Functional characterization of a putative Arabidopsis thaliana phosphatidylinositol transfer protein which increases Al tolerance in yeast

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Aluminum (Al) toxicity is a major limiting factor for crop production worldwide. The proposed research builds on the discovery of an Arabidopsis cDNA, AtAlr54, which encodes a protein homologous to the yeast Sec14p phosphatidyl inositol transfer protein (PITP) and whose expression causes increased Al tolerance in yeast. Because AtAlr54 shares homology with yeast Sec14p and complements the growth defect of a yeast sec14-1ts mutant, AtAlr54 is itself likely a PITP. Subsequent genetic experiments with yeast mutants defective in the metabolism of phosphoinositides suggested that AtAlr54-mediated increased Al tolerance might act by regulating amount or distribution of phosphoinositides. The proposed research addresses the biochemical characterization of AtAlr54 as a first step towards investigating the function of PITPs in Al tolerance. This characterization will include comprehensive biochemical analyses of AtAlr54 lipid binding and transfer properties, metabolic experiments designed to measure how AtAlr54 affects phosphoinositide and soluble inositol phosphate metabolism in vivo, genetic and mutagenesis experiments to determine what lipid signaling pathways are involved in AtAlr54-mediated Al tolerance, and imaging experiments to monitor phosphoinositide distribution in response to metal stress. This research will employ yeast as a system to get a picture of how AtAlr54 functions, and will guide functional analyses in plants engineered for altered AtAlr54 expression.