Search

Floral mimics deceive their pollinators by developing visual and olfactory resemblance to various models. We report a flower that exhibits phenotypes like aerial litter and deceives an aerial litter specialist beetle to achieve pollination. We assessed the floral phenology and the effective pollinators of an Australian understorey treelet, Meiogyne heteropetala (Annonaceae). The similarities of morphology, colour and odour between the flowers and co‐occurring aerial litter were investigated. The terpene synthase involved in floral scent emission was identified by expression patterns and product profile. The behavioural responses of the pollinator to various odours were assessed using bioassays. The erotylid beetle Loberus sharpi is the most likely effective pollinator because it was the only pollen‐laden visitor during the pistillate phase. Loberus sharpi was exclusively found in aerial litter and M. heteropetala flowers. The flowers offer an honest shelter reward. The beetle also oviposits there, but most larvae eventually perished as the petals dropped onto the forest floor. The morphology and spectral reflectance of the flowers overlap with aerial litter. The floral scent was dominated by monoterpenes, especially 1,8‐cineole. The cineole synthase MhCINS was the only highly expressed floral terpene synthase and possessed a highly similar product profile to the floral scent composition. The volatile composition of M. heteropetala flowers is distinct from other congeners and highly similar to aerial litter, indicating advergence to aerial litter. Visual and odour resemblance, coupled with low larval survivorship, provides evidence that the beetles were deceived into pollinating the flowers. Behavioural experiments showed that the pollinator was attracted to both aerial litter and M. heteropetala flowers. The beetles were also attracted to 1,8‐cineole and synthetic mixes of floral odour and MhCINS products. The beetles were unable to distinguish floral scent from MhCINS products nor from 1,8‐cineole, suggesting MhCINS alone sufficed to attract the pollinator olfactorily. The beetles, however, preferred aerial litter over flowers. The beetles likely categorised the flower as a general, but not the most preferred, brood substrate. Synthesis. This study reports the first case of floral mimicry of aerial litter and characterises the biochemical process responsible for olfactory mimicry. [ABSTRACT FROM AUTHOR]

Terpene synthases (TPSs) are crucial for the diversification of terpenes, catalyzing the formation of a wide variety of terpenoid compounds. However, genome-wide systematic characterization of TPS genes in Cymbidium ensifolium has not been reported. Within the genomic database of C. ensifolium, we found 30 CeTPS genes for this investigation. CeTPS genes were irregularly distributed throughout the seven chromosomes and primarily expanded through tandem duplications. The CeTPS proteins were classified into three TPS subfamilies, including 17 TPS-b members, 8 TPS-a members, and 5 TPS-c members. Conserved motif analysis showed that most CeTPSs contained DDxxD and RRX8W motifs. Cis-element analysis of CeTPS gene promoters indicated regulation primarily by plant hormones and stress. Transcriptome analysis revealed that CeTPS1 and CeTPS18 had high expression in C. ensifolium flowers. qRT-PCR results showed that CeTPS1 and CeTPS18 were predominantly expressed during the flowering stage. Furthermore, CeTPS1 and CeTPS18 proteins were localized in the chloroplasts. These results lay the theoretical groundwork for future research on the functions of CeTPSs in terpenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Background: Phalaenopsis bellina, an orchid native to Borneo, is renowned for its unique appearance. It releases distinct fragrances, which have been linked to the presence of terpenoids. However, the identification and study of sesquiterpene synthase in P. bellina remain limited. In this study, we examines the functional characterisation of terpene synthase (TPS) from P. bellina, known as PbTS, through recombinant protein expression and its manifestation in the flower. Methods and Results: Gene annotation of PbTS revealed that the inferred peptide sequence of PbTS comprises 1,680 bp nucleotides encoding 559 amino acids with an estimated molecular mass of 65.2 kDa and a pI value of 5.4. A similarity search against GenBank showed that PbTS shares similarities with the previously published partial sequence of P. bellina (ABW98504.1) and Phalaenopsis equestris (XP_020597359.1 and ABW98503.1). Intriguingly, the phylogenetic analysis places the PbTS gene within the TPS-a group. In silico analysis of PbTS demonstrated stable interactions with farnesyl pyrophosphate (FPP), geranyl pyrophosphate (GPP), and geranylgeranyl pyrophosphate (GGPP). To verify this activity, an in vitro enzyme assay was performed on the PbTS recombinant protein, which successfully converted FPP, GPP, and GGPP into acyclic sesquiterpene β-farnesene, yielding approximately 0.03 mg/L. Expressional analysis revealed that the PbTS transcript was highly expressed in P. bellina, but its level did not correlate with β-farnesene levels across various flowering time points and stages. Conclusion: The insights gained from this study will enhance the understanding of terpenoid production in P. bellina and aid in the discovery of novel fragrance-related genes in other orchid species. [ABSTRACT FROM AUTHOR]

Abstract Background Ashbya gossypii is a filamentous fungus widely utilized for industrial riboflavin production and has a great potential as a microbial chassis for synthesizing other valuable metabolites such as folates, biolipids, and limonene. Engineered strains of A. gossypii can effectively use various waste streams, including xylose-rich feedstocks. Notably, A. gossypii has been identified as a proficient biocatalyst for producing limonene from xylose-rich sources. This study aims to investigate the capability of engineered A. gossypii strains to produce various plant monoterpenes using agro-industrial waste as carbon sources. Results We overexpressed heterologous terpene synthases to produce acyclic, monocyclic, and bicyclic monoterpenes in two genetic backgrounds of A. gossypii. These backgrounds included an NPP synthase orthogonal pathway and a mutant erg20 F95W allele with reduced FPP synthase activity. Our findings demonstrate that A. gossypii can synthesize linalool, limonene, pinene, and sabinene, with terpene synthases showing differential substrate selectivity for NPP or GPP precursors. Additionally, co-overexpression of endogenous HMG1 and ERG12 with heterologous NPP synthase and terpene synthases significantly increased sabinene yields from xylose-containing media. Using mixed formulations of corn-cob lignocellulosic hydrolysates and either sugarcane or beet molasses, we achieved limonene and sabinene productions of 383 mg/L and 684.5 mg/L, respectively, the latter representing a significant improvement compared to other organisms in flask culture mode. Conclusions Engineered A. gossypii strains serve as a suitable platform for assessing plant terpene synthase functionality and substrate selectivity in vivo, which are crucial to understand monoterpene bioproduction. The NPP synthase pathway markedly enhances limonene and sabinene production in A. gossypii, achieving levels comparable to those of other industrial microbial producers. Furthermore, these engineered strains offer a novel approach for producing monoterpenes through the valorization of agro-industrial wastes.

Timber trees are targets of herbivorous attacks. The identification of genes associated with pest resistance can be accomplished through differential expression analysis using transcriptomes. We reported the de novo assembly of cedar (Cedrela odorata L.) transcriptome and the differential expression of genes involved in herbivore resistance. The assembly and annotation of the transcriptome were obtained using RNAseq from healthy cedar plants and those infested with Chrysobothris yucatanensis. A total of 325.6 million reads were obtained, and 127,031 (97.47%) sequences were successfully assembled. A total of 220 herbivory-related genes were detected, of which 170 genes were annotated using GO terms, and 161 genes with 245 functions were identified—165, 75, and 5 were molecular functions, biological processes, and cellular components, respectively. To protect against herbivorous infestation, trees produce toxins and volatile compounds which are modulated by signaling pathways and gene expression related to molecular functions and biological processes. The limited number of genes identified as cellular components suggests that there are minimal alterations in cellular structure in response to borer attack. The chitin recognition protein, jasmonate ZIM-domain (JAZ) motifs, and response regulator receiver domain were found to be overexpressed, whereas the terpene synthase, cytochrome P450, and protein kinase domain gene families were underexpressed. This is the first report of a cedar transcriptome focusing on genes that are overexpressed in healthy plants and underexpressed in infested plants. This method may be a viable option for identifying genes associated with herbivore resistance.

Abstract Background Several members of the Lamiaceae family of plants produce large amounts of essential oil [EO] that find extensive applications in the food, cosmetics, personal hygiene, and alternative medicine industries. There is interest in enhancing EO metabolism in these plants. Main body Lavender produces a valuable EO that is highly enriched in monoterpenes, the C10 class of the isoprenoids or terpenoids. In recent years, substantial effort has been made by researchers to study terpene metabolism and enhance lavender EO through plant biotechnology. This paper reviews recent advances related to the cloning of lavender monoterpene biosynthetic genes and metabolic engineering attempts aimed at improving the production of lavender monoterpenes in plants and microbes. Conclusion Metabolic engineering has led to the improvement of EO quality and yield in several plants, including lavender. Furthermore, several biologically active EO constituents have been produced in microorganisms.

The diverse class of plant diterpenoid metabolites serves important functions in mediating growth, chemical defence, and ecological adaptation. In major monocot crops, such as maize (Zea mays), rice (Oryza sativa), and barley (Hordeum vulgare), diterpenoids function as core components of biotic and abiotic stress resilience. Switchgrass (Panicum virgatum) is a perennial grass valued as a stress‐resilient biofuel model crop. Previously we identified an unusually large diterpene synthase family that produces both common and species‐specific diterpenoids, several of which accumulate in response to abiotic stress. Here, we report discovery and functional characterization of a previously unrecognized monofunctional class I diterpene synthase (PvKSL1) via in vivo co‐expression assays with different copalyl pyrophosphate (CPP) isomers, structural and mutagenesis studies, as well as genomic and transcriptomic analyses. In particular, PvKSL1 converts ent‐CPP into ent‐abietadiene, ent‐palustradiene, ent‐levopimaradiene, and ent‐neoabietadiene via a 13‐hydroxy‐8(14)‐ent‐abietene intermediate. Notably, although featuring a distinct ent‐stereochemistry, this product profile is near‐identical to bifunctional (+)‐levopimaradiene/abietadiene synthases occurring in conifer trees. PvKSL1 has three of four active site residues previously shown to control (+)‐levopimaradiene/abietadiene synthase catalytic specificity. However, mutagenesis studies suggest a distinct catalytic mechanism in PvKSL1. Genome localization of PvKSL1 distant from other diterpene synthases, and its phylogenetic distinctiveness from known abietane‐forming diterpene synthases, support an independent evolution of PvKSL1 activity. Albeit at low levels, PvKSL1 gene expression predominantly in roots suggests a role of diterpenoid formation in belowground tissue. Together, these findings expand the known chemical and functional space of diterpenoid metabolism in monocot crops. [ABSTRACT FROM AUTHOR]

[Objective] Terpenes play important roles in plant physiology and defense. The terpene synthase gene (TPS) is a key enzyme in the terpene biosynthetic pathway. The stems of the tropical oil-secreting tree species Sindora glabra contain a large amount of terpenoid resin oil. This study explores the role of S. glabra SgTPS in terpene synthesis and abiotic stress response. These will provide important genetic resources for the production of terpenes through synthetic biology, and help expand the understanding of plant terpene biosynthesis pathways and regulatory mechanisms. [Method] The SgTPS7 gene was cloned from the stem tissue of S. glabra using genome and transcriptome data, and bioinformatics analysis was performed on it. The SgTPS7 protein was expressed and purified and its catalytic function was identified using GC-MS, and RT-qPCR technology was used to analyze the expression pattern of SgTPS7. [Result] The full length sequence of the coding region of SgTPS7 of S. glabra was 1 650 bp. Homology comparison analysis found that SgTPS7 had the highest similarity with CoTPS2 of Copaifera officinalis, at 92.3%. It was distantly related to plants of other families and genera, and it belonged to the unique TPS genes of Caesalpinioideae. In vitro enzyme activity experiments showed that SgTPS7 mainly catalyzed farnesyl pyrophosphate to produce germacrene D, and catalyzed geranyl pyrophosphate to produce linalool. The expression of SgTPS7 gene was significantly different under different stress treatment conditions. After treatment with high-temperature stress, the expression of SgTPS7 in the leaves and stems reached the peak at 24 h, while the expression in the roots showed a downward trend. Long-term drought treatment significantly affected the expression of SgTPS7, and the expression of SgTPS7 in the leaves and stems reached the highest after 5 days of treatment, while the expression of SgTPS7 in the roots reached the highest after 10 d. [Conclusion] SgTPS7 is a multifunctional terpene synthase gene, which plays an important role in responding to abiotic stresses such as high temperature and drought. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we screened a MYB transcription factor controlling the synthesis of linanol in Freesia hybrida. This transcription factor is functionally conservative and can play a role in regulating the expression of the linanol synthase gene in the model plant Arabidopsis, but the interaction with another bHLH type transcription factor eventually leads to different regulatory effects of the MYB regulator in Freesia hybrida and Arabidopsis. Acting as the most abundant and widely distributed volatile secondary metabolites in plants, terpenoids play crucial roles in diverse physiological regulations and metabolic processes. Terpene synthases play a decisive role in determining the composition and diversity of terpenoids. Though the regulation of terpene synthases has been extensively investigated across various plant species, limited studies have focused on the upstream transcriptional regulation of terpene synthases. In this study, we have identified linalool as the predominant volatile compound that is released gradually from Freesia hybrida flowers throughout flower blooming. In the context of the transcriptome, a typical MYB transcription factor, FhMYB108, was screened based on homologous gene comparison. FhMYB108 is capable of regulating the expression of FhTPS1, and both their expression levels showed gradual increase during flower opening. Moreover, FhMYB108 exerts a stimulatory effect on the transcription of Arabidopsis thaliana AtTPS14, while no significant increase in AtTPS14 expression is observed upon the stabilization of FhMYB108 in A. thaliana. The highly expressed AtMYC2 in A. thaliana could interact with FhMYB108 to suppress the activation of AtTPS14 by FhMYB108. The present study not only elucidates the regulatory mechanism underlying linalool synthesis but also discovers the synergistic effect of MYB and bHLH transcription factors in governing the biosynthesis of volatile terpenoids. [ABSTRACT FROM AUTHOR]

Background: Several members of the Lamiaceae family of plants produce large amounts of essential oil [EO] that find extensive applications in the food, cosmetics, personal hygiene, and alternative medicine industries. There is interest in enhancing EO metabolism in these plants. Main body: Lavender produces a valuable EO that is highly enriched in monoterpenes, the C10 class of the isoprenoids or terpenoids. In recent years, substantial effort has been made by researchers to study terpene metabolism and enhance lavender EO through plant biotechnology. This paper reviews recent advances related to the cloning of lavender monoterpene biosynthetic genes and metabolic engineering attempts aimed at improving the production of lavender monoterpenes in plants and microbes. Conclusion: Metabolic engineering has led to the improvement of EO quality and yield in several plants, including lavender. Furthermore, several biologically active EO constituents have been produced in microorganisms. [ABSTRACT FROM AUTHOR]

Terpenoids are structurally diverse natural products that have been widely used in the pharmaceutical, food, and cosmetic industries. Research has shown that fungi produce a variety of terpenoids, yet fungal terpene synthases remain not thoroughly explored. In this study, the tps1 gene, a crucial component of the terpene synthetic pathway, was isolated from Trichoderma atroviride HB20111 through genome mining. The function of this gene in the terpene synthetic pathway was investigated by constructing tps1-gene-deletion- and overexpression-engineered strains and evaluating the expression differences in the tps1 gene at the transcript level. HS-SPME-GC-MS analysis revealed significant variations in terpene metabolites among wild-type, tps1-deleted (Δtps1), and tps1-overexpressed (Otps1) strains; for instance, most sesquiterpene volatile organic compounds (VOCs) were notably reduced or absent in the Δtps1 strain, while nerolidol, β-acorenol, and guaiene were particularly produced by the Otps1 strain. However, both the Δtps1 and Otps1 strains produced new terpene metabolites compared to the wild-type, which indicated that the tps1 gene played an important role in terpene synthesis but was not the only gene involved in T. atroviride HB20111. The TPS1 protein encoded by the tps1 gene could function as a sesquiterpene cyclase through biological information and evolutionary tree analysis. Additionally, fungal inhibition assay and wheat growth promotion assay results suggested that the deletion or overexpression of the tps1 gene had a minimal impact on fungal inhibitory activity, plant growth promotion, and development, as well as stress response. This implies that these activities of T. atroviride HB20111 might result from a combination of multiple metabolites rather than being solely dependent on one specific metabolite. This study offers theoretical guidance for future investigations into the mechanism of terpenoid synthesis and serves as a foundation for related studies on terpenoid metabolic pathways in fungi. [ABSTRACT FROM AUTHOR]

This article presents a chromosome-level genome assembly of Ajuga decumbens, a flowering plant used in traditional Chinese medicine. The genome assembly was achieved using PacBio HiFi sequencing and Hi-C sequencing, resulting in a high-quality assembly. The genome annotation revealed a high repeat content and identified protein-coding genes, non-coding RNAs, and unique gene families. Phylogenetic analysis showed the evolutionary relationship of A. decumbens with other angiosperm species. This genome resource will contribute to the exploration of medicinal resources and the cultivation of new varieties in the Ajuga genus. The text discusses the significance of the research on Ajuga decumbens, highlighting its whole genome duplication and its medicinal and economic value. It emphasizes the importance of genomics in understanding genetic diversity, evolutionary relationships, and functional genomics in A. decumbens. The study used various methods to analyze the genomic data and provides access to the data in public repositories. The document is a compilation of references to scientific articles and journals related to plant biotechnology and genomics, providing library patrons with further research options on specific topics. [Extracted from the article]

Main conclusion: CaTPS2 and CaTPS3 were significantly expressed in flowers of Curcuma alismatifolia ‘Shadow’ and demonstrated bifunctional enzyme activity, CaTPS2 generated linalool and nerolidol as products, and CaTPS3 catalyzed β-myrcene and β-farnesene formation. This study presents the discovery and functional characterization of floral terpene synthase (TPS) genes in Curcuma alismatifolia ‘Shadow’, a cultivar renowned for its unique fragrance. Addressing the gap in understanding the genetic basis of floral scent in this species, we identified eight TPS genes through comprehensive transcriptome sequencing. Among these, CaTPS2 and CaTPS3 were significantly expressed in floral tissues and demonstrated bifunctional enzyme activity corresponding to the major volatile compounds detected in ‘Shadow’. Functional analyses, including in vitro assays complemented with rigorous controls and alternative identification methods, elucidated the roles of these TPS genes in terpenoid biosynthesis. In vitro studies were conducted via heterologous expression in E. coli, followed by purification of the recombinant protein using affinity chromatography, enzyme assays were performed with GPP/FPP as the substrate, and volatile products were inserted into the GC–MS for analysis. Partially purified recombinant protein of CaTPS2 catalyzed GPP and FPP to produce linalool and nerolidol, respectively, while partially purified recombinant protein of CaTPS3 generated β-myrcene and β-farnesene with GPP and FPP as substrates, respectively. Real-time quantitative PCR further validated the expression patterns of these genes, correlating with terpenoid accumulation in different plant tissues. Our findings illuminate the molecular mechanisms underpinning floral fragrance in C. alismatifolia and provide a foundation for future genetic enhancements of floral scent in ornamental plants. This study, therefore, contributes to the broader understanding of terpenoid biosynthesis in plant fragrances, paving the way for biotechnological applications in horticulture plant breeding. [ABSTRACT FROM AUTHOR]

We report the complete telomere-to-telomere genome assembly of Oldenlandia diffusa which renowned in traditional Chinese medicine, comprising 16 chromosomes and spanning 499.7 Mb. The assembly showcases 28 telomeres and minimal gaps, with a total of only five. Repeat sequences constitute 46.41% of the genome, and 49,701 potential protein-coding genes have been predicted. Compared with O. corymbosa , O. diffusa exhibits chromosome duplication and fusion events, diverging 20.34 million years ago. Additionally, a total of 11 clusters of terpene synthase have been identified. The comprehensive genome sequence, gene catalog, and terpene synthase clusters of O. diffusa detailed in this study will significantly contribute to advancing research in this species' genetic, genomic, and pharmacological aspects. [ABSTRACT FROM AUTHOR]

Sesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii, each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC.

Terpenoids are the largest family of natural products, but prokaryotes are vastly underrepresented in this chemical space. However, genomics supports vast untapped biosynthetic potential for terpenoids in bacteria. We discovered the first trans-eunicellane terpene synthase (TS), AlbS from Streptomyces albireticuli NRRL B-1670, in nature. Mutagenesis, deuterium labeling studies, and quantum chemical calculations provided extensive support for its cyclization mechanism. In addition, parallel stereospecific labeling studies with Bnd4, a cis-eunicellane TS, revealed a key mechanistic distinction between these two enzymes. AlbS highlights bacteria as a valuable source of novel terpenoids, expands our understanding of the eunicellane family of natural products and the enzymes that biosynthesize them, and provides a model system to address fundamental questions about the chemistry of 6,10-bicyclic ring systems.

Terpenoids are one of the largest class of natural products with diverse structures and activities. This enormous diversity is embedded in enzymes called terpene synthases (TSs), which generate diverse terpene skeletons via sophisticated cyclization cascades. In addition to the many highly selective TSs, there are many promiscuous TSs that accept multiple prenyl substrates, or even noncanonical ones, with 6, 7, 8, 11, and 16 carbon atoms, synthesized via chemical approaches, C-methyltransferases, or engineered lepidopteran mevalonate pathways. The substrate promiscuity of TSs not only expands the structural diversity of terpenes but also highlights their potential for the discovery of novel terpenoids via combinatorial biosynthesis. In this review, we focus on the current knowledge on multisubstrate terpene synthases (MSTSs) and highlight their potential applications.

Sea sponges are the largest marine source of small-molecule natural products described to date. Sponge-derived molecules, such as the chemotherapeutic eribulin, the calcium-channel blocker manoalide, and antimalarial compound kalihinol A, are renowned for their impressive medicinal, chemical, and biological properties. Sponges contain microbiomes that control the production of many natural products isolated from these marine invertebrates. In fact, all genomic studies to date investigating the metabolic origins of sponge-derived small molecules concluded that microbes-not the sponge animal host-are the biosynthetic producers. However, early cell-sorting studies suggested the sponge animal host may play a role particularly in the production of terpenoid molecules. To investigate the genetic underpinnings of sponge terpenoid biosynthesis, we sequenced the metagenome and transcriptome of an isonitrile sesquiterpenoid-containing sponge of the order Bubarida. Using bioinformatic searches and biochemical validation, we identified a group of type I terpene synthases (TSs) from this sponge and multiple other species, the first of this enzyme class characterized from the sponge holobiome. The Bubarida TS-associated contigs consist of intron-containing genes homologous to sponge genes and feature GC percentage and coverage consistent with other eukaryotic sequences. We identified and characterized TS homologs from five different sponge species isolated from geographically distant locations, thereby suggesting a broad distribution amongst sponges. This work sheds light on the role of sponges in secondary metabolite production and speaks to the possibility that other sponge-specific molecules originate from the animal host.

In maize (Zea mays), fungal-elicited immune responses include the accumulation of terpene synthase (TPS) and cytochrome P450 monooxygenases (CYP) enzymes resulting in complex antibiotic arrays of sesquiterpenoids and diterpenoids, including α/β-selinene derivatives, zealexins, kauralexins and dolabralexins. To uncover additional antibiotic families, we conducted metabolic profiling of elicited stem tissues in mapping populations, which included B73 × M162W recombinant inbred lines and the Goodman diversity panel. Five candidate sesquiterpenoids associated with a chromosome 1 locus spanning the location of ZmTPS27 and ZmTPS8. Heterologous enzyme co-expression studies of ZmTPS27 in Nicotiana benthamiana resulted in geraniol production while ZmTPS8 yielded α-copaene, δ-cadinene and sesquiterpene alcohols consistent with epi-cubebol, cubebol, copan-3-ol and copaborneol matching the association mapping efforts. ZmTPS8 is an established multiproduct α-copaene synthase; however, ZmTPS8-derived sesquiterpene alcohols are rarely encountered in maize tissues. A genome wide association study further linked an unknown sesquiterpene acid to ZmTPS8 and combined ZmTPS8-ZmCYP71Z19 heterologous enzyme co-expression studies yielded the same product. To consider defensive roles for ZmTPS8, in vitro bioassays with cubebol demonstrated significant antifungal activity against both Fusarium graminearum and Aspergillus parasiticus. As a genetically variable biochemical trait, ZmTPS8 contributes to the cocktail of terpenoid antibiotics present following complex interactions between wounding and fungal elicitation.

• Secondary metabolites are compounds that play an important role in a wide range of fields, from medicine to agriculture. • For the first time, a detailed investigation of the secondary metabolite biosynthetic gene cluster was conducted in citrus sinensis. • The analyses indicated the presence of medically valuable compounds such as alkaloids, terpenes, lignans, polyketides, etc. in C. sinensis. • Terpene synthases (TPS) in C. sinensis are predominantly found in the TPS-a subfamily. Plants synthesize primary and secondary metabolites through their biochemical processes. Secondary metabolites are substances with diverse functions, ranging from stress response to pharmaceutical production. Terpenes belong to the category of secondary metabolites. The purpose of this article is to predict the biosynthetic gene clusters of secondary metabolites in the Citrus sinensis , to identify the terpene synthases present in terpene clusters, and to elucidate their functions. Additionally, another goal is to identify the enzymes in orthologous species that are counterparts to these enzymes. The plantiSMASH tool was utilized for the prediction of secondary metabolite biosynthetic gene clusters. Later, enzymes with defined functions in the terpene synthase category were selected, ID numbers were assigned to them, and they were listed as CsTS1-CsTS23. The phylogenetic tree of enzymes belonging to the terpene synthase category was generated using the MEGA11 program and the Interactive Tree Of Life v6.7.3 software. For ortholog analysis, the OrthoDB v11 software was utilized. Following the conducted analyses, a total of 11 putative, 3 alkaloid, 3 lignan, 12 saccharide, 6 terpene, 1 terpene-alkaloid, 2 polyketide, 1 saccharide-terpene, and 1 terpene-saccharide-polyketide gene cluster was identified in the C. sinensis. Furthermore, within the phylogenetic tree of terpene synthases, 3 distinct groups were observed. It was determined that the first group had the highest number of members. In addition, orthologs for CsTS9, CsTS10, CsTS12, CsTS19 , and CsTS22 could not be identified in the example species. In conclusion, the determination of gene clusters for secondary metabolites will contribute to studies in diverse fields such as medicine, pharmacy, and agriculture. [ABSTRACT FROM AUTHOR]

Lilium ‘Siberia’, an Oriental hybrid, is renowned as both a cut flower and garden plant, prized for its color and fragrance. The fragrance comprises volatile organic compounds (VOCs), primarily monoterpenes found in the plant. While the primary terpene synthases in Lilium ‘Siberia’ were identified, the transcriptional regulation of these terpene synthase (TPS) genes remains unclear. Thus, understanding the regulatory mechanisms of monoterpene biosynthesis is crucial for breeding flower fragrance, thereby improving ornamental and commercial values. In this study, we isolated a nuclear-localized LiNAC100 transcription factor from Lilium ‘Siberia’. The virus-induced gene silencing (VIGS) of LiNAC100 was found to down-regulate the expression of linalool synthase gene (LiLiS) and significantly inhibit linalool synthesis. Conversely, transient overexpression of LiNAC100 produced opposite effects. Additionally, yeast one-hybrid and dual-luciferase assays confirmed that LiNAC100 directly activates LiLiS expression. Our findings reveal that LiNAC100 plays a key role in monoterpene biosynthesis in Lilium ‘Siberia’, promoting linalool synthesis through the activation of LiLiS expression. These results offer insights into the molecular mechanisms of terpene biosynthesis in Lilium ‘Siberia’ and open avenues for biotechnological enhancement of floral scent. [ABSTRACT FROM AUTHOR]

Trees form symbioses with ectomycorrhizal (ECM) fungi, maintained in part through mutual benefit to both organisms. Our understanding of the signaling events leading to the successful interaction between the two partners requires further study. This is especially true for understanding the role of volatile signals produced by ECM fungi. Terpenoids are a predominant class of volatiles produced by ECM fungi. While several ECM genomes are enriched in the enzymes responsible for the production of these volatiles (i.e., terpene synthases (TPSs)) when compared to other fungi, we have limited understanding of the biochemical products associated with each enzyme and the physiological impact of specific terpenes on plant growth. Using a combination of phylogenetic analyses, RNA sequencing, and functional characterization of five TPSs from two distantly related ECM fungi (Laccaria bicolor and Pisolithus microcarpus), we investigated the role of these secondary metabolites during the establishment of symbiosis. We found that despite phylogenetic divergence, these TPSs produced very similar terpene profiles. We focused on the role of P. microcarpus terpenes and found that the fungus expressed a diverse array of mono-, di-, and sesquiterpenes prior to contact with the host. However, these metabolites were repressed following physical contact with the host Eucalyptus grandis. Exposure of E. grandis to heterologously produced terpenes (enriched primarily in γ -cadinene) led to a reduction in the root growth rate and an increase in P. microcarpus–colonized root tips. These results support a very early putative role of fungal-produced terpenes in the establishment of symbiosis between mycorrhizal fungi and their hosts. [ABSTRACT FROM AUTHOR]

Insect‐induced plant volatile organic compounds (VOCs) may function as either direct defence molecules to deter insects or indirect defence signals to attract the natural enemies of the invading insects. Tea (Camellia sinensis L.), an important leaf‐based beverage crop, is mainly infested by Ectropis obliqua which causes the most serious damage. Here, we report a mechanistic investigation of tea plant‐derived VOCs in an indirect defence mechanism against E. obliqua. Parasitoid wasp Parapanteles hyposidrae, a natural enemy of E. obliqua, showed strong electrophysiological response and selection behaviour towards S‐linalool and β‐ocimene, two monoterpenes with elevated emission from E. obliqua‐damaged tea plants. Larvae frass of E. obliqua, which also released S‐linalool and β‐ocimene, was found to attract both mated female or male Pa. hyposidrae according to gas chromatography‐electroantennogram detection and Y‐tube olfactometer assays. In a field setting, both S‐linalool and β‐ocimene were effective in recruiting both female and male Pa. hyposidrae wasps. To understand the molecular mechanism of monoterpenes‐mediated indirect defence in tea plants, two novel monoterpene synthase genes, CsLIS and CsOCS‐SCZ, involved in the biosynthesis of S‐linalool or β‐ocimene, respectively, were identified and biochemically characterised. When the expression of these two genes in tea plants was inhibited by antisense oligodeoxynucleotide, both volatile emission and attraction of wasps were reduced. Furthermore, gene expression analysis suggested that the expression of CsLIS and CsOCS‐SCZ is regulated by the jasmonic acid signalling pathway in the tea plant. Summary statement: S‐linalool and β‐ocimene elevated emission from Ectropis obliqua‐damaged tea plants. The two monoterpenes were key chemical cues, aroused parasitoid wasp Parapanteles hyposidrae, a dominant natural enemy of E. obliqua, strong electrophysiological response, selection behaviour, and aggregated the wasp in the field. Two novel Terpene synthases CsLIS and CsOCS‐SCZ, involved in the biosynthesis of S‐linalool and β‐ocimene were identified, when they were inhibited in tea plants by antisense oligonucleotides, both volatile emission and attraction of wasps were reduced. [ABSTRACT FROM AUTHOR]

Blumea balsamifera (L.) DC., an important economic and medicinal herb, has a long history of being used as a traditional Chinese medicine. Its leaves have always been used as a raw material for the extraction of essential oils, comprising large amounts of terpenoids, which have good therapeutic effects on many diseases, such as eczema, bacterial infection, and hypertension. However, the genetic basis of terpenoid biosynthesis in this plant is virtually unknown on account of the lack of genomic data. Here, a combination of next-generation sequencing (NGS) and full-length transcriptome sequencing was applied to identify genes involved in terpenoid biosynthesis at five developmental stages. Then, the main components of essential oils in B. balsamifera were identified using GC–MS. Overall, 16 monoterpenoids and 20 sesquiterpenoids were identified and 333,860 CCS reads were generated, yielding 65,045 non-redundant transcripts. Among these highly accurate transcripts, 59,958 (92.18%) transcripts were successfully annotated using NR, eggNOG, Swissprot, KEGG, KOG, COG, Pfam, and GO databases. Finally, a total of 56 differently expressed genes (DEGs) involved in terpenoid biosynthesis were identified, including 38 terpenoid backbone genes and 18 TPSs, which provide a significant amount of genetic information for B. balsamifera. These results build a basis for resource protection, molecular breeding, and the metabolic engineering of this plant. [ABSTRACT FROM AUTHOR]

Main conclusion: The study demonstrated thatArtemisia pallensroots can be a source of terpene-rich essential oil and root-specific ApTPS1 forms germacrene A contributing to major root volatiles. Davana (Artemisia pallens Bess) is a valuable aromatic herb within the Asteraceae family, highly prized for its essential oil (EO) produced in the aerial parts. However, the root volatile composition, and the genes responsible for root volatiles have remained unexplored until now. Here, we show that A. pallens roots possess distinct oil bodies and yields ~ 0.05% of EO, which is primarily composed of sesquiterpenes β-elemene, neryl isovalerate, β-selinene, and α-selinene, and trace amounts of monoterpenes β-myrcene, D-limonene. This shows that, besides aerial parts, roots of davana can also be a source of unique EO. Moreover, we functionally characterized a terpene synthase (ApTPS1) that exhibited high in silico expression in the root transcriptome. The recombinant ApTPS1 showed the formation of β-elemene and germacrene A with E,E-farnesyl diphosphate (FPP) as a substrate. Detailed analysis of assay products revealed that β-elemene was the thermal rearrangement product of germacrene A. The functional expression of ApTPS1 in Saccharomyces cerevisiae confirmed the in vivo germacrene A synthase activity of ApTPS1. At the transcript level, ApTPS1 displayed predominant expression in roots, with significantly lower level of expression in other tissues. This expression pattern of ApTPS1 positively correlated with the tissue-specific accumulation level of germacrene A. Overall, these findings provide fundamental insights into the EO profile of davana roots, and the contribution of ApTPS1 in the formation of a major root volatile. [ABSTRACT FROM AUTHOR]

Perilla is a characteristic medicinal and edible plant. According to the different monoterpenes in Perilla leaves, Perilla can be divided into many chemical types. In this study, four Perilla cultivars of different chemical types, including perillaldehyde (PA), perillaketone (PK), perillene (PL) and piperitenone (PT) were selected for metabolome and transcriptome analysis. In total, 35 terpene compounds were identified and main monoterpenes were consistent with corresponding chemical types, respectively. Combined with transcriptomes, 5,920 differentially expressed genes were obtained among them. In total, 69 genes referred to MVA (Mevalonate) and MEP (2-C-methyl-D-erythritol-4-phosphate) pathways and 109 PfTPSs encoded genes were identified, which possessed partial differentially expressed in four chemical types. The core PfTPSs in co-expression analysis were functionally characterized. Three PfTPSs were identified as linalool synthase and one PfTPSs was identified as geranyl synthase. This research analyzed the monoterpene biosynthesis and functional characterization of TPSs in Perilla, which can provide a foundation for in-depth research of the Perilla chemotype metabolic mechanism.

Jasmine (Jasminum sambac Aiton) is a well-known cultivated plant species for its fragrant flowers used in the perfume industry and cosmetics. However, the genetic basis of its floral scent is largely unknown. In this study, using PacBio, Illumina, 10× Genomics and high-throughput chromosome conformation capture (Hi-C) sequencing technologies, a high-quality chromosome-level reference genome for J. sambac was obtained, exploiting a double-petal phenotype cultivar ‘Shuangbanmoli’ (JSSB). The results showed that the final assembled genome of JSSB is 580.33 Mb in size (contig N50 = 1.05 Mb; scaffold N50 = 45.07 Mb) with a total of 39 618 predicted protein-coding genes. Our analyses revealed that the JSSB genome has undergone an ancient whole-genome duplication (WGD) event at 91.68 million years ago (Mya). It was estimated that J. sambac diverged from the lineage leading to Olea europaea and Osmanthus fragrans about 28.8 Mya. On the basis of a combination of genomic, transcriptomic and metabolomic analyses, a range of floral scent volatiles and genes were identified involved in the benzenoid/phenylpropanoid and terpenoid biosynthesis pathways. The results provide new insights into the molecular mechanism of its fragrance biosynthesis in jasmine.

Abstract Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light‐driven biofactories for the production of high‐value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene—an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g−1 total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate‐limiting enzymes in the 2‐C‐methyl‐ d‐erythritol 4‐phosphate pathway, Coleus forskohlii 1‐deoxy‐ d‐xylulose‐5‐phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g−1 DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.

Terpene synthases (TPSs) are crucial for the diversification of terpenes, catalyzing the formation of a wide variety of terpenoid compounds. However, genome-wide systematic characterization of TPS genes in Cymbidium ensifolium has not been reported. Within the genomic database of C. ensifolium, we found 30 CeTPS genes for this investigation. CeTPS genes were irregularly distributed throughout the seven chromosomes and primarily expanded through tandem duplications. The CeTPS proteins were classified into three TPS subfamilies, including 17 TPS-b members, 8 TPS-a members, and 5 TPS-c members. Conserved motif analysis showed that most CeTPSs contained DDxxD and RRX8W motifs. Cis-element analysis of CeTPS gene promoters indicated regulation primarily by plant hormones and stress. Transcriptome analysis revealed that CeTPS1 and CeTPS18 had high expression in C. ensifolium flowers. qRT-PCR results showed that CeTPS1 and CeTPS18 were predominantly expressed during the flowering stage. Furthermore, CeTPS1 and CeTPS18 proteins were localized in the chloroplasts. These results lay the theoretical groundwork for future research on the functions of CeTPSs in terpenoid biosynthesis.

Terpenes are important mediators of plant chemical response to environmental cues. Here, we describe the genome-wide identification and biochemical characterization of TPS-a members in Medicago truncatula, a model legume crop. Genome mining identified thirty-nine full-length terpene synthases with a significant number predicted to produce monoterpenes and sesquiterpenes. Biochemical characterization of the TPS-a subfamily associated with sesquiterpene biosynthesis revealed such compounds, that exhibit substantial biological activity in other plants. Gene expression analysis using qPCR and the Medicago gene atlas illustrated distinct tissue and time-based variation in expression in leaves and roots. Together our work establishes the gene-tometabolite relationships for sesquiterpene synthases in M. truncatula. Understanding the biosynthetic capacity is a foundational step to defining the ecological roles of this important family of compounds. [ABSTRACT FROM AUTHOR]

Zephyranthes candida is a frequently cultivated ornamental plant containing several secondary metabolites, including alkaloids, flavonoids, and volatile organic compounds (VOCs). However, extensive research has been conducted only on non-VOCs found in the plant, whereas the production of VOCs and the molecular mechanisms underlying the biosynthesis of terpenes remain poorly understood. In this study, 17 volatile compounds were identified from Z. candida flowers using gas chromatography–mass spectrometry (GC-MS), with 16 of them being terpenoids. Transcriptome sequencing resulted in the identification of 17 terpene synthase (TPS) genes; two TPS genes, ZcTPS01 and ZcTPS02, had high expression levels. Biochemical characterization of two enzymes encoded by both genes revealed that ZcTPS02 can catalyze geranyl diphosphate (GPP) into diverse products, among which is β-ocimene, which is the second most abundant compound found in Z. candida flowers. These results suggest that ZcTPS02 plays a vital role in β-ocimene biosynthesis, providing valuable insights into terpene biosynthesis pathways in Z. candida. Furthermore, the expression of ZcTPS02 was upregulated after 2 h of methyl jasmonate (MeJA) treatment and downregulated after 4 h of the same treatment. [ABSTRACT FROM AUTHOR]

Conifers have evolved sophisticated terpenoid defenses for protection against herbivores and pathogens. Pinus massoniana Lamb. is the most widely distributed pioneer afforestation and resin tree species in China, but is seriously harmed by pine wilt disease. Monochamus alternatus is the main vector of pine wilt disease in China. Monoterpenes, sesquiterpenes and diterpenes, the main secondary defensive compounds of P. massoniana, are catalyzed by different terpene synthases (TPSs), which participate in the important defense pathways against external biotic and abiotic stresses. Here, we aimed to identify the terpene synthases (TPSs) in P. massoniana, responding to the feeding of M. alternatus, and to characterize the functions and products of the mono-TPSs. We identified six differentially expressed TPS genes in the P. massoniana fed upon by M. alternatus, including four mono-TPS and two sesqui-TPS genes. The functions of the four mono-TPSs were verified by analysis of the main product and by-products of these mono-TPSs. (+)-α-Pinene, (−)-α-pinene, and limonene were the major products of TPS (+)-α-pinene, TPS (−)-α-pinene, and TPS limonene, respectively, but TPS (−)-β-pinene only catalyzed a trace amount of (−)-β-pinene in the products. Our findings shed light on the potential relationships between the structure of terpene synthases and their corresponding products. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Shenyang Agricultural University is the property of Journal of Shenyang Agricultural University Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Chia (Salvia hispanica L.) is one of the most popular nutrition-rich foods and pseudocereal crops of the family Lamiaceae. Chia seeds are a rich source of proteins, polyunsaturated fatty acids (PUFAs), dietary fibers, and antioxidants. In this study, we present the assembly of the chia reference genome, which spans 303.6 Mb and encodes 48,090 annotated protein-coding genes. Our analysis revealed that ~42% of the chia genome harbors repetitive content, and identified ~3 million single nucleotide polymorphisms (SNPs) and 15,380 simple sequence repeat (SSR) marker sites. By investigating the chia transcriptome, we discovered that ~44% of the genes undergo alternative splicing with a higher frequency of intron retention events. Additionally, we identified chia genes associated with important nutrient content and quality traits, such as the biosynthesis of PUFAs and seed mucilage fiber (dietary fiber) polysaccharides. Notably, this is the first report of in-silico annotation of a plant genome for protein-derived small bioactive peptides (biopeptides) associated with improving human health. To facilitate further research and translational applications of this valuable orphan crop, we have developed the Salvia genomics database (SalviaGDB), accessible at https://salviagdb.org. [ABSTRACT FROM AUTHOR]

Litsea cubeba , the core species of the Lauraceae family, is valuable for the production of essential oils due to its high concentration of monoterpenes (90%). The key monoterpene synthase and metabolic regulatory network of monoterpene biosynthesis have provided new insights for improving essential oil content. However, there are few studies on the regulation mechanism of monoterpenes in L. cubeba. In this study, we investigated LcTPS32 , a member of the TPS-b subfamily, and identified its function as an enzyme for the synthesis of monoterpenes, including geraniol, α-pinene, β-pinene, β-myrcene, linalool and eucalyptol. The quantitative real-time PCR analysis showed that LcTPS32 was highly expressed in the fruits of L. cubeba and contributed to the characteristic flavor of its essential oil. Overexpression of LcTPS32 resulted in a significant increase in the production of monoterpenes in L. cubeba by activating both the MVA and MEP pathways. Additionally, the study revealed that LcMYB106 played a negative regulatory role in monoterpenes biosynthesis by directly binding to the promoter of LcTPS32. Our study indicates that LcMYB106 could serve as a crucial target for metabolic engineering endeavors, aiming at enhancing the monoterpene biosynthesis in L. cubeba. [ABSTRACT FROM AUTHOR]

Salvia officinalis plant produce various terpene compounds have many roles and useful biological properties in plants. In different salvia species structure of sesquiterpenes was remarked to be the main group of compounds at various tissues and development stages. However, some genes responsible for sesquiterpene synthese in S. officinalis remain unclear. In this study, we clone the full-length of sesquiterpene synthase (SoTPS3) gene from S. officinalis. The full-length cDNA of SoTPS3 contains a 1668-bp open reading frame that putatively encoded a protein of 555 amino acids which resembled a sesquiterpene synthase in sequence. The deduced SoTPS3 shared an overall homology with other known sesquiterpene genes from other plants. Analysis of SoTPS3 amino acid sequence revealed that it contained two domains and four types of motifs such as, DDxxD, NSE/DTE, RRx8W and RXR. Bioinformatics and phylogenetic analyses revealed that SoTPS3 clustered in clade subfamily TPS-a, and the genes that belong to this subfamily can encode mono- and sesquiterpene. Functional complementation of SoTPS3 in Arabidopsis thaliana demonstrates that SoTPS3 is responsible for the production of Germacrene D-4α-ol. Overexpression of SoTPS3 in A. thaliana enhanced flower formation in transgenic plants compared with wild type. Our study will provide a basis for understanding the role of SoTPS3 in the biosynthesis of sesquiterpene in S. officinalis. [ABSTRACT FROM AUTHOR]

Acting as the most abundant and widely distributed volatile secondary metabolites in plants, terpenoids play crucial roles in diverse physiological regulations and metabolic processes. Terpene synthases play a decisive role in determining the composition and diversity of terpenoids. Though the regulation of terpene synthases has been extensively investigated across various plant species, limited studies have focused on the upstream transcriptional regulation of terpene synthases. In this study, we have identified linalool as the predominant volatile compound that is released gradually from Freesia hybrida flowers throughout flower blooming. In the context of the transcriptome, a typical MYB transcription factor, FhMYB108, was screened based on homologous gene comparison. FhMYB108 is capable of regulating the expression of FhTPS1, and both their expression levels showed gradual increase during flower opening. Moreover, FhMYB108 exerts a stimulatory effect on the transcription of Arabidopsis thaliana AtTPS14, while no significant increase in AtTPS14 expression is observed upon the stabilization of FhMYB108 in A. thaliana. The highly expressed AtMYC2 in A. thaliana could interact with FhMYB108 to suppress the activation of AtTPS14 by FhMYB108. The present study not only elucidates the regulatory mechanism underlying linalool synthesis but also discovers the synergistic effect of MYB and bHLH transcription factors in governing the biosynthesis of volatile terpenoids.

Terpenoids are structurally diverse natural products that have been widely used in the pharmaceutical, food, and cosmetic industries. Research has shown that fungi produce a variety of terpenoids, yet fungal terpene synthases remain not thoroughly explored. In this study, the tps1 gene, a crucial component of the terpene synthetic pathway, was isolated from Trichoderma atroviride HB20111 through genome mining. The function of this gene in the terpene synthetic pathway was investigated by constructing tps1-gene-deletion- and overexpression-engineered strains and evaluating the expression differences in the tps1 gene at the transcript level. HS-SPME-GC-MS analysis revealed significant variations in terpene metabolites among wild-type, tps1-deleted (Δtps1), and tps1-overexpressed (Otps1) strains; for instance, most sesquiterpene volatile organic compounds (VOCs) were notably reduced or absent in the Δtps1 strain, while nerolidol, β-acorenol, and guaiene were particularly produced by the Otps1 strain. However, both the Δtps1 and Otps1 strains produced new terpene metabolites compared to the wild-type, which indicated that the tps1 gene played an important role in terpene synthesis but was not the only gene involved in T. atroviride HB20111. The TPS1 protein encoded by the tps1 gene could function as a sesquiterpene cyclase through biological information and evolutionary tree analysis. Additionally, fungal inhibition assay and wheat growth promotion assay results suggested that the deletion or overexpression of the tps1 gene had a minimal impact on fungal inhibitory activity, plant growth promotion, and development, as well as stress response. This implies that these activities of T. atroviride HB20111 might result from a combination of multiple metabolites rather than being solely dependent on one specific metabolite. This study offers theoretical guidance for future investigations into the mechanism of terpenoid synthesis and serves as a foundation for related studies on terpenoid metabolic pathways in fungi.

The eunicellane diterpenoids are a unique family of natural products seen in marine organisms, plants, and bacteria. We used a series of biochemical, bioinformatics, and theoretical experiments to investigate the mechanism of the first diterpene synthase known to form the eunicellane skeleton. Deuterium labeling studies and quantum chemical calculations support that Bnd4, from Streptomyces sp. (CL12-4), forms the 6,10-bicyclic skeleton through a 1,10-cyclization, 1,3-hydride shift, and 1,14-cyclization cascade. Bnd4 also demonstrated sesquiterpene cyclase activity and the ability to prenylate small molecules. Bnd4 possesses a unique D94 NxxxD motif and mutation experiments confirmed an absolute requirement for D94 as well as E169.

Abstract Background Terpenoids play essential roles in plant defense against biotic stresses. In Citrus species, the monoterpene linalool mediates resistance against citrus canker disease caused by the gram-negative bacteria Xanthomonas citri subsp. citri (Xcc). Previous work had associated linalool contents with resistance; here we characterize transcriptional responses of linalool synthase genes. Results Leaf linalool contents are highly variable among different Citrus species. “Dongfang” tangerine (Citrus reticulata), a species with high linalool levels was more resistant to Xcc than “Shatian” pummelo (C. grandis) which accumulates only small amounts of linalool. The coding sequences of the major leaf-expressed linalool synthase gene (STS4) are highly conserved, while transcript levels differ between the two Citrus species. To understand this apparent differential transcription, we isolated the promoters of STS4 from the two species, fused them to a GUS reporter and expressed them in Arabidopsis. This reporter system revealed that the two promoters have different constitutive activities, mainly in trichomes. Interestingly, both linalool contents and STS4 transcript levels are insensitive to Xcc infestation in citrus plants, but in these transgenic Arabidopsis plants, the promoters are activated by challenge of a bacterial pathogen Pseudomonas syringae, as well as wounding and external jasmonic acid treatment. Conclusions Our study reveals variation in linalool and resistance to Xcc in citrus plants, which may be mediated by different promoter activities of a terpene synthase gene in different Citrus species.

This review presents a systematic analysis of the studies on volatiles in Dendrobium. Among the various components, aromatic terpenes are a crucial component in the development of the aromatic characteristics of Dendrobium and other plants. Recent advancements in detection and sequencing technology have resulted in a considerable rise in research on the biosynthetic processes of aromatic terpenes in Dendrobium and other flowering plants. Nevertheless, the inquiry into the precise means by which plants regulate the proportion of diverse aromatic terpenes in their floral scent, thereby preserving their olfactory traits, requires further investigation. A conjecture on the botanical perfumer mechanism, which condensed the findings of earlier studies, was put forward to address this area of interest. Specific transcription factors likely govern the coordinated expression of multiple key terpene synthase (TPS) genes during the flowering stage of plants, thereby regulating the proportional biosynthesis of diverse aromatic terpenes and sustaining the distinctive aromatic properties of individual plants. This review serves as a significant theoretical reference for further investigations into aromatic volatile compounds in Dendrobium.

Terpenes are important mediators of plant chemical response to environmental cues. Here, we describe the genome-wide identification and biochemical characterization of TPS-a members in Medicago truncatula, a model legume crop. Genome mining identified thirty-nine full-length terpene synthases with a significant number predicted to produce monoterpenes and sesquiterpenes. Biochemical characterization of the TPS-a subfamily associated with sesquiterpene biosynthesis revealed such compounds, that exhibit substantial biological activity in other plants. Gene expression analysis using qPCR and the Medicago gene atlas illustrated distinct tissue and time-based variation in expression in leaves and roots. Together our work establishes the gene-to-metabolite relationships for sesquiterpene synthases in M. truncatula. Understanding the biosynthetic capacity is a foundational step to defining the ecological roles of this important family of compounds.

Dendrobium officinale Kimura et Migo is a tonic plant that has both ornamental and medicinal properties. Terpenoids are significant and diverse secondary metabolites in plants, and are one of the important natural active ingredients in D. officinale. The AP2/ERF gene family plays a major role in primary and secondary metabolism. However, the AP2/ERF transcription factor family has not been identified in D. officinale, and it is unclear if it is involved in the regulation of terpenoid biosynthesis. This study identified a sesquiterpene synthetase-β-patchoulene synthase (DoPAES) using transcriptome and terpenic metabolic profile analyses. A total of 111 members of the AP2/ERF family were identified through the whole genome of D. officinale. The tissue-specific expression and gene co-expression pattern of the DoAP2/ERF family members were analyzed. The results showed that the expression of DoPAES was highly correlated with the expression of DoAP2/ERF89 and DoAP2/ERF47. The yeast one-hybrid (Y1H) assays and dual-luciferase experiments demonstrated that DoAP2/ERF89 and DoAP2/ERF47 could regulate the expression of DoPAES. The transcriptional regulatory effects were examined using homologous transient expression of DoAP2/ERF89 in protocorms of D. officinale. DoAP2/ERF89 positively regulated the biosynthesis of β-patchoulene. This study showed that DoAP2/ERF89 can bind to the promoter region of DoPAES to control its expression and further regulate the biosynthesis of β-patchoulene in D. officinale. These results provide new insights on the regulation of terpenoid biosynthesis.

Chia (Salvia hispanica L.) is one of the most popular nutrition-rich foods and pseudocereal crops of the family Lamiaceae. Chia seeds are a rich source of proteins, polyunsaturated fatty acids (PUFAs), dietary fibers, and antioxidants. In this study, we present the assembly of the chia reference genome, which spans 303.6 Mb and encodes 48,090 annotated protein-coding genes. Our analysis revealed that ~42% of the chia genome harbors repetitive content, and identified ~3 million single nucleotide polymorphisms (SNPs) and 15,380 simple sequence repeat (SSR) marker sites. By investigating the chia transcriptome, we discovered that ~44% of the genes undergo alternative splicing with a higher frequency of intron retention events. Additionally, we identified chia genes associated with important nutrient content and quality traits, such as the biosynthesis of PUFAs and seed mucilage fiber (dietary fiber) polysaccharides. Notably, this is the first report of in-silico annotation of a plant genome for protein-derived small bioactive peptides (biopeptides) associated with improving human health. To facilitate further research and translational applications of this valuable orphan crop, we have developed the Salvia genomics database (SalviaGDB), accessible at https://salviagdb.org.

Boswellia tree bark exudes oleo-gum resin in response to wounding, which is rich in terpene volatiles. But, the molecular and biochemical basis of wound-induced formation of resin volatiles remains poorly understood. Here, we combined RNA-sequencing (RNA-seq) and metabolite analysis to unravel the terpene synthase (TPS) family contributing to wound-induced biosynthesis of resin volatiles in B. serrata, an economically-important Boswellia species. The analysis of large-scale RNA-seq data of bark and leaf samples representing more than 600 million sequencing reads led to the identification of 32 TPSs, which were classified based on phylogenetic relationship into various TPSs families found in angiosperm species such as TPS-a, b, c, e/f, and g. Moreover, RNA-seq analysis of bark samples collected at 0–24 h post-wounding shortlisted 14 BsTPSs that showed wound-induced transcriptional upregulation in bark, suggesting their important role in wound-induced biosynthesis of resin volatiles. Biochemical characterization of a bark preferentially-expressed and wound-inducible TPS (BsTPS2) in vitro and in planta assays revealed its involvement in resin terpene biosynthesis. Bacterially-expressed recombinant BsTPS2 catalyzed the conversion of GPP and FPP into (S)-(+)-linalool and (E)-(−)-nerolidol, respectively, in vitro assays. However, BsTPS2 expression in Nicotiana benthamiana found that BsTPS2 is a plastidial linalool synthase. In contrast, cytosolic expression of BsTPS2 did not form any product. Overall, the present work unraveled a suite of TPSs that potentially contributed to the biosynthesis of resin volatiles in Boswellia and biochemically characterized BsTPS2, which is involved in wound-induced biosynthesis of (S)-(+)-linalool, a monoterpene resin volatile with a known role in plant defense. [ABSTRACT FROM AUTHOR]

Main conclusion: Santalum album (E,E)-α-farnesene synthase catalyzes FPP into (E,E)-α-farnesene. Overexpression of the SaAFS gene positively improved cold stress tolerance through JA biosynthesis and signaling pathways in Arabidopsis. Volatile terpenoids are released from plants that suffer negative effects following exposure to various biotic and abiotic stresses. Recent studies revealed that (E,E)-α-farnesene synthase (AFS) plays a significant role in a plant’s defence against biotic attack. However, little is known about whether AFS contributes to plant resistance to cold stress. In this study, a SaAFS gene was isolated from Indian sandalwood (Santalum album L.) and functionally characterized. The SaAFS protein mainly converts farnesyl diphosphate to (E,E)-α-farnesene. SaAFS was clustered into the AFS clade from angiosperms, suggesting a highly conserved enzyme. SaAFS displayed a significant response to cold stress and methyl jasmonate. SaAFS overexpression (OE) in Arabidopsis enhanced cold tolerance by increasing proline content, reducing malondialdehyde content, electrolyte leakage, and accumulating reactive oxygen species. Transcriptomic analysis revealed that upregulated genes related to stress response and JA biosynthesis and signaling were detected in SaAFS-OE lines compared with wild type plants that were exposed to cold stress. Endogenous JA and jasmonoyl-isoleucine content increased significantly in SaAFS-OE lines exposed to cold stress. Collectively considered, these results suggest that the SaAFS gene is a positive regulator during cold stress tolerance via JA biosynthesis and signaling pathways. [ABSTRACT FROM AUTHOR]

The functionalization of terpenes using cytochrome P450 enzymes is a versatile route to the production of useful derivatives that can be further converted to value-added products. Many terpenes are hydrophobic and volatile making their availability as a substrate for P450 enzymes significantly limited during microbial production. In this study, we developed a strategy to improve the accessibility of terpene molecules for the P450 reaction by linking terpene synthase and P450 together. As a model system, fusion proteins of 1,8-cineole synthase (CS) and P450cin were investigated and it showed an improved hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold. Structural analysis of the CS-P450cin fusion proteins by SEC-SAXS indicated a dimer formation with preferred orientations of the active sites of the two domains. We also applied the enzyme fusion strategy to the oxidation of a sesquiterpene epi-isozizaene and the fusion enzymes significantly improved albaflavenol production in engineered E. coli. From the analysis of positive and negative examples of the fusion strategy, we proposed key factors in structure-based prediction and evaluation of fusion enzymes. Developing fusion enzymes for terpene synthase and P450 presents an efficient strategy toward oxidation of hydrophobic terpene compounds. This strategy could be widely applicable to improve the biosynthetic titer of the functionalized products from hydrophobic terpene intermediates.

Terpene synthase (TPS) is a key enzyme in the synthesis of terpenoids in plants and plays an important role in the regulation of the synthesis of aromatic substances in plants. In this study, we treated Chrysanthemum indicum var. aromaticum as the experimental material to clone the CiTPS gene. The open reading frame of CiTPS is 1 818 bp, encoding a putative protein of 605 amino acids. There is a monoterpene synthase conserved domain and a highly conserved sequence, DDXXD, (N,D)D(L,I,V)X(S,T)XXXE and RRX8W, respectively, which belong to the monoterpene synthase family. The multiple sequence alignment and phylogenetic analysis showed that the CiTPS gene belongs to the TPS-b subfamily. The pBI121-TPS-GFP recombinant plasmid and the pBI121-GFP empty vector plasmid were introduced into Agrobacterium by electroporation and transferred into wild-type tobacco by the Agrobacterium-mediated method. Three transgenic CiTPS lines were screened, and the secretions of wild-type tobacco and transgenic tobacco T1 leaves in the vigorous growth period were analysed. The volatiles of the transgenic lines were similar to those of wild-type tobacco, but their monoterpenes increased significantly, and the sesquiterpenoids and diterpenoids decreased or remained unchanged, indicating that the CiTPS gene is related to the synthesis of the monoterpenes.