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Lavender species are of significant economic value being cultivated extensively worldwide for their essential oils (EOs), which include terpenes that play crucial roles in the cosmetic, personal care, and pharmaceutical industries. The terpene synthases in lavender, such as Lavandula angustifolia linalool synthase (LaLINS), limonene synthase (LaLIMS), and bergamotene synthase (LaBERS), are key enzymes in terpene biosynthesis. However, the functional mechanisms underlying these enzymes remain poorly understood. Here, we used AlphaFold2 to predict the three-dimensional structures of LaLINS, LaLIMS, and LaBERS. The hydrodynamic radii of LaLINS, LaLIMS, and LaBERS were 5.7 ± 0.2, 6.2 ± 0.3, and 5.4 ± 0.2 nm, respectively. Mutations D320A or D324A led to a complete loss of activity in LaLINS compared to the wild-type (WT) enzyme; similarly, mutations D356A or D360A abolished activity in LaLIMS, and D291A or D295A eliminated activity in LaBERS. Furthermore, the genes LaLINS , LaLIMS , and LaBERS exhibited significantly higher expression levels in leaves compared to stems and flowers, with peak expression occurring at 8:00 a.m. Our findings contribute to a deeper understanding of terpene biosynthesis in lavender and offer insights for improving essential oil production through genetic engineering. [ABSTRACT FROM AUTHOR]

The root of Paeonia lactiflora pall. is a significant component of traditional Chinese medicine, with terpenoids and their glycosides, such as paeoniflorins, serving as key active ingredients known for their anti-inflammatory, hepatoprotective, and analgesic properties. By generating a transcriptome and functionally characterizing 32 terpene synthases (TPSs) from P. lactiflora, we successfully constructed 24 pESC-Trp-PlTPS expression vectors. Through expression in Saccharomyces cerevisiae engineered strains, we identified four mono-TPSs and five sesqui-TPSs that produce 18 compounds, including eight monoterpenes and ten sesquiterpenes in vitro. This includes a bifunctional enzyme (PlTPS22). Additionally, PlTPS21 was characterized as a pinene synthase with α-pinene as its main product. The expression pattern of PlTPS21 aligns closely with the accumulation patterns of paeoniflorins and α-pinene in the plant, suggesting that PlTPS21 is a key enzyme in the biosynthesis of paeoniflorin. [ABSTRACT FROM AUTHOR]

Lauraceae, an important family of Angiospermae, comprises over 2500 species widely distributed in tropical and subtropical evergreen broad-leaved forests. This family is renowned for its rich resource of terpenoids, particularly monoterpenes, sesquiterpenes, and diterpenes. These compounds not only impart specific scents to Lauraceae species but also play crucial roles in plant growth, development, and environmental adaptation. These compounds also possess extensive bioactivities, such as antioxidant, antibacterial, anti-inflammatory, and neuroprotective effects, making them valuable in the fields of perfumery, cosmetics, food, and medicine, and thus holding significant economic value. Recent advancements in high-throughput technologies, especially genomics, transcriptomics, and metabolomics, have significantly advanced our knowledge of the chemical constituents and biosynthetic pathways of terpenoids in Lauraceae species. Such progress has also shed light on the diversity and functionality of the terpene synthases (TPSs) gene family, a key enzyme involved in terpenoid biosynthesis. This paper reviews the latest research findings on the biosynthetic pathways of terpenoids and their key enzyme-encoding gene families in Lauraceae plants. We also analyze the evolutionary patterns of TPS gene family members of four Lauraceae species at the whole-genome level and summarize their mechanisms of action in secondary metabolite synthesis. Furthermore, this paper highlights the current research challenges and proposes prospects, such as the complexity of gene families, the uncertainties in functional predictions, and unclear regulatory mechanisms. Our objective is to provide scientific foundations for the in-depth analysis of terpenoid biosynthesis mechanisms and the development and utilization of natural products in Lauraceae plants. [ABSTRACT FROM AUTHOR]

An unnatural tricyclic oxaterpenoid, obtained by treatment of the sesquiterpene synthase presilphiperfolan‐8β‐ol synthase (BcBOT2) with an unnatural farnesyl pyrophosphate ether derivative, itself obtained by chemical synthesis, was converted in oxidation studies as part of a broad screening program by selected unspecific peroxygenases (UPOs). Product analysis revealed that Agrocybe aegerita UPO, its mutant PaDa‐I, and two commercial ones, UPO54 and UPO49, provided new oxidation products with sufficient efficiency for subsequent upscaling that allowed product isolation and structure elucidation. As such new terpene‐based oxiranes and hemiacetals were formed by UPO‐mediated epoxidations and CH‐activation. The structure elucidation was further supported by comparison with products generated by chemical oxidation. [ABSTRACT FROM AUTHOR]

Volatile organic compounds (VOCs) and essential oils of conifers are widely used in the pharmaceutical industry. This work aimed to analyze the VOCs of 30 conifer species representing the Pinaceae and Cupressaceae families. Samples were collected from arboreta in Hungary, and their chemical composition was determined by gas chromatography (SPME-GC/MS); then, chemometric analyses were performed using multivariate methods to identify characteristic VOCs of conifers. Here, we present results for monoterpene and sesquiterpene profiles of the examined conifer samples. The most abundant compounds detected were α-pinene, bornyl acetate, limonene, β-pinene, β-caryophyllene, β-myrcene, δ-3-carene, and β-phellandrene. The results showed that the following volatiles were characteristic of the conifer groups: sabinene (RRT=6.0) for the cupressoid group (which includes the Cupressaceae species), longifolene (RRT=15.0) and β-pinene (RRT=6.1) were characteristic of the pinoid group (including Picea, Pinus, and Pseudotsuga species), and camphene (RRT=5.5) and bornyl acetate (RRT=12.6) were characteristic of the abietoid group (including Abies, Cedrus, and Tsuga species). Our results on VOCs in the Pinaceae and Cupressaceae families contribute to the elucidation of biodiversity patterns of conifer species and, in addition, may support the industrial application of terpenes. [ABSTRACT FROM AUTHOR]

Lavender species are of significant economic value being cultivated extensively worldwide for their essential oils (EOs), which include terpenes that play crucial roles in the cosmetic, personal care, and pharmaceutical industries. The terpene synthases in lavender, such as Lavandula angustifolia linalool synthase (LaLINS), limonene synthase (LaLIMS), and bergamotene synthase (LaBERS), are key enzymes in terpene biosynthesis. However, the functional mechanisms underlying these enzymes remain poorly understood. Here, we used AlphaFold2 to predict the three-dimensional structures of LaLINS, LaLIMS, and LaBERS. The hydrodynamic radii of LaLINS, LaLIMS, and LaBERS were 5.7 ± 0.2, 6.2 ± 0.3, and 5.4 ± 0.2 nm, respectively. Mutations D320A or D324A led to a complete loss of activity in LaLINS compared to the wild-type (WT) enzyme; similarly, mutations D356A or D360A abolished activity in LaLIMS, and D291A or D295A eliminated activity in LaBERS. Furthermore, the genes LaLINS, LaLIMS, and LaBERS exhibited significantly higher expression levels in leaves compared to stems and flowers, with peak expression occurring at 8:00 a.m. Our findings contribute to a deeper understanding of terpene biosynthesis in lavender and offer insights for improving essential oil production through genetic engineering.

Terpene synthases (TPSs) catalyze the first step in the formation of terpenoids, which comprise the largest class of natural products in nature. TPSs employ a family of universal natural substrates, composed of isoprenoid units bound to a diphosphate moiety. The intricate structures generated by TPSs are the result of substrate binding and folding in the active site, enzyme‐controlled carbocation reaction cascades, and final reaction quenching. A key unaddressed question in class I TPSs is the asymmetric nature of the diphosphate‐(Mg2+)3 cluster, which forms a critical part of the active site. In this asymmetric ion cluster, two diphosphate oxygen atoms protrude into the active site pocket. The substrate hydrocarbon tail, which is eventually molded into terpenes, can bind to either of these oxygen atoms, yet to which is unknown. Herein, we employ structural, bioinformatics, and EnzyDock docking tools to address this enigma. We bring initial data suggesting that this difference is rooted in evolutionary differences between TPSs. We hypothesize that this alteration in binding, and subsequent chemistry, is due to TPSs originating from plants or microorganisms. We further suggest that this difference can cast light on the frequent observation that the chiral products or intermediates of plant and bacterial terpene synthases represent opposite enantiomers. [ABSTRACT FROM AUTHOR]

Background: The fruity aromatic bouquet of coffee has attracted recent interest to differentiate high value market produce as specialty coffee. Although the volatile compounds present in green and roasted coffee beans have been extensively described, no study has yet linked varietal molecular differences to the greater abundance of specific substances and support the aroma specificity of specialty coffees. Results: This study compared four Arabica genotypes including one, Geisha Especial, suggested to generate specialty coffee. Formal sensory evaluations of coffee beverages stressed the importance of coffee genotype in aroma perception and that Geisha Especial-made coffee stood out by having fine fruity, and floral, aromas and a more balanced acidity. Comparative SPME–GC–MS analyses of green and roasted bean volatile compounds indicated that those of Geisha Especial differed by having greater amounts of limonene and 3-methylbutanoic acid in agreement with the coffee cup aroma perception. A search for gene ontology differences of ripening beans transcriptomes of the four varieties revealed that they differed by metabolic processes linked to terpene biosynthesis due to the greater gene expression of prenyl-pyrophosphate biosynthetic genes and terpene synthases. Only one terpene synthase (CaTPS10-like) had an expression pattern that paralleled limonene loss during the final stage of berry ripening and limonene content in the studied four varieties beans. Its functional expression in tobacco leaves confirmed its functioning as a limonene synthase. Conclusions: Taken together, these data indicate that coffee variety genotypic specificities may influence ripe berry chemotype and final coffee aroma unicity. For the specialty coffee variety Geisha Especial, greater expression of terpene biosynthetic genes including CaTPS10-like, a limonene synthase, resulted in the greater abundance of limonene in green beans, roasted beans and a unique citrus note of the coffee drink. [ABSTRACT FROM AUTHOR]

High-energy-density liquid fuels (HED fuels) are essential for volume-limited aerospace vehicles and could serve as energetic additives for conventional fuels. Terpene-derived HED biofuel is an important research field for green fuel synthesis. The direct extraction of terpenes from natural plants is environmentally unfriendly and costly. Designing efficient synthetic pathways in microorganisms to achieve high yields of terpenes shows great potential for the application of terpene-derived fuels. This review provides an overview of the current research progress of terpene-derived HED fuels, surveying terpene fuel properties and the current status of biosynthesis. Additionally, we systematically summarize the engineering strategies for biosynthesizing terpenes, including mining and engineering terpene synthases, optimizing metabolic pathways and cell-level optimization, such as the subcellular localization of terpene synthesis and adaptive evolution. This article will be helpful in providing insight into better developing terpene-derived HED fuels. [ABSTRACT FROM AUTHOR]

This article presents a study on the genome assembly of Syringa pinnatifolia, a shrub valued in traditional Chinese medicine for its volatile chemical constituents. The researchers aimed to elucidate the biosynthesis pathway of zerumbone, a compound found in S. pinnatifolia with various medicinal properties. Through genome sequencing and analysis, they identified the genes responsible for the production of key compounds in the pathway, including a terpene synthase (TPS) gene and a cytochrome P450 (CYP450) gene. The study provides insights into the biosynthesis of zerumbone in eudicots and expands our understanding of the functions of TPS and CYP450 enzymes. [Extracted from the article]

Diterpenoids form a diverse group of natural products, many of which are or could become pharmaceuticals or industrial chemicals. The modular character of diterpene biosynthesis and the promiscuity of the enzymes involved make combinatorial biosynthesis a promising approach to generate libraries of diverse diterpenoids. Here, we report on the combinatorial assembly in yeast of ten diterpene synthases producing (+)-copalyl diphosphate-derived backbones and four cytochrome P450 oxygenases (CYPs) in diverse combinations. This resulted in the production of over 200 diterpenoids. Based on literature and chemical database searches, 162 of these compounds can be considered new-to-Nature. The CYPs accepted most substrates they were given but remained regioselective with few exceptions. Our results provide the basis for the systematic exploration of the diterpenoid chemical space in yeast using sequence databases. Golden Gate assembly of 10 diterpene synthases (diTPS) and 4 cytochrome CYP oxygenases (CYPs) from diverse plant species in the yeast Saccharomyces cerevisiae leads to the production of more than 200 distinct diterpenoids. 132 of these diterpenoids are new-to-Nature indicating that combinatorial assembly of diTPS and CYPs in yeast is a promising approach to generate diverse libraries of diterpenoids in high-throughput. [Display omitted] • The biosynthesis of diterpenoids is modular. • Modularity allows the combinatorial assembly of diterpene synthases and cytochrome P450 oxygenases. • 10 diterpene synthases and 4 CYPs from different plants were combined using Golden Gate cloning and expressed in yeast. • In total more than 200 diterpenoids could be detected. • 162 of these diterpenoids are new-to-Nature. [ABSTRACT FROM AUTHOR]

A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin (1) and variecolactone (2) was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues (5 – 7). Analysis of the compounds' anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities, 5 was associated with significantly reduced toxic side effects in cancer-bearing mice, indicating its potentially broader therapeutic window. Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities. Heterologous production of bioactive fungal sesterterpenoid variecolin (1) and its unnatural analogues was achieved. One of the new analogues, compound 5 , exhibited an improved anticancer property in cancer-bearing mice. [Display omitted] [ABSTRACT FROM AUTHOR]

The root of Paeonia lactiflora pall. is a significant component of traditional Chinese medicine, with terpenoids and their glycosides, such as paeoniflorins, serving as key active ingredients known for their anti-inflammatory, hepatoprotective, and analgesic properties. By generating a transcriptome and functionally characterizing 32 terpene synthases (TPSs) from P. lactiflora, we successfully constructed 24 pESC-Trp-PlTPS expression vectors. Through expression in Saccharomyces cerevisiae engineered strains, we identified four mono-TPSs and five sesqui-TPSs that produce 18 compounds, including eight monoterpenes and ten sesquiterpenes in vitro. This includes a bifunctional enzyme (PlTPS22). Additionally, PlTPS21 was characterized as a pinene synthase with α-pinene as its main product. The expression pattern of PlTPS21 aligns closely with the accumulation patterns of paeoniflorins and α-pinene in the plant, suggesting that PlTPS21 is a key enzyme in the biosynthesis of paeoniflorin.

Terpenoids are important compounds associated with the pest and herbivore resistance mechanisms of plants; consequently, it is essential to identify and explore terpene synthase (TPS) genes in maize. In the present study, we identified 31 TPS genes based on a pan-genome of 26 high-quality maize genomes containing 20 core genes (present in all 26 lines), seven dispensable genes (present in 2 to 23 lines), three near-core genes (present in 24 to 25 lines), and one private gene (present in only 1 line). Evaluation of ka/ks values of TPS in 26 varieties revealed that TPS25 was subjected to positive selection in some varieties. Six ZmTPS had ka/ks values less than 1, indicating that they were subjected to purifying selection. In 26 genomes, significant differences were observed in ZmTPS25 expression between genes affected by structural variation (SV) and those not affected by SV. In some varieties, SV altered the conserved structural domains resulting in a considerable number of atypical genes. The analysis of RNA-seq data of maize Ostrinia furnacalis feeding revealed 10 differentially expressed ZmTPS, 9 of which were core genes. However, many atypical genes for these responsive genes were identified in several genomes. These findings provide a novel resource for functional studies of ZmTPS. [ABSTRACT FROM AUTHOR]

The Lecythidaceae family tree, Couroupita guianensis Aubl, popularly known as Nagpushpa, is a widely cultivated ornamental tree with several uses in traditional medicine. The tree is revered as highly sacred in Indian traditional culture due to its uniquely shaped, fragrant flowers. Considering the significance, we were prompted to carry out the metabolite and transcriptome analysis of Nagapushpa. The flower, petals, stamen, stem and leaf of C. guianensis were metabolically profiled, and it was discovered that the flower tissue contained the highest terpenoid reservoir. A number of terpenoid pathway transcripts were also found in the flower tissue after transcriptome profiling. KEGG pathway mapping was carried out to correlate transcript sequences with the biosynthesis of different types of terpenes. We were able to clone three full-length terpene synthase gene candidates, i.e. monoterpene ocimene synthase, diterpene ent-kaurene synthase and sesquiterpene farnesene synthase. The transcript expression of selected terpene synthase genes was also verified in flower tissue. These cloned sequences were used for in silico structural investigations and protein function prediction at the level of 3D structure. The data presented in this study provide a comprehensive resource for the metabolic and transcriptomic profiles of C. guianen sis. The study paves the way towards the elucidation of terpene biosynthetic pathway in C. guianensis and heterologous production of useful terpenoids in the future. [ABSTRACT FROM AUTHOR]

In response to herbivory, Capsicum annuum leaves adapt their specialized metabolome that may protect the plant against herbivore feeding either directly or indirectly through volatile metabolites acting as cues for natural enemies of the herbivore. The volatile blend of spider-mite infested leaves differs from non-challenged leaves predominantly by a higher contribution of mono- and sesquiterpenes. In addition to these terpenoids released into the headspace, the terpenoid composition of the leaves alters upon herbivory. All this suggests an important role for terpenoids and their biosynthetic machinery in the defence against herbivory. Here, we show that the C. annuum genome contains a terpene synthase (TPS) gene family of 103 putative members of which structural analysis revealed that 27 encode functional enzymes. Transcriptome analysis showed that several TPS loci were differentially expressed upon herbivory in leaves of two C. annuum genotypes, that differ in susceptibility towards spider mites. The relative expression of upstream biosynthetic genes from the mevalonate and the methylerythritol phosphate pathway also altered upon herbivory, revealing a shift in the metabolic flux through the terpene biosynthetic module. The expression of multiple genes potentially acting downstream of the TPSs, including cytochrome P450 monooxygenases, UDP-glucosyl transferases, and transcription factors strongly correlated with the herbivory-induced TPS genes. A selection of herbivory-induced TPS genes was functionally characterized through heterologous expression and the products that these enzymes catalysed matched with the volatile and non-volatile terpenoids induced in response to herbivory. Key message: Annotation of the Capsicum annuum terpene synthase gene family links terpene metabolism to the formation of volatile and non-volatile terpenoids important for direct and indirect defence. [ABSTRACT FROM AUTHOR]

Terpenes and terpenoids are key natural compounds for plant defense, development, and composition of plant oil. The synthesis and accumulation of a myriad of volatile terpenoid compounds in these plants may dramatically alter the quality and flavor of the oils, which provide great commercial utilization value for oil-producing plants. Terpene synthases (TPSs) are important enzymes responsible for terpenic diversity. Investigating the differentiation of the TPS gene family could provide valuable theoretical support for the genetic improvement of oil-producing plants. While the origin and function of TPS genes have been extensively studied, the exact origin of the initial gene fusion event - it occurred in plants or microbes - remains uncertain. Furthermore, a comprehensive exploration of the TPS gene differentiation is still pending. Here, phylogenetic analysis revealed that the fusion of the TPS gene likely occurred in the ancestor of land plants, following the acquisition of individual C- and Nterminal domains. Potential mutual transfer of TPS genes was observed among microbes and plants. Gene synteny analysis disclosed a differential divergence pattern between TPS-c and TPS-e/f subfamilies involved in primary metabolism and those (TPS-a/b/d/g/h subfamilies) crucial for secondary metabolites. Biosynthetic gene clusters (BGCs) analysis suggested a correlation between lineage divergence and potential natural selection in structuring terpene diversities. This study provides fresh perspectives on the origin and evolution of the TPS gene family. [ABSTRACT FROM AUTHOR]

Abstract Terpenoids are important compounds associated with the pest and herbivore resistance mechanisms of plants; consequently, it is essential to identify and explore terpene synthase (TPS) genes in maize. In the present study, we identified 31 TPS genes based on a pan-genome of 26 high-quality maize genomes containing 20 core genes (present in all 26 lines), seven dispensable genes (present in 2 to 23 lines), three near-core genes (present in 24 to 25 lines), and one private gene (present in only 1 line). Evaluation of ka/ks values of TPS in 26 varieties revealed that TPS25 was subjected to positive selection in some varieties. Six ZmTPS had ka/ks values less than 1, indicating that they were subjected to purifying selection. In 26 genomes, significant differences were observed in ZmTPS25 expression between genes affected by structural variation (SV) and those not affected by SV. In some varieties, SV altered the conserved structural domains resulting in a considerable number of atypical genes. The analysis of RNA-seq data of maize Ostrinia furnacalis feeding revealed 10 differentially expressed ZmTPS, 9 of which were core genes. However, many atypical genes for these responsive genes were identified in several genomes. These findings provide a novel resource for functional studies of ZmTPS.

High-energy-density liquid fuels (HED fuels) are essential for volume-limited aerospace vehicles and could serve as energetic additives for conventional fuels. Terpene-derived HED biofuel is an important research field for green fuel synthesis. The direct extraction of terpenes from natural plants is environmentally unfriendly and costly. Designing efficient synthetic pathways in microorganisms to achieve high yields of terpenes shows great potential for the application of terpene-derived fuels. This review provides an overview of the current research progress of terpene-derived HED fuels, surveying terpene fuel properties and the current status of biosynthesis. Additionally, we systematically summarize the engineering strategies for biosynthesizing terpenes, including mining and engineering terpene synthases, optimizing metabolic pathways and cell-level optimization, such as the subcellular localization of terpene synthesis and adaptive evolution. This article will be helpful in providing insight into better developing terpene-derived HED fuels.

Terpene synthases are responsible for the biosynthesis of terpenes, the largest family of natural products. Hydropyrene synthase generates hydropyrene and hydropyrenol as its main products along with two byproducts, isoelisabethatrienes A and B. Fascinatingly, a single active site mutation (M75L) diverts the product distribution towards isoelisabethatrienes A and B. In the current work, we study the competing pathways leading to these products using quantum chemical calculations in the gas phase. We show that there is a great thermodynamic preference for hydropyrene and hydropyrenol formation, and hence most likely in the synthesis of the isoelisabethatriene products kinetic control is at play.

Abstract Background Rhododendron molle (Ericaceae) is a traditional Chinese medicine, which has been used to treat rheumatism and relieve pain since ancient times. The characteristic grayanoids of this plant have been demonstrated to be the chemical basis for the analgesic activity. Moreover, unlike morphine, these diterpenoids are non-addictive. Grayanoids mainly distribute in the leaves, flowers, roots, and fruits of R. molle, with low content. Currently the research on the biosynthesis of grayanoids is hindered, partially due to lack of the genomic information. Results In the present study, a total of 744 Mb sequences were generated and assembled into 13 chromosomes. An ancient whole-genome duplication event (Ad-β) was discovered that occurred around 70 million years ago. Tandem and segmental gene duplications led to specific gene expansions in the terpene synthase and cytochrome P450 (CYP450) gene families. Two diterpene synthases were demonstrated to be responsible for the biosynthesis of 16α-hydroxy-ent-kaurane, the key precursor for grayanoids. Phylogenetic analysis revealed a species-specific bloom of the CYP71AU subfamily, which may involve the candidate CYP450s responsible for the biosynthesis of grayanoids. Additionally, three putative terpene biosynthetic gene clusters were found. Conclusions We reported the first genome assembly of R. molle and investigated the molecular basis underpinning terpenoids biosynthesis. Our work provides a foundation for elucidating the complete biosynthetic pathway of grayanoids and studying the terpenoids diversity in R. molle.

Terpene synthases (TPS) is a key enzyme in the synthesis of plant terpenoids. Studies on TPSs have not been reported in Gossypium barbadense and Gossypium arboreum. 260 TPSs were identified in Gossypium, including 71 in Gossypium hirsutum, 75 in Gossypium. barbadense, 60 in Gossypium. arboreum, and 54 in Gossypium raimondii. We systematically analyzed the TPS gene family of Gossypium from three aspects: gene structure, evolutionary process and gene function. (1) Gene structure: Based on the protein structure of two conserved domains (PF01397 and PF03936), the TPS gene family is divided into five clades: TPS -a, -b, -c, -e/f and -g. (2) Evolution: Whole genome duplication and segmental duplication are the main modes of TPS gene amplification. (3) Function: The abundance of cis-acting elements may reveal the functional diversity of TPSs in cotton. TPS gene has tissue specific expression in cotton. The hypomethylation of the exon of TPSs may help to enhance the adaptability of cotton to flooding stress. In conclusion, this study can broaden the understanding of structure-evolution-function of the TPS gene family, and provide reference for the mining and verification of new genes. [ABSTRACT FROM AUTHOR]

Caryopteris x Clandonensis, also known as bluebeard, is an ornamental plant containing a large variety of terpenes and terpene-like compounds. Four different cultivars were subjected to a principal component analysis to elucidate variations in terpenoid-biosynthesis and consequently, two representative cultivars were sequenced on a genomic level. Functional annotation of genes as well as comparative genome analysis on long read datasets enabled the identification of cultivar-specific terpene synthase and cytochrome p450 enzyme sequences. This enables new insights, especially since terpenoids in research and industry are gaining increasing interest due to their importance in areas such as food preservation, fragrances, or as active ingredients in pharmaceutical formulations. According to BUSCO assessments, the presented genomes have an average size of 355 Mb and about 96.8% completeness. An average of 52,090 genes could be annotated as putative proteins, whereas about 42 were associated with terpene synthases and about 1340 with cytochrome p450 enzymes. [ABSTRACT FROM AUTHOR]

Abstract Background Terpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their diverse bioactive properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana, which further enhances the scope for the manufacture of terpenes for industrial purposes. Results In this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l−1 and 395 mg l−1, respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected. Conclusion The presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis. Graphical Abstract

Background: Rhododendron molle (Ericaceae) is a traditional Chinese medicine, which has been used to treat rheumatism and relieve pain since ancient times. The characteristic grayanoids of this plant have been demonstrated to be the chemical basis for the analgesic activity. Moreover, unlike morphine, these diterpenoids are non-addictive. Grayanoids mainly distribute in the leaves, flowers, roots, and fruits of R. molle, with low content. Currently the research on the biosynthesis of grayanoids is hindered, partially due to lack of the genomic information. Results: In the present study, a total of 744 Mb sequences were generated and assembled into 13 chromosomes. An ancient whole-genome duplication event (Ad-β) was discovered that occurred around 70 million years ago. Tandem and segmental gene duplications led to specific gene expansions in the terpene synthase and cytochrome P450 (CYP450) gene families. Two diterpene synthases were demonstrated to be responsible for the biosynthesis of 16α-hydroxy-ent-kaurane, the key precursor for grayanoids. Phylogenetic analysis revealed a species-specific bloom of the CYP71AU subfamily, which may involve the candidate CYP450s responsible for the biosynthesis of grayanoids. Additionally, three putative terpene biosynthetic gene clusters were found. Conclusions: We reported the first genome assembly of R. molle and investigated the molecular basis underpinning terpenoids biosynthesis. Our work provides a foundation for elucidating the complete biosynthetic pathway of grayanoids and studying the terpenoids diversity in R. molle. [ABSTRACT FROM AUTHOR]

Plant volatiles play a major role in plant–insect interactions as defense compounds or attractants for insect herbivores. Recent studies have shown that endophytic fungi are also able to produce volatiles and this raises the question of whether these fungal volatiles influence plant–insect interactions. Here, we qualitatively investigated the volatiles released from 13 endophytic fungal species isolated from leaves of mature black poplar (Populus nigra) trees. The volatile blends of these endophytes grown on agar medium consist of typical fungal compounds, including aliphatic alcohols, ketones and esters, the aromatic alcohol 2-phenylethanol and various sesquiterpenes. Some of the compounds were previously reported as constituents of the poplar volatile blend. For one endophyte, a species of Cladosporium, we isolated and characterized two sesquiterpene synthases that can produce a number of mono- and sesquiterpenes like (E)-β-ocimene and (E)-β-caryophyllene, compounds that are dominant components of the herbivore-induced volatile bouquet of black poplar trees. As several of the fungus-derived volatiles like 2-phenylethanol, 3-methyl-1-butanol and the sesquiterpene (E)-β-caryophyllene, are known to play a role in direct and indirect plant defense, the emission of volatiles from endophytic microbial species should be considered in future studies investigating tree-insect interactions.

Terpenes, volatile compounds known for their aromatic and therapeutic properties, play a pivotal role in shaping the overall chemical profile of Cannabis sativa L. Their biosynthesis in planta occurs in trichomes and involves the 2-C-methyl-D-erythritol 4-phosphate (MEP) and the mevalonic acid (MVA) pathways, responsible for producing the substrates utilized by a family of enzymes, the terpene synthases (TPS), for terpene production. In this work, a comprehensive approach combining chemical analyses of the volatile compounds characterizing the aroma of the inflorescences three C. sativa genotypes collected at three stages of maturity and the transcriptional analyses of key genes involved in the terpene biosynthesis was adopted to study this pathway. The results revealed different terpene profiles among genotypes, which were characterized by peculiar compounds belonging to the sesqui- (CINBOL and Fibrante) or monoterpene (Ermo) categories. Both structural and putative regulatory genes were analysed by RT-qPCR, revealing distinct transcriptional profiles of Terpene Synthases , contributing to the diversity of mono and sesquiterpenes synthesized. Furthermore, the research delved into potential regulatory genes associated with trichome formation, a crucial factor influencing terpene accumulation. This integrated approach highlighted complex mechanisms governing terpene accumulation in cannabis, while also offering potential regulators putatively involved in this pathway. Up to 56 compounds belonging to the mono- or sesquiterpenes categories were identified in three Cannabis sativa inflorescences, with a few major compounds able to differentiate the genotypes. Some structural and regulatory genes were analysed by RT-qPCR, revealing distinct, genotype-specific transcriptional profiles contributing to the diversity of terpenes produced. [Display omitted] • Mono- and sesquiterpene composition varies among cannabis genotypes. • Selina-3,7(11)-diene is a peculiar compound of the drug-type variety. • All TPS genes were differentially expressed in genotypes and during time. • CsMYB5 , CsMYB45 , CsbHLH112 , and CsbHLH114 revealed a genotype- and time-dependent expression. [ABSTRACT FROM AUTHOR]

Significant research progress has recently been made on establishing the roles of tps46 in rice defense. (E)-β-farnesene (Eβf) is a major product of tps46 activity but its physiological functions and potential mechanisms against Chilo suppressalis have not yet been clarified. In the present study, C. suppressalis larvae were artificially fed a diet containing 0.8 g/kg Eβf and the physiological performance of the larvae was evaluated. In response to Eβf treatment, the average 2nd instar duration significantly increased from 4.78 d to 6.31 d while that of the 3rd instar significantly increased from 5.70 d to 8.00 d compared with the control. There were no significant differences between the control and Eβf-fed 4th and 5th instars in terms of their durations. The mortalities of the 2nd and 3rd Eβf-fed instars were 21.00-fold and 6.39-fold higher, respectively, than that of the control. A comparative transcriptome analysis revealed that multiple differentially expressed genes are involved in insect hormone biosynthesis. An insect hormone assay on the 3rd instars disclosed that Eβf disrupted the balance between the juvenile hormone and ecdysteroid levels. Eβf treatment increased the juvenile hormones titers but not those of the ecdysteroids. The qPCR results were consistent with those of the RNA-Seq. The foregoing findings suggested that Eβf impairs development and survival in C. suppressalis larvae by disrupting their hormone balance. Moreover, Eβf altered the pathways associated with carbohydrate and xenobiotic metabolism as well as those related to cofactors and vitamins in C. suppressalis larvae. The discoveries of this study may contribute to the development and implementation of an integrated control system for C. suppressalis infestations in rice. [ABSTRACT FROM AUTHOR]

Background: Terpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their diverse bioactive properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana, which further enhances the scope for the manufacture of terpenes for industrial purposes. Results: In this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l−1 and 395 mg l−1, respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected. Conclusion: The presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis. [ABSTRACT FROM AUTHOR]

Significant research progress has recently been made on establishing the roles of tps46 in rice defense. (E)-β-farnesene (Eβf) is a major product of tps46 activity but its physiological functions and potential mechanisms against Chilo suppressalis have not yet been clarified. In the present study, C. suppressalis larvae were artificially fed a diet containing 0.8 g/kg Eβf and the physiological performance of the larvae was evaluated. In response to Eβf treatment, the average 2nd instar duration significantly increased from 4.78 d to 6.31 d while that of the 3rd instar significantly increased from 5.70 d to 8.00 d compared with the control. There were no significant differences between the control and Eβf-fed 4th and 5th instars in terms of their durations. The mortalities of the 2nd and 3rd Eβf-fed instars were 21.00-fold and 6.39-fold higher, respectively, than that of the control. A comparative transcriptome analysis revealed that multiple differentially expressed genes are involved in insect hormone biosynthesis. An insect hormone assay on the 3rd instars disclosed that Eβf disrupted the balance between the juvenile hormone and ecdysteroid levels. Eβf treatment increased the juvenile hormones titers but not those of the ecdysteroids. The qPCR results were consistent with those of the RNA-Seq. The foregoing findings suggested that Eβf impairs development and survival in C. suppressalis larvae by disrupting their hormone balance. Moreover, Eβf altered the pathways associated with carbohydrate and xenobiotic metabolism as well as those related to cofactors and vitamins in C. suppressalis larvae. The discoveries of this study may contribute to the development and implementation of an integrated control system for C. suppressalis infestations in rice.

Terpenes are organic compounds and play important roles in plant development and stress response. Terpene synthases (TPSs) are the key enzymes for the biosynthesis of terpenes. For Rosaceae species, terpene composition represents a critical quality attribute, but limited information is available regarding the evolution and expansion occurring in the terpene synthases gene family. Here, we selected eight Rosaceae species with sequenced and annotated genomes for the identification of TPSs, including three Prunoideae, three Maloideae, and two Rosoideae species. Our data showed that the TPS gene family in the Rosaceae species displayed a diversity of family numbers and functions among different subfamilies. Lineage and species-specific expansion of the TPSs accompanied by frequent domain loss was widely observed within different TPS clades, which might have contributed to speciation or environmental adaptation in Rosaceae. In contrast to Maloideae and Rosoideae species, Prunoideae species owned less TPSs, with the evolution of Prunoideae species, TPSs were expanded in modern peach. Both tandem and segmental duplication significantly contributed to TPSs expansion. Ka/Ks calculations revealed that TPSs genes mainly evolved under purifying selection except for several pairs, where the divergent time indicated TPS-e clade was diverged relatively anciently. Gene function classification of TPSs further demonstrated the function diversity among clades and species. Moreover, based on already published RNA-Seq data from NCBI, the expression of most TPSs in Malus domestica, Prunus persica, and Fragaria vesca displayed tissue specificity and distinct expression patterns either in tissues or expression abundance between species and TPS clades. Certain putative TPS-like proteins lacking both domains were detected to be highly expressed, indicating the underlying functional or regulatory potentials. The result provided insight into the TPS family evolution and genetic information that would help to improve Rosaceae species quality. [ABSTRACT FROM AUTHOR]

Summary: Terpenoids play important roles in flavour, pollinator attraction and defence of plants. In cucumber (Cucumis sativus) they are important components of the herbivore‐induced plant volatile blend that attracts natural enemies of herbivores.We annotated the cucumber TERPENE SYNTHASE gene (CsTPS) family and characterized their involvement in the response towards herbivores with different feeding guilds using a combined molecular and biochemical approach.Transcripts of multiple CsTPS genes were upregulated in leaves upon herbivory and the products generated by the expressed proteins match the terpenoids recorded in the volatile blend released by herbivore‐damaged leaves. Spatial and temporal analysis of the promoter activity of CsTPS genes showed that cell content‐feeding spider mites (Tetranychus urticae) and thrips (Frankliniella occidentalis) induced promoter activity of CsTPS9 and CsTPS19 within hours after initiation of infestation, while phloem‐feeding aphids (Myzus persicae) induced CsTPS2 promoter activity.Our findings offer detailed insights into the involvement of the TPS gene family in the dynamics and fine‐tuning of the emission of herbivore‐induced plant volatiles in cucumber, and open a new avenue to understand molecular mechanisms that affect plant–herbivore interactions. [ABSTRACT FROM AUTHOR]

Galling insects gain food and shelter by inducing specialized anatomical structures in their plant hosts. Such galls often accumulate plant defensive metabolites protecting the inhabiting insects from predation. We previously found that, despite a marked natural chemopolymorphism in natural populations of Pistacia palaestina, the monoterpene content in Baizongia pistaciae-induced galls is substantially higher than in leaves of their hosts. Here we show a general up-regulation of key structural genes in both the plastidial and cytosolic terpene biosynthetic pathways in galls as compared with non-colonized leaves. Novel prenyltransferases and terpene synthases were functionally expressed in Escherichia coli to reveal their biochemical function. Individual Pistacia trees exhibiting chemopolymorphism in terpene compositions displayed differential up-regulation of selected terpene synthase genes, and the metabolites generated by their gene products in vitro corresponded to the monoterpenes accumulated by each tree. Our results delineate molecular mechanisms responsible for the formation of enhanced monoterpene in galls and the observed intraspecific monoterpene chemodiversity displayed in P. palaestina. We demonstrate that gall-inhabiting aphids transcriptionally reprogram their host terpene pathways by up-regulating tree-specific genes, boosting the accumulation of plant defensive compounds for the protection of colonizing insects. We identified specific host plant genes responsible for the enhanced terpene biosynthesis and monoterpene chemodiversity observed in insect-induced galls, providing chemical defenses for the gall inhabitants. [ABSTRACT FROM AUTHOR]

Caryopteris x Clandonensis, also known as bluebeard, is an ornamental plant containing a large variety of terpenes and terpene-like compounds. Four different cultivars were subjected to a principal component analysis to elucidate variations in terpenoid-biosynthesis and consequently, two representative cultivars were sequenced on a genomic level. Functional annotation of genes as well as comparative genome analysis on long read datasets enabled the identification of cultivar-specific terpene synthase and cytochrome p450 enzyme sequences. This enables new insights, especially since terpenoids in research and industry are gaining increasing interest due to their importance in areas such as food preservation, fragrances, or as active ingredients in pharmaceutical formulations. According to BUSCO assessments, the presented genomes have an average size of 355 Mb and about 96.8% completeness. An average of 52,090 genes could be annotated as putative proteins, whereas about 42 were associated with terpene synthases and about 1340 with cytochrome p450 enzymes.

Tomato produces a number of terpenes in their glandular trichomes that contribute to host plant resistance against pests. While glandular trichomes of cultivated tomato Solanum lycopersicum primarily accumulate a blend of monoterpenes, those of the wild tomato species Solanum habrochaites produce various sesquiterpenes. Recently, we have identified two groups of sesquiterpenes in S. habrochaites accessions that negatively affect the performance and choice behavior of the potato aphid (Macrosiphum euphorbiae). Aphids are piercing-sucking herbivores that use their mouthpart to penetrate and probe plant tissues in order to ultimately access vascular tissue and ingest phloem sap. Because secondary metabolites produced in glandular trichomes can affect the initial steps of the aphid feeding behavior, introducing the formation of defensive terpenes into additional plant tissues via metabolic engineering has the potential to reduce tissue penetration by aphids and in consequence virus transmission. Here, we have developed two multicistronic expression constructs based on the two sesquiterpene traits with activity toward M. euphorbiae previously identified in S. habrochaites. Both constructs are composed of sequences encoding a prenyl transferase and a respective S. habrochaites terpene synthase, as well as enhanced green fluorescent protein as a visible marker. All three coding sequences were linked by short nucleotide sequences encoding the foot-and-mouth disease virus 2A self-processing oligopeptide which allows their co-expression under the control of one promoter. Transient expression of both constructs under the epidermis-specific Arabidopsis CER5 -promoter in tomato leaves demonstrated that formation of the two sets of defensive sesquiterpenes, β-caryophyllene/α-humulene and (−)- endo -α-bergamotene/(+)-α-santalene/(+)- endo -β-bergamotene, can be introduced into new tissues in tomato. The epidermis-specific transgene expression and terpene formation were verified by fluorescence microscopy and tissue fractionation with subsequent analysis of terpene profiles, respectively. In addition, the longevity and fecundity of M. euphorbiae feeding on these engineered tomato leaves were significantly reduced, demonstrating the efficacy of this novel aphid control strategy. [ABSTRACT FROM AUTHOR]

Tomato produces a number of terpenes in their glandular trichomes that contribute to host plant resistance against pests. While glandular trichomes of cultivated tomato Solanum lycopersicum primarily accumulate a blend of monoterpenes, those of the wild tomato species Solanum habrochaites produce various sesquiterpenes. Recently, we have identified two groups of sesquiterpenes in S. habrochaites accessions that negatively affect the performance and choice behavior of the potato aphid (Macrosiphum euphorbiae). Aphids are piercing-sucking herbivores that use their mouthpart to penetrate and probe plant tissues in order to ultimately access vascular tissue and ingest phloem sap. Because secondary metabolites produced in glandular trichomes can affect the initial steps of the aphid feeding behavior, introducing the formation of defensive terpenes into additional plant tissues via metabolic engineering has the potential to reduce tissue penetration by aphids and in consequence virus transmission. Here, we have developed two multicistronic expression constructs based on the two sesquiterpene traits with activity toward M. euphorbiae previously identified in S. habrochaites. Both constructs are composed of sequences encoding a prenyl transferase and a respective S. habrochaites terpene synthase, as well as enhanced green fluorescent protein as a visible marker. All three coding sequences were linked by short nucleotide sequences encoding the foot-and-mouth disease virus 2A self-processing oligopeptide which allows their co-expression under the control of one promoter. Transient expression of both constructs under the epidermis-specific Arabidopsis CER5-promoter in tomato leaves demonstrated that formation of the two sets of defensive sesquiterpenes, β-caryophyllene/α-humulene and (−)-endo-α-bergamotene/(+)-α-santalene/(+)-endo-β-bergamotene, can be introduced into new tissues in tomato. The epidermis-specific transgene expression and terpene formation were verified by fluorescence microscopy and tissue fractionation with subsequent analysis of terpene profiles, respectively. In addition, the longevity and fecundity of M. euphorbiae feeding on these engineered tomato leaves were significantly reduced, demonstrating the efficacy of this novel aphid control strategy.

Ganoderma tsugae is an endemic medicinal mushroom in Northeast China, providing important source of pharmaceutical product. Comparing with other Ganoderma species, wild G. tsugae can utilize coniferous wood. However, functional genes related to medicinal component synthesis and the genetic mechanism of conifer substrate utilization is still obscure. Here, we assembled a high-quality G. tsugae genome with 18 contigs and 98.5% BUSCO genes and performed the comparative genomics with other Ganoderma species. G. tsugae diverged from their common ancestor of G. lingzhi and G. sinense about 21 million years ago. Genes in G. tsugae-specific and G. tsugae-expanded gene families, such as salh, phea, cyp53a1, and cyp102a, and positively selected genes, such as glpk and amie, were functionally enriched in plant-pathogen interaction, benzoate degradation, and fanconi anemia pathway. Those functional genes might contribute to conifer substrate utilization of G. tsugae. Meanwhile, gene families in the terpene synthesis were identified and genome-wide SNP variants were detected in population. Finally, the study provided valuable genomic resources and offered useful hints for the functional gene mapping and investigation of key gene contributing to conifer cultivation substrate utilization and medicinal component biosynthesis.

Ganoderma tsugae is an endemic medicinal mushroom in Northeast China, providing important source of pharmaceutical product. Comparing with other Ganoderma species, wild G. tsugae can utilize coniferous wood. However, functional genes related to medicinal component synthesis and the genetic mechanism of conifer substrate utilization is still obscure. Here, we assembled a high-quality G. tsugae genome with 18 contigs and 98.5% BUSCO genes and performed the comparative genomics with other Ganoderma species. G. tsugae diverged from their common ancestor of G. lingzhi and G. sinense about 21 million years ago. Genes in G. tsugae -specific and G. tsugae -expanded gene families, such as salh , phea , cyp53a1 , and cyp102a , and positively selected genes, such as glpk and amie , were functionally enriched in plant-pathogen interaction, benzoate degradation, and fanconi anemia pathway. Those functional genes might contribute to conifer substrate utilization of G. tsugae. Meanwhile, gene families in the terpene synthesis were identified and genome-wide SNP variants were detected in population. Finally, the study provided valuable genomic resources and offered useful hints for the functional gene mapping and investigation of key gene contributing to conifer cultivation substrate utilization and medicinal component biosynthesis. [ABSTRACT FROM AUTHOR]

Chrysanthemum is a significant genus belonging to one of the largest plant families, the Asteraceae. Among the over 40 recognized species, C. morifolium is best-known for its long history of cultivation and countless varieties. Like some other genera of this family, many Chrysanthemum species are producers of high levels of secondary metabolites. This review focuses on the largest class of plant secondary metabolites, namely terpenoids produced by Chrysanthemum species. An extensive literature search revealed a total of 390 terpenoids from these species, which include 183 monoterpenoids and 207 sesquiterpenoids. While some terpenes are produced by almost all of the investigated varieties/species of Chrysanthemum, many terpenoids show specificity to certain varieties or species, indicating species-specific functions. The enzymes and genes participating in the pathways for terpene biosynthesis are also described. In addition to a general overview, in this review we focus on isoprenyl diphosphate synthases and terpene synthases, two enzyme classes pivotal for generating the chemical diversity of terpenes, as established by our analysis of two sequenced genomes of Chrysanthemum. While our knowledge about the biological functions of terpenoids in Chrysanthemum is limited, we discuss their functions in chemical defenses against insects and microbial pathogens. Finally, we present the prospects of understanding the molecular basis of terpene biosynthesis in Chrysanthemum and elucidating its biological significance, as well as the prospects of translational research on terpene-based applications. [ABSTRACT FROM AUTHOR]

Sinocalycanthus chinensis , a diploid (2 n = 22) deciduous shrub, belongs to the Calycanthaceae family of magnoliids and is rich secondary metabolites, such as terpenoids. However, the regulation of terpenoid biosynthesis in S. chinensis is largely unknown. In this study, comparative transcriptome analyses were performed in the bark, branches, leaves, and flowers. KEGG enrichment analysis revealed that the terpenoid biosynthesis and cytochrome P450 pathways were significantly enriched in the four tissues. Twelve terpenoid backbone biosynthesis-related genes were identified, and eight terpene synthases (TPSs) were reassembled based on independent transcriptomes from the four tissues. Phylogenetic analysis of the TPSs showed high sequence similarity between S. chinensis and Arabidopsis, and these TPSs were classified into three subfamilies. Moreover, 39 phytohormone response-related genes, including 5 abscisic acid (ABA) receptors, 25 auxin response factors, 3 gibberellin (GA) response genes, 5 ethylene response genes, and 1 jasmonic acid (JA) response gene were analyzed. Most phytohormone pathway-related genes were upregulated in the flowers and downregulated in the leaves. The endogenous indole acetic acid (IAA) content was higher in the flowers than in the other comparisons. Our results provide an opportunity to reveal the regulation of terpenoid biosynthesis in S. chinensis. • Twelve terpenoid backbone biosynthesis-related genes and eight terpene synthases were identified in Sinocalycanthus chinensis. • Terpenoid biosynthesis-related genes showed the highest expression levels in flowers. • Terpenoid biosynthesis are related to ABA, GA, JA, auxin, and ethylene signaling pathway. [ABSTRACT FROM AUTHOR]

Terpene synthases (TPSs) are pivotal enzymes for the production of diverse terpenes, including monoterpenes, sesquiterpenes, and diterpenes. In our recent studies, dictyostelid social amoebae, also known as cellular slime molds, were found to contain TPS genes for making volatile terpenes. For comparison, here we investigated Physarum polycephalum, a plasmodial slime mold also known as acellular amoeba. Plasmodia of P. polycephalum grown on agar plates were found to release a mixture of volatile terpenoids consisting of four major sesquiterpenes (α-muurolene, (E)-β-caryophyllene, two unidentified sesquiterpenoids) and the monoterpene linalool. There were no qualitative differences in terpenoid composition at two stages of young plasmodia. To understand terpene biosynthesis, we analyzed the transcriptome and genome sequences of P. polycephalum and identified four TPS genes designated PpolyTPS1–PpolyTPS4. They share 28–73% of sequence identities. Full-length cDNAs for the four TPS genes were cloned and expressed in Escherichia coli to produce recombinant proteins, which were tested for sesquiterpene synthase and monoterpene synthase activities. While neither PpolyTPS2 nor PpolyTPS3 was active, PpolyTPS1 and PpolyTPS4 were able to produce sesquiterpenes and monoterpenes from the respective substrates farnesyl diphosphate and geranyl diphosphate. By comparing the volatile profile of P. polycephalum plasmodia and the in vitro products of PpolyTPS1 and PpolyTPS4, it was concluded that most sesquiterpenoids emitted from P. polycephalum were attributed to PpolyTPS4. Phylogenetic analysis placed the four PpolyTPSs genes into two groups: PpolyTPS1 and PpolyTPS4 being one group that was clustered with the TPSs from the dictyostelid social amoeba and PpolyTPS2 and PpolyTPS3 being the other group that showed closer relatedness to bacterial TPSs. The biological role of the volatile terpenoids produced by the plasmodia of P. polycephalum is discussed.

Agriophyllum squarrosum is a folk Mongolian medicine with pleiotropic pharmacological and ecological economic importance endemic to Asian temperature deserts. Terpenoids play critical roles in biotic and abiotic stresses due to their anti-oxidative activities. Based on non-targeted metabolomic analysis, we detected eight terpenoids enriched in the above-ground tissues of A. squarrosum, however, the molecular mechanism underlying terpenoids biosynthesis in this desert medicinal plant is rarely understood. Here, a comparative transcriptome analysis of different tissues in A. squarrosum was conducted to identify 84 unigenes encoding key enzymes in the upstream backbone biosynthesis and 53 unigenes encoding the downstream enzymes for terpenoid diversification. Most of the upstream genes exhibited significant high expression levels in leaf, and some of which were validated by qRT-PCR. Phylogenetic analysis showed that two downstream gene families OSCs (oxidosqualene cyclases) and TPSs (mainly in terpene synthases -g subfamily) had undergone notable gene expansions in A. squarrosum comparing with the other Amaranthaceae plant species and Arabidopsis. Nevertheless, most members from these two gene families showed the tissue-specific expression in A. squarrosum, which supported the diversification and tissue-specific enrichment of terpenoids across above-ground tissues. Considering to the habitat characteristics of A. squarrosum, we proposed that the enrichment of terpenoids and the functional diversification of terpenoids biosynthesis enzymes were more or less involved into its adaptation to stressful environments of deserts. These results expand the available genetic information underlying terpenoid biosynthesis in A. squarrosum, and contribute to deeper researches on pharmaceutical and eco-agricultural applications in this desert medicinal plant. [ABSTRACT FROM AUTHOR]

Terpenes are organic compounds and play important roles in plant development and stress response. Terpene synthases (TPSs) are the key enzymes for the biosynthesis of terpenes. For Rosaceae species, terpene composition represents a critical quality attribute, but limited information is available regarding the evolution and expansion occurring in the terpene synthases gene family. Here, we selected eight Rosaceae species with sequenced and annotated genomes for the identification of TPSs, including three Prunoideae, three Maloideae, and two Rosoideae species. Our data showed that the TPS gene family in the Rosaceae species displayed a diversity of family numbers and functions among different subfamilies. Lineage and species-specific expansion of the TPSs accompanied by frequent domain loss was widely observed within different TPS clades, which might have contributed to speciation or environmental adaptation in Rosaceae. In contrast to Maloideae and Rosoideae species, Prunoideae species owned less TPSs, with the evolution of Prunoideae species, TPSs were expanded in modern peach. Both tandem and segmental duplication significantly contributed to TPSs expansion. Ka/Ks calculations revealed that TPSs genes mainly evolved under purifying selection except for several pairs, where the divergent time indicated TPS-e clade was diverged relatively anciently. Gene function classification of TPSs further demonstrated the function diversity among clades and species. Moreover, based on already published RNA-Seq data from NCBI, the expression of most TPSs in Malus domestica, Prunus persica, and Fragaria vesca displayed tissue specificity and distinct expression patterns either in tissues or expression abundance between species and TPS clades. Certain putative TPS-like proteins lacking both domains were detected to be highly expressed, indicating the underlying functional or regulatory potentials. The result provided insight into the TPS family evolution and genetic information that would help to improve Rosaceae species quality.

SUMMARY: Terpenoid metabolism plays vital roles in stress defense and the environmental adaptation of monocot crops. Here, we describe the identification of the terpene synthase (TPS) gene family of the panicoid food and bioenergy model crop foxtail millet (Setaria italica). The diploid S. italica genome contains 32 TPS genes, 17 of which were biochemically characterized in this study. Unlike other thus far investigated grasses, S. italica contains TPSs producing all three ent‐, (+)‐ and syn‐copalyl pyrophosphate stereoisomers that naturally occur as central building blocks in the biosynthesis of distinct monocot diterpenoids. Conversion of these intermediates by the promiscuous TPS SiTPS8 yielded different diterpenoid scaffolds. Additionally, a cytochrome P450 monooxygenase (CYP99A17), which genomically clustered with SiTPS8, catalyzes the C19 hydroxylation of SiTPS8 products to generate the corresponding diterpene alcohols. The presence of syntenic orthologs to about 19% of the S. italica TPSs in related grasses supports a common ancestry of selected pathway branches. Among the identified enzyme products, abietadien‐19‐ol, syn‐pimara‐7,15‐dien‐19‐ol and germacrene‐d‐4‐ol were detectable in planta, and gene expression analysis of the biosynthetic TPSs showed distinct and, albeit moderately, inducible expression patterns in response to biotic and abiotic stress. In vitro growth‐inhibiting activity of abietadien‐19‐ol and syn‐pimara‐7,15‐dien‐19‐ol against Fusarium verticillioides and Fusarium subglutinans may indicate pathogen defensive functions, whereas the low antifungal efficacy of tested sesquiterpenoids supports other bioactivities. Together, these findings expand the known chemical space of monocot terpenoid metabolism to enable further investigations of terpenoid‐mediated stress resilience in these agriculturally important species. Significance Statement: The diverse class of terpenoids serves important functions in inter‐organismal and environmental interactions in many plant species. The discovery of terpenoid metabolic pathways in the model crop foxtail millet (Setaria italica) provides foundational knowledge of the diversity of terpenoid metabolism in poaceous food and bioenergy crops for possible future application of these natural defense mechanisms for improving crop traits. [ABSTRACT FROM AUTHOR]

We explore sequence determinants of enzyme activity and specificity in a major enzyme family of terpene synthases. Most enzymes in this family catalyze reactions that produce cyclic terpenes—complex hydrocarbons widely used by plants and insects in diverse biological processes such as defense, communication, and symbiosis. To analyze the molecular mechanisms of emergence of terpene cyclization, we have carried out in-depth examination of mutational space around (E)-β-farnesene synthase, an Artemisia annua enzyme which catalyzes production of a linear hydrocarbon chain. Each mutant enzyme in our synthetic libraries was characterized biochemically, and the resulting reaction rate data were used as input to the Michaelis–Menten model of enzyme kinetics, in which free energies were represented as sums of one-amino-acid contributions and two-amino-acid couplings. Our model predicts measured reaction rates with high accuracy and yields free energy landscapes characterized by relatively few coupling terms. As a result, the Michaelis–Menten free energy landscapes have simple, interpretable structure and exhibit little epistasis. We have also developed biophysical fitness models based on the assumption that highly fit enzymes have evolved to maximize the output of correct products, such as cyclic products or a specific product of interest, while minimizing the output of byproducts. This approach results in nonlinear fitness landscapes that are considerably more epistatic. Overall, our experimental and computational framework provides focused characterization of evolutionary emergence of novel enzymatic functions in the context of microevolutionary exploration of sequence space around naturally occurring enzymes. [ABSTRACT FROM AUTHOR]

Abstract: (E)-β-Caryophyllene is a sesquiterpene compound widely distributed in plants and functions in plant defence. However, little is known about the sequence and function of (E)-β-caryophyllene synthase in lima bean (Phaseolus lunatus). Here, we report a new full-length cDNA (PlCAHS) encoding (E)-β-caryophyllene synthase, a possible key enzyme of plant defence. The cDNA of PlCAHS contains an open reading frame of 1 761 bp, encoding a protein of 586 amino acids with a predicted mass of 67.95 kDa. The deduced amino acid sequence shows 52% identity with sesquiterpene synthase MtCAHS of Medicago truncatula. Based on phylogenetic analysis, PlCAHS is classified as the terpene synthases (TPS)-a subfamily. The recombinant enzyme, expressed in Escherichia coli, catalysed the formation of a major product (E)-β-caryophyllene (82%) and a minor product α-humulene (18%) from farnesyl diphosphate. Real-time quantitative PCR (qRT-PCR) analysis found that the PlCAHS transcript was significantly up-regulated in leaves after treatment with spider mites and alamethicin (ALA), suggesting its ecological function in plant defence.

Terpenoids comprise tens of thousands of small molecule natural products that are widely distributed across all domains of life. Plants produce by far the largest array of terpenoids with various roles in development and chemical ecology. Driven by selective pressure to adapt to their specific ecological niche, individual species form only a fraction of the myriad plant terpenoids, typically representing unique metabolite blends. Terpene synthase (TPS) enzymes are the gatekeepers in generating terpenoid diversity by catalyzing complex carbocation-driven cyclization, rearrangement, and elimination reactions that enable the transformation of a few acyclic prenyl diphosphate substrates into a vast chemical library of hydrocarbon and, for a few enzymes, oxygenated terpene scaffolds. The seven currently defined clades (a-h) forming the plant TPS family evolved from ancestral triterpene synthase- and prenyl transferase–type enzymes through repeated events of gene duplication and subsequent loss, gain, or fusion of protein domains and further functional diversification. Lineage-specific expansion of these TPS clades led to variable family sizes that may range from a single TPS gene to families of more than 100 members that may further function as part of modular metabolic networks to maximize the number of possible products. Accompanying gene family expansion, the TPS family shows a profound functional plasticity, where minor active site alterations can dramatically impact product outcome, thus enabling the emergence of new functions with minimal investment in evolving new enzymes. This article reviews current knowledge on the functional diversity and molecular evolution of the plant TPS family that underlies the chemical diversity of bioactive terpenoids across the plant kingdom.

Terpenoids comprise tens of thousands of small molecule natural products that are widely distributed across all domains of life. Plants produce by far the largest array of terpenoids with various roles in development and chemical ecology. Driven by selective pressure to adapt to their specific ecological niche, individual species form only a fraction of the myriad plant terpenoids, typically representing unique metabolite blends. Terpene synthase (TPS) enzymes are the gatekeepers in generating terpenoid diversity by catalyzing complex carbocation-driven cyclization, rearrangement, and elimination reactions that enable the transformation of a few acyclic prenyl diphosphate substrates into a vast chemical library of hydrocarbon and, for a few enzymes, oxygenated terpene scaffolds. The seven currently defined clades (a-h) forming the plant TPS family evolved from ancestral triterpene synthase- and prenyl transferase–type enzymes through repeated events of gene duplication and subsequent loss, gain, or fusion of protein domains and further functional diversification. Lineage-specific expansion of these TPS clades led to variable family sizes that may range from a single TPS gene to families of more than 100 members that may further function as part of modular metabolic networks to maximize the number of possible products. Accompanying gene family expansion, the TPS family shows a profound functional plasticity, where minor active site alterations can dramatically impact product outcome, thus enabling the emergence of new functions with minimal investment in evolving new enzymes. This article reviews current knowledge on the functional diversity and molecular evolution of the plant TPS family that underlies the chemical diversity of bioactive terpenoids across the plant kingdom. [ABSTRACT FROM AUTHOR]