Tetrahydrocannabinolic Acid Synthase, the Enzyme Controlling Marijuana Psychoactivity, is Secreted into the Storage Cavity of the Glandular Trichomes

Plant Cell Physiol. 46(9): 1578–1582 (2005) doi:10.1093/pcp/pci166, available online at www.pcp.oupjournals.org JSPP © 2005 Short Communication Tetrahydrocannabinolic Acid Synthase, the Enzyme Controlling Marijuana Psychoactivity, is Secreted into the Storage Cavity of the Glandular Trichomes Supaart Sirikantaramas 1, Futoshi Taura *, Yumi Tanaka, Yu Ishikawa, Satoshi Morimoto and Yukihiro Shoyama Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582 Japan Keywords: Biosynthesis — Glandular trichome — Localization — Marijuana — Tetrahydrocannabinol — Tetrahydrocannabinolic acid synthase. Abbreviations: CBGA, cannabigerolic acid; GFP, green fluorescent protein; THC, tetrahydrocannabinol; THCA, tetrahydrocannabinolic acid; THCAS, tetrahydrocannabinolic acid synthase gene. Marijuana (Cannabis sativa L.) is an annual herb that has been cultivated and used for thousands of years (Fairbairn 1976). It contains unique secondary metabolites called cannabinoids, a group of terpenophenolics. To date, about 70 cannabinoids have been isolated from marijuana (Mechoulam and Ben-Shabat 1999) including ∆1-tetrahydrocannabinol (THC), a well-known psychoactive component. Besides its psychoactivity, THC possesses analgesic, anti-inflammatory, appetite stimulant and anti-emetic properties, making this cannabinoid a very promising drug for therapeutic purposes (Baker et al. 2003, Guzman 2003). 1 * It is known that cannabinoids such as THC are originally generated in acidic forms. In fresh tissues, the concentration of THC is much lower than that of ∆1-tetrahydrocannabinolic acid (THCA). THCA is later decarboxylated by a non-enzymatic reaction during storage or smoking (Yamauchi et al. 1967). Despite the long history of marijuana research, the cannabinoid biosynthetic pathway was elucidated only recently. Studies on biosynthetic enzymes over the last decade have revealed the THCA biosynthetic pathway as shown in Fig. 1. Cannabigerolic acid (CBGA), a precursor of THCA, is the product of the alkylation of olivetolic acid with geranyl pyrophosphate by an enzyme called geranylpyrophosphate:olivatolate geranyltransferase (Fellermeier and Zenk 1998). Then, CBGA is converted into THCA by a novel enzyme called THCA synthase (Taura et al. 1995a). THCA synthase catalyzes a unique oxidative cyclization of the geranyl group of CBGA. Recently, we have successfully cloned THCA synthase and characterized its structural and functional properties (Sirikantaramas et al. 2004). Biochemical characterization demonstrated that THCA synthase is a flavinylated oxidase that requires molecular oxygen and produces THCA and hydrogen peroxide. In addition, we have already constructed transgenic tobacco hairy roots expressing THCA synthase that can produce THCA upon feeding of CBGA, suggesting a strategy for the biotechnological production of THC (Sirikantaramas et al. 2004). However, the metabolic engineering of the cannabinoid pathway requires a detailed understanding of the biosynthetic mechanism including regulation and trafficking of the enzymes involved in the pathway. In this study, we describe the cell-specific localization and possible physiological function of THCA synthase, the enzyme responsible for THC production. With regard to the localization of cannabinoids in C. sativa, it has been reported that THC is accumulated only in the secretory cavity of the glandular trichomes (Fujita et al. 1967, Fairbairn 1972). In addition, we have reported that the leaf bud tissue, which is rich in glandular trichomes, contains a potent THCA synthase activity (Taura et al. 1995a). Thus it appears possible that THCA is biosynthesized in the glandular tri- Present address: Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 263–8522 Japan. Corresponding author: E-mail, ; Fax, +81-92-642-6582. 1578 ; Tetrahydrocannabinolic acid (THCA) synthase is the enzyme responsible for the production of tetrahydrocannabinol (THC), the psychoactive component of marijuana (Cannabis sativa L.). We suggest herein that THCA is biosynthesized in the storage cavity of the glandular trichomes based on the following observations. (i) The exclusive expression of THCA synthase was confirmed in the secretory cells of glandular trichomes by reverse transcription–PCR (RT–PCR) analysis. (ii) THCA synthase activity was detected in the storage cavity content. (iii) Transgenic tobacco expressing THCA synthase fused to green fluorescent protein showed fluorescence in the trichome head corresponding to the storage cavity. These results also showed that secretory cells of the glandular trichomes secrete not only metabolites but also biosynthetic enzyme. Cannabinoid biosynthesis in glandular trichome 1579 Fig. 2 Semi-quantitative RT–PCR analysis of the expression of THCA synthase gene in the glandular trichome. SC, seed coats after removal of the glandular trichomes; G, glandular trichomes. 18S rRNA was amplified to verify the amount of template cDNA. chomes of Cannabis plants. To investigate this possibility, we performed a semi-quantitative reverse transcription–PCR (RT– PCR), using THCA synthase-specific primers, with cDNA samples from the glands and the tissue after gland removal. We used seed coats as a starting material since they provide a large Fig. 1 Biosynthetic pathway of THC. CBGA is a product of the alkylation of olivetolic acid with geranyl pyrophosphate. THCA synthase catalyzes the oxidative cyclization of the monoterpene moiety of CBGA to form THCA. THCA is decarboxylized to THC by a non-enzymatic reaction. The responsible enzymes are: geranylpyrophosphate:olivetolate geranyltransferase (1) and THCA synthase (2). number of glands. As a result, a much larger amount of PCR product, amplified by THCA synthase-specific primers, was detected for the sample from glandular trichomes (Fig. 2). In contrast, only a trace amount of PCR product was detected with the cDNA from gland-removed tissue. The control 18S rRNA gene fragment was amplified equally for both samples. These results suggested that THCA synthase is expressed exclusively in the glandular trichome. We previously have reported the characteristics of the amino acid sequence of THCA synthase (Sirikantaramas et al. 2004). The sequence contains a 28 amino acid signal peptide and eight putative N-glycosylation sites. In addition, a recombinant THCA synthase from an insect cell culture was secreted into the medium, suggesting the trafficking of THCA synthase from the endoplasmic reticulum to the outside of the cell. However, the destination of the protein in plants might be different from that in insect cells. Therefore, we performed a stable transformation to produce transgenic tobacco BY-2 cells harboring THCAS to confirm whether THCA synthase is secreted from plant cells. It is noted that we used tobacco as a host because it is difficult to transform C. s (...truncated)