Cannabidiolic-acid (CBDA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic-acid into CBDA, the dominant cannabinoid constituent of the fiber-type Cannabis sativa. We cloned a novel cDNA encoding CBDA synthase by reverse transcription and polymerase chain reactions with degenerate and gene-specific primers. Biochemical characterization of the recombinant enzyme demonstrated that CBDA synthase is a covalently flavinylated oxidase. The structural and functional properties of CBDA synthase are quite similar to those of tetrahydrocannabinolic-acid (THCA) synthase, which is responsible for the biosynthesis of THCA, the major cannabinoid in drug-type Cannabis plants.

Metabolic adjustments are a significant, but poorly understood, part of the response of plants to oxidative stress. In a previous study (Baxter et al., 2007), the metabolic response of Arabidopsis cells in culture to induction of oxidative stress by menadione was characterized. An emergency survival strategy was uncovered in which anabolic primary metabolism was largely down-regulated in favour of catabolic and antioxidant metabolism. The response in whole plant tissues may be different and we have therefore investigated the response of Arabidopsis roots to menadione treatment, analyzing the transcriptome, metabolome and key metabolic fluxes with focus on primary as well as secondary metabolism. Using a redox-sensitive GFP, it was also shown that menadione causes redox perturbation, not just in the mitochondrion, but also in the cytosol and plastids of roots. In the first 30 min of treatment, the response was similar to the cell culture: there was a decrease in metabolites of the TCA cycle and amino acid biosynthesis and the transcriptomic response was dominated by up-regulation of DNA regulatory proteins. After 2 and 6 h of treatment, the response of the roots was different to the cell culture. Metabolite levels did not remain depressed, but instead recovered and, in the case of pyruvate, some amino acids and aliphatic glucosinolates showed a steady increase above control levels. However, no major changes in fluxes of central carbon metabolism were observed and metabolic transcripts changed largely independently of the corresponding metabolites. Together, the results suggest that root tissues can recover metabolic activity after oxidative inhibition and highlight potentially important roles for glycolysis and the oxidative pentose phosphate pathway.

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.

⌬ 1 -Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of ⌬ 1 -tetrahydrocannabinol. We cloned a novel cDNA (GenBank TM accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.

Plants produce a variety of toxic compounds, which are often used as anticancer drugs. The self-resistance mechanism to these toxic metabolites in the producing plants, however, remains unclear. The plant-derived anticancer alkaloid camptothecin (CPT) induces cell death by targeting DNA topoisomerase I (Top1), the enzyme that catalyzes changes in DNA topology. We found that CPTproducing plants, including Camptotheca acuminata, Ophiorrhiza pumila, and Ophiorrhiza liukiuensis, have Top1s with point mutations that confer resistance to CPT, suggesting the effect of an endogenous toxic metabolite on the evolution of the target cellular component. Three amino acid substitutions that contribute to CPT resistance were identified: Asn421Lys, Leu530Ile, and Asn722Ser (numbered according to human Top1). The substitution at position 722 is identical to that found in CPT-resistant human cancer cells. The other mutations have not been found to date in CPT-resistant human cancer cells; this predicts the possibility of occurrence of these mutations in CPT-resistant human cancer patients in the future. Furthermore, comparative analysis of Top1s of CPT-producing and nonproducing plants suggested that the former were partially primed for CPT resistance before CPT biosynthesis evolved. Our results demonstrate the molecular mechanism of self-resistance to endogenously produced toxic compounds and the possibility of adaptive coevolution between the CPT production system and its target Top1 in the producing plants.evolution ͉ Ophiorrhiza pumila ͉ Camptotheca acuminata P lants produce a vast array of secondary metabolites to overcome environmental stress and defend themselves against their natural enemies. The broad metabolic diversity represents a process of adaptation that has been subjected to natural selection during evolution (1). Furthermore, humans have benefited from plant-derived compounds that are used as medicines. These compounds, including vincristine, vinblastine, taxol, and camptothecin (CPT), are currently prescribed as anticancer drugs, which function by disrupting basic biological processes in human cells. The fact that toxic metaboliteproducing plants are insensitive to the metabolites suggests the presence of a species-specific resistance mechanism to prevent cytotoxicity. How these plants have evolved a mechanism to prevent self-toxicity while simultaneously producing toxic compounds is a long-standing intriguing question that has not been clearly answered to date.CPT, a monoterpene indole alkaloid, is produced in many distantly related plants, including Ophiorrhiza pumila, Ophiorrhiza liukiuensis, and Camptotheca acuminata. It induces cell death by stabilizing a covalent complex between DNA topoisomerase I (Top1) and the nicked DNA, leading to a DNA lesion (2-4). Although CPT is toxic to most eukaryotic organisms, including higher plants, CPT-producing plants can avoid self-toxicity, thereby suggesting the presence of a species-specific resistance mechanism (5, 6). In contrast, the involvement of either a CPT tran...