Functional characterization of terpene synthases SmTPS1 involved in floral scent formation in Salvia miltiorrhiza.

Plants attract beneficial insects and promote pollination by releasing floral scents. Salvia miltiorrhiza, as an insect-pollinated flowering plant, which has been less studied for its floral aroma substances. This study revealed that S. miltiorrhiza flowers produce various volatile terpenoids, including five monoterpenes and ten sesquiterpenes, with the sesquiterpene compound (E)-ß-caryophyllene being the most abundant, accounting for 28.1% of the total volatile terpenoids. Y-tube olfactometer experiments were conducted on the primary pollinator of S. miltiorrhiza, the Apis ceranas. The results indicated that (E)-ß-caryophyllene compound had an attractive effect on the Apis ceranas. By comparing the homologous sequences with the genes of (E)-ß-caryophyllene terpene synthases in other plants, the SmTPS1 gene was selected for further experiment. Subcellular localization experiments showed SmTPS1 localized in the cytoplasm, and its in vitro enzyme assay revealed that it could catalyze FPP into ß-Elemene, (E)-ß-caryophyllene and α-Humulene. Overexpression of SmTPS1 in S. miltiorrhiza resulted in a 5.29-fold increase in gene expression. The GC-MS analysis revealed a significant increase in the concentration of (E)-ß-caryophyllene in the transgenic plants, with levels 2.47-fold higher compared to the empty vector plants. Furthermore, Y-tube olfactometer experiments showed that the transgenic plants were significantly more attractive to Apis ceranas compared to the empty vector plants. Co-expression analysis suggested that four SmMYCs (SmMYC1, SmMYC5, SmMYC10, and SmMYC11) may be involved in the transcriptional regulation of SmTPS1. The yeast one-hybrid screen and the Dual luciferase assay indicated that SmMYC10 positively regulates the expression of SmTPS1. In conclusion, this study lays a foundation for the functional analysis and transcriptional regulation of terpene synthase genes in S. miltiorrhiza.

Screening Universal Stress-Response Terpenoids and Their Biosynthetic Genes via Volatile and Transcriptomic Profiling in Citrus.

Volatile terpenoids accumulate in citrus and play important roles in plant defense against various stressors. However, the broad-spectrum response of terpenoid biosynthesis to ubiquitous stressors in citrus has not been comparatively investigated. In this study, volatile terpenoids were profiled under six stressors: high temperature, citrus miner, citrus red mite, citrus canker, Alternaria brown spot, and huanglongbing (HLB). Significant content changes in 15 terpenoids, including ß-ocimene, were observed in more than four of the six stressors, implying their possibly universal stress-response effects. Notably, the emission of terpenoids, including ß-caryophyllene, ß-ocimene, and nerolidol glucoside, was significantly increased by HLB in HLB-tolerant "Shatian" pomelo leaves. The upregulation of CgTPS1 and CgTPS2 and their characterization in vivo identified them as mono- or sesquiterpenoid biosynthetic genes. This study provides a foundation for determining stress resistance mechanisms in citrus and biopesticide designations for future industrial applications.

[Identification of terpene synthase gene family members in Rhododendron and its relationship with terpenoid metabolism].

Terpene synthase (TPS) plays important roles in the synthesis of terpenoids which are the main fragrances in Rhododendron flowers. To understand the function of TPS genes in terpenoid metabolism in relation to flower aroma formation, we identified all TPS gene family members in Rhododendron by analyzing its genome database. We then used a transcriptomic approach to analyze the differential gene expression patterns of TPS gene family members in the scented flower Rhododendron fortunei compared to the non-scented flower Rhododendron 'Nova Zembla'. The contents of terpenoid compounds in petals of the above two Rhododendron species at different developmental stages were also measured by using qRT-PCR and head space-solid phase micro-extraction combined with gas chromatography-mass spectrometry. Our results showed that a total of 47 RsTPS members, with individual lengths ranged from 591 to 2 634 bp, were identified in the Rhododendron genome. The number of exons in RsTPS gene ranged from 3 to 12, while the length of each protein encoded ranged from 196 to 877 amino acids. Members of the RsTPS family are mainly distributed in the chloroplast and cytoplasm. Phylogenetic analysis showed that RsTPS genes can be clustered into 5 subgroups. Seven gene family members can be functionally annotated as TPS gene family since they were temporally and spatially expressed as shown in the transcriptome data. Notably, TPS1, TPS10, TPS12 and TPS13 in Rhododendron fortunei were expressed highly in flower buds reached the peak in the full blossoming. Correlation analysis between gene expression levels and terpenoid content indicates that the expression levels of TPS1, TPS4, TPS9, TPS10, TPS12 and TPS13 were positively correlated with the content of terpenoids in the petals of R. fortunei at all flower developmental stages, suggesting that these six genes might be involved in the aroma formation in R. fortunei.

Mining, expression, and phylogenetic analysis of volatile terpenoid biosynthesis-related genes in different tissues of ten Elsholtzia species based on transcriptomic analysis.

We sequenced the leaf and inflorescence transcriptomes of 10 Elsholtzia species to mine genes related to the volatile terpenoid metabolic pathway. A total of 184.68 GB data and 1,231,162,678 clean reads were obtained from 20 Elsholtzia samples, and 333,848 unigenes with an average length of at least 1440 bp were obtained by Trinity assembly. KEGG pathway analysis showed that there were three pathways related to volatile terpene metabolism: terpenoid backbone biosynthesis (No. ko00900), monoterpenoid biosynthesis (No. ko00902), and sesquiterpenoid and triterpenoid biosynthesis (No. ko00909), with 437, 125, and 121 related unigenes, respectively. The essential oil content and composition in 20 Elsholtzia samples were determined by gas chromatography-mass spectrometry. The results showed that there were obvious interspecific differences among the 10 Elsholtzia species, but there were no significant differences between the different tissues among species. The expression levels of seven candidate genes involved in volatile terpenoid biosynthesis in Elsholtzia were further analyzed by quantitative real-time PCR. The results showed that HMGS had the highest expression among all genes, followed by GGPS4. In addition, there was not a significant correlation between the seven genes and the components with high essential oil contents. Combined with the essential oil components detected in this study, the possible biosynthetic pathway of the characteristic components in Elsholtzia plants was speculated to be a metabolic pathway with geraniol as the starting point and elsholtzione as the end product. Phylogenetic analysis was conducted using the nucleotide sequences of the geranyl diphosphate synthase candidate genes, and the results showed that genes related to the volatile terpenoid biosynthetic pathway may be more suitable gene fragments for resolving the Elsholtzia phylogeny.

Terpene Synthase Gene OtLIS Confers Wheat Resistance to Sitobion avenae by Regulating Linalool Emission.

Sitobion avenae (Fabricius) is a major insect pest of wheat worldwide that reduces crop yield and quality annually. Few germplasm resources with resistant genes to aphids have been identified and characterized. Here, octoploid Trititrigia, a species used in wheat distant hybridization breeding, was found to be repellent to S. avenae after 2 year field investigations and associated with physiological and behavioral assays. Linalool monoterpene was identified to accumulate dominantly in plants in response to S. avenae infestation. We cloned the resistance gene OtLIS by assembling the transcriptome of aphid-infested or healthy octoploid Trititrigia. Functional characterization analysis indicated that OtLIS encoded a terpene synthase and conferred resistance to S. avenae by linalool emission before and after aphid feeding. Our study suggests that the octoploid Trititrigia with the aphid-resistant gene OtLIS may have potential as a target resource for further breeding aphid-resistant wheat cultivars.

Characterization of trans-Nerolidol Synthase from Celastrus angulatus Maxim and Production of trans-Nerolidol in Engineered Saccharomyces cerevisiae.

Volatile terpenoids are a large group of important secondary metabolites and possess many biological activities. The acyclic sesquiterpene trans-nerolidol is one of the typical representatives and widely used in cosmetics and agriculture. Here, the accumulation of volatile terpenes in different tissues of Celastrus angulatus was investigated, and two trans-nerolidol synthases, CaNES1 and CaNES2, were identified and characterized by in vitro enzymatic assays. Both genes are differentially transcribed in different tissues of C. angulatus. Next, we constructed a Saccharomyces cerevisiae cell factory to enable high-level production of trans-nerolidol. Glucose was the sole carbon source to sequentially control gene expression between the competitive squalene and trans-nerolidol pathways. Finally, the trans-nerolidol production of recombinant strain LWG003-CaNES2 was 7.01 g/L by fed-batch fermentation in a 5 L bioreactor. The results clarify volatile terpenoid biosynthesis in C. angulatus and provide a promising potential for industrial production of trans-nerolidol in S. cerevisiae.

Time-series transcriptome provides insights into the gene regulation network involved in the volatile terpenoid metabolism during the flower development of lavender.

BACKGROUND: Essential oils (EOs) of Lavandula angustifolia, mainly consist of monoterpenoids and sesquiterpenoids, are of great commercial value. The multi-flower spiciform thyrse of lavender not only determines the output of EOs but also reflects an environmental adaption strategy. With the flower development and blossom in turn, the fluctuation of the volatile terpenoids displayed a regular change at each axis. However, the molecular mechanism underlying the regulation of volatile terpenoids during the process of flowering is poorly understood in lavender. Here, we combine metabolite and RNA-Seq analyses of flowers of five developmental stages at first- and second-axis (FFDSFSA) and initial flower bud (FB0) to discover the active terpenoid biosynthesis as well as flowering-related genes. RESULTS: A total of 56 mono- and sesquiterpenoids were identified in the EOs of L. angustifolia 'JX-2'. FB0' EO consists of 55 compounds and the two highest compounds, ß-trans-ocimene (20.57%) and (+)-R-limonene (17.00%), can get rid of 74.71 and 78.41% aphids in Y-tube olfactometer experiments, respectively. With sequential and successive blossoms, temporally regulated volatiles were linked to pollinator attraction in field and olfaction bioassays. In three characteristic compounds of FFDSFSA' EOs, linalyl acetate (72.73%) and lavandulyl acetate (72.09%) attracted more bees than linalool (45.35%). Many transcripts related to flowering time and volatile terpenoid metabolism expressed differently during the flower development. Similar metabolic and transcriptomic profiles were observed when florets from the two axes were maintained at the same maturity grade. Besides both compounds and differentially expressed genes were rich in FB0, most volatile compounds were significantly correlated with FB0-specific gene module. Most key regulators related to flowering and terpenoid metabolism were interconnected in the subnetwork of FB0-specific module, suggesting the cross-talk between the two biological processes to some degree. CONCLUSIONS: Characteristic compounds and gene expression profile of FB0 exhibit ecological value in pest control. The precise control of each-axis flowering and regular emissions at transcriptional and metabolic level are important to pollinators attraction for lavender. Our study sheds new light on lavender maximizes its fitness from "gene-volatile terpenoid-insect" three layers.

An Integrative Volatile Terpenoid Profiling and Transcriptomics Analysis for Gene Mining and Functional Characterization of AvBPPS and AvPS Involved in the Monoterpenoid Biosynthesis in Amomum villosum.

Amomum villosum, also known as Fructus Amomi, has been used to treat digestive diseases such as abdominal pain, vomiting, and dysentery. Volatile terpenoids are the active metabolites in the essential oil of Fructus Amomi. Nevertheless, downstream genes responsible for activating metabolites biosynthesis in A. villosum still remain unclear. Here, we report the use of an integrative volatile terpenoid profiling and transcriptomics analysis for mining the corresponding genes involved in volatile terpenoid biosynthesis. Ten terpene synthase (TPS) genes were discovered, and two of them were cloned and functionally characterized. AvTPS1 (AvPS: pinene synthase) catalyzed GPP to form α-pinene and ß-pinene; AvTPS3 (AvBPPS: bornyl diphosphate synthase) produced bornyl diphosphate as major product and the other three monoterpenoids as minor products. Metabolite accumulation and gene expression pattern combined with AvPS biochemical characterization suggested that AvPS might play a role in biotic defense. On the other hand, the most active ingredient, bornyl acetate, was highly accumulated in seeds and was consistent with the high expression of AvBPPS, which further indicated that AvBPPS is responsible for the biosynthesis of bornyl acetate, the final metabolite of bornyl diphosphate in A. villosum. This study can be used to improve the quality of A. villosum through metabolic engineering, and for the sustainable production of bornyl acetate in heterologous hosts.

Implementation of CsLIS/NES in linalool biosynthesis involves transcript splicing regulation in Camellia sinensis.

Volatile terpenoids produced in tea plants (Camellia sinensis) are airborne signals interacting against other ecosystem members, but also pleasant odorants of tea products. Transcription regulation (including transcript processing) is pivotal for plant volatile terpenoid production. In this study, a terpene synthase gene CsLIS/NES was recovered from tea plants (C. sinensis cv. "Long-Men Xiang"). CsLIS/NES transcription regulation resulted in 2 splicing forms: CsLIS/NES-1 and CsLIS/NES-2 lacking a 305 bp-fragment at N-terminus, both producing (E)-nerolidol and linalool in vitro. Transgenic tobacco studies and a gene-specific antisense oligo-deoxynucleotide suppression applied in tea leaves indicated that CsLIS/NES-1, localized in chloroplasts, acted as linalool synthase, whereas CsLIS/NES-2 localized in cytosol, functioned as a potential nerolidol synthase, but not linalool synthase. Expression patterns of the 2 transcript isoforms in tea were distinctly different and responded differentially to the application of stress signal molecule methyl jasmonate. Leaf expression of CsLIS/NES-1, but not CsLIS/NES-2, was significantly induced by methyl jasmonate. Our data indicated that distinct transcript splicing regulation patterns, together with subcellular compartmentation of CsLIS/NE-1 and CsLIS/NE-2 implemented the linalool biosynthesis regulation in tea plants in responding to endogenous and exogenous regulatory factors.

Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering.

MAIN CONCLUSION: Terpenoids play several physiological and ecological functions in plant life through direct and indirect plant defenses and also in human society because of their enormous applications in the pharmaceutical, food and cosmetics industries. Through the aid of genetic engineering its role can by magnified to broad spectrum by improving genetic ability of crop plants, enhancing the aroma quality of fruits and flowers and the production of pharmaceutical terpenoids contents in medicinal plants. Terpenoids are structurally diverse and the most abundant plant secondary metabolites, playing an important role in plant life through direct and indirect plant defenses, by attracting pollinators and through different interactions between the plants and their environment. Terpenoids are also significant because of their enormous applications in the pharmaceutical, food and cosmetics industries. Due to their broad distribution and functional versatility, efforts are being made to decode the biosynthetic pathways and comprehend the regulatory mechanisms of terpenoids. This review summarizes the recent advances in biosynthetic pathways, including the spatiotemporal, transcriptional and post-transcriptional regulatory mechanisms. Moreover, we discuss the multiple functions of the terpene synthase genes (TPS), their interaction with the surrounding environment and the use of genetic engineering for terpenoid production in model plants. Here, we also provide an overview of the significance of terpenoid metabolic engineering in crop protection, plant reproduction and plant metabolic engineering approaches for pharmaceutical terpenoids production and future scenarios in agriculture, which call for sustainable production platforms by improving different plant traits.

Transcriptome analysis of Cinnamomum longepaniculatum by high-throughput sequencing

Background: Cinnamomum longepaniculatum is an important commercial crop and the main source of volatile terpenoids. The biosynthesis of key bioactive metabolites of C. longepaniculatum is not well understood because of the lack of available genomic and transcriptomic information. To address this issue, we performed transcriptome sequencing of C. longepaniculatum leaves to identify factors involved in terpenoid metabolite biosynthesis. Results: Transcriptome sequencing of C. longepaniculatum leaves generated over 56 million raw reads. The transcriptome was assembled using the Trinity software and yielded 82,061 unigenes with an average length of 879.43 bp and N50 value of 1387 bp. Furthermore, Benchmarking Universal Single-Copy Orthologs analysis indicated that our assembly is 91% complete. The unigenes were used to query the nonredundant database depending on sequence similarity; 42,809 unigenes were homologous to known genes in different species, with an annotation rate of 42.87%. The transcript abundance and Gene Ontology analyses revealed that numerous unigenes were associated with metabolism, while others were annotated in functional categories including transcription, signal transduction, and secondary metabolism. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that 19,260 unigenes were involved in 385 metabolic pathways, with 233 unigenes found to be involved in terpenoid metabolism. Moreover, 23,463 simple sequence repeats were identified using the microsatellite identification tool. Conclusion: This is the first detailed transcriptome analysis of C. longepaniculatum. The findings provide insights into the molecular basis of terpenoid biosynthesis and a reference for future studies on the genetics and breeding of C. longepaniculatum.

Enantiomeric and quantitative analysis of volatile terpenoids in different teas (Camellia sinensis).

Volatile terpenoids play important roles in the formation of tea aroma quality due to their pleasant scents and low odor thresholds. Most volatile terpenoids contain stereogenic centers, which results in various stereo distributions of their enantiomers and diastereoisomers in different types of tea. However, the distribution characteristics of terpenoid enantiomers in teas were still unclear, which poses an obstacle to the scientific understanding of tea aroma. In this work, a new and efficient analysis approach based on headspace solid phase microextraction (HS-SPME)-chiral gas chromatography-mass spectrometry (GC-MS) was established to analyze 12 pairs of familiar terpenoid enantiomers in different teas. The extraction efficiency of the HS-SPME method to extract volatile terpenoids in teas was the greatest when using CAR-DVB-PDMS (50/30µm) fibers and 1:10 proportions between tea and boiling water at a 50°C extraction temperature for 40min, and the stability observation of enantiomeric ratios of the terpenoids well proved the feasibility of the extraction method. The favorable limits of detection, limits of quantitation, repeatability, linearity, and concentration ranges of each terpenoid enantiomer demonstrated the repeatability and reliability of the analytical approach. The enantiomeric and quantitative analyses indicated that S-limonene, S-linalool, (2S, 5S)-linalool oxide A, (2S, 5R)-linalool oxide B, R-4-terpineol, (2S, 5R)-linalool oxide C, (2S, 5S)-linalool oxide D, S-α-terpineol, R-α-ionone, peak 1 of theaspirane A and peak 2 of theaspirane B were the major terpenoid components in most Chinese teas; instead, higher proportions of the opposite enantiomers of the above terpenoids were frequently detected in black teas with large leaf origin and Indonesia white teas. Besides, great diversities of enantiomeric ratios and concentrations among different teas were observed. Furthermore, partial least-squares discriminant analyses were performed to distinguish the concentration differences of the terpenoid enantiomers among different teas; the analysis results indicated that highly significant concentration differences existed between large and small leaf origins of black teas, and significant differences of the concentrations of linalool oxides A-C were observed between green, white and dark teas. The successful application of this chiral analysis technique of tea aroma will lay a scientific foundation for further quality assessment, botanical origin determination and authenticity assessment of teas.

Methyl Jasmonate Affects Metabolism and Gene Transcription of Volatile Terpenoids from Amomum villosum Lour / 世界科学技术-中医药现代化

This study was aimed to reveal the effects and molecular regulation mechanism of methyl jasmonate (Me-JA) on volatile terpenoids from Amomum villosum Lour. After the leaves and fruits of A momum villosum Lour. were treated with different concentrations of MeJA, the volatile terpenoids of fresh fruits from A . villosum Lour. were ex-tracted with microwave method and analyzed by GC-MS. Then, leaves and fruits treated with MeJA were sequenced by Illumina. The transcriptome data was analyzed by bioinformatic methods. The results showed that there were 20 and 33 volatile terpenoids detected in peels and seed groups, respectively. Contents of volatile terpenoids in peels and seed groups were both improved after 600 μmol·L-1 MeJA treating fruits for 24 h, such as bornyl acetate, cam-phor, borneol, and etc. While 200 μmol·L-1 MeJA treating different parts for 24 h can regulate the biosynthesis of some volatile terpenoids in peels differently. And 200 μmol·L-1 MeJA treating fruits can improve the content of ma-jor volatile terpenoids in seed groups. A total of 68 168 unigenes were obtained with de novo assembly, and 48 627 unigenes were annotated after comparison with public protein databases. Analysis of functional annotation against KEGG database showed that there were 208 unigenes closely related with metabolism of volatile terpenoids and 22 u-nigenes related with MYC2 transcription factors. It was concluded that MeJA can effectively regulate the metabolism of volatile terpenoids from A . villosum Lour. There were a lot of candidate genes related with the biosynthesis of volatile terpenoids obtained by analyzing the transcriptome data which also provided a large amount of data for the discovery and regulation of functional genes related with the biosynthesis of volatile terpenoids from A . villosum Lour.