Volatile Terpenes Research Articles

Organotransition Metal Chemistry of Terpenes: Syntheses, Structures, Reactivity and Molecular Rearrangements.

The impact of organometallic chemistry on the terpene field only really blossomed in the 1960s and 1970s with the realisation that carbon-carbon bond formation under mild conditions could be achieved by using nickel or iron carbonyls as synthetic reagents. Concomitantly, the development of palladium derivatives capable of the controlled coupling of isoprene units attracted the attention of numerous highly talented researchers, including future Nobel laureates. We discuss briefly how early work on the syntheses of simple monoterpenes soon progressed to sesquiterpenes and diterpenes of increasing complexity, such as humulene, flexibilene, vitamin A, or pheromones of commercial value, in particular those used in perfumery (muscone, lavandulol), or grandisol and red scale pheromone as replacements for harmful pesticides. As the field progressed, there has been more emphasis on developing organometallic routes to enantiopure rather than racemic products, as well as gaining precise mechanistic data on the transformations, notably the course of metal-promoted molecular rearrangements that have long been a feature of terpene chemistry. We note the impact of the enormously enhanced analytical techniques, high-field NMR spectroscopy and X-ray crystallography, and their use to re-examine the originally proposed structures of terpenes and their organometallic derivatives. Finally, we highlight the very recent ground-breaking use of the crystalline sponge method to acquire structural data on low-melting or volatile terpenes. The literature cited herein covers the period 1959 to 2023.

Identification and expression analysis of TPS family gene in Cannabis sativa L

Terpene synthases (TPSs) regulate plant growth, development, and stress response. TPS genes have been identified in Arabidopsis thaliana and Zea mays. Cannabis sativa TPS genes were identified and analyzed using bioinformatics. Genomic data were downloaded from Plant Transcription Factor Database and National Center for Biotechnology Information database, and TPS genes were predicted, analyzed, and visualized using ExPASy, PlantCare, and other online websites along with TBtools, MEGA software, and other software. To verify its role, quantitative real-time polymerase chain reaction (qRT-PCR) tests were conducted. The Cannabis sativa TPS family comprises 41 elements distributed over 8 chromosomes and a single scaffold segment. The isoelectric point varied between 4.96 and 7.03, while the molecular weight spanned from 20705.90 to 102324.64 Da. The majority of genes were found in the cytoplasm and chloroplasts, with the remainder situated in the peroxisome, nucleus, plasma membrane, and mitochondria. Several cis-acting components associated with stress response were present in the gene's upstream promoter region. Data from RNA sequencing and qRT-PCR revealed specific expression of TPS genes in all five organs of female Cannabis sativa plants. Collinearity analysis showed 4 homologous gene pairs between the Cannabis sativa and Arabidopsis thaliana, with many pairs of homologous genes in other species, which was consistent with the dicotyledons evolutionary relationship. Furthermore, some genes may participate in Cannabis sativa growth and development and play a role in secondary metabolite synthesis. Therefore, bioinformatics analysis of the Cannabis sativa TPS gene family provides a theoretical basis for future research on the volatile terpene compounds of Cannabis sativa.

Method development for an untargeted HS-SPME-GC–MS analysis of terpenes and cannabinoids for the geographical sourcing of Marijuana

Despite growing decriminalization of Cannabis sativa (i.e., marijuana) possession throughout the United States of America, there remains to be an ongoing interest in the detection of unlawfully possessed and transported marijuana. This issue has resulted in an increasing interest regarding the generalization and specification related to the canine detection of marijuana. More specifically, canine trainers have expressed concerns on whether canines can generalize on the odor of marijuana regardless of the origin of their training materials. This research aims to differentiate multiple marijuana headspace samples from three regions in the USA based solely on the volatile organic compounds (VOCs) found in their odor profiles. In this study, a heated headspace solid phase micro-extraction (SPME) technique was optimized and implemented for the collection of both volatile terpenes and cannabinoids from marijuana. The headspace samples were analyzed using two full-scan, untargeted, optimized methods on a gas chromatograph coupled to a mass spectrometer (GC–MS), and a variety of chemometric tools were applied to the data to enable differentiation and potential classification between sample populations. Principal component analysis and sparse partial least squares discriminant analysis (sPLS-DA) employed in this study have demonstrated a disparity between marijuana varieties based on geography using the VOCs extracted from their odor profiles. With this research, it is intended to determine some fundamental differences between Cannabis of different geographical origins and set a foundation for the development and advancement of instrumental applications for other non-contact marijuana detection techniques in support of the improvement of illicit substance detection technology.

Ultrahigh-Performance Supercritical Fluid Chromatography-Multimodal Ionization-Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts.

Complex analysis of plant extracts usually requires a combination of several analytical approaches. Therefore, in this study, we developed a holistic two-injection approach for plant extract analysis, which is carried out within one instrument without the need for any manual intervention during the analysis. Ultrahigh-performance supercritical fluid chromatography (UHPSFC) was employed for the analysis of 17 volatile terpenes on a porous graphitic carbon column within 7.5 min, followed by analysis on short diol column where flavonoids, phenolic acids, and terpenoic acids were analyzed within 15.5 min. A multimodal ionization source combining electrospray and atmospheric pressure chemical ionization (ESCi) was selected for mass spectrometry detection as a simultaneous ionization of both lipophilic and polar compounds was required. The quantitative aspects of the final UHPSFC-ESI/ESCi-MS/MS two-injection approach were determined, and it was applied to the analysis of Eucalyptus sp. extracts prepared by supercritical fluid extraction. Current methods reported in the literature typically require a labor-intensive combination of liquid and gas chromatography for the complex analysis of plant extracts. We present for the first time a new UHPSFC approach requiring only a single instrument that provides an alternative approach to the analysis of complex plant extracts.

Identification of Volatile Compounds and Terpene Synthase (TPS) Genes Reveals ZcTPS02 Involved in β-Ocimene Biosynthesis in Zephyranthes candida.

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.

Functional genomics reveals functions of terpene synthases for volatile terpene formation in peach

Functional genomics reveals functions of terpene synthases for volatile terpene formation in peach

Maize terpene synthase 1 impacts insect behavior via the production of monoterpene volatiles β-myrcene and linalool

Plant-derived volatiles are important mediators of plant-insect interactions as they can provide cues for host location and quality, or act as direct or indirect defense molecules. The volatiles produced by Zea mays (maize) include a range of terpenes, likely produced by several of the terpene synthases (TPS) present in maize. Determining the roles of specific terpene volatiles and individual TPSs in maize-insect interactions is challenging due to the promiscuous nature of TPSs in vitro and their potential for functional redundancy. In this study, we used metabolite GWAS of a sweetcorn diversity panel infested with Spodoptera frugiperda (fall armyworm) to identify genetic correlations between TPSs and individual volatiles. This analysis revealed a correlation between maize terpene synthase 1 (ZmTPS1) and emission of the monoterpene volatiles linalool and β-myrcene. Electroantennogram assays showed gravid S. frugiperda could detect both linalool and β-myrcene. Quantification of headspace volatiles in a maize tps1 loss-of-function mutant confirmed that ZmTPS1 is an important contributor to linalool and β-myrcene emission in maize. Furthermore, pairwise choice assays between tps1 mutant and wild-type plants showed that ZmTPS1, and by extension its volatile products, aid host location in the chewing insect S. frugiperda, yet repel the sap-sucking pest, Rhopalosiphum maidis (corn leaf aphid). On the other hand, ZmTPS1 had no impact on indirect defense via the recruitment of the parasitoid Cotesia marginiventris. ZmTPS1 is therefore an important mediator of the interactions between maize and its insect pests.

Enhancing the taxonomic delimitation of Lippia origanoides Kunth (Verbenaceae) by analyzing volatile terpenes and molecular markers in micropropagated accessions

Enhancing the taxonomic delimitation of Lippia origanoides Kunth (Verbenaceae) by analyzing volatile terpenes and molecular markers in micropropagated accessions

Combining with volatilomic profiling and chemometrics to explore the volatile characteristics in five different dried Zanthoxylum bungeanum maxim

Owing to the short picking period of the fresh Zanthoxylum bungeanum, the postharvest drying has become an essential operation before the storage and transportation of Z. bungeanum. To explore the effects of drying methods on volatile characteristics, the volatilomic profiling of five different dried Z. bungeanum was investigated by E-nose, HS-SPME-GC/MS, GC-IMS in combination with chemometrics. The results indicated that W1W, W2W and W5S sensors within E-nose analysis showed the strongest responses in both fresh and dried Z. bungeanum. According to the identification of volatile organic compounds (VOCs), terpenes, esters and alcohols played the major roles in the volatile formation of the fresh and dried Z. bungeanum. The samples derived from hot air drying showed the relatively similar features with the fresh sample based on the relative abundances of these major VOCs. According to the results of multiple factor analysis (MFA), GC-IMS showed the strongest ability in distinguishing the fresh and different dried samples. Compared with the high levels of terpenes in fresh group, the significant increasement of terpene alcohols and terpene esters from the degradation and transformation of bound terpenoids was the main characteristics of all dried Z. bungeanum. Using the GC-IMS datasets, a weighted correlation network analysis (WCNA) model was constructed to clarify the VOC characteristics in all detetected samples. Thereinto, 6 significantly correlated modules were identified in fresh and five different dried samples. Additionally, a total of 23 hub VOCs can be recognized as the potential biomarkers for better distinguishing the fresh and five different dried Z. bungeanum.

WRKY Transcription Factors in Jasminum sambac: An Insight into the Regulation of Aroma Synthesis.

WRKY transcription factors are one of the largest families of transcription regulators that play essential roles in regulating the synthesis of secondary metabolites in plants. Jasmine (Jasminum sambac), renowned for its aromatic nature and fragrant blossoms, possesses a significant abundance of volatile terpene compounds. However, the role of the WRKY family in terpene synthesis in jasmine remains undetermined. In this study, 72 WRKY family genes of J. sambac were identified with their conserved WRKY domains and were categorized into three main groups based on their structural and phylogenetic characteristics. The extensive segmental duplications contributed to the expansion of the WRKY gene family. Expression profiles derived from the transcriptome data and qRT-PCR analysis showed that the majority of JsWRKY genes were significantly upregulated in fully bloomed flowers compared to buds. Furthermore, multiple correlation analyses revealed that the expression patterns of JsWRKYs (JsWRKY27/33/45/51/55/57) were correlated with both distinct terpene compounds (monoterpenes and sesquiterpenes). Notably, the majority of jasmine terpene synthase (JsTPS) genes related to terpene synthesis and containing W-box elements exhibited a significant correlation with JsWRKYs, particularly with JsWRKY51, displaying a strong positive correlation. A subcellular localization analysis showed that JsWRKY51 was localized in the nucleus. Moreover, transgenic tobacco leaves and jasmine calli experiments demonstrated that overexpression of JsWRKY51 was a key factor in enhancing the accumulation of β-ocimene, which is an important aromatic terpene component. Collectively, our findings suggest the roles of JsWRKY51 and other JsWRKYs in regulating the synthesis of aromatic compounds in J. sambac, providing a foundation for the potential utilization of JsWRKYs to facilitate the breeding of fragrant plant varieties with an improved aroma.

Single versus dual inoculation with indigenous Saccharomyces cerevisiae strains in winemaking.

Indigenous Saccharomyces cerevisiae strains and their combinations may be used to diversify wines and add complexity to sensory profiles. Here, two S. cerevisiae strains that represent regional genetic and phenotypic specificities for two major winegrowing areas of Greece were used in single- and mixed-culture fermentations. The kinetics and metabolic activities of the strains were analysed to evaluate the influence of each strain individually or in combination on wine quality. The two strains differentially affected the kinetics and the outcome of fermentation. They showed significant differences in the production of important metabolites that strongly affect the organoleptic profile of wines, such as volatile acidity, acetaldehyde, certain esters, and terpenes. Furthermore, the chemical and sensory profiles of wines produced by single cultures were different from those fermented by mixed culture inoculum. The concentration of certain metabolites was enhanced (e.g. isoamyl acetate, 1-heptanol), while others were suppressed (e.g. hexyl acetate, octyl acetate). Results highlight the potential worth of indigenous S. cerevisiae strains to differentiate local wines. The mixed culture S. cerevisiae inoculum was shown to generate novel wine characteristics, as compared to single cultures, thus offering alternatives to further diversify wines and increase their complexity.

Unraveling the terpene synthase family and characterization of BsTPS2 contributing to (S)-( +)-linalool biosynthesis in Boswellia.

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 0-24h 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.

Single-cell RNA sequencing reveals a hierarchical transcriptional regulatory network of terpenoid biosynthesis in cotton secretory glandular cells

Plants can synthesize a wide range of terpenoids in response to various environmental cues. However, the specific regulatory mechanisms governing terpenoid biosynthesis at the cellular level remain largely elusive. In this study, we employed single-cell RNA sequencing to comprehensively characterize the transcriptome profile of cotton leaves and established a hierarchical transcriptional network regulating cell-specific terpenoid production. We observed substantial expression levels of genes associated with the biosynthesis of both volatile terpenes (such as β-caryophyllene and β-myrcene) and non-volatile gossypol-type terpenoids in secretory glandular cells. Moreover, two novel transcription factors, namely GoHSFA4a and GoNAC42, are identified to function downstream of the Gossypium PIGMENT GLAND FORMATION genes. Both transcription factors could directly regulate the expression of terpenoid biosynthetic genes in secretory glandular cells in response to developmental and environmental stimuli. For convenient retrieval of the single-cell RNA sequencing data generated in this study, we developed a user-friendly web server (https://www.cottonleafatlas.com). Our findings not only offer valuable insights into the precise regulation of terpenoid biosynthesis genes in cotton leaves but also provide potential targets for cotton breeding endeavors.

Integration of transcriptome, volatile and non-volatile metabolite profile reveals characteristic aroma formation in Toona sinensis

Toona sinensis is renowned for its unique aroma, but the formation mechanism remains unclear. In this study, volatile and non-volatile metabolites were combined with transcriptomes to investigate the potential mechanism of aroma formation in T. sinensis buds (TSB) and microgreens (TSM). Volatile sulfur compounds (VSCs) and terpenes were the main volatiles of TSM and TSB, respectively. 20 volatiles were identified as potential biomarkers, mainly VSCs and terpenes. In VSC biosynthesis pathways, cysteine was primarily synthesized from serine transformation in TSM. S-(trans-l-propenyl)-l-cysteine was likely to be the main precursor of VSC biosynthesis in T. sinensis. Higher expression of lachrymatory-factor synthase (LFS) consuming more precursor (1-propenyl sulfenic acid) in TSB led to reduced accumulation of VSCs. Isopentenyl diphosphate isomerase (IDI) and mevalonate diphosphate decarboxylase (MPDC) might play crucial roles in T. sinensis terpene biosynthesis. This study provided valuable insights into the formation of characteristic aromas in T. sinensis.

Transcriptome Analyses Reveal the Aroma Terpeniods Biosynthesis Pathways of Primula forbesii Franch. and the Functional Characterization of the PfDXS2 Gene.

Primula forbesii Franch. is a unique biennial herb with a strong floral fragrance, making it an excellent material for studying the aroma characteristics of the genus Primula. The floral scent is an important ornamental trait that facilitates fertilization. However, the molecular mechanism regulating the floral scent in Primula is unknown. In order to better understand the biological mechanisms of floral scents in this species, this study used RNA sequencing analysis to discuss the first transcriptome sequence of four flowering stages of P. forbesii, which generated 12 P. forbesii cDNA libraries with 79.64 Gb of clean data that formed 51,849 unigenes. Moreover, 53.26% of the unigenes were annotated using public databases. P. forbesii contained 44 candidate genes covering all known enzymatic steps for the biosynthesis of volatile terpenes, the major contributor to the flower's scent. Finally, 1-deoxy-d-xylulose 5-phosphate synthase gene of P. forbesii (PfDXS2, MK370094), the first key enzyme gene in the 2-c-methyl-d-erythritol 4-phosphate (MEP) pathway of terpenoids, was cloned and functionally verified using virus-induced gene silencing (VIGs). The results showed that PfDXS2-silencing significantly reduced the relative concentrations of main volatile terpenes. This report is the first to present molecular data related to aroma metabolites biosynthesis pathways and the functional characterization of any P. forbesii gene. The data on RNA sequencing provide comprehensive information for further analysis of other plants of the genus Primula.

Volatile Profile of Strawberry Fruits and Influence of Different Drying Methods on Their Aroma and Flavor: A Review.

Strawberries are the most popular berry fruit in the world, due to their distinctive aroma, flavor, and known health properties. Because volatile substances play a large role in strawberry flavor, even little alterations can have a big impact on how the fruit tastes. Strawberries are thought to have a complex aroma. Fresh strawberry fruits contain more than 360 volatile compounds, including esters, furans, terpenes, alcohols, aldehydes, ketones, and sulfur compounds. Despite having far lower concentrations than esters, terpenoids, furanones, and sulfur compounds, all have a considerable impact on how people perceive the aroma of strawberries. With a focus on the active aroma components and the many analytical methods used to identify them, including gas chromatography, electronic nose sensing, and proton-transfer- reaction mass spectrometry, the present review's aim was to provide a summary of the relevant literature. Additionally, strawberry fruits are frequently dried to create a powder in order to increase their shelf life. Consequently, the impact of various drying techniques on strawberries' volatile profile was investigated in the current review. This review can be considered a good reference for research concerning the aroma profile of strawberries. It helps to better understand the complex aroma and flavor of strawberries and provides a guide for the effects of drying processing.

Cross-validation of the peppermint benchmarking experiment across three analytical platforms

The Peppermint Experiment is a breath analysis benchmarking initiative that seeks to address the lack of inter-comparability of outcomes across independent breath biomarker studies. In this experiment, the washout profiles of volatile terpene constituents of encapsulated peppermint oil (mainly α-pinene, β-pinene, limonene and 1,8-cineole) in exhaled breath are characterized through a series of measurements at defined sampling intervals up to 6 h after ingestion of the capsule. In the present work, the Peppermint Experiment was carried out on a cohort of volunteers (n= 11) that provided breath samples in three sittings on different days (i.e. triplicates per volunteer) for concurrent analysis by three different analytical platforms. These platforms were proton transfer reaction-time-of-flight-mass spectrometry (PTR-TOFMS) interfaced with a buffered end-tidal (BET) breath sampler, gas chromatography-ion mobility spectrometry (GC-IMS) in conjunction with a compatible handheld direct breath sampler, and thermal desorption comprehensive two-dimensional gas chromatography-time-of-flight-mass spectrometry (TD-GC×GC-TOFMS) with a Respiration Collection for in-vitro Analysis (ReCIVA) system for trapping breath volatiles onto adsorbent tubes. Regression analysis yielded mean washout times across the cohort of 448 min (PTR-TOFMS and GC-IMS) and 372 min (TD-GC×GC-TOFMS), which are in good alignment with published benchmark values. Large variations in washout profiles were observed at the individuals level, both between (inter-individual) and within (intra-individual) participants, indicating high variability in the degree of absorption, distribution, metabolism and excretion of volatile terpenes in the body within individuals and across the cohort. The comparably low inter-instrument variability indicates that differences in benchmark values from independent studies reported in the literature are driven by biological variability rather than different performances between sampling methods or analytical platforms.

Advances in the Biosynthesis of Terpenoids and Their Ecological Functions in Plant Resistance.

Secondary metabolism plays an important role in the adaptation of plants to their environments, particularly by mediating bio-interactions and protecting plants from herbivores, insects, and pathogens. Terpenoids form the largest group of plant secondary metabolites, and their biosynthesis and regulation are extremely complicated. Terpenoids are key players in the interactions and defense reactions between plants, microorganisms, and animals. Terpene compounds are of great significance both to plants themselves and the ecological environment. On the one hand, while protecting plants themselves, they can also have an impact on the environment, thereby affecting the evolution of plant communities and even ecosystems. On the other hand, their economic value is gradually becoming clear in various aspects of human life; their potential is enormous, and they have broad application prospects. Therefore, research on terpenoids is crucial for plants, especially crops. This review paper is mainly focused on the following six aspects: plant terpenes (especially terpene volatiles and plant defense); their ecological functions; their biosynthesis and transport; related synthesis genes and their regulation; terpene homologues; and research and application prospects. We will provide readers with a systematic introduction to terpenoids covering the above aspects.

Chemical profile and analysis of biosynthetic pathways and genes of volatile terpenes in Pityopsis ruthii, a rare and endangered flowering plant.

It is critical to gather biological information about rare and endangered plants to incorporate into conservation efforts. The secondary metabolism of Pityopsis ruthii, an endangered flowering plant that only occurs along limited sections of two rivers (Ocoee and Hiwassee) in Tennessee, USA was studied. Our long-term goal is to understand the mechanisms behind P. ruthii's adaptation to restricted areas in Tennessee. Here, we profiled the secondary metabolites, specifically in flowers, with a focus on terpenes, aiming to uncover the genomic and molecular basis of terpene biosynthesis in P. ruthii flowers using transcriptomic and biochemical approaches. By comparative profiling of the nonpolar portion of metabolites from various tissues, P. ruthii flowers were rich in terpenes, which included 4 monoterpenes and 10 sesquiterpenes. These terpenes were emitted from flowers as volatiles with monoterpenes and sesquiterpenes accounting for almost 68% and 32% of total emission of terpenes, respectively. These findings suggested that floral terpenes play important roles for the biology and adaptation of P. ruthii to its limited range. To investigate the biosynthesis of floral terpenes, transcriptome data for flowers were produced and analyzed. Genes involved in the terpene biosynthetic pathway were identified and their relative expressions determined. Using this approach, 67 putative terpene synthase (TPS) contigs were detected. TPSs in general are critical for terpene biosynthesis. Seven full-length TPS genes encoding putative monoterpene and sesquiterpene synthases were cloned and functionally characterized. Three catalyzed the biosynthesis of sesquiterpenes and four catalyzed the biosynthesis of monoterpenes. In conclusion, P. ruthii plants employ multiple TPS genes for the biosynthesis of a mixture of floral monoterpenes and sesquiterpenes, which probably play roles in chemical defense and attracting insect pollinators alike.

The hops (Humulus lupulus) genome contains a mid-sized terpene synthase family that shows wide functional and allelic diversity

BackgroundHops (Humulus lupulus L.) are a dioecious climbing perennial, with the dried mature “cones” (strobili) of the pistillate/female inflorescences being widely used as both a bittering agent and to enhance the flavour of beer. The glandular trichomes of the bract and bracteole flowering structures of the cones produce an abundance of secondary metabolites, such as terpenoids, bitter acids and prenylated phenolics depending on plant genetics, developmental stage and environment. More knowledge is required on the functional and allelic diversity of terpene synthase (TPS) genes responsible for the biosynthesis of volatile terpenes to assist in flavour-directed hop breeding.ResultsMajor volatile terpene compounds were identified using gas chromatography–mass spectrometry (GC–MS) in the ripe cones of twenty-one hop cultivars grown in New Zealand. All cultivars produced the monoterpene β-myrcene and the sesquiterpenes α-humulene and β-caryophyllene, but the quantities varied broadly. Other terpenes were found in large quantities in only a smaller subset of cultivars, e.g. β-farnesene (in seven cultivars) and α-pinene (in four). In four contrasting cultivars (Wakatu™, Wai-iti™, Nelson Sauvin™, and ‘Nugget’), terpene production during cone development was investigated in detail, with concentrations of some of the major terpenes increasing up to 1000-fold during development and reaching maximal levels from 50–60 days after flowering. Utilising the published H. lupulus genome, 87 putative full-length and partial terpene synthase genes were identified. Alleles corresponding to seven TPS genes were amplified from ripe cone cDNA from multiple cultivars and subsequently functionally characterised by transient expression in planta. Alleles of the previously characterised HlSTS1 produced humulene/caryophyllene as the major terpenes. HlRLS alleles produced (R)-(-)-linalool, whilst alleles of two sesquiterpene synthase genes, HlAFS1 and HlAFS2 produced α-farnesene. Alleles of HlMTS1, HlMTS2 and HlTPS1 were inactive in all the hop cultivars studied.ConclusionsAlleles of four TPS genes were identified and shown to produce key aroma volatiles in ripe hop cones. Multiple expressed but inactive TPS alleles were also identified, suggesting that extensive loss-of-function has occurred during domestication and breeding of hops. Our results can be used to develop hop cultivars with novel/improved terpene profiles using marker-assisted breeding strategies to select for, or against, specific TPS alleles.