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Terpene synthases from Cannabis sativa


Abstract and Figures

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-myr-cene, (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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Terpene synthases from Cannabis sativa
Judith K. Booth
, Jonathan E. Page
, Jo
¨rg Bohlmann
1Michael Smith Laboratories, University of British Columbia, East Mall, Vancouver, B.C., Canada, V6T 1Z4,
2Anandia Laboratories, Lower Mall, Vancouver, B.C., Canada, V6T 1Z4, 3Botany Department, University of
British Columbia, University Blvd, Vancouver, B.C., V6T 1Z4
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 dif-
ferent 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-myr-
cene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Tran-
scripts 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
Cannabis sativa, referred to here as cannabis, has been used for millennia as a medicine and
recreational intoxicant [1,2]. The species Cannabis sativa comprises both marijuana and
hemp [3,4,5]. Medicinal cannabis is highly valued for its pharmacologically active cannabi-
noids, a class of terpenophenolic metabolites unique to cannabis. These compounds are
primarily found in the resin produced in the glandular trichomes of pistillate (female) inflores-
cences. Cannabis resin also contains a variety of monoterpenes and sesquiterpenes (Fig 1),
which are responsible for much of the scent of cannabis flowers and contribute characteristi-
cally to the unique flavor qualities of cannabis products. Similarly, terpenes in hop (Humulus
lupulus), a close relative of cannabis, are an important flavoring component in the brewing
industry. Differences between the pharmaceutical properties of different cannabis strains have
been attributed to interactions (or an ‘entourage effect’) between cannabinoids and terpenes
[6,7]. For example, the sesquiterpene β-caryophyllene interacts with mammalian cannabinoid
receptors [8]. As a result, medicinal compositions have been proposed to incorporate blends of
cannabinoids and terpenes [9]. Terpenes may contribute anxiolytic, antibacterial, anti-inflam-
matory, and sedative effects [6].
PLOS ONE | March 29, 2017 1 / 20
Citation: Booth JK, Page JE, Bohlmann J (2017)
Terpene synthases from Cannabis sativa. PLoS
ONE 12(3): e0173911.
Editor: Bjo¨rn Hamberger, Michigan State
Received: January 9, 2017
Accepted: February 28, 2017
Published: March 29, 2017
Copyright: ©2017 Booth et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: GenBank accession
numbers are within the paper, under the "GenBank
Accessions" section.
Funding: This work was supported with funds to
JB from a Discovery Grant of the Natural Sciences
and Engineering Research Council (NSERC) of
Canada and an NSERC Graduate Scholarship to
JKB. JEP contributed to the study in his academic
capacity as an Adjunct Professor in the Department
of Botany at the University of British Columbia. JEP
is also the CEO and President of Anandia Labs Inc.,
which is hereby acknowledged. The funder
(NSERC) provided financial support in the form of
Terpene biosynthesis in plants involves two pathways to produce the general 5-carbon iso-
prenoid diphosphate precursors of all terpenes, the plastidial methylerythritol phosphate
(MEP) pathway and the cytosolic mevalonate (MEV) pathway. These pathways ultimately con-
trol the different substrate pools available for terpene synthases (TPS). The MEP pathway is
comprised of seven steps that convert pyruvate and glyceraldehyde-3-phosphate into isopente-
nyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (Fig 2A). Enzymes thought
to be critical for flux regulation through this pathway include the first two and final two
steps: 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductase,
4-hydroxy-3-methylbut-2-enyl diphosphate synthase, and 4-hydroxy-3-methylbut-2-enyl
diphosphate reductase [10,11]. The MEV pathway converts three units of acetyl-CoA to IPP,
which is then isomerized to DMAPP by IPP isomerase. A rate-limiting step in this six-step
pathway is 3-hydroxy-3-methylglutaryl-CoA reductase, which produces mevalonate [12]. IPP
and DMAPP are condensed into longer-chain isoprenoid diphosphates by prenyltransferases,
which include geranyl diphosphate (GPP) synthase (GPPS) and farnesyl diphosphate (FPP)
synthase (FPPS). GPPS and FPPS condense one unit of IPP and one or two units of DMAPP
to form 10- and 15-carbon linear trans-isoprenoid diphosphates, respectively. GPP is the
10-carbon precursor of monoterpenes and is typically derived from 5-carbon isoprenoid
diphosphate units of the MEP pathway. GPP is also a building block in the biosynthesis of
Fig 1. Glandular trichomes on the surface of pistillate inflorescences and leavesof Cannabis sativa
‘Finola’. The inflorescence (left) with a high density of glandular trichomes was at five weeks post onset
of flowering. Non-inflorescence leaves (right) have lower density of glandular trichomes. Structures of
representative cannabis resin components are shown in white: monoterpenes(top row), sesquiterpenes
(middle row), and cannabinoids (bottom row). GBGA = cannabigerolic acid; THCA = tetrahydrocannabinolic
acid; CBDA = cannabidiolic acid.
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 2 / 20
salary through a fellowship for JKB and research
materials. Anandia Labs provided in-kind support
in the form of plant materials. Anandia Labs did not
provide financial support for this project. The
funder (NSERC) and Anandia Labs did not play a
role in the study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. The specific roles of all authors are
articulated in the ‘author contributions’ section.
Competing interests: JEP is the CEO and President
of Anandia Labs Inc. JB is a consultant and adviser
to CannaRoyalty Corp. (since December 2016).
These affiliations do not alter our adherence to
PLOS ONE policies on sharing data and materials.
cannabinoids [13,14]. FPP is the 15-carbon precursor of sesquiterpenes and is commonly
produced from 5-carbon isoprenoid diphosphate units of the cytosolic mevalonate (MEV)
pathway. GPPSs exist as homo- or heterodimeric enzymes. In hops, the closest known relative
of cannabis, heterodimeric GPPSs can produce both GPP and the 20-carbon geranylgeranyl
diphosphate (GGPP), with the ratio of large to small G(G)PPS subunits controlling the product
outcome [15,16,17]. The linear isoprenoid diphosphates GPP and FPP are substrates for
monoterpene synthases (mono-TPS) and sesquiterpene synthases (sesqui-TPS), respectively,
which diversify these precursors into a large number of different mono- and sesquiterpenes.
TPS are typically encoded in large and diverse gene families in plants [18], where they con-
tribute to both general and specialized metabolism. The plant TPS gene family has been anno-
tated with six subfamilies. In angiosperms, the subfamily TPS-b is typically comprised of
mono-TPS and TPS-a enzymes are often sesquiterpene synthases. TPS produce cyclic and acy-
clic terpenes via carbocationic intermediates, formed by divalent metal co-factor dependent
elimination of the diphosphate. The reactive cationic intermediate can undergo cyclization
and rearrangements until the reaction is quenched by deprotonation or water capture [19].
Many TPS form multiple products from the same substrate.
The terpene composition of cannabis resin varies substantially based on genetic, environ-
mental, and developmental factors [20,21,22,23]. Concentrations and ratios of cannabinoids
are relatively predictable for different strains, but terpene profiles are often unknown or unpre-
dictable [20,23]. To select and improve cannabis strains with desirable terpene profiles, it is
necessary to identify genes responsible for terpene biosynthesis, which can be accomplished by
harnessing available cannabis transcriptome and genome resources. Draft genomes and tran-
scriptomes for the marijuana strain Purple Kush and the hemp variety ‘Finola’ have previously
Fig 2. Schematic of the plastidial methylerythritol phosphate pathway (MEP) and mevalonic acid
pathway (MEV) and transcript abundance in different parts of cannabis. Steps shown in bold (a) were
included in the qPCR analysis (b) of relative abundance of transcripts. Letters indicate significantly different
means between tissues (tested within each gene), Fisher’s LSD (alpha = 0.05). Abbreviations: Tr = trichome;
Le = leaf; Sf = stamenate flower; Ro = root; Sm = stem.
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 3 / 20
been published [24]. We used these resources to explore the expression of genes involved in all
stages of terpene biosynthesis. We identified nine TPS gene models in the ‘Finola’ transcrip-
tome. TPS genes and gene transcripts in the MEP and MEV pathways were highly expressed
in floral trichomes. We identified biochemical functions of TPS that are highly expressed in
‘Finola’. The TPS enzymes characterized account for most of the terpenes found in ‘Finola’
Materials and methods
Plant materials
Cannabis seeds, ‘Finola’, were obtained from Alberta Innovates Technology Futures (www. All plants were grown indoors in a growth chamber under a Health
Canada license. Seeds were germinated on filter paper, then transferred to 4:1 Sunshine Mix
#4 ( Daylight length was 16 h under fluorescent lights, and ambient
temperature 28˚C. About two weeks after germination, seedlings were transferred to larger
pots. After repotting, all plants were fertilized weekly with Miracle-Gro all-purpose plant food
(24-8-26) ( according to manufacturer’s instructions.
Terpene extraction
Pistillate inflorescences were collected and trimmed of leaves and stems. All flowers from
an individual plant were pooled. Tissue samples of ~0.2 g were weighed to determine fresh
weight. Three rounds of extraction in 1 ml of pentane were performed for 1 hour each at room
temperature with gentle shaking. Isobutyl benzene was added as an internal standard. After
three extractions, no terpenes were identified in a fourth solvent extraction. Floral tissue was
then dried overnight and weighed to determine dry weight. All three pentane extracts were
combined for a total volume of 3 ml for analysis.
Trichome isolation
The heads of glandular trichomes were isolated from whole inflorescences as previously
described [25] without XAD-4 and with the addition of 5 mM aurintricarboxilic acid in the
isolation buffer. Instead of a cell disruptor, floral tissue was vortexed with glass beads in a Fal-
con tube to remove trichome heads. After vortexing, trichomes were separated from beads and
green tissue by filtration through a 105 μm nylon mesh. Trichomes were concentrated by gen-
tle centrifugation in ice-cold buffer. The supernatant was removed with a pipette, and the pel-
let of trichomes was immediately frozen in liquid nitrogen.
Metabolite analysis
Gas chromatography (GC) analysis of floral extracts was performed on an Agilent (www. 7890A GC with a 7683B series autosampler and 7000A TripleQuad mass
spectrometer (MS) detector at 70 eV electrospray ionization with a flow rate of 1 ml min
The column was an Agilent VF-5MS or DB-5MS (30 m, 250 μm internal diameter, 0.25 μm
film). The following temperature program was used: 50˚C, then increase 150˚C min
320˚C, hold for 5 minutes. Injection was pulsed splitless at 250˚C. Compounds were identified
by comparison of retention index and mass spectra to authentic standards. Standards were
available for all monoterpenes and the following sesquiterpenes: β-caryophyllene, α-humulene,
farnesol, valencene, germacrene D, and alloaromadendrene. Tentative identifications for
all other sesquiterpenes were made by comparison of retention index and mass spectra
to National Institute of Standards and Technology (NIST) MS library. Identifications of
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 4 / 20
bergamotene, δ-selinene, and farnesene were strengthened by comparison to essential oils of
Citrus bergamia (Bergamot) and Pimenta racemose (Bay) ( TPS assay
products were analyzed by the same procedure described above for plant extracts, but with the
following temperature program: 50˚C for 3 minutes, then increase 15˚C min
to 280˚C, hold
for 2 minutes. Assay products were analyzed using Agilent HP-5 and DB-Wax columns (30 m
length, 250 μm internal diameter, 0.25 μm film). For cold injection of sesqui-TPS assay prod-
ucts, the following program was used on a DB-Wax column: 40˚C for 3 minutes, then increase
10˚C min
to 230˚C, hold for 7 minutes. Injection was at 40˚C with a 1:1 split ratio. Chiral
analysis of terpenes was done using a Cyclodex-B column (30 m length, 250 μm internal diam-
eter, 0.25 μm film). Injection was pulsed splitless, with the following program: 40˚C for 1 min-
ute, then increase 5˚C min
to 100˚C, then increase 15˚C min
to 250˚C, hold for 4 minutes.
Chirality was determined by retention index and comparison with authentic standards.
cDNA cloning and characterization of TPS genes
Total RNA was isolated from ‘Finola’ flowers, leaves, stem, and roots using Invitrogen PureLink
Plant RNA reagent ( RNA quality and concentration was measured
with a Bioanalyzer 2100 RNA Nanochip assay ( cDNA was synthesized with the
Superscript III reverse transcriptase kit (Thermo Fisher). Full length and N-terminally truncated
cDNAs without transit peptides where applicable [26] were amplified from cDNA using gene-
specific primers (S1 Table) designed from published transcriptomic data [24]. N-terminal transit
peptides were predicted based on sequence alignments [27] and using the TargetP and ChloroP
servers [28]. PCR amplified ‘Finola’ cDNAs were ligated into pJET vector (
for sequence verification, and subcloned into expression vectors pET28b+ (www.endmillipore.
ca) or pASK-IBA37 ( in the case of CsTPS5FN.
High-confidence full-length TPS cDNA candidates from Purple Kush (CsTPS13PK,
CsTPS30PK, and CsTPS33PK) were synthesized by GenScript ( into
pET28b+. For this purpose, putative TPS sequences from Purple Kush transcriptome data
were verified by comparison to genomic sequences [24].
Plasmids were transformed into E.coli strain BL21DE3-C43 for heterologous protein
expression, as previously described [29]. Recombinant protein was purified using the GE
healthcare His SpinTrap kit ( according to manufacturer’s instruc-
tions. Binding buffer for purification was 20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-
sulfonic acid (HEPES) (pH 7.5), 500 mM NaCl, 25 mM imidazole, and 5% glycerol. Cells were
lysed in binding buffer supplemented with Roche complete protease inhibitor tablets (life- and 0.1 mg ml
lysozyme. Elution buffer was 20 mM HEPES (pH 7.5), 500
mM NaCl, 350 mM imidazole, and 5% glycerol. Purified protein was desalted through Sepha-
dex into TPS assay buffer. In vitro assays were performed in 500 μl volume by incubating puri-
fied protein with isoprenoid diphosphate substrates (Sigma) as previously described [30],
except that the TPS assay buffer was 25 mM HEPES (pH 7.3), 100 mM KCl, 10 mM MgCl
, 5%
glycerol, and 5 mM DTT. Isoprenoid diphosphate substrates were dissolved in 50% methanol
and added to the assay at final concentrations of 32 μM (GPP) and 26 μM (FPP). Enzyme con-
centrations were variable ranging from 20 to 100 μg per 500 μl assay volume. Assays were over-
laid with 400 μl hexane or pentane, with 2.5 μM isobutyl benzene as internal standard.
Nicotiana benthamiana transformation and transient expression
The CsTPS5FN coding sequence was inserted into the Golden Gate plant expression vector
pEAQ-GG, which contains a CaMV 35S promoter. This construct and the suppressor-of-
silencing gene p19 were transformed into Agrobacterium tumefasciens strain AGL1. For
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 5 / 20
infiltration, A.tumefasciens was grown overnight as previously described [31], then pelleted
and resuspended in 10 mM 2-(N-morpholine)-ethanesulphonic acid (MES) buffer, pH 5.8, 10
mM MgCl
, 20 μM acetosyringone to OD
0.5. Equal volumes of bacteria, 25 ml each, con-
taining TPS5 and p19 were infiltrated into the abaxial side of 4-week-old N.benthamiana
plants. Infiltrated plants were grown for three days in the dark. Infiltrated leaves were har-
vested and ground in TPS assay buffer, and enzyme activity assays were conducted as above.
RT-qPCR analysis of transcript abundance
cDNA for qPCR was synthesized using the Maxima First Strand cDNA synthesis kit (Thermo
Fisher) according to manufacturer’s instructions. qPCR reactions were done in 15 μl volumes
with SsoFast EvaGreen supermix (Bio-Rad), 4 μl template (2 ng), and 0.3 μM primers. Primers
(S2 Table) were designed using Primer3 software [32]. Reference genes were chosen by geN-
orm [33], analyzed with qBase+ software ( Reference genes used for RT-
qPCR of early isoprenoid biosynthesis across different plant organs were actin and CDK3. For
RT-qPCR of TPS transcripts in trichomes, reference genes were CDK3 and GAPDH. RT-qPCR
analyses were done with four biological and two technical replicates for the early isoprenoid
biosynthetic transcripts in different organs. For TPS transcript analysis in trichomes, three bio-
logical and three technical replicates were performed. Gene expression was analyzed using
qBase+. Statistical analysis was performed by ANOVA on log-transcript abundance, with Bon-
ferroni correction.
TPS gene prediction and phylogeny
‘Finola’ genome and transcriptome assemblies [24] were downloaded from the cannabis
genome browser ( These assemblies were
used as the subject of a tBLASTn search using 71 TPS genes (S3 Table) downloaded from Gen-
Bank and Phytozome. Gene and splice site prediction was performed on scaffolds containing
regions with similarity to TPS sequences using the Exonerate gene prediction algorithm [34].
A preliminary Purple Kush genome assembly based on PacBio ( sequencing
data was also used. Predicted genes were manually curated against earlier Purple Kush
sequence data, and examined to establish open reading frames, start codons, and stop codons.
A maximum likelihood phylogeny was built using [35]. The alignment used for
input was built using the MUSCLE algorithm with all translated amino acid sequences from
the predicted TPS gene models from cannabis and the 71 published TPS sequences listed
above. Alignments were curated using the Gblocks algorithm, and tree construction was per-
formed using PhyML 3.0 with 100 bootstrap replicates.
Terpene profiles of cannabis inflorescences
We used the C.sativa oilseed hemp variety ‘Finola’ to investigate terpene profiles of pistillate
flowers. ‘Finola’ was chosen because reference draft genome and transcriptome assemblies
were published [24]. Pistillate flowers, which have the highest density of glandular trichomes
relative to other parts of the plant (Fig 1), were sampled to cover early to mid-stage inflores-
cences between three and eight weeks post onset of flowering, where onset of flowering is
defined as the first appearance of pistils. Independent of the stage of inflorescence, the most
abundant monoterpenes were myrcene, (+)-α-pinene, (-)-limonene, (+)-β-pinene, terpino-
lene, and (E)-β-ocimene (Table 1). The most abundant sesquiterpenes were β-caryophyllene,
α-humulene, bergamotene, and farnesene. Terpene profiles showed considerable variations
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 6 / 20
between individual plants as indicated with the relatively high standard deviation (Table 1).
No trends were observed for individual metabolites as a function of inflorescence develop-
ment, but total monoterpenes increased compared to sesquiterpenes as inflorescences
matured. Mid-stage flowers (~4 weeks post onset of flowering) had a mean monoterpene con-
tent of 389 μg g
DW (SE = 44, n = 9), and a mean sesquiterpene content of 34 μg g
(SE = 6.3, n = 9).
Transcriptome mining of early isoprenoid biosynthesis genes
We queried the ‘Finola’ transcriptome for transcripts involved in the early stages of isoprenoid
biosynthesis. We combined four transcriptome sets downloaded from the Cannabis Genome
Browser (, including transcripts from
developing seeds, mature pistillate flowers, stamenate (male) flowers, and whole seedlings. The
tBLASTn algorithm was used to search translated ‘Finola’ nucleotide sequences, using amino
acid sequences from Vitis vinifera and Arabidopsis thaliana, and an e-value cut-off of 1
At least one full-length or nearly full-length (>95%) transcript was found for each of the
core genes in the MEP and MEV pathways, and linear isoprenoid diphosphate prenyltrans-
ferases (Fig 2B). The genes included in the analysis of the MEP pathway were 1-deoxy-D-xylu-
lose 6-phosphate (DOXP) synthase (DXS), DOXP reductoisomerase (DXR), 2-C-methyl-D-
erythritol cytidyltransferase (MCT), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase
(CMK), 4-hydroxy-3-methyl-but-2-enyl diphosphate (HMB-PP) synthase (HDS), and HMB-
PP reductase (HDR). Two versions of DXS, CsDXS1 and CsDXS2, were found, which are
62.8% identical at the amino acid level. In a phylogeny, CsDXS1 clusters with members of
the DXS subfamily DXS-I of other plant species, and CsDXS2 clusters with members of the
DXS-II subfamily (S1 Fig).
The genes included in the MEV pathway analysis were 3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA) synthase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK),
phospho-mevalonate kinase (PMK), mevalonate-5-phosphate decarboxylase (MPDC), and
IPP isomerase (IDI). At least one transcript was found corresponding to each enzyme. Two
transcripts were found for HMGR, HMGR1 and HMGR2, which are 72.7% identical at the
amino acid level.
As candidate prenyltransferases, we found transcripts of a heterodimeric GPPS system sim-
ilar to that characterized in hop [17], with a GPPS large subunit (GPPS.lsu) and a GPPS small
subunit (GPPS.ssu). Two transcripts were identified corresponding to FPPS, 80.3% identical to
one another at the amino acid level.
Table 1. Relative composition of terpene profiles in C.sativa ’Finola’ pistillate flowers. Twenty two individual plants were sampled. Contribution of indi-
vidual terpenes is expressed as a proportion of the total terpenes within a given class (i.e., monoterpenes or sesquiterpenes).
Metabolite Percent Proportion (mean ±st. dev) Terpene class
(+)-α-Pinene 23 ±17 Monoterpene
(+)-β-Pinene 8.6 ±4.6 Monoterpene
Myrcene 27 ±21 Monoterpene
(-)-Limonene 12 ±10 Monoterpene
(E)-β-Ocimene 10 ±6.5 Monoterpene
Terpinolene 18 ±14 Monoterpene
β-Caryophyllene 46 ±13 Sesquiterpene
Bergamotene 3.6 ±3.0 Sesquiterpene
Farnesene 4.4 ±3.6 Sesquiterpene
α-Humulene 19 ±7.6 Sesquiterpene
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 7 / 20
RT-qPCR expression analysis of isoprenoid biosynthetic transcripts
To assess gene expression of isoprenoid biosynthesis across different parts of the cannabis
plant, we used qRT-PCR to examine the transcript abundance of prenyltransferases and key
genes in the MEP and MEV pathways. We selected genes for three steps in the MEP pathway,
DXS,DXR, and HDR, as well as two MEV pathway genes, HMGR and IDI. We also included
the two FPPS genes. In heterodimeric GPPS, the rate of GPP biosynthesis is governed by ratios
of small to large subunits, with higher ratios of small to large subunits leading to higher GPP
formation [17]. We therefore measured transcript levels of the GPPS.ssu gene. Transcript levels
were determined in ‘Finola’ leaves, stems, roots, staminate flowers, and glandular trichomes
from pistillate flowers. Pistillate flowers were harvested between 10–12 weeks post germination.
CsDXS1 was expressed in all samples, with no significant differences between different parts
of the plant (Fig 2B). CsDXS2 was also expressed in all samples. Levels of CsDXS1 and CsDXS2
transcripts were not significantly different except in roots, where average CsDXS2 levels were
14-fold more abundant than CsDXS1.HMGR1 and HMGR2 were both expressed in every sam-
ple. Their transcript abundances were not significantly different, except in leaves and roots
where HMGR2 was significantly more highly expressed. Abundances of FPPS transcripts in
roots and staminate flowers were significantly different between the two FPPS genes. The MEP
pathway genes DXR and HDR were significantly more highly expressed in trichomes and
leaves. Cannabis leaves bear glandular trichomes, but much less densely than flowers. Genes in
the MEP pathway were also more highly expressed in trichomes and leaves than genes of the
MEV pathway. Transcripts of GPPS.ssu were very highly abundant (>25 fold higher) in tri-
chomes compared to other tissues.
Members of the cannabis TPS gene family
In the ‘Finola’ (FN) trichome transcriptome we identified nine full-length or nearly full-length
(predicted >95% of amino acid length) and six partial putative TPS genes (CsTPS FN). A maxi-
mum likelihood phylogeny of the nine full-length CsTPSFN predicted protein sequences and rep-
resentative TPS from other plant species placed the CsTPSFN most closely with each other and
with TPS from hop (HlSTS1 and HlSTS2) indicating a recent expansion of TPS genes in the Can-
nabaceae (Fig 3). Five of the nine CsTPSFN (CsTPS1FN, CsTPS2FN, CsTPS3FN, CsTPS5FN,
CsTPS6FN) clustered with members of the TPS-b subfamily, and the remaining four (CsTPS4FN,
CsTPS7FN, CsTPS8FN, CsTPS9FN) clustered with the TPS-a subfamily. Two of the CsTPSFN
TPS-b genes, CsTPS1FN and CsTPS2FN, encode predicted proteins that were 98.7% and 96.8%
identical to CsTPS1 and CsTPS2 previously reported [36] and identified there as (-)-limonene
synthase (CsTPS1) and (+)-α-pinene synthase (CsTPS2) from the C.sativa strain ‘Skunk’.
Functional characterization of CsTPSFN TPS-b subfamily members
CsTPSFN were cloned as cDNAs from ‘Finola’ pistillate flowers or synthesized for heterologous
expression and identification of product profiles of the encoded enzymes. We cloned four TPS-
b family members, CsTPS1FN,CsTPS2FN,CsTPS5FN, and CsTPS6FN, from cDNA. CsTPS3FN
could not be cloned from cDNA and was obtained as a synthetic cDNA. Three TPS-b sequences
from the Purple Kush (PK) trichome transcriptome, CsTPS13PK,CsTPS30PK, and CsTPS33PK,
were also synthesized for comparison. These five CsTPS from ‘Finola’ and three from Purple
Kush were expressed as recombinant proteins and then tested for activity with GPP and FPP
and products identified by GC-MS analysis (Fig 4,Table 2,S2 and S3 Figs).
The major product of CsTPS1FN was (-)-limonene, with minor products of (+)-α-pinene,
camphene, (+)-β-pinene, and myrcene (Fig 4). CsTPS2FN produced mostly (+)-α-pinene,
with minor amounts of (+)-β-pinene, myrcene, (-)-limonene, β-phellandrene and a monoterpene
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 8 / 20
Fig 3. Maximum likelihood phylogeny of CsTPS. Within the TPS-a and TPS-b subfamilies, TPS from the Cannabaceae, including cannabis and hops, are
more closely related to one another than to TPS from other angiosperms. Cannabis TPS are in bold. The cannabis strain or variety of origin is indicated by two
letters following the TPS#: FN: ‘Finola’, SK: ‘Skunk’, PK: Purple Kush. Branches with bootstrap values >80% (100 repetitions) are indicated with a grey dot.
TPS of other species included are from Pp: Physcomitrella patens, Os: Oryza sativa, Cm: Cucurbita maxima, At: Arabidopsis thaliana, Cb: Clarkia breweri,
Ag: Abies grandis, Pa: Picea abies, Fa: Fragaria ananassa, Am: Antirrhinum majus, Mp: Mentha x piperita, Rc: Ricinus communis, Ci: Cichorium intybus, Sl:
Solanum lycopersicum, Nt: Nicotiana tabacum, Le: Lycopersicum esculentum, Ga: Gossypium arboreum, St: Solanum tuberosum, Vv: Vitis vinifera, Hl:
Humulus lupulus, So: Salvia officinalis, Cl: Citrus limon, Ms: Mentha spicata, Pf: Perilla frutescens. ‘S’ suffix = synthase.
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 9 / 20
tentatively identified as isoterpinolene (Fig 4). CsTPS3FN produced myrcene as a single detectable
product when incubated with GPP (Fig 4). CsTPS30PK also produced only myrcene when
tested with GPP (S2 Fig). These two single-product myrcene synthases share only 54.5% amino
acid identity. CsTPS5FN also produced myrcene as its most abundant monoterpene product
(37%) (Fig 4), but unlike CsTPS3FN and CsTPS30PK, CsTPS5FN produced four additional
Fig 4. Representative GC-MS traces showing products of CsTPSFN TPS-b subfamily members. Black
traces show GC-MS total ion chromatogram from CsTPS assays with GPP. Green trace, dotted line, is a
representative terpene profile from a ‘Finola’ inflorescence. (a) shows representative chromatograms from six
TPS and a ‘Finola’ floral extract run on an HP-5 GC column. (b) shows the representative chromatogram from
CsTPS2FN run on a DB-Wax GC column. Peaks: a) α-pinene, b) camphene, c) sabinene, d) β-pinene, e)
myrcene, f) α-terpinene, g) limonene, h) (Z)- β-ocimene, i) (E)-β-ocimene, j) γ-terpinene, k) terpinolene, l) β
-phellandrene, m) isoterpinolene. i.s. = internal standard
Table 2. Functionally characterized CsTPS enzymes.
Functional gene ID Nearest ’PK’ TPS gene model Major products Strain of origin Activity on GPP Activity on FPP
CsTPS1FN CsTPS1PK (-)-limonene Finola +++ np
CsTPS1PK (-)-limonene Skunk nd nd
CsTPS2FN CsTPS2PK (+)-α-pinene Finola +++ np
CsTPS2PK (+)-α-pinene Skunk nd nd
CsTPS3FN CsTPS3PK β-myrcene Finola +++ np
CsTPS4FN CsTPS9PK alloaromadendrene Finola + +++
CsTPS5FN CsTPS5PK β-myrcene, (-)-α-pinene Finola +++ +
CsTPS30PK CsTPS30PK β-myrcene Purple Kush +++ +
CsTPS6FN CsTPS6PK (E)-β-ocimene Finola +++ np
CsTPS7FN CsTPS7PK δ-selinene*Finola + +++
CsTPS8FN CsTPS8PK γ-eudesmol*, valencene Finola + +++
CsTPS9FN CsTPS9PK β-caryophyllene, α-humulene Finola + +++
CsTPS13PK CsTPS13PK (Z)-β-ocimene Purple Kush +++ +
CsTPS33PK CsTPS33PK α-terpinene, γ-terpinene Purple Kush +++ np
Published in Gunnewich et al., 2008
*Product not compared to authentic standard.
np, no product detected. nd, no data available.
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 10 / 20
monoterpenes (-)-α-pinene (23%), (-)-limonene (17%), sabinene (15%), and (-)-β-pinene (8%).
The same product profile was identified when CsTPS5FN was transiently expressed in N.
benthamiana (S4 Fig). CsTPS5FN was somewhat unusual among TPS-b members in lacking any
obvious N-terminal plastidial targeting sequence. CsTPS5FN also produced minor amounts of
farnesene when incubated with FPP, making it the only member of the TPS-b subfamily to pro-
duce detectable sesquiterpenes. CsTPS6FN produced 97% (E)-β-ocimene with GPP, and the
remaining 3% of product was (Z)-β-ocimene. A TPS sequence found in Purple Kush,
CsTPS13PK, shares 95.5% amino acid sequence identity with CsTPS6FN. CsTPS13PK produces
94% (Z)-β-ocimene. A third TPS from Purple Kush, CsTPS33PK, produced two different mono-
terpenes, α-terpinene (61%) and γ-terpinene (39%) (Fig 4).
Functional characterization of CsTPSFN TPS-a subfamily members
Four TPS-a family members cloned as cDNAs from ‘Finola’, CsTPS4FN,CsTPS7FN,
CsTPS8FN, and CsTPS9FN, were expressed as recombinant proteins, proteins tested with GPP
and FPP and products identified by GC-MS (Fig 5A,Table 2). CsTPS4FN produced mostly
alloaromadendrene (52.3% of total products) with FPP (Fig 5C). The remaining products are a
mixture of five sesquiterpene olefins and two alcohols, including valencene, α-humulene, and
a product tentatively identified as palustrol. CsTPS4FN was also active with GPP, producing
minor amounts of myrcene (S5 Fig). CsTPS7FN produced 21 sesquiterpene olefins and two
sesquiterpene alcohols. Of these, products tentatively identified as δ-selinene and selina-6-en-
Fig 5. Representative GC-MS traces showing products of CsTPSFN TPS-a subfamily members. Black
traces show GC-MS total ion chromatogram (TIC) from CsTPS assays with FPP. Green trace, dotted line, in
(a) is representative terpene profiles from ‘Finola’ inflorescences. The right-hand region of the ‘Finola’ terpene
profile has been amplified 30-fold to facilitate comparison with the products of CsTPS7FN. (b) shows the trace
of CsTPS8FN after cold injection (40˚C inlet) onto a DB-wax column. (c) shows the trace for CsTPS4FN on an
HP-5 column. Peaks: n) β-caryophyllene, o) α-humulene, p) δ-selinene, q) selina-6-en-4-ol, r) valencene, s) γ-
eudesmol, t) alloaromadendrene, u) palustrol.
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 11 / 20
4-ol make up 20.5% and 13.9% of the product profile, respectively. Very few of ‘Finola’ individ-
uals tested contained minor amounts of the products of CsTPS7FN (Fig 5A). The remaining
minor products each make up <10% of total sesquiterpene products. When incubated with
GPP, CsTPS7FN produced myrcene and limonene (S5 Fig). The most abundant product of
CsTPS8FN was initially identified as β-elemol (S6 Fig), which is often an artifact of heat-
induced rearrangement. Using a lower injection temperature of 40˚C, the β-elemol product
was no longer detected and was replaced by peaks corresponding to 11 sesquiterpene olefins
and three sesquiterpene alcohols. Of these, two of the major products were identified as γ-
eudesmol (19.8%) and valencene (19.6%) (Fig 5B). When CsTPS8FN was incubated with GPP
limonene and myrcene were detected (S5 Fig).
CsTPS9FN produced β-caryophyllene and α-humulene from FPP (Fig 5A). These two ter-
penes are always the most abundant sesquiterpenes in cannabis resin terpene profiles. The
CsTPS9FN enzyme produces these two sesquiterpenes in a ratio of approximately 2.5 to 1,
which is similar to the ratio of 2.4 +/- 0.2 to 1 observed in ‘Finola’ terpene profiles.
CsTPS transcripts are highly abundant in pistillate inflorescences
To determine to what extent the CsTPS genes described above contribute to the trichome ter-
pene profile, we performed RT-qPCR on transcripts of five CsTPS in glandular trichomes iso-
lated from pistillate flowers. Transcript levels of CsTPS1FN,CsTPS2FN,CsTPS3FN,CsTPS6FN,
and CsTPS9FN were examined in trichomes (S7 Fig) isolated from eight ‘Finola’ individuals
between two and four weeks post onset of flowering. These five CsTPS were chosen because
they have a single or at most two products, thus it was deemed more likely to be possible to
attempt correlating metabolite abundance with transcript abundances than would be possible
with the multiproduct CsTPS.
Of the eight individual plants, seven showed typical inflorescence terpene metabolite pro-
files. Surprisingly, one individual had no detectable inflorescence monoterpenes except for
traces of (E)-β-ocimene, although it did contain cannabinoids and sesquiterpenes in floral tri-
chomes (S8 Fig). In Fig 6A, metabolite levels of the target compounds are expressed as a pro-
portion of the total mono- or sesquiterpenes in each floral terpene extract. CsTPS2FN was the
most abundant of the six different TPS transcripts measured, and its major product, (+)-α-
pinene, was also the most abundant monoterpene on average in the eight plants examined.
Similarly, (E)-β-ocimene and the (E)-β-ocimene synthase CsTPS6FN were the least abundant
of all metabolites and transcripts measured, respectively. However, within transcript/metabo-
lite pairs, only the correlation between (-)-limonene and CsTPS1FN transcript level was sig-
nificant (Fig 6A). Correlation between metabolite level and transcript abundance was not
significant for any of the other metabolite/transcript pairs.
In addition, we measured the transcript abundance of the multiproduct monoterpene
synthase CsTPS5FN, to assess if its expression may contribute to terpene profiles in the resin.
CsTPS5FN transcripts were highly abundant in some individuals, comparable to the highest
transcript levels of any other CsTPS tested (Fig 6B). Transcript levels of this gene did not ex-
plain any of the lack of correlations between the five terpene-metabolite pairs tested above.
Additionally, plant X, which had no detectable monoterpenes, had moderate levels of CsTPS5FN
transcript. It appears that CsTPS5FN, while highly expressed, does not contribute to terpene accu-
mulation in ‘Finola’.
The resin of C.sativa is rich in mono- and sesquiterpenes, which are of interest for their puta-
tive contributions to cannabis pharmacology [6]. Most studies of terpenes in cannabis have
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 12 / 20
focused on phytochemical composition for forensics and breeding, while less research has
gone into the molecular biology of terpene formation in cannabis. Knowledge of the genomics
and gene functions of terpene biosynthesis may facilitate genetic improvement of cannabis for
desirable terpene profiles. Using the hemp strain ‘Finola’ and its genome and transcriptome
resources [24], we identified early isoprenoid pathway genes as well as specific CsTPS genes
and their enzymes involved in the biosynthesis of nearly all of the different monoterpenes
identified in extracts of the cannabis inflorescences, which are densely covered with terpene
and cannabinoid accumulating glandular trichomes (Fig 1). One exception is terpinolene, for
which a CsTPS has not yet been identified. The terpene profiles of cannabis can be explained
Fig 6. Correlation analysis of metabolite abundance in inflorescence and transcript abundance for five CsTPS in isolated trichomes. (a) Data
are shown for five CsTPS/metabolite pairs each in eight ‘Finola’ individuals. Metabolites given with their relative abundance were those that match the
product of the corresponding CsTPS. Plant ‘X’ was not included in the left-most panel. Metabolite abundances are expressed as a proportion of thetotal
mono- or sesquiterpenes for each individual. Transcript abundances are calibrated normalized values comparedto two reference genes.
rho = Spearman rank correlation between transcript and metabolite abundances, p value indicates significance. (b) Transcript abundance of CsTPS5FN
in eight ‘Finola’ individuals.
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 13 / 20
by activities of both single-product and multi-product CsTPS. Individual ‘Finola’ plants
showed substantial variation in their profiles of mono- and sesquiterpenes. ‘Finola’ has few
monoterpene alcohols or ethers, such as linalool or geraniol, which are common in some can-
nabis strains.
It is reasonable to expect that there are additional CsTPS not described in this work, such as
aCsTPS that encodes a terpinolene synthase. Characterization of further TPS may also clarify
the poor correlation between TPS and the abundance of their products shown in Fig 6A. A
search of a new assembly of the Purple Kush genome, to which we recently had pre-publica-
tion access (Dr. Jonathan Page, personal communication), identified a total of 33 complete
CsTPSPK gene models and additional partial sequences (Fig 7). Purple Kush is a marijuana
strain which requires special research licensing to grow. Thus, characterization of this more
Fig 7. Maximum likelihood phylogeny for 33 TPS translated from gene models identified in the Cannabis
sativa Purple Kush genomic sequences. 41 published TPS sequences from other organisms were included for
comparison. Names of cannabis genes identified in this study are in bold. Gene names from Purple Kush followed
by an asterisk (*) represent biochemically characterized enzymes from Purple Kush transcriptome data. Their
nearest homologue in the genome was assigned the same gene ID when the sequences had >95% amino acid
identity. Branches with greater than 80% boostrap support are identified with a grey circle.
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 14 / 20
comprehensive set of CsTPSPK will have to be completed in future work as it requires synthe-
sized genes.
Fig 7 indicates a set of putatively orthologous CsTPSFN and CsTPSPK genes, which may
contribute to overlapping terpene profiles in hemp and marijuana varieties. However, some
orthologous genes may have evolved different functions in different strains, and non-ortholo-
gous CsTPS may contribute to some of the same terpene products in different cannabis strains.
For example, α-pinene is a major component of strains reported as Purple Kush [37], but no
obvious orthologue of the α-pinene synthase CsTPS2 as identified in the ‘Finola’ and ‘Skunk’
strains was found in the Purple Kush genome (Fig 7). Another example is the set of apparently
non-orthologous single-product myrcene synthases, CsTPS3FN and CsTPS30PK identified in
‘Finola’ and Purple Kush, which only share 52.5% amino acid identity but produce the same
monoterpene. Also, not all CsTPS are expected to contribute to terpene accumulation in the
resin of cannabis inflorescences and some may function in a different context of the plant biol-
ogy. For example, CsTPS5FN is expressed in inflorescences and the recombinant enzyme pro-
duces a mixture of monoterpenes, but does not contribute substantially to the terpene profile
of the resin. This gene appears most closely related to MTS1 from hops (Fig 3) where the
encoded protein was inactive in vitro [38].
Cannabis inflorescences are densely covered with glandular trichomes, which are special-
ized to produce and accumulate terpenes [11]. Transcripts of several CsTPS genes (Fig 6A) are
abundant in trichomes isolated from mid-stage ‘Finola’ inflorescences. Transcripts associated
with early isoprenoid biosynthesis and especially the MEP pathway, which feeds into both
monoterpene and cannabionoid biosynthesis, were also abundant in trichomes (Fig 2B). Ses-
quiterpenes have been reported to be most abundant in early floral stages [39], and thus MEV
pathway transcripts may be more abundant at earlier stages of flower development. Different
DXS and HMGR genes were differentially expressed in roots relative to other parts of the
plant. Terpenes in the roots, if present in cannabis, may contribute to defense as reported in
other plant species [10]. In plants, DXS genes generally fall into two clades, of which DXS I
members are generally involved in primary metabolism, and DXS II members are often
induced in defense responses [40,41,42]. Abundance of cannabis DXS2 transcripts, which
clusters with the DXS II subfamily (S1 Fig), suggests defense related terpenoids in cannabis
roots and warrants future work on the cannabis root metabolome. We also observed high
FPPS transcript abundance in stamenate flowers and roots, resembling a previous finding that
Arabidopsis FPS1 was primarily expressed in flowers and roots compared to AtFPS2 [43].
Domestication and selective breeding can result in changes in terpene profiles and abun-
dance. For example, domestication can lead to a decrease in the quantity or variability of ter-
penes [44,45,46]. Cannabis, especially marijuana, has been domesticated for thousands of
years for increased resin volume and potency [2,47] and as a result profiles and ecological
roles of terpenes in ancestral (i.e., undomesticated) cannabis are unknown. The present study
highlights the large number of CsTPS genes and the diverse products of the encoded TPS
enzyme activities, which contribute to the complex terpene profiles of cannabis. The knowl-
edge of multigene nature of the CsTPS family and the often multiple products of the encoded
enzymes will be critical when selecting or breeding, or improving plants by genome editing,
for particular terpene profiles for standardized cannabis varieties. While cannabinoid-free
individuals have occasionally been reported [48], we are not aware of any reports in the litera-
ture of terpene-free cannabis. In this study, we observed a single monoterpene-free individual,
which however still contained cannabinoids and sesquiterpenes. This observation implies that
biosynthesis of the different classes of terpenoid metabolites are independently regulated. The
fact that terpenes have persisted throughout domestication as a substantial and diverse compo-
nent of cannabis resin highlights their significance for human preferences.
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 15 / 20
GenBank accessions
GenBank accession numbers for the terpene synthases described in this paper are CsTPS1FN:
KY014557, CsTPS2FN: KY014565, CsTPS3FN: KY014561, CsTPS4FN: KY014564, CsTPS5FN:
KY014560, CsTPS6FN: KY014563, CsTPS7FN: KY014554, CsTPS8FN: KY014556, CsTPS9FN:
KY014555, CsTPS11FN: KY014562, CsTPS12PK: KY014559, CsTPS13PK: KY014558. Acces-
sion numbers for genes in the MEP pathway are CsDXS1: KY014576, CsDXS2: KY014577,
CsDXR: KY014568, CsMCT: KY014578, CsCMK: KY014575, CsHDS: KY014570, CsHDR:
KY014579. Accession numbers for genes in the MEV pathway are CsHMGS: KY014582,
CsHMGR1: KY014572, CsHMGR2: KY014553, CsMK: KY014574, CsPMK: KY014581,
CsMPDC: KY014566, CsIDI: KY014569. Prenyltransferase accession numbers are CsGPPS.
ssu1: KY014567, CsGPPS.ssu2: KY014583, CsFPPS1: KY014571, CsFPPS2: KY014580. Acces-
sion numbers for genomic regions containing putative terpene synthases from Purple Kush are
CsTPS1PK: KY624372, CsTPS4PK: KY624361, CsTPS5PK: KY624374, CsTPS6PK: KY624363,
CsTPS7PK: KY624368, CsTPS8PK: KY624352, CsTPS9PK: KY624366, CsTPS10PK: KY624347,
CsTPS11PK: KY624348, CsTPS12PK: KY624349, CsTPS13PK: KY624350, CsTPS14PK:
KY624351, CsTPS15PK: KY624353, CsTPS16PK: KY624354, CsTPS17PK: KY624355,
CsTPS18PK: KY624356, CsTPS19PK: KY624357, CsTPS20PK: KY624358, CsTPS21PK:
KY624360, CsTPS22PK: KY624360, CsTPS23PK: KY624362, CsTPS24PK and CsTPS25PK:
KY624364, CsTPS26PK and CsTPS27PK: KY624365, CsTPS30PK: KY624367, CsTPS31PK:
KY624369, CsTPS32PK: KY624370, CsTPS33PK: KY624371, CsTPS34PK: KY624373,
CsTPS35PK: KY624375.
Supporting information
S1 Table. Primers used to clone TPS genes.
S2 Table. qPCR primers.
S3 Table. Accession numbers of TPS sequences used in tblastn and to construct phylog-
S1 Fig. DXS Phylogeny. Maximum likelihood phylogeny of DXS enzymes. Cannabis sativa
genes are in bold. DXS of other species included are from: At: Arabidopsis thaliana; Pt: Populus
trichocarpa; Os: Oryza sativa; Cr: Chlamydomonas reinhardtii; Mt: Medicago truncatula; Pa:
Picea abies.
S2 Fig. Representative GC-MS traces of myrcene synthase products.
S3 Fig. Mass spectra of TPS products. Labels “Peak a” through “Peak u” correspond to peaks
labeled in Figs 4and 5.
S4 Fig. Products of CsTPS5FN expressed in E.coli and Nicotiana benthamiana.Black trace
represents products of recombinant enzyme expressed in E.coli, green trace represents prod-
ucts of recombinant enzyme expressed in N.benthamiana. Leaf images (right) show GFP posi-
tive expression control.
Terpene synthases from Cannabis sativa
PLOS ONE | March 29, 2017 16 / 20
S5 Fig. Products of CsTPS with alternative substrates. Members of TPS-a with GPP as sub-
strate are on the left-hand side. Members of TPS-b with FPP as substrate are on the right.
S6 Fig. Hot vs. cold injection of CsTPS8FN products. Top panel represents total ion chro-
matogram (TIC) with the injection port at 250˚C on a DB-Wax column. The bottom panel
represents TIC with the injection port at 40˚C, using the same program and the same column.
S7 Fig. Isolated glandular trichome heads.
S8 Fig. Terpene chemotypes of ‘Finola’ flowers. Abundance of five metabolites or metabolite
pairs is measured relative to floral weight and an internal standard. Error bars indicate the
standard deviation of five metabolite samples taken from each individual.
S9 Fig. Amino acid sequence alignment of functionally characterized CsTPS enzymes.
We thank We thank Mr. Jan Slaski (Alberta Innovates Technology Futures) for hemp seeds;
Mr. Mack Yuen (UBC), Ms. Lina Madilao (UBC), and Dr. Melissa Mageroy (UBC) for techni-
cal advice; and Dr. Justin Whitehill (UBC) and Dr. Sandra Irmisch (UBC) for comments on
the manuscript.
Author Contributions
Conceptualization: JB JEP JKB.
Formal analysis: JKB.
Funding acquisition: JB JKB.
Investigation: JB JKB.
Project administration: JB JKB.
Resources: JB JEP.
Supervision: JB JEP.
Writing – original draft: JB JKB.
Writing – review & editing: JB JEP JKB.
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... Functions of several structural genes involved in the biosynthesis of cannabinoids have been systematically identified, including the terpene synthase (CsTPs) family (Booth et al. 2020;Luo et al. 2019). Cannabinoids are synthesized via the plastid methylerythritol phosphate (MEP), GPP pathway, and hexanoate pathway (Booth et al. 2020(Booth et al. , 2017. MEP pathway produces isopentenyl diphosphate (IPP), the precursor of all terpenes. ...
... It is known 1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first rate-limiting enzyme in the MEP pathway. It converts pyruvate and glyceraldehyde 3-phosphate (G3P) to form 1-deoxy-d-xylose 5-phosphate (DXP) (Fig. 7) (Booth et al. 2017). After a series of catalytic reactions, GPP, an intermediate product for CBD and Δ9-THC synthesis, is finally formed (Wróbel et al. 2018) In type II, two circRNAs (CiR0159 and CiR0044)were generated from DXS1(PG0159) potentially involved in the biosynthesis of cannabinoids, in which ciR0159 showed a significant positive correlation with CBN (C7) (Fig. 6a, Table S12). ...
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Main conclusion We identified circRNAs in the Cannabis sativa L. genome and examined their association with 28 cannabinoids in three tissues of C. sativa. Nine circRNAs are potentially involved in the biosynthesis of six cannabinoids. Abstract Cannabis sativa L. has been widely used in the production of medicine, textiles, and food for over 2500 years. The main bioactive compounds in C. sativa are cannabinoids, which have multiple important pharmacological actions. Circular RNAs (circRNAs) play essential roles in growth and development, stress resistance, and the biosynthesis of secondary metabolites. However, the circRNAs in C. sativa remain unknown. In this study, to explore the role of circRNAs in cannabinoid biosynthesis, we performed RNA-Seq and metabolomics analysis on the leaves, roots, and stems of C. sativa. We identified 741 overlapping circRNAs by three tools, of which 717, 16, and 8 circRNAs were derived from exonic, intronic, and intergenic, respectively. Functional enrichment analysis indicated that the parental genes (PGs) of circRNAs were enriched in many processes related to biological stress responses. We found that most of the circRNAs showed tissue-specific expression and 65 circRNAs were significantly correlated with their PGs (P < 0.05, |r|≥ 0.5). We also determined 28 cannabinoids by High-performance liquid chromatography-ESI-triple quadrupole-linear ion trap mass spectrometry. Ten circRNAs, including ciR0159, ciR0212, ciR0153, ciR0149, ciR0016, ciR0044, ciR0022, ciR0381, ciR0006, and ciR0025 were found to be associated with six cannabinoids by weighted gene co-expression network analysis. Twenty-nine of 53 candidate circRNAs, including 9 cannabinoids related were validated successfully using PCR amplification and Sanger sequencing. Taken together, all these results would help to enhance our acknowledge of the regulation of circRNAs, and lay the foundation for breeding new C. sativa cultivars with high cannabinoids through manipulating circRNAs.
... C. sativa includes a large number of extremely volatile terpenoids which act as insect repellent and are regularly used in insect repellent cream (Booth et al., 2017). When THC is added to cannabis oil, neurobehavioral symptoms like delusions, agitation, irritability, apathy, lack of sleep, and caregiver stress go down by a massive proportion (Verma et al., 2021). ...
Cannabis sativa L. is a flowering plant in the family Cannabaceae, and has been cultivated since ancient times for its fibres, oils, resins, dried inflorescences, and leaves. It can be used for a variety of industrial purposes. Over the years, the therapeutic and pharmacological efficacy of its phytoconstituents is shown in a variety of human diseases and health. The use and exploitation of the plant have sparked controversy; however, there are recent legalizations of its use for medical and other purposes in many countries within the corresponding legislative framework. In addition to this legalization, C. sativa is encouraging the very rapid growth of the cannabis oriented pharmaceutical industry. This chapter summarized recent developments in the science of C. sativa and its products about their industrial application, while also addressing gaps in the existing knowledge and future research directions for this high-value multi-use, and potential industrial plant with universal benefits.
... The twelve TPS genes identified for S. officinalis match the range of TPS numbers reported for other genomes (six: Arabidopsis thaliana (Aubourg et al., 2002), eight: Cannabis sativa (Booth et al., 2017), 16: Ocimum sanctum (Kumar et al., 2018), 6: Cinnamomum burmannii (Ma et al., 2022), 7: Pinus nigra subsp. laricio (Alicandri et al., 2022), 19: Vitis vinifera (Martin et al., 2010)). ...
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The diversity of plant monoterpenes is largely based on the catalytic activity of monoterpene synthases. Additionally, copy number variation of monoterpene synthase genes may contribute to the quantity of transcripts and hence to the essential oil profile. This study used whole-genome sequencing and digital PCR for the measurement of copy number variation and quantification of gene expression in three closely related Salvia species, namely Salvia officinalis, Salvia pomifera and Salvia fruticosa. Twelve, 13 and 15 monoterpene synthase-encoding open-reading frames were predicted for Salvia officinalis, Salvia pomifera and Salvia fruticosa, respectively. In Salvia officinalis, one of the open reading frames was disrupted indicating a pseudogene. Monoterpene synthase genes were generally single copy per haploid genome, only a few were double or triple copy genes. Expression levels of monoterpene synthases in leaves corresponded generally well with essential oil composition. In some cases, a higher expression level of a certain monoterpene synthase could be explained by its duplication or triplication. The very high content of thujones in Salvia pomifera, for example, was accompanied by gene duplication and increased gene expression of (+)-sabinene synthase responsible for the thujone precursor sabinene. In Salvia officinalis, three individuals different in their essential oil profile showed significant differences in their monoterpene synthase expression levels corresponding roughly to the profile of the essential oils. Transcript expression of monoterpene synthase genes were measured in leaf, calyx and corolla. The corolla differed significantly from leaves, while calyces usually showed a profile intermediary between leaf and corolla.
Cannabis sativa (Hemp) is an herbaceous species used in foods and beverages, cosmetics, pharmaceuticals, nutraceuticals, etc. It has two active constituents: Tetrahydrocannabinol and Cannabidiol. Cannabinoids suppress uncontrolled growth of cancerous cells, provide pain relief to HIV patients, and help with neurogenic symptoms. Cannabis extracts have exhibited more effectiveness against various bacteria and yeast. Improvement in skin hydration and skin elasticity was observed. Excessive sebum production and proliferation of sebocytes is normalized. Hemp extracts arrest the anagen phase of the hair cycle to cause reversal of the temporal and vertex thinning in Androgenetic alopecia. It has antioxidant potential as well as an anti-ageing effect. Owing to the variability in legislation in different countries, Cannabis sativa poses challenges in research. This chapter discusses the diverse applications of this double-edged herb.
Lilacs have high ornamental value due to their strong aroma. However, the molecular regulatory mechanisms of aroma biosynthesis and metabolism in lilac were largely unclear. In this study, two varieties with distinct aroma, Syringa oblata 'Zi Kui' (faint aroma) and Syringa vulgaris 'Li Fei' (strong aroma), were used for exploring the regulation mechanism of aroma difference. Via GC-MS analysis, a total of 43 volatile components were identified. Terpene volatiles was the most abundant volatiles constituting the aroma of two varieties. Notably, 3 volatile secondary metabolites were unique in 'Zi Kui' and 30 volatile secondary metabolites were unique in 'Li Fei'. Then, a transcriptome analysis was performed to clarify the regulation mechanism of aroma metabolism difference between these two varieties, and identified 6411 differentially expressed genes (DEGs). Interestingly, ubiquinone and other terpenoid-quinone biosynthesis genes were significantly enriched in DEGs. We further conducted a correlation analysis between the volatile metabolome and transcriptome and found that TPS, GGPPS, and HMGS genes might be the key contributors to the differences in floral fragrance composition between the two lilac varieties. Our study improves the understanding in the regulation mechanism of Lilac aroma and would help improve the aroma of ornamental crops by metabolic engineering.
Cannabis sativa is widely used for fiber, medicinal, and other purposes, and many cultivars exist, yielding varying proportions of cannabinoids and terpenes. There is considerable interest in characterizing genomes and transcriptomes of C. sativa. Alternative splicing (AS) is a fundamental aspect of gene expression that results in multiple types of mRNAs produced by differential splicing. Transcriptome-wide identification of AS events in drug-type cultivars of C. sativa has not been reported. Here we identified AS events using a transcriptome dataset derived from five drug-type cultivars with divergent chemotypes. Intron retention is the most common event type, followed by alternative acceptor, alternative donor, and skipped exons. We also sought to assess conservation of AS events among cultivars. We found 547 events (5%) unique to a single cultivar, 2661 (25%) shared by 2-4 cultivars, and 7569 (70%) common to all five cultivars. Genes with AS events in each set were analyzed for gene ontology enrichment, showing that genes with AS unique to a single cultivar are enriched for molecular functions related to interactions with ATP, and processes involving transport within cells and across membranes. These results provide insights into the conservation and variation of AS events in multiple cultivars of C. sativa.
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Orchids are widely used in traditional medicine for the treatment of a whole range of different health conditions, and representatives of the Neotropical subtribe Maxillariinae are not an exception. They are utilized, for instance, for their spasmolytic and anti-inflammatory activities. In this work, we analyze the literature concerning the chemical composition of the plant extracts and secretions of this subtribe's representatives published between 1991 and 2022. Maxillariinae is one of the biggest taxa within the orchid family; however, to date, only 19 species have been investigated in this regard and, as we report, they produce 62 semiochemicals of medical potential. The presented review is the first summary of biologically active compounds found in Maxillariinae.
Cannabis glandular trichomes produce and store an abundance of lipidic specialised metabolites (e.g. cannabinoids and terpenes) that are consumed by humans for medicinal and recreational purposes. Due to a lack of genetic resources and inherent autofluorescence of cannabis glandular trichomes, our knowledge of cannabinoid trafficking and secretion is limited to transmission electron microscopy (TEM). Advances in cryofixation methods has resulted in ultrastructural observations closer to the 'natural state' of the living cell, and recent reports of cryofixed cannabis trichome ultrastructure challenge the long-standing model of cannabinoid trafficking proposed by ultrastructural reports using chemically fixed samples. Here, we compare the ultrastructural morphology of cannabis glandular trichomes preserved using conventional chemical fixation and ultrarapid cryofixation. We show that chemical fixation results in amorphous metabolite inclusions surrounding the organelles of glandular trichomes that were not present in cryofixed samples. Vacuolar morphology in cryofixed samples exhibited homogenous electron density, while chemically fixed samples contained a flocculent electron dense periphery and electron lucent lumen. In contrast to the apparent advantages of cryopreservation, fine details of cell wall fibre orientation could be observed in chemically fixed glandular trichomes that were not seen in cryofixed samples. Our data suggest that chemical fixation results in intracellular artefacts that impact the interpretation of lipid production and trafficking, while enabling greater detail of extracellular polysaccharide organisation.
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Considering the large number of volatile molecules that characterize Cannabis sativa L., adequate investigation supported by the application of robust and effective analytical methods is essential to better understand the impact of these low- and medium-molecular-weight molecules on the entire phytocomplex. This work aimed to characterize the volatile fraction of the chemical profile of three different cultivars of Cannabis sativa L. pollen, grown in Italy, which were thoroughly investigated by the application of two complementary techniques: SPME-GC-MS and PTR-ToF-MS. Furthermore, in order to provide more information on the chemical profile of the matrices under study, the cannabinoid content of the hexane extracts was also measured by GC-MS. Until now, no similar study, in terms of survey techniques applied, has been performed on C. sativa pollen. The obtained results showed a high content of volatile molecules, which differentiated the three matrices. The data relating to the content of cannabinoids were also interesting as they showed that one of the three cultivars was richer than the others. Finally, an in-depth statistical survey was performed to better compare the investigated samples and identify the molecules that most contribute to differentiating them. The findings of this study may be useful for integrating the compositional information on C. sativa L.
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The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one from chemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and flowers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantified using GC-FID and verified via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and III needed more time to reach peak production of THCA, CBDA, and monoterpenes. Differences in the cannabigerolic acid development among the different chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of different chemotypes were clearly differentiated by their terpene content, and characteristic terpenes of each chemotype were identified.
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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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Background: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. Results: We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. Conclusions: The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
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In this paper, we present principal component analysis (PCA) results from a dataset containing 494 cannabis flower samples and 170 concentrate samples analyzed for 31 compounds. A continuum of chemical composition amongst cannabis strains was found instead of distinct chemotypes. Our data shows that some strains are much more reproducible in chemical composition than others. Strains labeled as indica were compared with those labeled as sativa and no evidence was found that these two cultivars are distinctly different chemotypes. PCA of “OG” and “Kush” type strains found that “OG” strains have relatively higher levels of α-terpineol, fenchol, limonene, camphene, terpinolene and linalool where “Kush” samples are characterized mainly by the compounds trans-ocimene, guaiol, β-eudesmol, myrcene and α-pinene. The composition of concentrates and flowers were compared as well. Although the absolute concentration of compounds in concentrates is much higher, the relative composition of compounds between flowers and concentrates is similar.
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Cannabis sativa is an economically important source of durable fibers, nutritious seeds, and psychoactive drugs but few economic plants are so poorly understood genetically. Marijuana and hemp were crossed to evaluate competing models of cannabinoid inheritance and to explain the predominance of tetrahydrocannabinolic acid (THCA) in marijuana compared with cannabidiolic acid (CBDA) in hemp. Individuals in the resulting F2 population were assessed for differential expression of cannabinoid synthase genes and were used in linkage mapping. Genetic markers associated with divergent cannabinoid phenotypes were identified. Although phenotypic segregation and a major quantitative trait locus (QTL) for the THCA/CBDA ratio were consistent with a simple model of codominant alleles at a single locus, the diversity of THCA and CBDA synthase sequences observed in the mapping population, the position of enzyme coding loci on the map, and patterns of expression suggest multiple linked loci. Phylogenetic analysis further suggests a history of duplication and divergence affecting drug content. Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to enhance psychoactivity. An unlinked QTL for cannabinoid quantity may also have played a role in the recent escalation of drug potency. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.
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The enzyme farnesyl-diphosphate synthase (FPS; EC catalyzes the synthesis of farnesyl diphosphate (FPP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This reaction is considered to be a rate-limiting step in isoprenoid biosynthesis. Southern blot analysis indicates that Arabidopsis thaliana contains at least 2 genes (FPS1 and FPS2) encoding FPS. The FPS1 and FPS2 genes have been cloned and characterized. The two genes have a very similar organization with regard to intron positions and exon sizes and share a high level of sequence similarity, not only in the coding region but also in the intronic sequences. Northern blot analysis showed that FPS1 and FPS2 have a different pattern of expression. FPS1 mRNA accumulates preferentially in roots and inflorescences, whereas FPS2 mRNA is predominantly expressed in inflorescences. The cDNA corresponding to the FPS1 gene was isolated by functional complementation of a mutant yeast strain deffective in FPS activity (Delourme, D., Lacroute, F., and Karst, F.(1994) Plant Mol. Biol. 26, 1867-1873). By using a reverse transcription-polymerase chain reaction strategy we have cloned the cDNA corresponding to the FPS2 gene. Analysis of the FPS2 cDNA sequence revealed an open reading frame encoding a protein of 342 amino acid residues with a predicted molecular mass of 39,825 Da. FPS1 and FPS2 isoforms share an overall amino acid identity of 90.6%. Arabidopsis FPS2 was able to rescue the lethal phenotype of an ERG20-disrupted yeast strain. We demonstrate that FPS2 catalyzes the two successive condensations of IPP with both DMAPP and geranyl diphosphate leading to FPP. The significance of the occurrence of different FPS isoforms in plants is discussed in the context of the complex organization of the plant isoprenoid pathway.
Although primarily used today as one of the most prevalent illicit leisure drugs, the use of Cannabis sativa L., commonly referred to as marijuana, for medicinal purposes has been reported for more than 5000 years. Marijuana use has been shown to create numerous health problems, and, consequently, the expanding use beyond medical purposes into recreational use (abuse) resulted in control of the drug through international treaties. Much research has been carried out over the past few decades following the identification of the chemical structure of THC in 1964. The purpose of Marijuana and the Cannabinoids is to present in a single volume the comprehensive knowledge and experience of renowned researchers and scientists. Each chapter is written independently by an expert in his/her field of endeavor, ranging from the botany, the constituents, the chemistry and pharmacokinetics, the effects and consequences of illicit use on the human body, to the therapeutic potential of the cannabinoids.
Cannabis sativa has been employed for thousands of years, primarily as a source of a stem fiber (both the plant and the fiber termed “hemp”) and a resinous intoxicant (the plant and its drug preparations commonly termed “marijuana”). Studies of relationships among various groups of domesticated forms of the species and wild-growing plants have led to conflicting evolutionary interpretations and different classifications, including splitting C. sativa into several alleged species. This review examines the evolving ways Cannabis has been used from ancient times to the present, and how human selection has altered the morphology, chemistry, distribution and ecology of domesticated forms by comparison with related wild plants. Special attention is given to classification, since this has been extremely contentious, and is a key to understanding, exploiting and controlling the plant. Differences that have been used to recognize cultivated groups within Cannabis are the results of disruptive selection for characteristics selected by humans. Wild-growing plants, insofar as has been determined, are either escapes from domesticated forms or the results of thousands of years of widespread genetic exchange with domesticated plants, making it impossible to determine if unaltered primeval or ancestral populations still exist. The conflicting approaches to classifying and naming plants with such interacting domesticated and wild forms are examined. It is recommended that Cannabis sativa be recognized as a single species, within which there is a narcotic subspecies with both domesticated and ruderal varieties, and similarly a non-narcotic subspecies with both domesticated and ruderal varieties. An alternative approach consistent with the international code of nomenclature for cultivated plants is proposed, recognizing six groups: two composed of essentially non-narcotic fiber and oilseed cultivars as well as an additional group composed of their hybrids; and two composed of narcotic strains as well as an additional group composed of their hybrids.
Secretory structures in terrestrial plants appear to have first emerged as intracellular oil bodies in liverworts. In vascular plants, internal secretory structures, such as resin ducts and laticifers, are usually found in conjunction with vascular bundles, whereas subepidermal secretory cavities and epidermal glandular trichomes generally have more complex tissue distribution patterns. The primary function of plant secretory structures is related to defense responses, both constitutive and induced, against herbivores and pathogens. The ability to sequester secondary (or specialized) metabolites and defense proteins in secretory structures was a critical adaptation that shaped plant-herbivore and plant-pathogen interactions. Although this review places particular emphasis on describing the evolution of pathways leading to terpenoids, it also assesses the emergence of other metabolite classes to outline the metabolic capabilities of different plant lineages. Expected final online publication date for the Annual Review of Plant Biology Volume 66 is April 29, 2015. Please see for revised estimates.