Monoterpene Synthase Research Articles

A monoterpene synthase gene cluster of tea plant (Camellia sinensis) potentially involved in constitutive and herbivore-induced terpene formation

Monoterpenes and sesquiterpenes are the most abundant volatiles in tea plants and have dual functions in aroma quality formation and defense responses in tea plants. Terpene synthases (TPS) are the key enzymes for the synthesis of terpenes in plants; however, the functions of most of them in tea plants are still unknown. In this study, six putative terpene biosynthesis gene clusters were identified from the tea plant genome. Then we cloned three new TPS-b subfamily genes, CsTPS08, CsTPS10 and CsTPS58. In vitro enzyme assays showed that CsTPS08 and CsTPS58 are two multiple-product terpene synthases, with the former synthesizing linalool as the main product, and β-myrcene, α-phellandrene, α-terpinolene, D-limonene, cis-β-ocimene, trans-β-ocimene and (4E,6Z)-allo-ocimene as minor products are also detected, while the latter catalyzing the formation of α-pinene and D-limonene using GPP as the substrate. No product of CsTPS10 was detected in the prokaryotic expression system, but geraniol production was detected when transiently expressed in tobacco leaves. CsTPS08 and CsTPS10 are two functional members of a monoterpene synthase gene cluster, which were significantly induced during both Ectropis oblique feeding and fresh leaf spreading treatments, suggesting that they have dual functions involved in tea plant pest defense and tea aroma quality regulation. In addition, the differences in their expression levels in different tea plant cultivars provide a possibility for the subsequent screening of tea plant resources with a specific aroma flavor. Our results deepen the understanding of terpenoid synthesis in tea plants.

Isolation of Monoterpene Synthase Gene (NsTPS2) and Evaluation of Terpenoid Compounds in Black Cumin Medicinal Plant (Nigella sativa L.)

Isolation of Monoterpene Synthase Gene (NsTPS2) and Evaluation of Terpenoid Compounds in Black Cumin Medicinal Plant (Nigella sativa L.)

Monoterpene Synthase Genes and Monoterpene Profiles in Pinus nigra subsp. laricio.

In the present study, we carried out a quantitative analysis of the monoterpenes composition in different tissues of the non-model conifer Pinus nigra J.F. Arnold subsp. laricio Palib. ex Maire (P. laricio, in short). All the P. laricio tissues examined showed the presence of the same fourteen monoterpenes, among which the most abundant were β-phellandrene, α-pinene, and β-pinene, whose distribution was markedly tissue-specific. In parallel, from the same plant tissues, we isolated seven full-length cDNA transcripts coding for as many monoterpene synthases, each of which was found to be attributable to one of the seven phylogenetic groups in which the d1-clade of the canonical classification of plants’ terpene synthases can be subdivided. The amino acid sequences deduced from the above cDNA transcripts allowed to predict their putative involvement in the biosynthesis of five of the monoterpenes identified. Transcripts profiling revealed a differential gene expression across the different tissues examined, and was found to be consistent with the corresponding metabolites profiles. The genomic organization of the seven isolated monoterpene synthase genes was also determined.

Molecular Determinants of Carbocation Cyclisation in Bacterial Monoterpene Synthases.

Monoterpene synthases are often promiscuous enzymes, yielding product mixtures rather than pure compounds due to the nature of the branched reaction mechanism involving reactive carbocations. Two previously identified bacterial monoterpene synthases, a linalool synthase (bLinS) and a cineole synthase (bCinS), produce nearly pure linalool and cineole from geranyl diphosphate, respectively. We used a combined experimental and computational approach to identify critical residues involved in bacterial monoterpenoid synthesis. Phe77 is essential for bCinS activity, guiding the linear carbocation intermediate towards the formation of the cyclic α‐terpinyl intermediate; removal of the aromatic ring results in variants that produce acyclic products only. Computational chemistry confirmed the importance of Phe77 in carbocation stabilisation. Phe74, Phe78 and Phe179 are involved in maintaining the active site shape in bCinS without a specific role for the aromatic ring. Phe295 in bLinS, and the equivalent Ala301 in bCinS, are essential for linalool and cineole formation, respectively. Where Phe295 places steric constraints on the carbocation intermediates, Ala301 is essential for bCinS initial cyclisation and activity. Our multidisciplinary approach gives unique insights into how carefully placed amino acid residues in the active site can direct carbocations down specific paths, by placing steric constraints or offering stabilisation via cation‐π interactions.

Duplication and Specialization of NUDX1 in Rosaceae Led to Geraniol Production in Rose Petals.

Nudix hydrolases are conserved enzymes ubiquitously present in all kingdoms of life. Recent research revealed that several Nudix hydrolases are involved in terpenoid metabolism in plants. In modern roses, RhNUDX1 is responsible for formation of geraniol, a major compound of rose scent. Nevertheless, this compound is produced by monoterpene synthases in many geraniol-producing plants. As a consequence, this raised the question about the origin of RhNUDX1 function and the NUDX1 gene evolution in Rosaceae, in wild roses or/and during the domestication process. Here, we showed that three distinct clades of NUDX1 emerged in the Rosoidae subfamily (Nudx1-1 to Nudx1-3 clades), and two subclades evolved in the Rosa genus (Nudx1-1a and Nudx1-1b subclades). We also showed that the Nudx1-1b subclade was more ancient than the Nudx1-1a subclade, and that the NUDX1-1a gene emerged by a trans-duplication of the more ancient NUDX1-1b gene. After the transposition, NUDX1-1a was cis-duplicated, leading to a gene dosage effect on the production of geraniol in different species. Furthermore, the NUDX1-1a appearance was accompanied by the evolution of its promoter, most likely from a Copia retrotransposon origin, leading to its petal-specific expression. Thus, our data strongly suggest that the unique function of NUDX1-1a in geraniol formation was evolved naturally in the genus Rosa before domestication.

Unveiling Monoterpene Biosynthesis in Taiwania cryptomerioides via Functional Characterization.

Taiwania cryptomerioides is a monotypic species, and its terpenoid-rich property has been reported in recent years. To uncover monoterpene biosynthesis in T. cryptomerioides, this study used transcriptome mining to identify candidates with tentative monoterpene synthase activity. Along with the phylogenetic analysis and in vitro assay, two geraniol synthases (TcTPS13 and TcTPS14), a linalool synthase (TcTPS15), and a β-pinene synthase (TcTPS16), were functionally characterized. Via the comparison of catalytic residues, the Cys/Ser at region 1 might be crucial in determining the formation of α-pinene or β-pinene. In addition, the Cupressaceae monoterpene synthases were phylogenetically clustered together; they are unique and different from those of published conifer species. In summary, this study aimed to uncover the ambiguous monoterpenoid network in T. cryptomerioide, which would expand the landscape of monoterpene biosynthesis in Cupressaceae species.

Transcriptome sequencing reveals terpene biosynthesis pathway genes accounting for volatile terpene of tree peony.

Transcriptomic and volatile component analyses showed that high expression levels of genes from the terpenoid backbone biosynthesis pathway and the monoterpene metabolic pathway can strengthen the floral fragrance of tree peony. Floral fragrance is a crucial ornamental trait whose improvement is one of the primary objectives of tree peony breeding. So far, exploration of the floral fragrance of tree peony has focused on the identification of its volatile components, but the molecular mechanisms responsible for their formation remain unclear. Here, we identified 128 volatile components from the petals of tree peony and found that they consisted primarily of terpenes, alcohols, and esters. Based on the distribution pattern of these major fragrance components, 24 tree peony cultivars were classified into 4 types: grassy scent (ocimene), woody scent (longifolene), lily of the valley scent (linalool), and fruity scent (2-ethyl hexanol). We used RNA-seq to explore the mechanistic basis of terpenoid metabolism in tree peony petals with various scents. The expression levels of AACT, HMGR, PMK, DXS, DXR, HDS, HDR, and GGPS, which encode key enzymes of terpenoid backbone biosynthesis, were upregulated in 'Huangguan' (strong fragrance) compared to 'Fengdan' (faint fragrance). Moreover, the transcript abundance of LIS and MYS, two monoterpene synthase genes, was also enhanced in petals of 'Huangguan' compared to those of 'Fengdan'. Together, these results demonstrate that differences in the expression of genes from the monoterpene synthesis and terpenoid backbone pathways are associated with differences in the fragrance of tree peony. This research provides crucial genetic resources for fragrance improvement and also lays a foundation for further clarification of the mechanisms that underlie tree peony fragrance.

Key Enzymes Involved in the Synthesis of Hops Phytochemical Compounds: From Structure, Functions to Applications.

Humulus lupulus L. is an essential source of aroma compounds, hop bitter acids, and xanthohumol derivatives mainly exploited as flavourings in beer brewing and with demonstrated potential for the treatment of certain diseases. To acquire a comprehensive understanding of the biosynthesis of these compounds, the primary enzymes involved in the three major pathways of hops’ phytochemical composition are herein critically summarized. Hops’ phytochemical components impart bitterness, aroma, and antioxidant activity to beers. The biosynthesis pathways have been extensively studied and enzymes play essential roles in the processes. Here, we introduced the enzymes involved in the biosynthesis of hop bitter acids, monoterpenes and xanthohumol derivatives, including the branched-chain aminotransferase (BCAT), branched-chain keto-acid dehydrogenase (BCKDH), carboxyl CoA ligase (CCL), valerophenone synthase (VPS), prenyltransferase (PT), 1-deoxyxylulose-5-phosphate synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), Geranyl diphosphate synthase (GPPS), monoterpene synthase enzymes (MTS), cinnamate 4-hydroxylase (C4H), chalcone synthase (CHS_H1), chalcone isomerase (CHI)-like proteins (CHIL), and O-methyltransferase (OMT1). Furthermore, research advancements of each enzyme in terms of reaction conditions, substrate recognition, enzyme structures, and use in engineered microbes are described in depth. Hence, an extensive review of the key enzymes involved in the phytochemical compounds of hops will provide fundamentals for their applications in beer production.

Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

Identification and Molecular Characterization of Geraniol and Linalool Synthase Genes Related to Monoterpene Biosynthesis in Damask Rose (Rosa damascena Mill.), Several Genes for Little Scent

Rosa damascena Mill. (Damask rose) is a famous traditional flower that is widely appreciated for its valuable essential oil and high-quality fragrance used in the perfume and cosmetics industries. Rose oil contains more than 300 volatile constituents, and the main constituents geraniol, linalool, nerol, and citronellol determine the essential oil quality. In the present study, we reported the cloning and characterization of three rose monoterpene synthase genes related to rose oil quality. The genes encoding geranyl diphosphate synthase (GDPS) designated as RdGDP (accession No. MN650820), geraniol synthase (GES) as RdGES (accession No. MN639696), and linalool synthase (LIS) as RdLIS (accession No. MN639697) and involved in the monoterpene synthases pathways were successfully cloned from R. damascena and their expression profiles in different flower developmental stages were determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The deduced RdGDP, RdGES, and RdLIS proteins contain 383, 603, and 562 amino acids, and the corresponding size of their open-reading frames is 1152, 1812, and 1689 bp, respectively. Expression analysis revealed that these genes demonstrated persistent expressions from the budding to the full opening stages of R. damascena flower development, and the expression levels of RdLIS were lower than that of RdGDP and RdGES. These results suggest that the GDP and GES can regulate the biosynthesis of floral scents in Damask rose flowers. The cloning of rose genes, including RdGDP, RdGES, and RdLIS, could have far-reaching implications for improving the quality of R. damascena essential oils through metabolic engineering.

Metabolism and transcriptome profiling provides insight into the genes and transcription factors involved in monoterpene biosynthesis of borneol chemotype of Cinnamomum camphora induced by mechanical damage.

BackgroundThe borneol chemotype of Cinnamomum camphora (BCC), a monoterpene-rich woody plant species, is the sole source prescribed by the Chinese Pharmacopoeia for the production of natural D-borneol, a major monoterpene in BCC used for millennia as a topical analgesic in China. Nevertheless, the possible gene-regulatory roles of transcription factors (TFs) in BCC’s monoterpenoid biosynthesis remained unknown. Here, a joint analysis of the transcriptome and terpenoid metabolome of BCC induced by mechanical damage (MD) was used to comprehensively explore the interaction between TFs and terpene synthase (TPS) unigenes that might participate in monoterpene biosynthesis in BCC.ResultsGas chromatography–mass spectrometry analysis detected 14 monoterpenes and seven sesquiterpenes. All but two monoterpenes underwent a significantly increased accumulation after the MD treatment. RNA sequencing data revealed that 10 TPS, 82 MYB, 70 AP2/ERF, 38 BHLH, 31 WRKY, and 29 bZIP unigenes responded to the MD treatment. A correlation analysis revealed that three monoterpene synthase genes (CcTPS1, CcTPS3, CcTPS4) highly correlated with multiple monoterpenes, namely D-borneol, camphor, and bornyl acetate, which could be responsible for monoterpenoid biosynthesis in BCC. Furthermore, five WRKY, 15 MYB, 10 ERF/AP2, five bZIP, and two BHLH genes had strong, positive correlations with CcTPS1 or CcTPS4, judging by their high coefficient values (R2 > 0.8). The bioinformatics results were verified by quantitative real-time PCR.ConclusionThis study provides insight into the genes involved in the biosynthesis and regulation of monoterpene in BCC and thus provides a pool of candidate genes for future mechanistic analyses of how monoterpenes accumulate in BCC.

Biochemical and functional characterization of two microbial type terpene synthases from moss Stereodon subimponens

Terpenes constitute a large class of plant secondary metabolites. Usually, there is only one type of terpene synthase in seed plants, which is called typical plant terpene synthase. Currently, as a new family of plant terpene synthases, microbial terpene synthase-like (MTPSL) is identificated in nonseed plants. However, our knowledge about the biological function of most MTPSLs in nonseed plants is very limited. Here, we investigated the biochemical and functional characterization of the enzymes encoded by two MTPSLs from moss Stereodon subimponens, SsMTPSL1 and SsMTPSL2. A phylogenetic tree analysis showed that SsMTPSL1 and SsMTPSL2 are homologous to AaMTPSL1, AaMTPSL3, ApMTPSL1, and ApMTPSL3 from hornworts. The enzyme activity experiment demonstrated that SsMTPSL1 has monoterpene synthase and sesquiterpene synthase activity, and SsMTPSL2 has monoterpene synthase activity. Next, we selected SsMTPSL1 to study its biochemical functions. Anti-bacterial activity test in vitro showed that the products of SsMTPSL1 have an anti-bacterial effect on Escherichia coli, Pseudomonas syringae pv. Tomato DC3000 (Pst DC3000), and Staphylococcus aureus. To further understand the function of SsMTPSL1, the transgenic Arabidopsis thaliana plant of SsMTPSL1 is inoculated by Pst DC3000, and the result showed that SsMTPSL1 enhances the resistance of A. thaliana to Pst DC3000. All in all, this study provides new information about the functions of moss MTPSLs.

Combining Metabolic and Monoterpene Synthase Engineering for de Novo Production of Monoterpene Alcohols in Escherichia coli

The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli. We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production. Further, through site-directed mutation of LdtBPPS based on the structural simulation, we screened multiple variants that markedly elevated the production of acyclic nerol or bicyclic borneol, of which the LdtBPPSS488T mutant outperformed the wild-type LdtBPPS on borneol synthesis and the LdtBPPSF612A variant was superior to GmNES on nerol production. Subsequently, we overexpressed the endogenous Nudix hydrolase NudJ to facilitate the dephosphorylation of precursors and boosted the production of nerol and borneol from glucose. Finally, after the optimization of the fermentation process, the engineered strain ENO2 produced 966.55 mg/L nerol, and strain ENB57 generated 87.20 mg/L borneol in a shake flask, achieving the highest reported titers of nerol and borneol in microbes to date. This work shows a combinatorial engineering strategy for microbial production of natural terpene alcohols.

Monoterpene geraniol produced by rice terpene synthase 21 suppresses the expression of cell-division related genes in the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae

Plant volatile compounds such as monoterpenes and sesquiterpenes represent some of the classes of plant volatile compounds and they are involved in the plant defense responses. They are synthesized by terpene synthases (TPSs). The rice genome has reportedly more than 30 TPSs genes and several of them are regulated by a plant hormone, jasmonic acid (JA). In the present study, we analyzed a rice terpene synthase, OsTPS21, and investigated its potential antibacterial effects against Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial blight. OsTPS21 was localized in the chloroplast and produced a monoterpene geraniol as a monoterpene synthase. In addition, OsTPS21 did not exhibit any activity as a sesquiterpene synthase. The amount of geraniol in rice increased along with the upregulation of OsTPS21 after JA treatment. Geraniol had a negative effect on the growth of Xoo. The growth inhibition of Xoo by geraniol was caused by the suppression of expression of the cell-division-related genes of Xoo and not by damage to Xoo cell membranes. The results suggest that geraniol plays an important role in JA-induced resistance against Xoo and that it functions as an antibacterial compound in rice.

Volatile metabolic profiling and functional characterization of four terpene synthases reveal terpenoid diversity in different tissues of Chrysanthemum indicum L

Chrysanthemum indicum has long been used in traditional Chinese medicine for its health-promoting benefits. Studies on C. indicum have mainly focused on the flowers. Terpenoid distribution in various parts of the plant and characterization of terpene synthases remain unclear. In this study, volatile metabolic profiling was performed to compare the composition and quantity of terpenoids distributed in the root, stem, leaf, flower bud and flower of C. indicum. The potential for extracting active ingredients from the root, stem, and leaf was also examined. In total, 17 monoterpenoids and 27 sesquiterpenoids were identified. Transcriptome data were used to clone two monoterpene synthases and two sesquiterpene synthases highly expressed in the root. The recombinant proteins of full-length and truncated versions of C. indicum terpene synthase (CiTPS1) produced α-pinene, but the truncated one was catalytically more efficient than the full-length version. No product could be detected when full-length version of CiTPS2 was used for catalyzing GPP, but the truncated one can produce a minor amount of α-pinene. CiTPS3 contributed to the production of three sesquiterpenoids, namely β-farnesene, petasitene, and α-bisabolene. CiTPS4 acted as a difunctional enzyme, contributing to the production of four monoterpenoids and three sesquiterpenoids, including petasitene. The evidence suggests that petasitene and the genes responsible for its biosynthesis were first found in the genus Chrysanthemum. The present findings provide insights into the composition, formation, and regulation of these bioactive compounds.

Mining of trait specific gene candidates through mRNA sequencing emphasizing on expression study of terpenoid biosynthesis genes in betelvine cash crop

Betelvine (Piper betle L., Family: Piperaceae) is one of the important cultivated cash crop and medicinal plant of India, lacking sufficient molecular resources in various databases for exploration of biosynthetic pathways involved in terpenoid synthesis. Molecular markers like SSRs (Simple Sequence Repeats) and SNPs (Single Nucleotide Polymorphisms) are generally used for genetic diversity study. However, limited number of nucleotides and absence of SSRs as well as SNPs restrict molecular study of betelvine landraces. These problems also minimise molecular characterization of novel genes that are involved in terpenoid biosynthesis. To elucidate the biochemical pathways of terpenoid biosynthesis, transcriptome profiling of RNA sample was performed from leaves of betelvine. The pair end transcriptome profiling generated 6.7 G bases (4.4Gb) of data containing 33,235,667 raw reads for each end of RNA Seq. After removing contaminants, 30,104,811 clean reads were subjected to de novo transcriptome assembling, which subsequently produced 46,656 clean transcripts. The clean transcripts were annotated and their functions were identified using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Eukaryotic Orthologous Groups (KOG), PlantCyc, Swissprot, and TrEMBL database. These annotations resulted mining of various trait specific genes including important terpenoid biosynthesis genes. Presence of 43 terpenoid compounds in essential oil of four betelvine landraces has also been confirmed through GCMS. Further, gene expression analysis of five terpenoid biosynthesis genes were carried out by real time polymerase chain reaction (RT PCR). The expression of monoterpene synthase genes (limonene synthase, linalool synthase, and α-terpeneol synthase) and sesquiterpene synthase genes (germacrene synthase and sesquiterpene) were validated using four landraces of betelvine. Landraces specific high quality 4181 SSRs and 350,676 SNPs were identified for further genetic diversity study. In this study, terpenoid biosynthesis specific transcription factors and 22 miRNAs having 33 target sites were also predicted. Overall, this study will enrich the molecular repository of betelvine for further improvement of the taxa and to develop new genotype of betelvine crop with quality traits.

A microfactory in yeast peroxisomes

Synthetic Biology Considerable synthetic biology efforts are focused on engineering yeast to produce valuable metabolites. Production in the cytosol can be challenging because of toxicity or crosstalk with cellular pathways. Dusseaux et al. harnessed the yeast peroxisome to produce geranyl diphosphate (GPP), a precursor to monoterpenoids, monoterpene indole alkoids, and cannabinoids. Targeting the entire pathway for GPP synthesis, along with an enzyme that converts GPP to the monoterpene limonene, to the peroxisome gives a 125-fold improvement in yield of limonene compared with production in the cytoplasm. Additional monoterpenes can be synthesized with the same platform using the appropriate monoterpene synthase. Peroxisomal production also facilitates downstream processing involving oxidation by cytochrome P450 enzymes. Peroxisome microfactories could be used for the modular assembly and optimization of other complex pathways. Proc. Natl. Acad. Sci. U.S.A. 117 , 31789 (2020).

Five TPSs are responsible for volatile terpenoid biosynthesis in Albizia julibrissin

Silk tree, Albizia julibrissin Duraz, is an old ornamental plant and extensively cultivated in Asia. Previous works have discovered that the terpenoids were the dominating compounds in the floral VOC of A. julibrissin, however the biosynthesis of these terpenoids was poorly understood so far. Here, 11 terpene synthase genes (TPSs) were identified by transcriptome sequencing that fell into TPS-a, TPS-b and TPS-g subfamilies. The enzymatic activity tests showed that five genes were functional: AjTPS2 was a sesquiterpene synthase and produced α-farnesene and (Z, E)-β-farnesene; AjTPS5 was able to catalyze the formation of five monoterpenes and nine sesquiterpenes; AjTPS7, AjTPS9 and AjTPS10 were dedicated monoterpene synthases, as AjTPS7 and AjTPS10 formed the single product β-ocimene and linalool, respectively, and AjTPS9 produced γ-terpinene with other three monoterpenes. More importantly, the main catalytic products of the characterized AjTPSs were consistent with the terpenoids observed in A. julibrissin volatiles. Combining terpene chemistry, TPSs biochemical activities and gene expression analysis, we demonstrate that AjTPS2, AjTPS5, AjTPS7, AjTPS9 and AjTPS10 are responsible for the volatile terpenoids biosynthesis in A. julibrissin.

The carrot monoterpene synthase gene cluster on chromosome 4 harbours genes encoding flavour-associated sabinene synthases

In plants, low molecular weight terpenes produced by terpene synthases (TPS) contribute to multiple ecologically and economically important traits. The present study investigates a carrot terpene synthase gene cluster on chromosome 4 associated with volatile monoterpene production. Two carrot mutants, yellow and cola, which are contrasting in the content of low molecular weight terpenes, were crossed to develop an F2 mapping population. The mapping analysis revealed overlapping QTLs on chromosome 4 for sabinene, α-thujene, α-terpinene, γ-terpinene, terpinen-4-ol and 4-carene. The genomic region of this locus includes a cluster of five terpene synthase genes (DcTPS04, DcTPS26, DcTPS27, DcTPS54 and DcTPS55). DcTPS04 and DcTPS54 displayed genotype- and tissue-specific variation in gene expression. Based on the QTL mapping results and the gene expression patterns, DcTPS04 and DcTPS54 were selected for functional characterization. In vitro enzyme assays showed that DcTPS54 is a single-product enzyme catalysing the formation of sabinene, whereas DcTPS04 is a multiple-product terpene synthase producing α-terpineol as a major product and four additional products including sabinene, β-limonene, β-pinene and myrcene. Furthermore, we developed a functional molecular marker that could discriminate carrot genotypes with different sabinene content in a set of 85 accessions.

Monoterpene synthases responsible for the terpene profile of anther glands in Eucalyptus polybractea R.T. Baker (Myrtaceae).

Research on terpene biosynthesis in the genus Eucalyptus (Myrtaceae) is poorly developed, but recently large numbers of terpene synthase (TPS) genes have been identified. Few of these have been characterized or their expression localized to specific tissues. A prime candidate for detailed examination of TPS gene expression is the bisexual eucalypt flower-composed of male and female reproductive organs, and vegetative tissues that may express different TPS genes. We aimed to characterize and compare the terpene profile and TPS genes expressed in anthers and gynoecia in the high oil-yielding Eucalyptus polybractea R.T. Baker. We hypothesized that gynoecia will produce greater amounts of defensive terpenes, whereas anthers will have a terpene profile that is biased towards a role in pollination. Microscopy of isolated anthers showed them to possess a single, prominent oil gland. Chemical analysis of whole floral structures at different stages of development showed total oil per unit dry mass increased as flower buds expanded, with highest concentrations in mature flower buds just prior to flower opening. The oil profile of gynoecia was dominated by the monoterpene 1,8-cineole, whereas that of isolated anthers were enriched with the monoterpene α-pinene. Through transcriptomic analysis and recombinant protein expression, we were able to identify monoterpene synthases responsible for the different profiles. Synthases for α-pinene and 1,8-cineole were expressed in each tissue type, but the relative expression of the former was higher in anthers. Sequence comparison and site-directed mutagenesis of the α-pinene synthase allowed us to identify amino acids that influence the α-pinene to β-pinene ratio of the product profile. We suggest the terpene constituents of anthers may have multiple roles including attracting pollinators through emission of volatile α-pinene, deterrence of palynivores through emission of volatile 1,8-cineole and adhesion of pollen to pollinators via the release of sticky α-pinene onto the anther surface.