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.

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.

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 CPT-producing 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.

Cannabinoids, consiting of alkylresorcinol and monoterpene groups, are the unique secondary metabolites that are found only in Cannabis sativa. Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) are well known cannabinoids and their pharmacological properties have been extensively studied. Recently, biosynthetic pathways of these cannabinoids have been successfully established. Several biosynthetic enzymes including geranylpyrophosphate: olivetolate geranyltransferase, tetrahydrocannabinolic acid (THCA) synthase, cannabidiolic acid (CBDA) synthase and cannabichromenic acid (CBCA) synthase have been purified from young rapidly expanding leaves of C. sativa. In addition, molecular cloning, characterization and localization of THCA synthase have been recently reported. THCA and cannabigerolic acid (CBGA), its substrate, were shown to be apoptosis-inducing agents that might play a role in plant defense. Transgenic tobacco hairy roots expressing THCA synthase can produce THCA upon feeding of CBGA. These results open the way for biotechnological production of cannabinoids in the future.

Camptothecin (CPT) and its derivatives have been received considerable attention recently. Two semi-synthetic derivatives, topotecan and irinotecan, are currently prescribed as anticancer drugs. Several more are now in clinical trial. CPT is produced in many plants belonging to unrelated orders of angiosperms. At present, CPT supplied for pharmaceutical use is extracted from the plants, Camptotheca acuminata and Nothapodytes foetida. Several efforts have been made to sustain a stable production of CPT by in vitro cell cultures of C. acuminata, N. foetida and Ophiorrhiza pumila. Recent report showed that plants are not the only sources that produce CPT. CPT was reported to be produced from the endophytic fungus isolated from the inner bark of N. foetida. The hairy root cultures of C. acuminata and O. pumila produce and secrete CPT into the medium in large quantities. These reports suggest the possibility to develop large-scale production of CPT. In addition, recent advance in the cloning and characterization of biosynthetic enzymes involved in CPT biosynthetic pathway provides valuable information for developing genetically engineered CPT-producing plants.