Transport and detoxification of cadmium, copper and zinc in the Cd/Zn hyperaccumulator plant Thlaspi caerulescens

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

Summary
In 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 caerulescens

First, 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 detoxification

Not 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.

Zusammenfassung in einer weiteren Sprache

In der hier vorliegenden Arbeit wurden verschiedene Aspekte der Metal-Akkumulation durch die Hyperakkumulatorpflanze Thlaspi caerulescens untersucht. Thlaspi caerulescens gehört zur Familie der Brassicaceen und hyperakkumuliert Zink. Der Ökotyp Ganges, entdeckt auf dem Gebiet einer ehemaligen Zinkmine in Südfrankreich, nimmt zusätzlich auch aktiv Cadmium auf. Aus früheren Studien ist bekannt, dass Hyperakkumulatoren Metalltransporter überexprimieren und dass der Großteil dieser Pflanzen das aufgenommene Metall in den Vakuolen von großen Epidermiszellen speichern.


Cd Akklimation und Translokation in Thlaspi caerulescens

Zunächst wurde die Langzeit-Reaktion von T. caerulescens auf Cadmium untersucht. Dazu wurden Pflanzen sechs Monate lang auf einer Nährlösung, die Cadmium beinhaltete, angezogen. Zuerst zeigten die Pflanzen Vergiftungssymptome wie eine Gelbfärbung der Blätter, wuchsen aber weiter. Nach zwei Monaten des Wachstums begannen die Pflanzen sich zu akklimatisieren und die Vergiftungssymptome verschwanden nahezu vollständig. Mithilfe von Chlorophyll-Fluoreszenz-Kinetik Messungen konnte gezeigt werden, dass während der Akklimatisierung nicht alle Zellen gleich von Toxizität betroffen waren. Die Verteilung von Cadmium in den Blättern war heterogen, manche Mesophyllzellen nahmen mehr Cadmium auf als andere. Mit der Einlagerung des Metalls in die Vakuole verschwand diese Heterogenität langsam. Desweiteren wurde entdeckt, dass Cadmium die photosynthetischen Lichtreaktionen stärker hemmt als den Calvin-Zyklus und dass es im/am Photosystem II mindestens zwei verschiedene Angriffsstellen für Cadmium gibt.
Mit Hilfe eines für Cadmium spezifischen Fluoreszenzfarbstoffes und Protoplasten aus Thlaspi Blättern konnte die Aufnahme von Cadmium in Mesophyll- sowie große und kleinere Epidermiszellen gezeigt werden. Die Aufnahmeraten in große Epidermiszellen waren dabei deutlich höher als die Aufnahmeraten, die für die beiden anderen Zelltypen gemessen wurden. Nachdem für die Messungen isolierte Protoplasten verwendet wurden ist es offensichtlich, dass diese Unterschiede nicht durch den Transpirationsstrom herbeigeführt werden können sondern durch eine unterschiedliche Expression von Metalltransportern in den verschiedenen Zelltypen zustande kommen.
Kurz nach der Zugabe von Cadmium zum Messmedium erschien ein heller Ring an der Innenseite der Zellen und war dort eine Weile lang zu beobachten. Erst im weiteren Verlauf wurde langsam die gesamte Zelle hell, was zeigte, dass die Translokation von Cadmium in die Vakuole ein zeitlimitierender Schritt bei der Hyperakkumulation in T. caerulescens ist.

Metalltransport und Detoxifizierung

Bis heute ist nicht viel bekannt über Metalltransporter in Pflanzen, insbesondere nicht über metall-transportierende ATPasen. Nachdem ATPasen eine wichtige Rolle in der Hyperakkumulation spielen, wurde TcHMA4, eine P1B-Typ ATPase, die in die Metallbeladung des Xylems involviert sein soll, aus Thlaspi-Wurzeln aufgereinigt und charakterisiert. Weil das Protein sehr reich an Cysteinen ist, war die Instabilität des aufgereinigten Proteins hier ein großes Problem. Daher mussten sämtliche Experimente zur Charakterisierung sofort nach der Reinigung durchgeführt werden und für Wiederholungen und neue Experimente musste frisches Protein gereingt werden. Die Identität und Reinheit der jeweiligen Proteincharge wurde mittels SDS Gelen und Western Blots überprüft und es wurden ATPase-Aktivitätstests in der Gegenwart von verschiedenen Metallen in unterschiedlichen Konzentrationen durchgeführt. Diese zeigten, dass TcHMA4 nicht nur von Zink und Cadmium, sondern auch von Kupfer aktiviert wird. Auch mit der höchsten Konzentration (10µM) an Zink und Cadmium war die ATPase noch aktivierbar, während die absolute Phosphatkonzentration mit Zugabe von 3µM Kupfer bereits abnahm. Dies ist vermutlich nicht durch eine Hemmung zu erklären, sondern durch ein Gleichgewicht zwischen ATPase- und ATP-Synthase-Aktivität. Da auch die Temperaturabhängigkeit der Proteinaktivität gemessen wurde, konnte mittels Arrheniusgraphen die Aktivierungsenergie in der Gegenwart von Metall bestimmt werden. TcHMA4 zeigte in der Gegenwart von Zink keinerlei Änderungen der Aktivierungsenergie, während sie mit ansteigender Kupferkonzentration zunahm.

Mit der Anwendung von Röntgenabsorptions-Spektroskopie (EXAFS) an proteingebundenem Cadmium konnte gezeigt werden, dass Cadmium in TcHMA4 hauptsächlich von Schwefel, also von Cysteinen gebunden wird, und zu einem weitaus geringeren Anteil von Histidin. Dies war ein sehr interessanter Befund, da in der Vergangenheit immer wieder diskutiert wurde, welchen Beitrag Histidin zur Bindung von Cadmium in TcHMA4 leisten könnte.

EXAFS wurde auch für die Kupferanalyse in gefrorenen Blättern von T. caerulescens verwendet. Eine wichtige Entdeckung war hierbei, dass sich innerhalb einer Population einige wenige Individuen als kupferresistent erwiesen, während der Großteil der Pflanzen sehr empfindlich auf Kupferzugabe reagierte. Besonders bei den resistenten Individuen wurde eine Interaktion von Kupfer mit anderen Kupferatomen entdeckt, die auf Biomineralisation als Cu-Oxalat hinweist. Dieses Mineral wurde bereits in früheren Arbeiten an Pilzen nachgewiesen. Zusätzlich wurde in allen Pflanzen, etwas verstärkt in den resistenten Individuen, ein starkes Schwefelsignal festgestellt, welches auf Metallothionein als Kupferligand hinweist. Dies war ein weiterer interessanter Befund, da sowohl Zink als auch Cadmium in T. caerulescens hauptsächlich von Sauerstoffliganden gebunden werden und nicht von Metallothioneinen. Hier zeigt sich ein weiteres Mal die erstaunliche Fähigkeit von Hyperakkumulatoren, zwischen dem hyperakkumulierten Metall und nicht-akkumulierten, manchmal sogar toxischen Metallen, zu unterscheiden.

Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Hyperakkumulation
Konferenz
Rezension
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Zitieren
ISO 690LEITENMAIER, Barbara, 2010. Transport and detoxification of cadmium, copper and zinc in the Cd/Zn hyperaccumulator plant Thlaspi caerulescens [Dissertation]. Konstanz: University of Konstanz
BibTex
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  year={2010},
  title={Transport and detoxification of cadmium, copper and zinc in the Cd/Zn hyperaccumulator plant Thlaspi caerulescens},
  author={Leitenmaier, Barbara},
  address={Konstanz},
  school={Universität Konstanz}
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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.&lt;br /&gt;&lt;br /&gt;&lt;br /&gt;Metal transport and detoxification&lt;br /&gt;&lt;br /&gt;Not 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. 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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.&lt;br /&gt;&lt;br /&gt;EXAFS has also been used for the analysis of copper in frozen leaf tissue of T. caerulescens. 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