A functional genomics approach to the study of alkaloid biosynthesis and metabolism in Nicotiana tabacum and Hyoscyamus muticus cell cultures: Dissertation

The aim of this work was to improve understanding of the regulation of alkaloid biosynthesis in two Solanaceae plants, Nicotiana tabacum (tobacco) and Hyoscyamus muticus (Egyptian henbane). In order to map the biosynthetic genes involved in the tobacco alkaloid pathway, a functional genomics-based technology was established by combining genome-based transcript profiling (cDNA-AFLP) with targeted metabolite analysis. Altogether 459 genes were found to be differentially expressed in methyl jasmonate-elicited N. tabacum BY-2 cells. Homology searches performed with these genes revealed that 58 % of the genes displayed similarity with genes having known functions, whereas no sequence similarity was found with 26 % of the genes, suggesting that some of them may take part in unknown steps in tobacco alkaloid biosynthesis. Alkaloids accumulated 12 hours after methyl jasmonate application, with varying kinetic patterns. For the first time the alkaloid anatalline was shown to accumulate in Nicotiana cell cultures, and together with anatabine they formed the main alkaloid pool. Anatalline was further characterized structurally as being present in two isomeric forms, anatalline and trans-2,4-di(3-pyridyl)piperidine. Contrary to the case in whole tobacco plants, nicotine was only a minor alkaloid accumulating in elicited cells, whereas the production of a precursor methylputrescine was highly induced. Based on these results, it was suggested that the limiting step in nicotine biosynthesis occurred between methylputrescine and nicotine. Altogether 34 methyl jasmonate-modulated genes were selected for further functional testing in BY-2 cell cultures using Agrobacterium-mediated gene transformation. Six genes caused a lower alkaloid accumulation compared to the control when assayed in cell cultures, whereas three genes elevated the production of one or several alkaloids. One of the genes causing enhanced alkaloid accumulation was found to possess high sequence similarity with lysine decarboxylase, a gene responsible for the conversion of lysine in early anabasine biosynthesis. However, since lysine decarboxylase activity was not shown by the corresponding protein, the exact nature of this gene requires further elucidation. The selected genes were also assayed in hairy roots, which constitutively produce alkaloids. Two highly homologous genes were found, which showed divergent effects on alkaloid biosynthesis. These genes were suggested to function in auxin homeostasis. The other gene also resulted in marked increase in nicotine accumulation. Tropane and tobacco alkaloids share a common biosynthetic origin, and therefore it was of interest to study whether Nicotiana genes could have a role in the formation of tropane alkaloids in a related species H. muticus. It was observed that the same gene which elevated nicotine contents in Nicotiana showed a positive effect on tropane alkaloid intermediate in H. muticus, suggesting a possible conserved role of this gene in Solanaceae species. On the other hand, when a known tropane alkaloid pathway gene, hyoscyamine-6?-hydroxylase (H6H), was overexpressed in N. tabacum hairy roots, a 45 % conversion of hyoscyamine into scopolamine took place when hyoscyamine was supplied to the cultures. Furthermore, up to 85 % of the produced scopolamine was secreted out of the cells. Besides being able to uptake and convert a foreign substrate, an altered tobacco alkaloid production in roots was observed after hyoscyamine feeding, suggesting highly complex regulation of the production of these defence-related compounds. In order to improve the understanding of alkaloid transport and secretion, the function of a yeast ATP-binding cassette transporter was investigated and it was shown to attribute enhanced tolerance of tropane alkaloids in N. tabacum cell cultures. Combined with the information of the regulation of the biosynthesis, transporters can be exploited to design novel tools to enhance the yield and diversity of alkaloids.