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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite its cultivation as a source of food, fibre and medicine, and its global status as the most used illicit drug, the genus Cannabis has an inconclusive taxonomic organization and evolutionary ...history. Drug types of Cannabis (marijuana), which contain high amounts of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC), are used for medical purposes and as a recreational drug. Hemp types are grown for the production of seed and fibre, and contain low amounts of THC. Two species or gene pools (C. sativa and C. indica) are widely used in describing the pedigree or appearance of cultivated Cannabis plants. Using 14,031 single-nucleotide polymorphisms (SNPs) genotyped in 81 marijuana and 43 hemp samples, we show that marijuana and hemp are significantly differentiated at a genome-wide level, demonstrating that the distinction between these populations is not limited to genes underlying THC production. We find a moderate correlation between the genetic structure of marijuana strains and their reported C. sativa and C. indica ancestry and show that marijuana strain names often do not reflect a meaningful genetic identity. We also provide evidence that hemp is genetically more similar to C. indica type marijuana than to C. sativa strains.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we ...describe a combined physical and genetic map derived from a cross between the drug-type strain Purple Kush and the hemp variety "Finola." The map reveals that cannabinoid biosynthesis genes are generally unlinked but that aromatic prenyltransferase (
), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (
) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis.
and
(which determine the drug vs. hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly nonhomologous between drug- and hemp-type alleles and are furthermore embedded within ∼40 Mb of minimally recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant.
The terpene synthase gene family contributes to variations in cannabis metabolite profiles.
Abstract
Cannabis (Cannabis sativa) resin is the foundation of a multibillion dollar medicinal and ...recreational plant bioproducts industry. Major components of the cannabis resin are the cannabinoids and terpenes. Variations of cannabis terpene profiles contribute much to the different flavor and fragrance phenotypes that affect consumer preferences. A major problem in the cannabis industry is the lack of proper metabolic characterization of many of the existing cultivars, combined with sometimes incorrect cultivar labeling. We characterized foliar terpene profiles of plants grown from 32 seed sources and found large variation both within and between sets of plants labeled as the same cultivar. We selected five plants representing different cultivars with contrasting terpene profiles for clonal propagation, floral metabolite profiling, and trichome-specific transcriptome sequencing. Sequence analysis of these five cultivars and the reference genome of cv Purple Kush revealed a total of 33 different cannabis terpene synthase (CsTPS) genes, as well as variations of the CsTPS gene family and differential expression of terpenoid and cannabinoid pathway genes between cultivars. Our annotation of the cv Purple Kush reference genome identified 19 complete CsTPS gene models, and tandem arrays of isoprenoid and cannabinoid biosynthetic genes. An updated phylogeny of the CsTPS gene family showed three cannabis-specific clades, including a clade of sesquiterpene synthases within the TPS-b subfamily that typically contains mostly monoterpene synthases. The CsTPSs described and functionally characterized here include 13 that had not been previously characterized and that collectively explain a diverse range of cannabis terpenes.
∆⁹-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid ...biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The psychoactive and analgesic cannabinoids (e.g. Δ9‐tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short‐chain fatty acyl‐coenzyme A (CoA) precursor hexanoyl‐CoA. Cannabinoids ...are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl‐CoA using LC‐MS/MS and found levels of 15.5 pmol g−1 fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end‐product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl‐activating enzyme (AAE) that synthesizes hexanoyl‐CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short‐ and medium‐chained fatty acids. This activity and the trichome‐specific expression of CsAAE1 suggest that it is the hexanoyl‐CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Key message
Three novel transcription factors were successfully identified and shown to interact with the trichome-specific THCAS promoter regulatory region.
Cannabinoids are important secondary ...metabolites present in
Cannabis sativa
L. (cannabis). One cannabinoid that has received considerable attention, 9-tetrahydrocannabinol (THC), is derived from Delta-9-Tetrahydrocannabinolic acid (THCA) and responsible for the mood-altering and pain-relieving effects of cannabis. A detailed understanding of transcriptional control of
THCA synthase
(
THCAS
) is currently lacking. The primary site of cannabinoid biosynthesis is the glandular trichomes that form on female flowers. Transcription factors (TFs) have been shown to play an important role in secondary-metabolite biosynthesis and glandular trichome formation in
Artemisia annua
,
Solanum lycopersicum and Humulus lupulus
. However, analogous information is not available for cannabis. Here, we characterize a 548 bp fragment of the
THCAS
promoter and regulatory region that drives trichome-specific expression. Using this promoter fragment in a yeast-one-hybrid screen, we identified 3 novel TFs (
CsAP2L1, CsWRKY1
and
CsMYB1
) and provided evidence that these 3 TFs regulate the
THCAS
promoter
in planta
. The O-Box element within the proximal region of the
THCAS
promoter is necessary for CsAP2L1-induced transcriptional activation of
THCAS
promoter. Similar to
THCAS
, the genes for all three TFs have trichome-specific expression, and subcellular localization of the TFs indicates that all three proteins are in the nucleus.
CsAP2L1
and
THCAS
exhibit a similar temporal, spatial and strain-specific gene expression profiles, while those expression patterns of
CsWRKY1
and
CsMYB1
are opposite from
THCAS.
Our results identify
CsAP2L1
playing a positive role in the regulation of
THCAS
expression, while
CsWRKY1
and
CsMYB1
may serve as negative regulators of
THCAS
expression.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Summary
The cannabis leaf is iconic, but it is the flowers of cannabis that are consumed for the psychoactive and medicinal effects of their specialized metabolites. Cannabinoid metabolites, together ...with terpenes, are produced in glandular trichomes. Superficially, stalked and sessile trichomes in cannabis only differ in size and whether they have a stalk. The objectives of this study were: to define each trichome type using patterns of autofluorescence and secretory cell numbers, to test the hypothesis that stalked trichomes develop from sessile‐like precursors, and to test whether metabolic specialization occurs in cannabis glandular trichomes. A two‐photon microscopy technique using glandular trichome intrinsic autofluorescence was developed which demonstrated that stalked glandular trichomes possessed blue autofluorescence correlated with high cannabinoid levels. These stalked trichomes had 12–16 secretory disc cells and strongly monoterpene‐dominant terpene profiles. In contrast, sessile trichomes on mature flowers and vegetative leaves possessed red‐shifted autofluorescence, eight secretory disc cells and less monoterpene‐dominant terpene profiles. Moreover, intrinsic autofluorescence patterns and disc cell numbers supported a developmental model where stalked trichomes develop from apparently sessile trichomes. Transcriptomes of isolated floral trichomes revealed strong expression of cannabinoid and terpene biosynthetic genes, as well as uncharacterized genes highly co‐expressed with CBDA synthase. Identification and characterization of two previously unknown and highly expressed monoterpene synthases highlighted the metabolic specialization of stalked trichomes for monoterpene production. These unique properties and highly expressed genes of cannabis trichomes determine the medicinal, psychoactive and sensory properties of cannabis products.
Significance Statement
The unique monoterpene‐rich cannabis stalked trichomes that determine the medicinal, psychoactive and sensory properties of cannabis flowers develop from sessile‐like intermediates.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
BACKGROUND: Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced ...cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored. RESULTS: We sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of Δ⁹-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why the psychoactive cannabinoid Δ⁹-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers of cannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp. CONCLUSIONS: The availability of the Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.
This review provides an overview of the chemistry, biological activities, and biotechnological aspects of xanthohumol, 8-prenylnaringenin, and other prenylated flavonoids from hops,
Humulus lupulus ...L.
Xanthohumol (3
′-3,3-dimethyl allyl-2
′,4
′,4-trihydroxy-6
′-methoxychalcone) is the principal prenylated flavonoid of the female inflorescences of the hop plant (`hops'), an ingredient of beer. Human exposure to xanthohumol and related prenylflavonoids, such as 8-prenylnaringenin and isoxanthohumol, is primarily through beer consumption. Xanthohumol has been characterized a `broad-spectrum' cancer chemopreventive agent in in vitro studies, while 8-prenylnaringenin enjoys fame as the most potent phytoestrogen known to date. These biological activities suggest that prenylflavonoids from hops have potential for application in cancer prevention programs and in prevention or treatment of (post-)menopausal `hot flashes' and osteoporosis. Xanthohumol and 8-prenylnaringenin are metabolized into many flavonoid derivatives with modified 3,3-dimethyl allyl (prenyl) moieties. Xanthohumol is formed in lupulin glands by a specialized branch of flavonoid biosynthesis that involves prenylation and O-methylation of the polyketide intermediate chalconaringenin. Although a lupulin gland-specific chalcone synthase is known, the aromatic prenyltransferase and
O-methyltransferase participating in xanthohumol have not been identified. The prenylflavonoid pathway is a possible target for breeding or biotechnological modification of hops with the aim of increasing xanthohumol levels for beer brewing and 8-prenylnaringenin levels for pharmaceutical production.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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