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

重金属超积累植物因其在污染土壤的植物修复和植物修复中的应用而备受关注，同时也是金属代谢基本机制的模型。我们的项目将从两个方面帮助理解超积累的生理和生化机制。1)我们将对Cd/Zn转运P1B-ATPase HMA3和HMA4的生化和生物物理特性进行研究。这类蛋白质在超积累植物中的强烈自然过度表达已经使我们能够从甘蓝假单胞菌中纯化HMA4，在那里它可能参与将金属装载到木质部。此外，我们已经成功地对这种蛋白质进行了初步的生化和生物物理表征。我们现在想以这一经验为起点，详细研究它的生物化学和生物物理，并与HMA3进行比较。后者可能将Cd隔离到液泡存储位置。这项工作产生的关于P1B-ATPase功能的生化/生物物理机制的知识也将有助于理解这种ATPase在其他生物体中的功能，包括。人类(例如，ATP7A和ATP7B，其故障导致门克斯和威尔逊病)。2)我们将利用自行开发的定量mRNA原位杂交方法，在细胞水平上研究这些转运蛋白表达的转录调控。以前对其他蛋白质家族(CDF和ZIPs)转运蛋白的研究揭示了不同细胞类型、个体发生阶段和对矿物质营养的反应之间的重要差异，这对于总体上理解金属代谢是重要的。到目前为止，在P1B-ATPase上还没有这样的工作。

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.