Transport und Entgiftung von Cadmium, Kupfer und Zink im Cd/Zn Hyperakkumulator Thlaspi caerulescens

Abstract

SummaryIn this thesis, various aspects on heavy metal accumulation by the hyperaccumulator plant Thlaspi caerulescens have been investigated. T. caerulescens belongs to the family of Brassicaceae and hyperaccumulates zinc. Its ecotype Ganges, originating from Southern France, additionally takes up cadmium actively. It is known from previous studies that hyperaccumulators have highly overexpressed metal transporters and that most of them store the metal in the vacuole of large epidermal cells.Cd acclimation and sequestration in Thlaspi caerulescensFirst, the long-term behaviour of T. caerulescens upon cadmium treatment has been studied. For this purpose, plants were grown for six months on a nutrient solution containing elevated concentrations of cadmium. First, they showed toxicity symptoms like yellowing of leaves, but continued growing. After two months, the plants started to acclimate and toxicity symptoms almost disappeared. Using chlorophyll fluorescence kinetic measurements it has been shown that during acclimation, not all cells are affected by cadmium. The distribution of cadmium within the leaves was heterogenous, some mesophyll cells took up much more metal than others. Slowly this heterogenity disappeared with the metal being sequestred into epidermal vacuoles. The study also showed that cadmium inhibits the photosynthetic light reactions more than the Calvin-Benson cycle and that at least two different targets in/around photosystem II are affected by cadmium. Using a fluorescent dye specific for cadmium and protoplasts from Thlaspi leaves, we were able to show cadmium uptake into mesophyll cells as well as normal sized and storage epidermal cells. The uptake rates into storage cells were significantly higher than the uptake rates into mesophyll or normal sized epidermal cells. This shows that the differential accumulation in leaf tissues is not due to differences in cell walls or transpiration stream (absent in protoplasts), but different expression levels of transport proteins. Shortly after addition of cadmium to the measuring medium, a bright ring inside the cells appeared and stayed there for some time. Very slowly the whole cell became bright, showing that the sequestration from the cytoplasm into the vacuole is one time limiting step in cadmium hyperaccumulation in T. caerulescens.Metal transport and detoxificationNot much is known about metal transporters in plants in general and about metal transporting ATPases in particular. As metal ATPases play an important role in hyperaccumulation, TcHMA4, a P1B-type ATPase that is suggested to pump cadmium and zinc out of root cells into the xylem, has been isolated and purified from T. caerulescens roots. As the protein is naturally rich in cysteins, stability was a major problem once the protein had been purified. Therefore, all characterisation steps had to be performed immediately after purification and for each new data set, fresh protein had to be purified. Identity and puritiy have been confirmed by SDS gels and western blots. ATPase activity assays in the presence of various metals in different concentrations have been conducted. These showed that TcHMA4 is not only acitivated by zinc and cadmium, but also by copper. Nevertheless, with cadmium and zinc up to a concentration of 10µM the ATPase acitivity was increased while using 3µM of copper, the absolute phosphate concentration generated by TcHMA4 decreased slightly. This suggests that not only ATPase activity, but also ATP synthase activity can be increased by addition of copper yielding an equilibrium of hydrolysis and synthesis of ATP. As also the temperature dependence of activity has been measured, it was possible to determine the energy of activation for different metals and concentrations using Arrhenius plots. TcHMA4 did not show any changes in activation energy in the presence of different concentrations of zinc. Towards higher concentrations of copper, the activation energy increased. Performing extended x-ray absorption fine structure (EXAFS) measurements on cadmium bound to the protein, the fourier transformed data showed a peak characteristic for sulfur. This suggests that cadmium in TcHMA4 is mainly bound to cysteins and less to histidine, which is also present in the sequence and has been discussed in several articles to be involved in metal binding in the protein.EXAFS has also been used for the analysis of copper in frozen leaf tissue of T. caerulescens. A very important finding was that within a population of T. caerulescens, a few individuals seem to be resistent to copper, while the majority of Thlaspi plants reacts very sensitively upon copper treatment. An interaction of copper with other copper atoms has been found, suggesting biomineralisation, a phenomenon that has been reported earlier for fungi. Additionally, all of our plants, especially the resistent ones, showed a high sulfur signal. The sulfur signal was most likely due to metallothioneins. This was a very interesting finding as in T. caerulescens, zinc and cadmium are both mainly bound by oxygen ligands and not by metallothioneins. Our finding once again shows how clearly hyperaccumulator plants can distinguish between a hyperaccumulated and a non-hyperaccumulated, probably even toxic, metal