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

重金属富集植物不仅可用于污染土壤的植物修复和植物矿化，而且可作为金属代谢基本机制的模型，因此引起了人们极大的兴趣。我们的项目将有助于从两个方面了解超积累的生理和生化机制。1)本论文以模式植物紫穗槐Thlaspi caerulescens和拟南芥Arabidopsis halleri为研究对象，研究了Cd/Zn转运P1 B-ATP酶HMA 3和HMA 4的生物化学和生物物理特性。这些蛋白质在高脂血症中的强烈天然过表达已经允许我们从T. caerulescens，其中它可能参与将金属装载到木质部中。此外，我们已经成功地对这种蛋白质进行了初步的生物化学和生物物理表征。我们现在想利用这一经验作为起点，详细研究其生物化学和生物物理学，并与HMA 3进行比较。后者推测螯合镉到液泡存储网站。通过这项工作产生的关于P1 B-ATP酶功能的生物化学/生物物理机制的知识也将有助于理解这种ATP酶在其他生物体中的功能，包括。人类（例如ATP 7A和ATP 7 B，其功能障碍导致门克斯和威尔逊病）。2)我们将使用一个自己开发的定量mRNA原位杂交的方法来研究这些转运蛋白的表达在细胞水平上的转录调控。以前的工作与这种技术的转运蛋白从其他蛋白质家族（CDF和ZIPs）揭示了重要的差异，不同的细胞类型，阶段的个体发育和响应矿物质营养，这是重要的了解一般的金属代谢。到目前为止，P1 B-ATP酶还没有这样的工作。

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.