ARS | Publication request: CLONING AND CHARACTERIZATION OF METAL TRANSPORTING P-TYPE ATPASE FROM THE HEAVY METAL HYPERACCUMULATOR, THLASPI CARULESCENS

Submitted to: American Society of Plant Biologists Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: March 19, 2004
Publication Date: July 24, 2004
Citation: Papoyan, A., Pineros, M., Kochian, L.V. 2004. Cloning and characterization of metal transporting p-type atpase from the heavy metal hyperaccumulator, thlaspi carulescens. American Society of Plant Biologists Annual Meeting. p. 268.

Technical Abstract: Thlaspi caerulescens is a Zn/Cd-hyperaccumulating plant species that can accumulate and tolerate up to 30,000ppm Zn and 10,000ppm Cd in the shoots without exhibiting toxicity symptoms. The research described here is focused on identifying gene(s) that may be responsible for extreme Cd tolerance exhibited by these plants. We have employed yeast functional complementation techniques to isolate these gene(s). We found that 100µM Cd is lethal for the wild type yeast strain DY1457. Subsequently, we complemented this yeast strain with a T. caerulescens cDNA library in the yeast expression vector, pFL61, and identified a number of colonies of transformed yeast that grow well on this restrictive level of Cd. Among 35 yeast colonies that were highly Cd tolerant, 5 of the colonies harbored a T. caerulescens cDNA with a high level of similarity to P-type heavy metal transporting ATPases. The T. caerulescens clone was 58% similar to the Arabidopsis thaliana P-type heavy metal transporting ATPase, hma4. We are in the process characterizing this ATPase in T. caerulescens, as well as conducting a functional characterization of its transport properties in yeast (Cd influx, efflux, and accumulation). In addition, we are studying the altered expression of this gene in Arabidopsis (overexpression of the Thlaspi gene and Arabidopsis hma4 knockouts). Finally, we are generating N and T-terminus GFP-ATPase chimeras and transfecting mammalian cells (HEK 293) to assay the subcellular localization of the ATPase protein and to elucidate the biophysical properties of this transporter. This work is supported by NSF Grant # IBN-0129844 to LVK.