Overexpression of a novel Arabidopsis gene related to putative zinc-transporter genes from animals can lead to enhanced zinc resistance and accumulation

Abstract

We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter). The protein encoded by the ZAT (Zn transporter of Arabidopsis thaliana) gene has 398 amino acid residues and is predicted to have six membrane-spanning domains. To obtain evidence for the postulated function of the Arabidopsis gene, transgenic plants with the ZAT coding sequence under control of the cauliflower mosaic virus 35S promoter were analyzed. Plants obtained with ZAT in the sense orientation exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better understanding of the mechanism of Zn homeostasis and resistance in plants.

Figure 1

The Arabidopsis ZAT nucleotide sequence is shown with the amino acid sequence below (one letter code). ATG initiation and TAA termination codons are shown in boldface type. The cDNA sequence did not end in a poly(A⁺) tail, but occurred just after the putative polyadenylation signal fused with a second cDNA sequence (not shown). The XbaI site used for the C-terminal deletion just after the sixth TM domain (see Fig. 3) is indicated in boldface.

Figure 2

Dendrogram of the amino acid sequences of several (putative) Zn transporters. CzcD, Alcaligenes eutrophus (accession no. D67044); YrdO (CzcD), Bacillus subtilis (accession no. U93876 [U62055]); CzcD-Bst, Bacillus stearothermophilus (accession no. D87026); ZnT-2, Rattus norvegicus (accession no. S70632; Palmiter et al., 1996a); ZnT-3, Homo sapiens (accession no. U76010; Palmiter et al., 1996b); ZCE, C. elegans (accession no. T18D3.3 in Z68119); ZnT-4, H. sapiens (accession no. AF025409; Huang and Gitschier, 1997); ZAT, Arabidopsis (this study); COT1, Saccharomyces cerevisiae (accession no. P32798; Conklin et al., 1992); ZRC1, S. cerevisiae (accession no. P20107; Kamizono et al., 1989); ZRC-Sp, ZRC1 homolog, Schizosaccharomyces pombe (accession no. D89236); ZnT-1, R. norvegicus (accession no. S54303; Palmiter et al., 1995); COT-Ce, COT1 homolog in C. elegans (accession no. U23529). The distance between the branches on the horizontal axis are proportional to the degree of relatedness according to the program used, whereas the distances along the vertical axis are without any meaning.

Figure 3

Multiple alignment of representatives of (putative) Zn transporters most closely related to ZAT. The six putative TM regions are overlined. Amino acid residues that are identical in four or five of the sequences are shown in boldface. See legend to Figure 2 for further details.

Figure 4

Nothern blots of RNA isolated from seedlings treated in liquid medium with increasing concentrations of ZnSO₄ for 6 and 24 h. Lanes C, Control seedlings (30 μm Zn already present in the medium); lanes 0.25, 0.50, and 1.00, seedlings with different millimolar concentrations of extra ZnSO₄ added. Exposure was for 2 d at −70°C with an intensifying screen.

Figure 5

Zn-resistance test of representative Arabidopsis transgenic lines. C, Control; S, sense. Seedlings were germinated and grown on solid medium (one-half-strength Murashige-Skoog medium). 0.5 MS, Control seedlings (30 μm Zn already present in the medium); 0.25, 0.50, and 1.00, seedlings with different millimolar concentrations of extra ZnSO₄ added.

Figure 6

Arabidopsis root growth during 72 h of exposure to Zn in hydroponic culture. •, Control; ○, ZAT-AS; ▪, ZAT-S; □, ZAT-dC. Data points are the means of 15 plants. Vertical bars represent ±se.

Figure 7

Arabidopsis root Zn content after 72 h of exposure to Zn in hydroponic culture. •, Control; ○, ZAT-AS; ▪, ZAT-S; □, ZAT-dC. Data points are the means of five pooled samples of three plants each. Vertical bars represent ±se. DW, Dry weight.

Figure 8

Northern blots probed with strand-specific RNA probes. A, Antisense probe (showing sense mRNA). B, Sense probe (showing antisense RNA). C, Ethidium-bromide-stained total RNA present on the original gels. Lane C24, Nontransformed C24 ecotype; lane C, vector-transformed control; lane AS, antisense construct line; lane S, sense construct line; lane dC, C-terminal deletion construct line. Exposure was for 1 d without an intensifying screen.