection with P. myriotylum Drechsler in ginger cultivar, Zingiber officinale cv. Varada and a wild congener, Z. zerumbet at varying zoospore concentrations (10⁴–10¹² spores/ml) revealed high disease severity (100%) in ginger cultivar whereas Z. zerumbet displayed resistance. Absence of positive correlation between Z. zerumbet resistance and polyphenolic content indicates role of polyketides and zerumbone in preventing pathogen ingress, as reported earlier. Towards elucidating this, Z. zerumbet specific polyketide synthase (PKS) and terpene synthase (TPS) gene sequences designated ZzTPS and ZzPKS respectively were characterised. Phylogenetic analysis clustered ZzTPS with TPS-b sub-family and ZzPKS with non-chalcone forming PKS. ZzTPS and ZzPKS showed biphasic expression with first at 6?hours post infection (hpi) and then at 8 hpi, indicative of rapid induction followed by reinforcement to sustain resistance mechanisms in the wild taxon.     

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.     

Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily

Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (-)-limonene synthase, (-)-alpha/beta-pinene synthase, and (-)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-alpha-farnesene synthase, and E-alpha-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.     

Isolation and characterization of terpene synthases in cotton (Gossypium hirsutum)

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.     

Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum.

A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi-aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes.     

Alteration of product formation by directed mutagenesis and truncation of the multiple-product sesquiterpene synthases delta-selinene synthase and gamma-humulene synthase

Two recombinant sesquiterpene synthases from grand fir, delta-selinene synthase and gamma-humulene synthase, each produce more than 30 sesquiterpene olefins from the acyclic precursor farnesyl diphosphate. These enzymes contain a pair of DDxxD motifs, on opposite lips of the presumptive active site, which are thought to be involved in substrate binding and could promote multiple orientations of the substrate alkyl chain from which multiple families of cyclic olefins could derive. Mutagenesis of the first aspartate of either DDxxD motif resulted in depressed k(cat), with lesser effect on K(m), for delta-selinene synthase and afforded a much simpler product spectrum composed largely of monocyclic olefins. Identical alterations in gamma-humulene synthase produced similar kinetic effects with a simplified product spectrum of mostly acyclic and monocyclic olefins. Although impaired in product diversity, none of the mutant synthases lost entirely the capacity to generate complex structures. These results confirm the catalytic significance of the DDxxD motifs and imply that they also influence permitted modes of cyclization. Deletion of an N-terminal arginine pair in delta-selinene synthase (an element potentially involved in substrate isomerization) altered kinetics without substantially altering product outcome. Finally, mutation of an active-site tyrosine residue thought to play a role in proton exchange had little influence; however, substitution of a nearby active site aspartate dramatically altered kinetics and product outcome.     

Demonstration and characterization of (E)-nerolidol synthase from maize: a herbivore-inducible terpene synthase participating in (3E)-4,8-dimethyl-1,3,7-nonatriene biosynthesis

Upon herbivore attack, maize (Zea mays L.) emits a mixture of volatile compounds that attracts herbivore enemies to the plant. One of the major components of this mixture is an unusual acyclic C₁₁ homoterpene, (3E )-4,8-dimethyl-1,3,7-nonatriene (DMNT), which is also emitted by many other species following herbivore damage. Biosynthesis of DMNT has been previously shown to proceed via the sesquiterpene alcohol, (E )-nerolidol. Here we demonstrate an enzyme activity that converts farnesyl diphosphate, the universal precursor of sesquiterpenes, to (3S)-(E )-nerolidol in cell-free extracts of maize leaves that had been fed upon by Spodoptera littoralis. The properties of this (E )-nerolidol synthase resemble those of other terpene synthases. Evidence for its participation in DMNT biosynthesis includes the direct incorporation of deuterium-labeled (E )-nerolidol into DMNT and the close correlation between increases in (E )-nerolidol synthase activity and DMNT emission after herbivore damage. Since farnesyl diphosphate has many other metabolic fates, (E )-nerolidol synthase may represent the first committed step of DMNT biosynthesis in maize. However, the formation of this unusual acyclic terpenoid appears to be regulated at both the level of (E )-nerolidol synthase and at later steps in the pathway. Received: 20 August 1999 / Accepted: 27 October 1999     

Terpene synthases and the regulation, diversity and biological roles of terpene metabolism

Terpene synthases are the primary enzymes in the formation of low-molecular-weight terpene metabolites. Rapid progress in the biochemical and molecular analysis of terpene synthases has allowed significant investigations of their evolution, structural and mechanistic properties, and regulation. The organization of terpene synthases in large gene families, their characteristic ability to form multiple products, and their spatial and temporal regulation during development and in response to biotic and abiotic factors contribute to the time-variable formation of a diverse group of terpene metabolites. The structural diversity and complexity of terpenes generates an enormous potential for mediating plant-environment interactions. Engineering the activities of terpene synthases provides opportunities for detailed functional evaluations of terpene metabolites in planta.     

Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence development

Despite the commercial importance of Lavandula angustifolia Mill. and L. x intermedia Emeric ex Loisel floral essential oils (EOs), no information is currently available on potential changes in individual volatile organic compound (VOC) content during inflorescence development. Calyces were found to be the main sites of VOC accumulation. The 20 most abundant VOCs could be separated into three sub‐groups according to their patterns of change in concentration The three groups of VOCs sequentially dominated the global scent bouquet of inflorescences, the transition between the first and second groups occurring around the opening of the first flower of the inflorescence and the one between the second and third groups at the start of seed set. Changes in calyx VOC accumulation were linked to the developmental stage of individual flowers. Leaves accumulated a smaller number of VOCs which were a subset of those seen in preflowering inflorescences. Their nature and content remained constant during the growing season. Quantitative real time polymerase chain reaction assessments of the expression of two terpene synthase (TPS) genes, LaLIMS and LaLINS, revealed similar trends between their patterns of expression and those of their VOC products. Molecular and chemical analyses suggest that changes in TPS expression occur during lavender inflorescence development and lead to changes in EO composition. Both molecular data and terpene analysis support the findings that changes in biosynthesis of terpene occurred during inflorescence development.     

Pseudomonas syringae elicits emission of the terpenoid (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene in Arabidopsis leaves via jasmonate signaling and expression of the terpene synthase TPS4

Volatile, low-molecular weight terpenoids have been implicated in plant defenses, but their direct role in resistance against microbial pathogens is not clearly defined. We have examined a possible role of terpenoid metabolism in the induced defense of Arabidopsis thaliana plants against leaf infection with the bacterial pathogen Pseudomonas syringae. Inoculation of plants with virulent or avirulent P. syringae strains induces the emission of the terpenoids (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT), beta-ionone and alpha-farnesene. While the most abundant volatile, the C16-homoterpene TMTT, is produced relatively early in compatible and incompatible interactions, emission of both beta-ionone and alpha-farnesene only increases in later stages of the compatible interaction. Pathogen-induced synthesis of TMTT is controlled through jasmonic acid (JA)-dependent signaling but is independent of a functional salicylic acid (SA) pathway. We have identified Arabidopsis T-DNA insertion lines with defects in the terpene synthase gene TPS4, which is expressed in response to P. syringae inoculation. The tps4 knockout mutant completely lacks induced emission of TMTT but is capable of beta-ionone and alpha-farnesene production, demonstrating that TPS4 is specifically involved in TMTT formation. The tps4 plants display at least wild type-like resistance against P. syringae, indicating that TMTT per se does not protect against the bacterial pathogen in Arabidopsis leaves. Similarly, the ability to mount SA-dependent defenses and systemic acquired resistance (SAR) is barely affected in tps4, which excludes a signaling function of TMTT during SAR. Besides P. syringae challenge, intoxication of Arabidopsis leaves with copper sulfate, a treatment that strongly activates JA biosynthesis, triggers production of TMTT, beta-ionone, and alpha-farnesene. Taken together, our data suggest that induced TMTT production in Arabidopsis is a by-product of activated JA signaling, rather than an effective defense response that contributes to resistance against P. syringae.     

Analysis of trehalose-6-phosphate synthase (TPS) gene family suggests the formation of TPS complexes in rice

Trehalose-6-phosphate (T6P), an intermediate in the trehalose biosynthesis pathway, is emerging as an important regulator of plant metabolism and development. T6P levels are potentially modulated by a group of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) homologues. In this study, we have isolated 11 TPS genes encoding proteins with both TPS and TPP domains, from rice. Functional complement assays performed in yeast tps1 and tps2 mutants, revealed that only OsTPS1 encodes an active TPS enzyme and no OsTPS protein possesses TPP activity. By using a yeast two-hybrid analysis, a complicated interaction network occurred among OsTPS proteins, and the TPS domain might be essential for this interaction to occur. The interaction between OsTPS1 and OsTPS8 in vivo was confirmed by bimolecular fluorescence complementation and coimmunoprecipitation assays. Furthermore, our gel filtration assay showed that there may exist two forms of OsTPS1 (OsTPS1a and OsTPS1b) with different elution profiles in rice. OsTPS1b was particularly cofractionated with OsTPS5 and OsTPS8 in the 360 kDa complex, while OsTPS1a was predominantly incorporated into the complexes larger than 360 kDa. Collectively, these results suggest that OsTPS family members may form trehalose-6-phosphate synthase complexes and therefore potentially modify T6P levels to regulate plant development.