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

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

Compartmentation and complexation of metals in hyperaccumulator plants

Hyperaccumulators are being intensely investigated. They are not only interesting in scientific context due to their “strange” behavior in terms of dealing with high concentrations of metals, but also because of their use in phytoremediation and phytomining, for which understanding the mechanisms of hyperaccumulation is crucial. Hyperaccumulators naturally use metal accumulation as a defense against herbivores and pathogens, and therefore deal with accumulated metals in very specific ways of complexation and compartmentation, different from non-hyperaccumulator plants and also non-hyperaccumulated metals. For example, in contrast to non-hyperaccumulators, in hyperaccumulators even the classical phytochelatin-inducing metal, cadmium, is predominantly not bound by such sulfur ligands, but only by weak oxygen ligands. This applies to all hyperaccumulated metals investigated so far, as well as hyperaccumulation of the metalloid arsenic. Stronger ligands, as they have been shown to complex metals in non-hyperaccumulators, are in hyperaccumulators used for transient binding during transport to the storage sites (e.g., nicotianamine) and possibly for export of Cu in Cd/Zn hyperaccumulators [metallothioneins (MTs)]. This confirmed that enhanced active metal transport, and not metal complexation, is the key mechanism of hyperaccumulation. Hyperaccumulators tolerate the high amount of accumulated heavy metals by sequestering them into vacuoles, usually in large storage cells of the epidermis. This is mediated by strongly elevated expression of specific transport proteins in various tissues from metal uptake in the shoots up to the storage sites in the leaf epidermis. However, this mechanism seems to be very metal specific. Non-hyperaccumulated metals in hyperaccumulators seem to be dealt with like in non-hyperaccumulator plants, i.e., detoxified by binding to strong ligands such as MTs

Cadmium uptake and sequestration kinetics in individual leaf cell protoplasts of the Cd/Zn hyperaccumulator Thlaspi caerulescens

Hyperaccumulators store accumulated metals in the vacuoles of large leaf epidermal cells (storage cells). For investigating cadmium uptake, we incubated protoplasts obtained from leaves of Thlaspi caerulescens (Ganges ecotype) with a Cd-specific fluorescent dye. A fluorescence kinetic microscope was used for selectively measuring Cd-uptake and photosynthesis in different cell types, so that physical separation of cell types was not necessary. Few minutes after its addition, cadmium accumulated in the cytoplasm before its transport into the vacuole. This demonstrated that vacuolar sequestration is the rate-limiting step in cadmium uptake into protoplasts of all leaf cell types. During accumulation in the cytoplasm, Cd-rich vesicle-like structures were observed. Cd uptake rates into epidermal storage cells were higher than into standard-sized epidermal cells and mesophyll cells. This shows that the preferential heavy metal accumulation in epidermal storage cells, previously observed for several metals in intact leaves of various hyperaccumulator species, is due to differences in active metal transport and not differences in passive mechanisms like transpiration stream transport or cell wall adhesion. Combining this with previous studies, it seems likely that the transport steps over the plasma and tonoplast membranes of leaf epidermal storage cells are driving forces behind the hyperaccumulation phenotype

Reversible coupling of individual phycobiliprotein isoforms during state transitions in the cyanobacterium Trichodesmium analysed by single-cell fluorescence kinetic measurements

In the non-heterocyst. marine cyanobacterium Trichodesmium nitrogen fixation is con fined to the photoperiod and occurs coevally with oxygenic photosynthesis although nitrogenase is irreversibly inactivated by oxygen. In previous studies it was found that regulation of photosynthesis for nitrogen fixation involves Mehler reaction and various activity states with reversible coupling of photosynthetic components. We now investigated these activity states in more detail. Spectrally resolved fluorescence kinetic measurements of single cells revealed that they were related to alternate uncoupling and coupling of phycobilisomes from and to the photosystems. changing the effective cross-section of PSI!. Therefore, we isolated and purified the phycobiliproteins of Trichodesmium via ion exchange chromatography and recorded their UV/VIS absorption. fluorescence excitation and fluorescence emission spectra. After describing these spectra by mathematical equations via the Gauss-Peak-Spectra method. we used them to deconvolute the in vivo fluorescence spectra of richodesmium cells. This revealed that the contribution of different parts of the phycobilisome antenna to fluorescence quenching changed during the daily activity cycle. and that individual phycobiliproteins can be reversibly coupled to the photosystems, while the expression levels of these proteins did not change much during the daily activity cycle. Thus we propose that variable phycobilisome coupling plays a key role in the regulation of photosynthesis for nitrogen fixation in Trichodesmium