• Häkkinen, Suvi

Abstract

The aim of this work was to improve understanding of theregulation of alkaloid biosynthesis in two Solanaceaeplants, Nicotiana tabacum (tobacco) and Hyoscyamusmuticus (Egyptian henbane). In order to map thebiosynthetic genes involved in the tobacco alkaloidpathway, a functional genomics-based technology wasestablished by combining genome-based transcriptprofiling (cDNA-AFLP) with targeted metabolite analysis.Altogether 459 genes were found to be differentiallyexpressed in methyl jasmonate-elicited N. tabacum BY-2cells. Homology searches performed with these genesrevealed that 58 % of the genes displayed similarity withgenes having known functions, whereas no sequencesimilarity was found with 26 % of the genes, suggestingthat some of them may take part in unknown steps intobacco alkaloid biosynthesis. Alkaloids accumulated 12hours after methyl jasmonate application, with varyingkinetic patterns. For the first time the alkaloidanatalline was shown to accumulate in Nicotiana cellcultures, and together with anatabine they formed themain alkaloid pool. Anatalline was further characterizedstructurally as being present in two isomeric forms,anatalline and trans-2,4-di(3-pyridyl)piperidine.Contrary to the case in whole tobacco plants, nicotinewas only a minor alkaloid accumulating in elicited cells,whereas the production of a precursor methylputrescinewas highly induced. Based on these results, it wassuggested that the limiting step in nicotine biosynthesisoccurred between methylputrescine and nicotine.Altogether 34 methyl jasmonate-modulated genes wereselected for further functional testing in BY-2 cellcultures using Agrobacterium-mediated genetransformation. Six genes caused a lower alkaloidaccumulation compared to the control when assayed in cellcultures, whereas three genes elevated the production ofone or several alkaloids. One of the genes causingenhanced alkaloid accumulation was found to possess highsequence similarity with lysine decarboxylase, a generesponsible for the conversion of lysine in earlyanabasine biosynthesis. However, since lysinedecarboxylase activity was not shown by the correspondingprotein, the exact nature of this gene requires furtherelucidation. The selected genes were also assayed inhairy roots, which constitutively produce alkaloids. Twohighly homologous genes were found, which showeddivergent effects on alkaloid biosynthesis. These geneswere suggested to function in auxin homeostasis. Theother gene also resulted in marked increase in nicotineaccumulation.Tropane and tobacco alkaloids share a common biosyntheticorigin, and therefore it was of interest to study whetherNicotiana genes could have a role in the formation oftropane alkaloids in a related species H. muticus. It wasobserved that the same gene which elevated nicotinecontents in Nicotiana showed a positive effect on tropanealkaloid intermediate in H. muticus, suggesting apossible conserved role of this gene in Solanaceaespecies. On the other hand, when a known tropane alkaloidpathway gene, hyoscyamine-6?-hydroxylase (H6H), wasoverexpressed in N. tabacum hairy roots, a 45 %conversion of hyoscyamine into scopolamine took placewhen hyoscyamine was supplied to the cultures.Furthermore, up to 85 % of the produced scopolamine wassecreted out of the cells. Besides being able to uptakeand convert a foreign substrate, an altered tobaccoalkaloid production in roots was observed afterhyoscyamine feeding, suggesting highly complex regulationof the production of these defence-related compounds.In order to improve the understanding of alkaloidtransport and secretion, the function of a yeastATP-binding cassette transporter was investigated and itwas shown to attribute enhanced tolerance of tropanealkaloids in N. tabacum cell cultures. Combined with theinformation of the regulation of the biosynthesis,transporters can be exploited to design novel tools toenhance the yield and diversity of alkaloids.

Topics

• compound