Terpene synthases from Cannabis sativa

Abstract

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

Fig 1. Glandular trichomes on the surface of pistillate inflorescences and leaves of 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.

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.

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.

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

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.

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 the total mono- or sesquiterpenes for each individual. Transcript abundances are calibrated normalized values compared to 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.

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.