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 microg g(-1) Zn and 10,000 microg g(-1) 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 P1B 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.

Figure 1.

Sequence alignment of TcHMA4 from T. caerulescens and the Arabidopsis homolog AtHMA4. Deduced amino acid sequences for TcHMA4 and AtHMA4 (accession no. 064474) are shown aligned using the ClustalW method. Asterisks indicate identical residues. A number of motifs common to P_1B-type ATPases are indicated, including the E1-E2 ATPase phosphorylation site (shaded in dark gray), the highly conserved CPx motif (boxed), and a putative N-terminal heavy metal binding site (underlined). Also, the numerous His and Cys residues in the C terminus are also highlighted.

Figure 2.

Cd tolerance test for wild-type (transformed with empty pFL61 vector) and TcHMA4-transformed yeast cells. Yeast cells were grown to an OD₆₀₀ of 1.0, serially diluted to an OD₆₀₀ of 0.1, 0.01, and 0.001, and then 20-μL drops spotted on SD plates containing 0, 75, and 90 μm CdCl₂. WT, Wild type.

Figure 3.

Cd and Pb accumulation by wild-type yeast cells (transformed with the empty pFL61 vector) and TcHMA4-transformed yeast cells. A, Yeast Cd accumulation for two time periods (30 and 70 min) in liquid SD media supplemented with 20 μm CdCl₂. B, Yeast Pb accumulation for two time periods (30 and 70 min) in liquid SD media supplemented with 10 μm PbCl₂. The error bars represent the mean of four replicate measurements ± the se of the mean. WT, Wild type.

Figure 4.

Radiotracer (¹⁰⁹Cd) Cd influx and efflux in wild-type yeast cells (transformed with the empty pFL61 vector) and TcHMA4-transformed yeast cells. The influx and efflux data are presented as ¹⁰⁹Cd CPM/10⁶ cells/ min. The error bars represent the mean of four replicate measurements ± the se of the mean.

Figure 5.

Northern analysis of TcHMA4 in roots of T. caerulescens plants grown under Zn-deficient, -sufficient, and high-Zn conditions, or under high-Cd conditions. Seedlings were grown on hydroponic media containing 0, 1, 5, 10, and 100 μm ZnCl₂, or 10 and 100 μm CdCl₂. The 25S ribosomal bands are shown as loading controls. Each specific northern was repeated at least twice, with the same results.

Figure 6.

A, Amino acid sequence alignment of the 384- and 141-amino acid partial peptides from the C terminus of TcHMA4 that were identified from the initial yeast complementation screen. The conserved His stretch, numerous Cys repeats, and single His residues in the C-terminal region of the peptide are indicated in light gray. B, Schematic representation of location of two partial peptides sequences relative to each other and to the full-length TcHMA4 protein.

Figure 7.

Cd tolerance test for yeast expressing the partial TcHMA4 peptides. Yeast cells were transformed either with the empty pFL61 vector (WT) or with the 384- or 141-amino acid peptides (see Fig. 6 for their sequence). The plates were set up as described in the legend for Figure 2. Metal tolerance was assayed by visualizing growth on SD plates containing 100 and 200 μm CdCl₂.

Figure 8.

Cd accumulation by yeast expressing the empty pFL61 vector (WT), compared with yeast expressing the 384- and 141-amino acid partial peptides from TcHMA4. Cd accumulation was conducted in liquid SD media supplemented with 20 μm CdCl₂. The error bars represent the mean of four replicate measurements ± the se of the mean.