Volatile Terpenoids Research Articles

Chemical profiling and clustering of various dried cannabis flowers revealed by volatilomics and chemometric processing

Cannabis flower scent is one of the key characteristics of the cannabis plant. The diverse scents impact user experiences and offer medicinal benefits. These scents originate from volatile compounds, particularly terpenes and terpenoids. This study characterized the volatile profile of 19 different dried cannabis flowers using gas chromatography-mass spectrometry coupled with headspace-solid phase microextraction (HS-SPME-GC-MS). A total of 75 compounds were identified, including alcohols, aldehydes, benzenes, esters, ketone, monoterpenes, monoterpenoids, sesquiterpenes, and sesquiterpenoids. Cluster analysis was able to group the 19 cannabis cultivars into five clusters based on volatile chemotypes using chemometric techniques of hierarchical cluster analysis (HCA) and principal component analysis (PCA). Potential discriminant markers of each cultivar were then analyzed using a supervised partial least squares discriminant analysis (PLS-DA) verified through Variable Importance in Projection values (VIP), identifying twenty discriminant markers. In addition, the correlations among 75 volatile compounds were also obtained. The findings of this study provide a valuable database of single cannabis cultivars, useful for identifying individual strains and verifying their quality. Clustering the cultivars by volatile chemotype can be used for the classification of cannabis in the market. The results of this study are expected to be a starting point for further cannabis breeding programs to expand knowledge of this plant. Furthermore, the proposed method is applicable to other aroma plants in the future.

A pivotal switch in β-domain determines the substrate selectivity of TPS genes involved in Gardenia jasminoides floral scent synthesis.

Plants have evolved a diverse array of secondary metabolites to enhance their adaptability to environmental stresses, with volatile terpenoids being a notable example. Gardenia (Gardenia jasminoides), celebrated for its unique fragrance, is a key natural source of volatile terpenoids. Using our chromosome-level genome and transcriptome data for G. jasminoides, we previously identified six terpene synthases (TPS) involved in the production of its floral scent. Here, we functionally characterized these six key TPS enzymes, aligning their product profiles with volatile organic compound (VOC) analysis. Notably, we identified two highly similar terpene synthases, GjTPS1 and GjTPS2, which share high sequence homology but differ in substrate selectivity. By leveraging AI-based predictions and site-directed mutagenesis, we pinpointed a single amino acid in the β-domain that acts as a "switch," modulating substrate selectivity-an unusual finding, as this residue is outside the active site region. Comparative genomic analyses with Coffea canephora and other eudicots revealed that G. jasminoides TPS genes primarily expanded through dispersed (DSD) and tandem duplications (TD). Our study is the first to reveal a "switch" in a previously deemed "non-functional" region, regulating substrate selectivity in TPS genes. These insights could facilitate breeding elite gardenia varieties and support the rational engineering of terpenoid synthases.

Characterization of primary aroma compounds in Pu‐erh raw tea sourced from various regions using gas chromatography‐mass spectrometry and headspace solid‐phase microextraction

AbstractThe assessment of aroma plays a crucial role in determining the characteristics and quality of Pu‐erh tea. The volatile compounds present in Pu‐erh raw tea sourced from five regions in Yunnan Province have been studied using gas chromatography‐mass spectrometry (GC‐MS) in conjunction with headspace solid phase microextraction (HS‐SPME). A total of 69 aroma‐active compounds were identified, with alcohols, ketones, and aldehydes being the predominant component types. Furthermore, notable variations were observed among four pairs of volatile terpenoid enantiomers. Significant discrimination was achieved using orthogonal partial least squares discrimination analysis (R(x2) = 0.926, R(y2) = 0.843, Q2 = 0.501). Moreover, 33 important differential compounds were identified based on variable importance in the projection values (VIP > 1.0) and one‐way ANOVA analysis (p < 0.05). Subsequently, a KEGG pathway analysis was conducted, revealing enrichment pathways primarily focused on the mevalonate pathway, steroid biosynthesis, and fatty acid biosynthesis. Ultimately, the key volatile compounds and major aroma‐contributing compounds were successfully identified.

Binding properties of olfactory proteins to host volatiles, free fatty acids and cuticular hydrocarbons in the termite Reticulitermes aculabialis

As eusocial insects prevalent in tropical and subtropical regions, termites are characterized by highly organized behaviors and exceptional adaptability, rooted in caste differentiation and chemical communication. These traits make them excellent models for studying insect social structures and ecological interactions. Investigating how termites use chemical signals to perceive and respond to their environment provides insights into their coordination and adaptation within complex ecosystems. This study delved into the chemosensory mechanisms of Reticulitermes aculabialis, examining the interactions of four olfactory proteins with 70 ligands, including host volatiles, cuticular hydrocarbons (CHCs), and free fatty acids (FFAs). Molecular docking simulations revealed varied affinities of the olfactory proteins for long-chain hydrocarbons (n-C23 to n-C28), suggesting a nuanced chemical communication system through specific hydrocarbon detection. RacuCSP1 and RacuCSP2 exhibited specific binding to linoleic acid and undecanoic acid, respectively, highlighting the significance of FFAs in the physiological and behavioral processes of termites. The four olfactory proteins showed a strong affinity for longifolene in fluorescence competitive binding experiments. Notably, RacuOBPs exhibited unique affinities for terpenoid volatiles such as β-lonone and neocembrene, while RacuCSPs specifically bound with terpenoids like 3-carene, myrtenol, α-pinene oxide and β-pinene indicating their critical roles in host detection. Behavioral observations following gene silencing revealed that RacuOBP5 was essential for recognizing longifolene and α-lonone recognition, while RacuCSP1 was key for detecting α-pinene in termites. These findings enhance our understanding of the termite chemosensory system and offer insights for developing precise pest management strategies.

The Effect of Reduced Nitrogen Fertilizer Application on japonica Rice Based on Volatile Metabolomics Analysis.

Nitrogen is critical for rice yield and quality, but its overuse can be detrimental to efficiency and the environment. To identify changes in the quality of rice in response to the reduced application of nitrogen fertilizer, we carried out a comprehensive metabolomics study of SuiJing 18 using volatile metabolomics methods. Our results showed that SuiJing 18 had a total of 358 volatile metabolites, mainly lipids (16.25%), terpenoids (15.41%), heterocyclic compounds (15.13%), and hydrocarbons (13.45%). SuiJing 18 underwent significant changes in response to the reduced application of nitrogen fertilizer. Key sweet volatile compounds such as 4-methyl-benzeneacetaldehyde, hexyl acetate, and 2-methylnaphthalene were present at significantly higher levels when nitrogen fertilizer was applied at a rate of 68 kg of pure nitrogen per hectare, and their flavor characteristics also differed significantly from the compounds resulting from the other two treatments. Focusing on 16 differential volatile metabolites, we further investigated their effects on flavor and quality, thus laying the foundation for a greater understanding of the biomarkers associated with changes in rice quality. This study contributes to a better understanding of the mechanisms underlying changes in rice quality after reduced nitrogen fertilizer application.

Study on dynamic alterations of volatile organic compounds reveals aroma development over enzymatic-catalyzed process of Tieguanyin oolong tea production

To elucidate the formation of characteristic aroma over enzymatic-catalyzed processes (ECP), GC–MS-based volatile-metabolomic combined with desorption-electrospray-ionization coupled mass-spectrometry-imaging (DESI-MSI) were employed to analyze the changes of volatile organic compounds (VOCs) in Tieguanyin tea. A total of 579 VOCs were obtained, from which 24 components involved in five pathways were identified as biomarkers. Among these, four VOCs including 2-furancarboxylic acid, 4-methylbenzaldehyde, N-benzylformamide, cuminaldehyde, were detected in both DESI-MSI and GC–MS analysis, exhibiting dynamic changes along processing steps. RNA-sequencing analysis indicated the genes referring to stress response were activated during tea processing, facilitating the accumulation of flora-fruity aroma in tea leaf. Metabolic pathways analysis revealed that the increase in floral-fruity related components such as volatile terpenoids, phenylpropanoids/benzenoids, indole, alongside a decrease in green leaf volatiles including (E)-2-Hexenal, (Z)-3-Hexenol, played a crucial role in development of characteristic aroma, which could be a feasible index for evaluating processing techniques or quality of oolong tea.

Genome-wide investigation of WRKY gene family in Lavandula angustifolia and potential role of LaWRKY57 and LaWRKY75 in the regulation of terpenoid biosynthesis.

The WRKY transcription factors are integral to plant biology, serving essential functions in growth, development, stress responses, and the control of secondary metabolism. Through the use of bioinformatics techniques, this research has effectively characterized 207 members of the WRKY family (LaWRKY) present in the entire genome of Lavandula angustifolia. Phylogenetic analysis classified LaWRKYs into three distinct categories based on conserved domains. Collinearity analysis revealed tandem repeats, segmental duplications, and whole genome duplications in LaWRKYs, especially for segmental duplication playing a significant role in gene family expansion. LaWRKYs displayed distinct tissue-specific expression profiles across six different tissues of L. angustifolia. Particularly noteworthy were 12 genes exhibiting high expression in flower buds and calyx, the primary sites of volatile terpenoid production, indicating their potential role in terpenoid biosynthesis in L. angustifolia. RT-qPCR analysis revealed a notable increase in the expression levels of most examined LaWRKY genes in flower buds in response to both intense light and low-temperature conditions, whereas the majority of these genes in leaves were primarily induced by drought stress. However, all genes exhibited downregulation following GA treatment in both flower buds and leaves. Overexpression of LaWRKY57 (La13G01665) and LaWRKY75 (La16G00030) in tobacco led to a reduction in the density of glandular trichomes on leaf surfaces, resulting in changes to the volatile terpenoid composition in the leaves. Specifically, the content of Neophytadiene was significantly elevated compared to wild-type tobacco, while compounds such as eucalyptol, cis-3-Hexenyl iso-butyrate, and D-Limonene were produced, which were absent in the wild type. These findings provide a valuable reference for future investigations into the biological functions of the WRKY gene family in L. angustifolia.

Using Headspace Gas Chromatography–Mass Spectrometry to Investigate the Volatile Terpenoids Released from the Liquidambar formosana Leaf and Its Essential Oil

Phytoncides and aromatherapy scents mainly derive from plant secondary metabolites and are now well known for their health benefits. In this study, we analyzed the chemical composition of the leaf-derived essential oil of Liquidambar formosana (Altingiaceae) using GC-MS; we also investigated the VOCs released from L. formosana leaves and the leaf essential oil at different temperatures by means of headspace gas chromatography–mass spectrometry (HS-GC-MS). Regarding the VOCs of the leaves, monoterpenes predominated the VOCs at both temperatures, mainly comprising sabinene, followed by γ-terpinene, α-terpinene, and α-pinene. The intensity of the leaf VOCs at 50 °C was nearly three times higher than that at 25 °C; the emission of monoterpenes significantly increases at higher environmental temperatures. The VOC emissions of oxygenated monoterpenes from the leaf essential oil increased at higher temperatures (50 °C), especially those of terpinen-4-ol. Our results reveal that HS-GC-MS can be used to conveniently and directly analyze the VOCs emitted from L. formosana leaves and their essential oils and to evaluate the influence of temperature on the composition of the VOCs of specimens. These VOC studies will assist in the sustainable development and utilization of L. formosana trees for forest therapy, as well as the use of their leaf essential oil for aromatherapy.

The complexity of volatile terpene biosynthesis in roses: Particular insights into β-citronellol production.

The fascinating scent of rose (Rosa genus) flowers has captivated human senses for centuries, making them one of the most popular and widely used floral fragrances. Despite much progress over the last decade, many biochemical pathways responsible for rose scents remain unclear. We analyzed the floral scent compositions from various rose varieties and selected the modern cultivar Rosa hybrida "Double Delight" as a model system to unravel the formation of rose dominant volatile terpenes, which contribute substantially to the rose fragrance. Key genes involved in rose terpene biosynthesis were functionally characterized. Cytosolic geranyl diphosphate (GPP) generated by geranyl/farnesyl diphosphate synthase (G/FPPS1) catalysis played a pivotal role in rose scent production, and terpene synthases in roses play an important role in the formation of most volatile terpenes, but not for geraniol, citral, or β-citronellol. Subsequently, a series of enzymes, including geraniol dehydrogenase, geranial reductase, 12-oxophytodienoate reductase, and citronellal reductase, were characterized as involved in the transformation of geraniol to β-citronellol in roses through three successive steps. Interestingly, the β-citronellol biosynthesis pathway appears to be conserved in other horticultural plants like Lagerstroemia caudata and Paeonia lactiflora. Our findings provide valuable insights into the biosynthesis of rose volatile terpenoid compounds and offer essential gene resources for future breeding and molecular modification efforts.

Plant-Derived Terpenoids: A Plethora of Bioactive Compounds with Several Health Functions and Industrial Applications-A Comprehensive Overview.

Terpenoids are a large class of natural secondary plant metabolites which are highly diverse in structure, formed from isoprene units (C-5), associated with a wide range of biological properties, including antioxidant, antimicrobial, anti-inflammatory, antiallergic, anticancer, antimetastatic, antiangiogenesis, and apoptosis induction, and are considered for potential application in the food, cosmetics, pharmaceutical, and medical industries. In plants, terpenoids exert a variety of basic functions in growth and development. This review gives an overview, highlighting the current knowledge of terpenoids and recent advances in our understanding of the organization, regulation, and diversification of core and specialized terpenoid metabolic pathways and addressing the most important functions of volatile and non-volatile specialized terpenoid metabolites in plants. A comprehensive description of different aspects of plant-derived terpenoids as a sustainable source of bioactive compounds, their biosynthetic pathway, the several biological properties attributed to these secondary metabolites associated with health-promoting effects, and their potential industrial applications in several fields will be provided, and emerging and green extraction methods will also be discussed. In addition, future research perspectives within this framework will be highlighted. Literature selection was carried out using the National Library of Medicine, PubMed, and international reference data for the period from 2010 to 2024 using the keyword "terpenoids". A total of 177,633 published papers were found, of which 196 original and review papers were included in this review according to the criteria of their scientific reliability, their completeness, and their relevance to the theme considered.

FhMYB108 Regulates the Expression of Linalool Synthase Gene 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.

Chromosome genome assembly of the Camphora longepaniculata (Gamble) with PacBio and Hi-C sequencing data.

Camphora longepaniculata, a crucial commercial crop and a fundamental component of traditional Chinese medicine, is renowned for its abundant production of volatile terpenoids. However, the lack of available genomic information has hindered pertinent research efforts in the past. To bridge this gap, the present study aimed to use PacBio HiFi, short-read, and highthroughput chromosome conformation capture sequencing to construct a chromosome-level assembly of the C. longepaniculata genome. With twelve chromosomes accounting for 99.82% (766.69 Mb) of the final genome assembly, which covered 768.10 Mb, it was very complete. Remarkably, the assembly's contig and scaffold N50 values are exceptional as well-41.12 and 63.78 Mb, respectively-highlighting its excellent quality and intact structure. Furthermore, a total of 39,173 protein-coding genes were predicted, with 38,766 (98.96%) of them being functionally annotated. The completeness of the genome was confirmed by the Benchmarking Universal Single-Copy Ortholog evaluation, which revealed 99.01% of highly conserved plant genes. As the first comprehensive assembly of the C. longepaniculata genome, it provides a crucial starting point for deciphering the complex pathways involved in terpenoid production. Furthermore, this excellent genome serves as a vital resource for upcoming research on the breeding and genetics of C. longepaniculata.

Functional characterization of geranyl/farnesyl diphosphate synthase in Wurfbainia villosa and Wurfbainia longiligularis

Wurfbainia villosa and Wurfbainia longiligularis are the two primary plant sources of Fructus Amomi, a traditional Chinese medicine. Both plants are rich in volatile terpenoids, including monoterpenes and sesquiterpenes, which are the primary medicinal components of Fructus Amomi. The trans-isopentenyl diphosphate synthase (TIDS) gene family plays a key part in determining terpenoid diversity and accumulation. However, the TIDS gene family have not been identified in W. villosa and W. longiligularis. This study identified thirteen TIDS genes in W. villosa and eleven TIDS genes in W. longiligularis, which may have expanded through segmental replication events. Based on phylogenetic analysis and expression levels, eight candidate WvTIDSs and five WlTIDSs were selected for cloning. Functional characterization in vitro demonstrated that four homologous geranyl diphosphate synthases (GPPSs) (WvGPPS1, WvGPPS2, WlGPPS1, WlGPPS2) and two geranylgeranyl diphosphate synthases (GGPPSs) (WvGGPPS and WlGGPPS) were responsible for catalyzing the biosynthesis of geranyl diphosphate (GPP), whereas two farnesyl diphosphate synthases (FPPSs) (WvFPPS and WlFPPS) catalysed the biosynthesis of the farnesyl diphosphate (FPP). A comparison of six proteins with identified GPPS functions showed that WvGGPPS and WlGGPPS exhibited the highest activity levels. These findings indicate that homologous GPPS and GGPPS together promote the biosynthesis of GPP in W. villosa and W. longiligularis, thus providing sufficient precursors for the synthesis of monoterpenes and providing key genetic elements for Fructus Amomi variety improvement and molecular breeding.

Volatile communication in plants relies on a KAI2-mediated signaling pathway.

Plants are constantly exposed to volatile organic compounds (VOCs) that are released during plant-plant communication, within-plant self-signaling, and plant-microbe interactions. Therefore, understanding VOC perception and downstream signaling is vital for unraveling the mechanisms behind information exchange in plants, which remain largely unexplored. Using the hormone-like function of volatile terpenoids in reproductive organ development as a system with a visual marker for communication, we demonstrate that a petunia karrikin-insensitive receptor, PhKAI2ia, stereospecifically perceives the (-)-germacrene D signal, triggering a KAI2-mediated signaling cascade and affecting plant fitness. This study uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).

Exogenous methyl jasmonate treatment induced the transcriptional responses and accumulation of volatile terpenoids in Oenanthe javanica (Blume) DC

Water dropwort is favored by consumers for its unique flavor and medicinal value. Terpenoids were identified as the main volatile compounds related to its flavor. In this study, water dropwort was treated with different concentrations of exogenous methyl jasmonate (MeJA). The contents of volatile terpenoids were determined under various MeJA treatments. The results indicated that 0.1 mM of MeJA most effectively promoted the biosynthesis of flavor-related terpenoids in water dropwort. Terpinolene accounted the highest proportion among terpene compounds in water dropwort. The contents of jasmonates in water dropwort were also increased after exogenous MeJA treatments. Transcriptome analysis indicated that DEGs involved in the terpenoid biosynthesis pathway were upregulated. The TPS family was identified from water dropwort, and the expression levels of Oj0473630, Oj0287510 and Oj0240400 genes in TPS-b subfamily were consistent with the changes of terpene contents under MeJA treatments. Oj0473630 was cloned from the water dropwort and designated as OjTPS3, which is predicted to be related to the biosynthesis of terpinolene in water dropwort. Subcellular localization indicated that OjTPS3 protein was localized in chloroplast. Protein purification and enzyme activity of OjTPS3 protein were conducted. The results showed that the purified OjTPS3 protein catalyzed the biosynthesis of terpinolene by using geranyl diphosphate (GPP) as substrate in vitro. This study will facilitate to further understand the molecular mechanism of terpenoid biosynthesis and provide a strategy to improve the flavor of water dropwort.

Complex floral morphology and scent chemistry mediate hawkmoth pollination of an African orchid in the diverse Habenaria clade

Abstract One explanation for the evolution of complex floral morphology in orchids is that it can function as a component of ‘lock-and-key’ mechanisms that ensure pollen transfer by specific flower visitors. We investigated the roles of floral morphology and scent in the pollination of Bonatea antennifera, an African orchid that is phylogenetically embedded in the hyper-diverse Habenaria clade. Using camera traps and direct observations, we established that B. antennifera is pollinated by an assemblage of short-tongued hawkmoths with proboscides ~4 cm in length, corresponding to the spur length of the orchid. Pollinaria are attached between the palps of these hawkmoths via an intricate mechanism involving a wedge-shaped rostellum chamber and a tooth in the floral spur that deflect the head and proboscis of the hawkmoth to either side of the spur entrance. This functional subdivision of the flower results in removal of a single pollinarium per visit. Scent production is synchronized with the peak activity of hawkmoths at dusk, and electrophysiology experiments showed that hawkmoth antennae respond to several of the dominant volatile terpenoid compounds [e.g. R-(−)-linalool, caryophyllene epoxide, and (E)-nerolidol] emitted by the flowers. These results illustrate the functional significance of complex floral morphology and scent for pollen transfer in orchids.

The push-pull intercrop Desmodium does not repel, but intercepts and kills pests.

Over two decades ago, an intercropping strategy was developed that received critical acclaim for synergizing food security with ecosystem resilience in smallholder farming. The push-pull strategy reportedly suppresses lepidopteran pests in maize through a combination of a repellent intercrop (push), commonly Desmodium spp., and an attractive, border crop (pull). Key in the system is the intercrop's constitutive release of volatile terpenoids that repel herbivores. However, the earlier described volatile terpenoids were not detectable in the headspace of Desmodium, and only minimally upon herbivory. This was independent of soil type, microbiome composition, and whether collections were made in the laboratory or in the field. Furthermore, in oviposition choice tests in a wind tunnel, maize with or without an odor background of Desmodium was equally attractive for the invasive pest Spodoptera frugiperda. In search of an alternative mechanism, we found that neonate larvae strongly preferred Desmodium over maize. However, their development stagnated and no larva survived. In addition, older larvae were frequently seen impaled and immobilized by the dense network of silica-fortified, non-glandular trichomes. Thus, our data suggest that Desmodium may act through intercepting and decimating dispersing larval offspring rather than adult deterrence. As a hallmark of sustainable pest control, maize-Desmodium push-pull intercropping has inspired countless efforts to emulate stimulo-deterrent diversion in other cropping systems. However, detailed knowledge of the actual mechanisms is required to rationally improve the strategy, and translate the concept to other cropping systems.

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)-β-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)-β-caryophyllene compound had an attractive effect on the Apis ceranas. By comparing the homologous sequences with the genes of (E)-β-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 β-Elemene, (E)-β-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)-β-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.

The identification and functional characterization of the LcMCT gene from Liriodendron chinense reveals its potenatial role in carotenoids biosyanthesis

Terpenoids are not only important component of plant floral scent, but also indispensable elements in the formation of floral color. The petals of Liriodendron chinense are rich in tetraterpene carotenoids and release large amounts of volatile monoterpene and sesquiterpene compounds during full blooming stage. However, the mechanism of terpenoid synthesis is not clear in L. chinense. In this study, we identified a LcMCT gene and characterized its potential function in carotenoids biosynthesis. A total of 2947 up-regulated differentially expressed genes (DEGs) were discerned from the transcriptomic data of L. chinense petals, with a significant enrichment of DEGs related to plant hormone signal transduction and terpenoid backbone biosynthesis. After comprehensive analysis on these DEGs, the LcMCT gene was selected for subsequent function characterization. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results showed that LcMCT was expressed at the highest level in the petals during full blooming stage, suggesting a possible role in carotenoids biosynthesis and volatile terpenoid biosynthesis. Subcellular localization showed that the LcMCT protein was localized in the chloroplast. Overexpression of LcMCT in Arabidopsis thaliana affected the expression levels of MEP pathway genes. Moreover, the MCT enzyme activity and carotenoids contents in transgenic A. thaliana were increased by 69.27% and 15.57%, respectively. These results suggest that LcMCT promotes the biosynthesis of terpenoid precursors via the MEP pathway. Our work lays a foundation for exploring the mechanism of terpenoid synthesis in L. chinense.

Functional characterization of LcTPS1 and LcTPS14 explains the biosynthesis of citronellol, citronellal and linalool in Lagerstroemia caudata

Lagerstroemia caudata, a native species in China with a remarkable floral aroma, had been demonstrated the monoterpenoids as the main fragrant volatiles and integrated into crape myrtle breeding programs to enhance the aesthetic qualities. Terpene synthases (TPSs) were critical enzymes in terpenoids biosynthesis, which had not been isolated and reported in Lagerstroemia genus. Nine TPS genes were scrutinized from transcriptome data of L. caudata flowers, two of which, LcTPS1 and LcTPS14, were found gene expression mode aligned with the release pattern of floral scents, with subcellular localization showing LcTPS1 was expressed in plastids, while LcTPS14 in cytoplasm. Heterologous expression in E. coli revealed that LcTPS14 employed either geranyl pyrophosphate (GPP) or farnesyl pyrophosphate (FPP) to generate linalool or E-nerolidol. Over-expressing LcTPS1 or LcTPS14 in Arabidopsis increased the volatile terpenoids in flowers. Moreover, after virus-induced gene silencing (VIGS), the quantities of citronellal and citronellol dropped drastically in TRV - LcTPS1 samples, and TRV - LcTPS14 plants exhibited diminished linalool compared to controls. Our findings suggested LcTPS1 and LcTPS14 were pivotal in monoterpene production and had potentials in further molecular modulation of crape myrtle floral scents.