Identification of Thlaspi caerulescens Genes That May Be Involved in Heavy Metal Hyperaccumulation and Tolerance. Characterization of a Novel Heavy Metal Transporting ATPase

Abstract

Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 mg g 21 Zn and 10,000 mg g 21 Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils. In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T. caerulescens. A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P 1B subfamily of purported heavy metal-translocating ATPases. A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead [Pb], and copper [Cu]) out of the cell. However, in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance. Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens. Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding. When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation. The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.There are a small number of terrestrial plant species that not only can tolerate high levels of toxic heavy metals in the soil but also can accumulate those metals to unusually high levels in their shoot biomass. These fascinating plant species, first coined hyperaccumulators by Brooks et al. (1977), are loosely categorized as plants that can accumulate metals in the shoot from 100-to 1,000-fold higher than normal, nonaccumulator plants (McGrath et al., 2002). Hyperaccumulating plant species have been identified for a number of heavy metals, including nickel (Ni), zinc (Zn), and cadmium (Cd), as well as for the metalloids selenium and arsenic. Probably the best known metal hyperaccumulator is Thlaspi caerulescens, a member of the Brassica family that has been the object of interest in the plant biology community for over a century, based on its ability to colonize calamine and serpentine soils containing naturally elevated levels of heavy metals...

Transition metal transport

“…In A. halleri, HMA4 proteins are encoded by more than one gene copy [44]. TcHMA4 was previously identified through screening of cDNA expression libraries for clones conferring Cd tolerance to yeast [56,57] and was shown to mediate the efflux of Cd from yeast cells [56]. In both hyperaccumulator species, HMA4 expression is approximately 2-3-fold higher in roots than in shoots.…”

“…In A. halleri, expression levels appear to be relatively constant over different external Zn concentrations. In T. caerulescens roots, HMA4 transcript levels are increased in response to high Zn and high Cd, and also under Zn deficiency [44,56,57].…”

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant Thlaspi caerulescens

“…The gene encoding TcHMA4 was originally isolated using functional complementation in yeast under Cd exposure ). The expression of TcHMA4 is induced in the roots by elevated Zn and Cd as well as by Zn deWciency, and it is proposed to play a role in the xylem loading of Zn in plant roots (Papoyan and Kochian 2004).…”

Characterization of a cDNA from Beta maritima that confers nickel tolerance in yeast

“…Recent studies on manganese tolerance clearly show the robustness of cDNA library based studies to identify heavy metal detoxification genes in plant species (Chen et al, 2013;Erbasol et al, 2013;Papoyan and Kochian, 2004;Pence et al, 2000). Apart from that, these studies have also shown the evolutionary importance and convergence of the secretory pathway proteins' significant involvement on the heavy metal detoxification (Chen et al, 2013;Erbasol et al, 2013;Peiter et al, 2007).…”