Monitoring Metabolite Profiles of Cannabis sativa L. Trichomes during Flowering Period Using ¹H NMR-Based Metabolomics and Real-Time PCR

Abstract

Cannabis sativa trichomes are glandular structures predominantly responsible for the biosynthesis of cannabinoids, the biologically active compounds unique to this plant. To the best of our knowledge, most metabolomic works on C. sativa that have been reported previously focused their investigations on the flowers and leaves of this plant. In this study, ¹H NMR-based metabolomics and real-time PCR analysis were applied for monitoring the metabolite profiles of C. sativa trichomes, variety Bediol, during the last 4 weeks of the flowering period. Partial least squares discriminant analysis models successfully classified metabolites of the trichomes based on the harvest time. Δ ⁹-Tetrahydrocannabinolic acid (1) and cannabidiolic acid (2) constituted the vital differential components of the organic preparations, while asparagine, glutamine, fructose, and glucose proved to be their water-extracted counterparts. According to RT-PCR analysis, gene expression levels of olivetol synthase and olivetolic acid cyclase influenced the accumulation of cannabinoids in the Cannabis trichomes during the monitoring time. Moreover, quantitative ¹H NMR and RT-PCR analysis of the Cannabis trichomes suggested that the gene regulation of cannabinoid biosynthesis in the C. sativa variety Bediol is unique when compared with other C. sativa varieties.

Key words

Cannabis sativa - Cannabaceae - trichomes - Metabolomics - RT-PCR - cannabinoid

• References

• 1 Schilmiller AL, Last RL, Pichersky E. Harnessing plant trichome biochemistry for the production of useful compounds. Plant J 2008; 54: 702-711
• 2 Gaoni Y, Mechoulam R. Isolation, structure, and partial synthesis of an active constituent of hashish. J Am Chem Soc 1964; 86: 1646-1647
• 3 Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990; 346: 561-564
• 4 Munro S, Thomas KL, Abushaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993; 365: 61-65
• 5 Muntendam R, Happyana N, Erkelens C, Bruining F, Kayser O. Time dependent metabolomics and transcriptional analysis of cannabinoid biosynthesis in Cannabis sativa var. Bedrobinol and Bediol grown under standardized condition and with genetic homogeneity. Online Int J Med Plant Res 2012; 1: 31-40
• 6 Fischedick JT, Hazekamp A, Erkelens T, Choi YH, Verpoorte R. Metabolic fingerprinting of Cannabis sativa L., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes. Phytochemistry 2010; 71: 2058-2073
• 7 Choi YH, Kim HK, Hazekamp A, Erkelens C, Lefeber AWM, Verpoorte R. Metabolomic differentiation of Cannabis sativa cultivars using ¹H NMR spectroscopy and principal component analysis. J Nat Prod 2004; 67: 953-957
• 8 Hur M, Campbell AA, Almeida-de-Macedo M, Li L, Ransom N, Jose A, Crispin M, Nikolau BJ, Wurtele ES. A global approach to analysis and interpretation of metabolic data for plant natural product discovery. Nat Prod Rep 2013; 30: 565-583
• 9 Lee YR, Wang X. Concise synthesis of biologically interesting (±)-cannabichromene, (±)-cannabichromenic acid, and (±)-daurichromenic acid. Bull Korean Chem Soc 2005; 26: 1933-1936
• 10 Kirk H, Cheng D, Choi YH, Vrieling K, Klinkhamer PG. Transgressive segregation of primary and secondary metabolites in F(2) hybrids between Jacobaea aquatica and J. vulgaris . Metabolomics 2012; 8: 211-219
• 11 Abreu IN, Choi YH, Sawaya AC, Eberlin MN, Mazzafera P, Verpoorte R. Metabolic alterations in different developmental stages of Pilocarpus microphyllus . Planta Med 2011; 77: 293-300
• 12 Ali K, Maltese F, Fortes AM, Pais MS, Choi YH, Verpoorte R. Monitoring biochemical changes during grape berry development in Portuguese cultivars by NMR spectroscopy. Food Chem 2011; 124: 1760-1769
• 13 Broyart C, Fontaine JX, Molinie R, Cailleu D, Terce-Laforgue T, Dubois F, Hirel B, Mesnard F. Metabolic profiling of maize mutants deficient for two glutamine synthetase isoenzymes using 1H-NMR-based metabolomics. Phytochem Anal 2010; 21: 102-109
• 14 Liu NQ, Cao M, Frederich M, Choi YH, Verpoorte R, van der Kooy F. Metabolomic investigation of the ethnopharmacological use of Artemisia afra with NMR spectroscopy and multivariate data analysis. J Ethnopharmacol 2010; 128: 230-235
• 15 Nuringtyas TR, Choi YH, Verpoorte R, Klinkhamer PG, Leiss KA. Differential tissue distribution of metabolites in Jacobaea vulgaris, Jacobaea aquatica and their crosses. Phytochemistry 2012; 78: 89-97
• 16 Zhi HJ, Qin XM, Sun HF, Zhang LZ, Guo XQ, Li ZY. Metabolic fingerprinting of Tussilago farfara L. using ¹H-NMR spectroscopy and multivariate data analysis. Phytochem Anal 2012; 23: 492-501
• 17 Westerhuis JA, Hoefsloot HCJ, Smit S, Vis DJ, Smilde AK, van Velzen EJJ, van Duijnhoven JPM, van Dorsten FA. Assessment of PLSDA cross validation. Metabolomics 2008; 4: 81-89
• 18 Taura F, Tanaka S, Taguchi C, Fukamizu T, Tanaka H, Shoyama Y, Morimoto S. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway. FEBS Lett 2009; 583: 2061-2066
• 19 Gagne SJ, Stout JM, Liu EW, Boubakir Z, Clark SM, Page JE. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides. Proc Natl Acad Sci U S A 2012; 109: 12811-12816
• 20 Sirikantaramas S, Morimoto S, Shoyama Y, Ishikawa Y, Wada Y, Shoyama Y, Taura F. The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Delta(1)-tetrahydrocannabinolic acid synthase from Cannabis sativa L. J Biol Chem 2004; 279: 39767-39774
• 21 Taura F, Morimoto S, Shoyama Y. Purification and characterization of cannabidiolic-acid synthase from Cannabis sativa L. Biochemical analysis of a novel enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid. J Biol Chem 1996; 271: 17411-17416
• 22 Happyana N, Kayser O. ¹H NMR-based metabolomics differentiation and real time PCR analysis of medicinal Cannabis organs. International Horticulture Congress, 18th–22nd of August, 2014. Brisbane, Australia: Acta Horticulture; 2014
• 23 Grlic L. Combined spectrophotometric differentiation of samples of Cannabis . Bull Narc 1968; 20: 25-29
• 24 Grlic L. A comparative-study on some chemical and biological characteristics of various samples of Cannabis resin. Bull Narc 1962; 14: 37-46
• 25 Grlic L, Andrec A. Content of acid fraction in cannabis resin of various age and provenance. Experientia 1961; 17: 325-326
• 26 Turner CE, Elsohly MA, Boeren EG. Constituents of Cannabis Sativa L. XVII. A review of the natural constituents. J Nat Prod 1980; 43: 169-234
• 27 Happyana N, Agnolet S, Muntendam R, Van Dam A, Schneider B, Kayser O. Analysis of cannabinoids in laser-microdissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR. Phytochemistry 2013; 87: 51-59
• 28 Yerger EH, Grazzini RA, Hesk D, Coxfoster DL, Craig R, Mumma RO. A rapid method for isolating glandular trichomes. Plant Physiol 1992; 99: 1-7
• 29 Hazekamp A, Choi YH, Verpoorte R. Quantitative analysis of cannabinoids from Cannabis sativa using ¹H-NMR. Chem Pharm Bull (Tokyo) 2004; 52: 718-721
• 30 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25: 402-408