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Abstract
The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.01–0.8% DW). There is therefore an urgent need to design new strategies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: artemisinin (8μg/g), artesunate (2.24μg/g), dihydroartemisinin (13.6μg/g) and artemether (12.8μg/g). Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin content in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS), cytochrome P450 (CYP71AV1), and aldehyde dehydrogenase 1 (ALDH1), with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1) showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism.
Highlights
Artemisinin (AN), a sesquiterpene lactone produced mainly in Artemisia annua as a secondary metabolite, is a highly effective natural product against malaria and other parasitic diseases, as well as an anti-cancer agent
Morphological variability was observed between wild type plants and transgenics bearing rol B and rol C genes
Similar findings were reported by another group who showed for the first time how rol genes induce ginsenoside overproduction in transformed cell cultures of P. ginseng, using plasmid DNA containing the individual rol genes from the TL-DNA of A. rhizogenes strain A4 [32]
Summary
Artemisinin (AN), a sesquiterpene lactone produced mainly in Artemisia annua as a secondary metabolite, is a highly effective natural product against malaria and other parasitic diseases, as well as an anti-cancer agent. Among different strategies used to improve secondary metabolite production, the recombinant DNA technology has made it possible to directly modify the expression of biosynthetic genes, and manipulate the pathways that lead to secondary plant compounds [7]. The rol C gene, which has cytokinin glucosidase activity, is capable of stimulating the production of many secondary compounds in transformed plants and cell cultures, such as tropane alkaloids, pyridine alkaloids, indole alkaloids, ginsenosides and anthraquinones [12,13,14]. Our previous work showed that Artemisia dubia, when transformed with rol ABC genes, produces 10 times more artemisinin and its derivatives [15]
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