SGER: Alkaloid Transport in Nicotiana Tabacum

Virginia Polytechnic Institute And State University, Blacksburg VA

Investigators

Abstract

The objective of this project is to characterize a Nicotiana tabacum membrane transporter that may be responsible for the transport of alkaloids from their initial site of biosynthesis in roots to xylem tissue and for their final accumulation in leaves. This will be accomplished by using a gene fragment previously isolated during transcriptional profiling experiments as a hybridization probe to isolate the full-length cDNA. With this cDNA in hand, an extensive effort will be made to determine the tissue and intercellular localization patterns of the putative alkaloid transport protein, using both RNase protection assays and transgenic reporter gene fusions. Biochemical confirmation of alkaloid transport activity will be accomplished by heterologous expression of the putative alkaloid transporter in Xenopus oocytes and directly measuring alkaloid transport kinetics. Finally, in vivo modulation of the putative alkaloid transporter gene expression levels will be achieved using overexpression and antisense transgenic hairy root cultures to assess the effect on overall alkaloid accumulation levels. These experiments may lead to the first identification and molecular characterization of a postulated alkaloid transporter that is responsible for mobilizing alkaloids between their site of biosynthesis in specific root cell types towards their site of action in the leaves. This would be a significant contribution to understanding both how and why the site of biosynthesis of many plant secondary metabolites is often spatially separated from the site of action of these important defense compounds.