Showing papers on "Terpene published in 2017"

Terpenes in the Aroma of Grapes and Wines: A Review

Abstract: This paper reviews some aspects of terpenes and terpene derivatives in grapes and wines, such as the identification of these compounds, their use in the identification of and differentiation between cultivars, the effect of grape maturity and wine-making techniques on terpene concentrations and wine quality, as well as transformations of terpenes in grapes and wine.

Terpene synthases from Cannabis sativa

Abstract: Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety ‘Finola’ revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono- and sesqui-TPS, whose products collectively comprise most of the terpenes of ‘Finola’ resin, including major compounds such as β-myrcene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.

Terpenes from Forests and Human Health.

Abstract: Forest bathing has beneficial effects on human health via showering of forest aerosols as well as physical relaxation. Terpenes that consist of multiple isoprene units are the largest class of organic compounds produced by various plants, and one of the major components of forest aerosols. Traditionally, terpene-containing plant oil has been used to treat various diseases without knowing the exact functions or the mechanisms of action of the individual bioactive compounds. This review categorizes various terpenes easily obtained from forests according to their anti-inflammatory, anti-tumorigenic, or neuroprotective activities. Moreover, potential action mechanisms of the individual terpenes and their effects on such processes, which are described in various in vivo and in vitro systems, are discussed. In conclusion, the studies that show the biological effectiveness of terpenes support the benefits of forest bathing and propose a potential use of terpenes as chemotherapeutic agents for treating various human diseases.

'Hidden' Terpenoids in Plants: Their Biosynthesis, Localization and Ecological Roles.

Abstract: Terpenoids are the largest group of plant specialized (secondary) metabolites. These naturally occurring chemical compounds are highly diverse in chemical structure. Although there have been many excellent studies of terpenoids, most have focused on compounds built solely of isoprene units. Plants, however, also contain many 'atypical' terpenoids, such as glycosylated volatile terpenes and composite-type terpenoids, the latter of which are synthesized by the coupling of isoprene units on aromatic compounds. This mini review describes these 'hidden' terpenoids, providing an overview of their biosynthesis, localization, and biological and ecological activities.

UV-B irradiation differentially regulates terpene synthases and terpene content of peach.

Abstract: Plants generate protective molecules in response to ultraviolet (UV) light. In laboratory experiments, 48 h UV-B irradiation of peach fruits and leaves reduced the flavour-related monoterpene linalool by 60%. No isoprene was detected, but other terpenoids increased significantly, including a threefold accumulation of the sesquiterpene (E,E)-α-farnesene, which was also increased by jasmonic acid treatment. RNA sequencing revealed altered transcript levels for two terpene synthases (TPSs): PpTPS1, a TPS-g subfamily member, decreased by 86% and PpTPS2, a TPS-b subfamily member, increased 80-fold. Heterologous expression in Escherichia coli and transient overexpression in tobacco and peach fruits showed PpTPS1 was localized in plastids and associated with production of linalool, while PpTPS2 was responsible for (E,E)-α-farnesene biosynthesis in the cytoplasm. Candidate regulatory genes for these responses were identified. Commercial peach production in Asia involves fruit bagging to maintain marketable yield and quality. TPS gene expression and volatile terpenoid production in field experiments, using bags transmitting high UV-B radiation, showed similar effects on peach volatiles to those from laboratory experiments. Bags transmitting less UV-B light ameliorated the reduction in the flavour volatile linalool, indicating that flavour components of peach fruits can be modulated by selecting an appropriate source of environmental screening material.

Analysis of terpenes and turpentines using gas chromatography with vacuum ultraviolet detection

Abstract: The separation and identification of natural mixtures of terpenes is challenging and laborious. A gas chromatographic method based on vacuum ultraviolet spectroscopic detection, which is characterized by full-scan absorption in the range of 125-240 nm, was developed and applied to analyze terpenes. In this study, the vacuum ultraviolet absorption spectra of 41 different standard terpenes were investigated and compared. The spectra were found to be highly featured and easily differentiated. Several commercial turpentine samples were analyzed and the vacuum ultraviolet detector demonstrated good specificity for qualitative identification of constituent terpenes. A total of 31 terpenes were detected in the four turpentine samples. α-Pinene was the predominant terpene ranging from 744.2 ± 9.7 to 917 ± 21 mg/mL. The other major constituents in the turpentines included β-pinene, δ-3-carene, camphene, and p-isopropyltoluene. Deconvolution of co-eluting signals of terpenes was achieved utilizing the data analysis software. The technique has been demonstrated to be a powerful tool for reliable and accurate qualitative and quantitative analysis of terpenes from complex natural mixtures.

Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

Abstract: Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial mon...

Genomic Analysis of Terpene Synthase Family and Functional Characterization of Seven Sesquiterpene Synthases from Citrus sinensis.

Abstract: Citrus aroma and flavour, chief traits of fruit quality, are derived from their high content in essential oils of most plant tissues, including leaves, stems, flowers and fruits. Accumulated in secretory cavities, most components of these oils are volatile terpenes. They contribute to defence against herbivores and pathogens, and perhaps also protect tissues against abiotic stress. In spite of their importance, our understanding of the physiological, biochemical and genetic regulation of citrus terpene volatiles is still limited. The availability of the sweet orange (Citrus sinensis L. Osbeck) genome sequence allowed us to characterize for the first time the terpene synthase (TPS) family in a citrus type. CsTPS is one of the largest angiosperm TPS families characterized so far, formed by 95 loci from which just 55 encode for putative functional terpene synthases. All TPS angiosperm families, TPS-a, TPS-b, TPS-c, TPS-e/f and TPS-g were represented in the sweet orange genome, with 28, 18, 2, 2 and 5 putative full length genes each. Additionally, sweet orange -farnesene synthase, (Z)--cubebene/-copaene synthase, two -caryophyllene synthases and three multiproduct enzymes yielding -cadinene/-copaene, -elemene and -cadinene/ledene/allo-aromandendrene as major products were identified and functionally characterized via in vivo recombinant E. coli assays.

Terpenoids as source of anti-inflammatory compounds

Abstract: Terpenoids accounts for the major class of secondary metabolites produced by plants. It shows defense activity against environmental stress and help to heal injuries. Medicinal plants are rich in monoterpenoids, diterpenoids, sesquiterpenes, triterpenes, tetraterpenes, and ceramide. A number of therapeutic applications such as antibacterial, antimicrobial, antitumor, anti-inflammatory activity have been identified. Terpenoids are compounds similar to terpenes derived from 5-carbon monomer isoprene units. The review puts and detail insight on different class of compounds isolated from natural source from 2000 to 2016 showing anti-inflammatory potential of pharmacologically interesting agent and their mechanism of action. Keywords: Anti-inflammatory, Mechanism of action, Natural source, Plant, Terpenoid, Bioactive compound.

Bioactive chemical compounds in Eremurus persicus (Joub. & Spach) Boiss. essential oil and their health implications.

Abstract: The genus Eremurus is native to Eastern Europe and temperate Asia. Particularly, Eremurus persicus (Joub. & Spach) Boiss. is highly valued in traditional foods and medicine. Scientific knowledge about E. persicus chemical composition and bioactivity is required. Therefore, the present study is aimed to determine the volatile composition of E. persicus essential oil (EO) by means of gas chromatography coupled to flame ionization/mass spectrometry detection. Moreover, the antioxidant, antimicrobial, anticancer, and acetylcholinesterase inhibitory activities of the EO were tested. Interestingly, the anti-dermatophyte potency was close to that of the drug griseofulvin, with minimum fungicidal concentration ranging between 0.7 and 4.5% depending on the fungi strain. The EO was also effective against hepatocellular carcinoma (Hep-G2) and breast adenocarcinoma (MCF-7) human cancer cell lines in a concentration (200-1500 ng/mL)-dependent manner, with a decrease of the cell viability up to 65% and 52%, respectively. The E. persicus EO was rich in terpenes and oxygenated terpene derivatives. Individually, limonene (16.25%), geranylgeraniol (15.23%), n-nonanal (9.48%), geranyl acetone (9.12%), benzene acetaldehyde (8.51%), linalool (7.93%), α-pinene (6.89%), and 1,8-cineol (5.22%) were the most abundant volatile compounds and could be chosen as analytical markers of this essential oil. In conclusion, our results suggested that this EO possesses a wide range of bioactive properties that could be useful in nutraceutical, functional foods and cosmeceutical formulations.

Potential Contribution of Fish Feed and Phytoplankton to the Content of Volatile Terpenes in Cultured Pangasius (Pangasianodon hypophthalmus) and Tilapia (Oreochromis niloticus)

Abstract: Geosmin and 2-methylisoborneol are the most recognized off-flavors in freshwater fish, but terpenes may also contribute off-flavor in fish. We identified six monoterpenes, 11 sesquiterpenes, and three terpene-related compounds in pangasius and tilapia from aquaculture farms in Bangladesh. The concentrations of most of the volatiles were below published odor thresholds, except for α-pinene, limonene, β-caryophyllene, α-humulene, and β-ionone in tilapia, and limonene and β-ionone in pangasius. To identify sources of the terpenes, terpene profiles of fish feed and phytoplankton in the ponds were analyzed. In feed and mustard cake (feed ingredient), five monoterpenes and two sesquiterpenes were identified, and five of these compounds were also detected in the fish. In phytoplankton, 11 monoterpenes were found and three also occurred in the fish. The higher number of terpenes common to both fish and feed, than to fish and phytoplankton, suggests that feed was a more abundant source of odor-active terpenes in t...

Volatile terpenoids as potential drug leads in Alzheimer’s disease

Abstract: Abstract Alzheimer’s disease (AD) is by far the most prevalent of all known forms of dementia. Despite wide-spread research, the main causes of emergence and development of AD have not been fully recognized. Natural, low-molecular, lipophilic terpenoids constitute an interesting group of secondary plant metabolites, that exert biological activities of possible use in the prevention and treatment of AD. In order to identify secondary metabolites possessing both antioxidant activity and the potential to increase the level of acetylcholine, selected terpenoids have been screened for possible acetylcholinesterase inhibitory activity by use of two methods, namely Marston (chromatographic assay) and Ellman (spectrophotometric assay). In order to describe the interaction between terpenes and AChE active gorge, molecular docking simulations were performed. Additionally, all analyzed terpenes were also evaluated for their cytotoxic properties against two normal cell lines using MTT assay. The obtained results show that: carvone (6), pulegone (8) and γ-terpinene (7) possess desirable AChE inhibitory activity. MTT assay revealed low or lack of cytotoxicity of these metabolites. Thus, among the investigated terpenes, carvone (6), pulegone (8) and y-terpinene (7) can be recognized as compounds with most promising activities in the development of multi-target directed ligands.

Biosynthetic studies on terpenoids produced by Streptomyces

Abstract: Terpenoids are a large and highly diverse group of natural products. All terpenoids are biosynthesized from isoprenyl diphosphate formed by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer dimethylallyl diphosphate (DMAPP). Two distinct biosynthetic pathways produce the essential primary metabolites IPP and DMAPP: the 2-C-methylerythritol 4-phosphate pathway and the mevalonate pathway. The isoprenyl substrates can be cyclized by terpene cyclase into single-ring or multi-ring products, which can be further diversified by subsequent modification reactions, such as hydroxylation and glycosylation. This review article describes the biosynthetic pathways of terpenoids produced by Streptomyces and their related novel enzymes.

Transcriptomic insight into terpenoid and carbazole alkaloid biosynthesis, and functional characterization of two terpene synthases in curry tree (Murraya koenigii).

Abstract: Curry tree (Murraya koenigii L.) is a rich source of aromatic terpenes and pharmacologically important carbazole alkaloids. Here, M. koenigii leaf transcriptome was generated to gain insight into terpenoid and alkaloid biosynthesis. Analysis of de novo assembled contigs yielded genes for terpene backbone biosynthesis and terpene synthases. Also, gene families possibly involved in carbazole alkaloid formation were identified that included polyketide synthases, prenyltransferases, methyltransferases and cytochrome P450s. Further, two genes encoding terpene synthases (MkTPS1 and MkTPS2) with highest in silico transcript abundance were cloned and functionally characterized to determine their involvement in leaf volatile formation. Subcellular localization using GFP fusions revealed the plastidial and cytosolic localization of MkTPS1 and MkTPS2, respectively. Enzymatic characterization demonstrated the monoterpene synthase activity of recombinant MkTPS1, which produced primarily (−)-sabinene from geranyl diphosphate (GPP). Recombinant MkTPS2 exhibited sesquiterpene synthase activity and formed (E,E)-α-farnesene as the major product from farnesyl diphosphate (FPP). Moreover, mRNA expression and leaf volatile analyses indicated that MkTPS1 accounts for (−)-sabinene emitted by M. koenigii leaves. Overall, the transcriptome data generated in this study will be a great resource and the start point for characterizing genes involved in the biosynthetic pathway of medicinally important carbazole alkaloids.

Inibitory effects of linalool on fungal pathogenicity of clinical isolates of Microsporum canis and Microsporum gypseum.

Abstract: Summary In humans worldwide, Microsporum sp. is a frequent agent of dermatophytsosis. When considering the emergence of resistant fungi and the clinical relevance of dermatophytosis, terpene antifungal activity is of great interest. Linalool is a monoterpene alcohol with pharmacological properties. In this study, antifungal in vitro activity of linalool and ketoconazole (as a positive control) were evaluated against clinical isolates of M. canis and M. gypseum. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of each drug were determined by broth microdilution. The effects of the drugs (1/2MIC, MIC, 2xMIC) on radial mycelial growth, conidial production and germination were analysed. The effect on the fungal cell membrane (release of intracellular material) was also investigated. Linalool (MIC: 128 μg/mL) and ketoconazole (MIC: 64 μg/mL) were effective in inhibiting all dermatophytes studied. The MFC values of linalool ranged between 128 and 256 μg/mL, whereas ketoconazole showed MFC values of from 64 to 256 μg/mL. Linalool (at MIC and 2xMIC) and ketoconazole (at 1/2MIC, MIC, 2xMIC) inhibited mycelial growth (P < 0.05). The drugs (1/2MIC, MIC, 2xMIC) were also active on conidiogenesis and conidia germination, causing complete inhibition (P < 0.05). Linalool caused leakage of intracellular material. Our study supports the use of linalool as a potential antifungal agent against M. canis and M. gypseum.

Antifeedant effects of common terpenes from Mediterranean aromatic plants on Leptinotarsa decemlineata

Abstract: Essential oil terpenes are secondary metabolites produced in different biological pathways as pollinator attraction signals, but also as defense mechanisms against insect pests, herbivores, microorganisms, competing plants, etc. In this context, we have evaluated a total of 24 terpenes commonly found in Mediterranean aromatic plants, including 8 monoterpene hydrocarbons, 9 oxygenated monoterpenes, 2 esterified monoterpenes, 3 sesquiterpene hydrocarbons and 2 oxygenated sesquiterpenes in order to determine their antifeedant effects on the Colorado potato beetle (Leptinotarsa decemlineata Say), a major pest of potato crops. Terpene hydrocarbons showed low antifeedant activity, whereas the oxygenated sesquiterpene (-)-α-bisabolol with an inhibition activity of 96.3% was the most active, followed by carvacrol (90.9%), (+)-Terpinen-4-ol (87.1%) and thymol (81.5%). Other terpenes like (1S)-(-)-verbenone (72.9%), (+/-)-camphor (63.4%) and linalyl acetate (60.7%) showed moderate activity. Subsequently, terpenes showing antifeedant activity against this insect were also tested in allelopathic assays to determine potential damage to the crops. Results showed phytotoxic effects for (1S)-(-)-verbenone on Lactuca sativa germination, and for carvacrol on both the seed germination and the leaf and root growth of Lolium perenne. In conclusion, (-)-α-bisabolol is the best option to develop natural antifeedant formulations against L. decemlineata on the basis of its high antifeedant and low phytotoxic activities.

System and method for producing a terpene-enhanced cannibinoid concentrate

Abstract: A system and method of producing a blended extract of cannabinoids and terpenes, which extracts terpenes using supercritical CO2, and extracts a cannabinoid concentrate from the residual material using a cold ethanol flush followed by distillation; the CO2-extracted terpenes are then added back to the cannabinoid concentrate in a final blending step Blending terpenes at the end of extraction may enhance the flavor and effectiveness of the cannabinoid concentrate By separately extracting terpenes and cannabinoids, optimal processes and parameters may be used for each step Blending may combine terpenes and cannabinoids in any desired ratio; for example, a terpene-to-cannabinoid ratio of approximately 1:10 may be used The ethanol used in the cold ethanol extraction of cannabinoids may be recovered and reused for subsequent batches Cannabinoid concentrates may be redistilled multiple times to enhance their concentration, followed by terpene blending

Intraspecific diversity of terpenes of Eucalyptus camaldulensis (Myrtaceae) at a continental scale

Abstract: Plants show a high degree of intraspecific variation in several traits including plant secondary metabolites This variation can be influenced by genetic and environmental factors that result in geographical structure in their distribution By growing plants from several populations in a controlled environment, we studied variation in foliar terpenes in Eucalyptus camaldulensis, which is the widest distributed eucalypt, with a large range both latitudinally and longitudinally We found that the concentration of terpenes is highly variable among subspecies We identified four chemotypes dominated by 1,8-cineole, γ-terpinene, α- and β-phellandrene While the 1,8-cineole chemotype is abundant in all populations, the other three chemotypes are rare in the central area and the north-east of Australia The γ-terpinene chemotype is mainly restricted to the north and west of Australia, whereas the α- and β-phellandrene chemotypes show an opposite distribution in the north and south of the continent The annual mean temperature and humidity of the source populations correlate with the abundance of the dominant terpenes We also tested the effects of elevated CO2 concentrations on the terpene concentration and found that elevated CO2 atmosphere reduces the overall accumulation of foliar terpenes The results suggest that variation in terpene composition in E camaldulensis can be influenced by environmental variables, mainly favouring the 1,8-cineole chemotype in arid locations

System for producing a terpene-enhanced cannabinoid concentrate

Abstract: A system for producing a blended extract of cannabinoids and terpenes, which extracts terpenes using supercritical CO2, and extracts a cannabinoid concentrate from the residual material using a cold ethanol flush followed by distillation; the CO2-extracted terpenes are then added back to the cannabinoid concentrate in a final blending step. Blending terpenes at the end of extraction may enhance the flavor and effectiveness of the cannabinoid concentrate. By separately extracting terpenes and cannabinoids, optimal processes and parameters may be used for each step. Blending may combine terpenes and cannabinoids in any desired ratio; for example, a terpene-to-cannabinoid ratio of approximately 1:10 may be used. The ethanol used in the cold ethanol extraction of cannabinoids may be recovered and reused for subsequent batches. Cannabinoid concentrates may be redistilled multiple times to enhance their concentration, followed by terpene blending.

Terpene-enriched cannabinoid composition

Abstract: Terpene-enriched cannabinoid composition. Cannabinoid compositions are described including at least one cannabinoid in a specific amount, a primary terpene in a specific amount, at least 5% by weight of a non-cannabinoid, non-terpene, carrier, optionally at least three secondary terpenes; less than 5% by weight glycol; and less than 20% by weight water, where said non-cannabinoid, non-terpene carrier comprises cellulose and the terpenes to cannabinoids weight/weight ratio in said composition is about 0.1 to about 1.0. Also described are the above compositions where said non-cannabinoid, non-terpene carrier comprise less than 5% by weight cellulose and the terpenes to cannabinoids weight/weight ratio in said compositions is about 0.05 to about 1.0, forming terpene-enriched cannabinoid compositions with enhanced therapeutic effect compared with that of a composition comprising the same cannabinoids amounts and one half the amount of said primary terpene.

Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems.

Abstract: With over 50.000 identified compounds terpenes are the largest and most structurally diverse group of natural products. They are ubiquitous in bacteria, plants, animals and fungi, conducting several biological functions such as cell wall components or defense mechanisms. Industrial applications entail among others pharmaceuticals, food additives, vitamins, fragrances, fuels and fuel additives. Central building blocks of all terpenes are the isoprenoid compounds isopentenyl diphosphate and dimethylallyl diphosphate. Bacteria like Escherichia coli harbor a native metabolic pathway for these isoprenoids that is quite amenable for genetic engineering. Together with recombinant terpene biosynthesis modules, they are very suitable hosts for heterologous production of high value terpenes. Yet, in contrast to the number of extracted and characterized terpenes, little is known about the specific biosynthetic enzymes that are involved especially in the formation of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized terpene producing Escherichia coli, this review shall give an insight in recent progresses regarding manipulation of mostly diterpene synthases.

Chemoenzymatic Synthesis of a Novel Borneol-Based Polyester.

Abstract: Terpenes are a class of natural compounds that have recently moved into the focus as bio-based resource for chemicals productions due to their abundance, their mostly cyclic structure and the presence of olefin or single hydroxyl groups. In order to apply this raw material in new industrial fields, a second hydroxyl group is inserted into borneol by cytochrome P450cam (CYP101) enzymes in a whole cell catalytic biotransformation with Pseudomonas putida KT2440. Next, a semi-continuous batch system was developed to produce 5-exo-hydroxyborneol with a final concentration of 0.54 g L-1. The bifunctional terpene was then used for the synthesis of an all bio-based polyesters by a solvent free polycondensation reaction. The resulting polymer showed a glass transition temperature around 70 °C and a molecular weight in the range of 2,000 - 4,000 g mol-1 (Mw). These results show that whole cell catalytic biotransformation of terpenes could lead to bio-based, higher-functionalized monomers, which might be basic raw materials for different fields of application, such as biopolymers.

Four terpene synthases contribute to the generation of chemotypes in tea tree (Melaleuca alternifolia)

Abstract: Terpene rich leaves are a characteristic of Myrtaceae. There is significant qualitative variation in the terpene profile of plants within a single species, which is observable as “chemotypes”. Understanding the molecular basis of chemotypic variation will help explain how such variation is maintained in natural populations as well as allowing focussed breeding for those terpenes sought by industry. The leaves of the medicinal tea tree, Melaleuca alternifolia, are used to produce terpinen-4-ol rich tea tree oil, but there are six naturally occurring chemotypes; three cardinal chemotypes (dominated by terpinen-4-ol, terpinolene and 1,8-cineole, respectively) and three intermediates. It has been predicted that three distinct terpene synthases could be responsible for the maintenance of chemotypic variation in this species. We isolated and characterised the most abundant terpene synthases (TPSs) from the three cardinal chemotypes of M. alternifolia. Functional characterisation of these enzymes shows that they produce the dominant compounds in the foliar terpene profile of all six chemotypes. Using RNA-Seq, we investigated the expression of these and 24 additional putative terpene synthases in young leaves of all six chemotypes of M. alternifolia. Despite contributing to the variation patterns observed, variation in gene expression of the three TPS genes is not enough to explain all variation for the maintenance of chemotypes. Other candidate terpene synthases as well as other levels of regulation must also be involved. The results of this study provide novel insights into the complexity of terpene biosynthesis in natural populations of a non-model organism.