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Thlaspi
caerulescens EST sequence analysis Mark G.M. Aarts1, Diana Rigola1, Peter Goossens2, Martijn Fiers2, Ana Assunção3 and Henk Schat3. 1Laboratory of Genetics, Wageningen University, Postbus 9101, 6700 HA Wageningen, The Netherlands; 2Plant Research International, Wageningen, The Netherlands; 3Dept. of Plant Ecology and Ecotoxicology, Vrije Universiteit, Amsterdam, The Netherlands. Thlaspi caerulescens is a well-studied natural heavy metal hyperaccumulating species, of which several accessions are known. The accession "La Calamine" (LC) originates from a calamine ore waste in Belgium. It accumulates zinc and cadmium and is also highly tolerant to these metal species. Accession "Monte Prinzera" (MP), originating from serpentine soils in Italy and is adapted to nickel exposure, whereas accession "Lellingen" (LE) was collected from non-metalliferous soil in Luxembourg. The genetic origin underlying and controlling heavy metal hyperaccumulation in plants is still largely unknown, but T. caerulescens appears to be a suitable species to study this phenomenon conveniently. In a previous study (Assunção et al., 2001), we identified three different metal transporters in T. caerulescens, with homologies to the ZIP and ZAT-type of metal transporters identified in Arabidopsis thaliana. These three genes were much higher expressed, even under elevated zinc-exposure conditions, in T. caerulescens, compared to the expression of their orthologues in the non-accumulating T. arvense and A. thaliana. This prompted us to see if there were other T. caerulescens cDNAs which were expressed at higher levels compared to their orthologues. In a pilot experiment, 106 random cDNA clones were picked from a LC root library (grown at 10 µM Zn2+) and their partial 5’ DNA sequence was determined. A relatively large portion of these showed homology to Arabidopsis genes which were at the time never reported to be expressed in Arabidopsis. Recently a new EU-project called PHYTAC has been started for which another 5000 ESTs will be determined. In order to perform genetic analyses of metal accumulation and tolerance traits reciprocal crosses have been made between the mentioned accessions. F3 populations are currently constructed and phenotyped. These populations will provide valuable material for the identification of intraspecific genetic variation contributing to zinc, cadmium or nickel tolerance and accumulation. Progress on the in silico analysis of EST sequences and the genetic analysis of populations will be presented and discussed.
The Arabidopsis genome contains seven genes encoding low molecular weight, cysteine-rich metallothionein (MT) proteins. These MTs can be divided into 4 classes based on amino acid sequence and similar MT gene families have been identified in other plants. The metal binding properties of MT proteins suggest they may play a role in metal ion homeostasis. Analysis of MT gene expression indicates that MTs are expressed in a variety of tissues including the phloem, suggesting that MTs may be involved in metal transport. Translational fusions between GFP and MTs are being used to further examine this possibility. MT gene expression is also induced during leaf senescence and in response to copper treatment and oxidative stress. Experiments are in progress to define the promoter elements responsible for copper-regulated expression of MT genes. Transgenic plants with reduced expression of MT genes have been developed. Some of these show increased sensitivity to copper, as would be predicted if MTs function in copper homeostasis. These lines also have reduced growth on media containing the copper chelator bathocuproinedisulfonic acid. These results suggest that plant MTs not only protect against copper toxicity but also serve as a reservoir for the metal under conditions of copper deficiency. Investigating the molecular physiology
of heavy metal accumulation and tolerance using Thlaspi caerulescens
as a model system Our research program has been focusing on the molecular biology and physiology of heavy metal transport and tolerance in the Zn/Cd hyperaccumulating plant species, Thlaspi caerulescens, which can accumulate extraordinarily high levels of Zn in the shoot (up to 3% Zn dry wt. without any toxicity symptoms). Physiological investigations of Thlaspi Zn (65Zn2+) transport have demonstrated that a number of Zn transport sites are stimulated or altered in T. caerulescens compared with a related nonaccumulator, T. arvense, contributing to the hyperaccumulation trait. The transport sites that were stimulated or altered include Zn influx into both root and leaf cells, Zn sequestration in root-cell vacuoles, and Zn loading into the xylem. Molecular studies have focused on the cloning and characterization of Zn transport genes in T. caerulescens. Complementation of a yeast Zn transport-defective mutant with a T. caerulescens cDNA library resulted in the cloning of a Zn transport cDNA, ZNT1. Sequence analysis of ZNT1 indicated it is a member of the ZIP family of micronutrient transporters. Expression of ZNT1 in yeast allowed for a physiological characterization of this transporter. It was shown to encode a high affinity Zn transporter that can also mediate low affinity Cd transport. Northern analysis of ZNT1 and its homologue in the two Thlaspi species indicated that this transporter is expressed at very high levels in roots and shoots of the hyperaccumulator. A study of ZNT1 expression and high affinity Zn2+ uptake in roots of the two Thlaspi species showed that alteration(s) in the regulation of ZNT1 gene expression by plant Zn status results in the over expression of this transporter and increased root and shoot Zn influx. Subsequent expression analysis using other members of the ZIP gene family, as well as other micronutrient transporter genes and other non-transporter genes implicated in heavy metal homeostasis in plants reveal a pattern of overexpression in T. caerulescens which is similar to that for ZNT1. This altered expression of a suite of genes relating to heavy metal transport/homeostasis in response to Zn status suggests that the Zn-dependent regulation of gene expression is an integral component of Zn hyperaccumulation in T. caerulescens. Thus, we are currently focusing efforts on the elucidation of the components of this Zn-dependent pathway of gene regulation in Thlaspi. Heavy metal and metalloid hyperaccumulating
plants: physiology and potential for phytoremediation This paper focuses on the physiology and phytoremediation potential of heavy metal and metalloid hyperaccumulator plants. Thlaspi caerulescens and Arabidopsis halleri are able to accumulate up to 30,000 mg Zn kg-1 in the shoot dry matter without suffering from phytotoxicity, and Pteris vitatta up to 10,000 mg As kg-1 in fronds. In T. caerulescens leaves, Zn and Cd are detoxified by preferential sequestration in the epidermal vacuoles, and in A. halleri in trichomes and mesophyll cells. Both species also accumulate Cd. However, there is a marked difference between different ecotypes of T. caerulescens in Cd accumulation, suggesting that the mechanism of Cd uptake may differ from that of Zn. We have studied the ecotypic differences in terms of Cd and Zn uptake kinetics and expression of metal transporter genes, and the results will be presented. The results suggest that there may exist a high-affinity Cd transporter in the high Cd accumulating ecotype, and there is substantial scope to screen and select efficient ecotypes of metal hyperaccumulators. We have also studied the rhizosphere processes associated with Zn/Cd hyperaccumulation in T. caerulescens, and found that root exudates and rhizosphere acidification were not involved. However, it has been shown that T. caerulescens was able to proliferate its roots in metal-rich zones, thus actively foraging metals from the soil. In P. vittata we found that arsenate was taken up by via the phosphate transporters, and reduced to arsenite before being transported to and sequestered in the fronds. Testing strategies for the engineered
phytoremediation of toxic mercury and arsenic pollution Mercury and arsenic contamination of the environment are serious problems resulting from a variety of industrial, mining, and agricultural practices. These toxic elemental pollutants affect the health of hundreds of millions of people world-wide. Elemental pollutants are a particular problem because they are immutable and cannot be chemically degraded like organic pollutants. Physical cleanup methods like excavation and reburial of contaminated sediments are too expensive and environmentally destructive to be used on the scale needed to clean the tens of thousands of polluted sites that exist world-wide. Plants can be used to extract, detoxify, and/or sequester toxic pollutants from soil, water, and air, in a process called phytoremediation. Our long-term goal is to engineer highly productive conservation plant species for the phytoremediation of toxic heavy metals and metalloids. We have developed strategies for the efficient phytoremediation of elemental pollutants like mercury and arsenic that are based on controlling their electrochemical state, chemical species, toxicity, transport, and aboveground sinks for sequestration. An initial strategy has been tested for mercury using a variety of bacterial and plant genes in the model plants Arabidopsis and tobacco. We have engineered plants to detoxify both methylmercury and ionic mercury and either volatilize the least toxic form metallic mercury or sequester ionic mercury in peptide thiol-complexes abovegound. Some of the steps in the process have been re-tested in potential field species like cottonwood, yellow poplar, rice, and canola. For arsenic a different strategy is being exployed, which involves blocking endogenous arsenate reduction in roots, reducing arsenate in leaves, and trapping arsenite aboveground in leaves for later harvest. Initial data on engineered arsenic remediation in model plants are vary promising. Plants have been engineered which resist five times higher levels of arsenic and accumulate three times more arsenic aboveground than controls. These genes are now being transformed into larger plants to prepare for eventual field testing and site remediation.
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Illustrations: Heavy Metal Plant cartoon by Sam
Day. Arabidopsis thaliana - the model plant (Philip Rea). Micrograph
of the leaf surface of the Ni-hyperaccumulator Alyssum lesbiacum (Ute
Kraemer). Arabidopsis halleri growing at the bottom of a heap of minewaste
(Ute Kraemer) Last updated: February 18, 2003 |
