SGER: Alkaloid Transport in Nicotiana Tabacum

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The objective of this project is to characterize a Nicotiana tabacum membrane transporter thatmay be responsible for the transport of alkaloids from their initial site of biosynthesis in roots toxylem tissue and for their final accumulation in leaves. This will be accomplished by using a genefragment previously isolated during transcriptional profiling experiments as a hybridizationprobe to isolate the full-length cDNA. With this cDNA in hand, an extensive effort will be madeto determine the tissue and intercellular localization patterns of the putative alkaloid transportprotein, using both RNase protection assays and transgenic reporter gene fusions. Biochemicalconfirmation of alkaloid transport activity will be accomplished by heterologous expression ofthe putative alkaloid transporter in Xenopus oocytes and directly measuring alkaloid transportkinetics. Finally, in vivo modulation of the putative alkaloid transporter gene expression levelswill be achieved using overexpression and antisense transgenic hairy root cultures to assess theeffect on overall alkaloid accumulation levels. These experiments may lead to the firstidentification and molecular characterization of a postulated alkaloid transporter that isresponsible for mobilizing alkaloids between their site of biosynthesis in specific root cell typestowards their site of action in the leaves. This would be a significant contribution tounderstanding both how and why the site of biosynthesis of many plant secondary metabolites isoften spatially separated from the site of action of these important defense compounds.