Cadmium ATPases in hyperaccumulator plants: biophysical and biochemical mechanism of their function and cellular expression regulation

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Heavy metal hyperaccumulators are of great interest because of their use in phytoremediation of contaminated soils and in phytomining, but also as models for fundamental mechanisms of metal metabolism. Our project will contribute to understanding of physiological and biochemical mechanisms of hyperaccumulation in two ways. 1) We will characterise the biochemistry and biophysics of the Cd/Zn transporting P1B-ATPases HMA3 and HMA4 in the model hyperaccumulators Thlaspi caerulescens and Arabidopsis halleri. The strong natural overexpression of such proteins in hyperaccumulators already allowed us to purify HMA4 from T. caerulescens, where it is presumably involved in loading of the metal into the xylem. Further, we were already successful with a preliminary biochemical and biophysical characterisation of this protein. We now want to use this experience as a starting point for a detailed investigation of its biochemistry and biophysics and comparison with HMA3. The latter presumably sequesters Cd into the vacuolar storage sites. The knowledge about biochemical/biophysical mechanisms of function of P1B-ATPases generated by this work will also help in understanding the function of such ATPases in other organisms, incl. humans (e.g. ATP7A and ATP7B, the malfunction of which causes Menkes and Wilson's disease). 2) We will use a self-developed method for quantitative mRNA in situ hybridisation to investigate the transcriptional regulation of the expression of these transporters on the cellular level. Previous work with this technique on transporters from other protein families (CDFs and ZIPs) revealed important differences between different cell types, stages of ontogenesis and response to mineral nutrition, which are important for understanding metal metabolism in general. No such work exists on P1B-ATPases so far.