Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency

Abstract

Millions of people worldwide suffer from nutritional imbalances of essential metals like zinc. These same metals, along with pollutants like cadmium and lead, contaminate soils at many sites around the world. In addition to posing a threat to human health, these metals can poison plants, livestock, and wildlife. Deciphering how metals are absorbed, transported, and incorporated as protein cofactors may help solve both of these problems. For example, edible plants could be engineered to serve as better dietary sources of metal nutrients, and other plant species could be tailored to remove metal ions from contaminated soils. We report here the cloning of the first zinc transporter genes from plants, the ZIP1, ZIP2, and ZIP3 genes of Arabidopsis thaliana. Expression in yeast of these closely related genes confers zinc uptake activities. In the plant, ZIP1 and ZIP3 are expressed in roots in response to zinc deficiency, suggesting that they transport zinc from the soil into the plant. Although expression of ZIP2 has not been detected, a fourth related Arabidopsis gene identified by genome sequencing, ZIP4, is induced in both shoots and roots of zinc-limited plants. Thus, ZIP4 may transport zinc intracellularly or between plant tissues. These ZIP proteins define a family of metal ion transporters that are found in plants, protozoa, fungi, invertebrates, and vertebrates, making it now possible to address questions of metal ion accumulation and homeostasis in diverse organisms.

Figure 1

Alignment of the amino acid sequences of seven full length Arabidopsis and Saccharomyces ZIP proteins (IRT1, ZIP1, ZIP2, ZIP3, ZIP4, ZRT1, and ZRT2). The eight transmembrane domains predicted for each these proteins are numbered I–VIII. Residues highlighted in blue are identical, and residues highlighted in green represent conservative substitutions. The histidines found in the variable region between transmembrane domains III and IV are highlighted in gold. Residues highlighted in pink are conserved histidines or acidic residues found within predicted transmembrane helices.

Figure 2

Biochemical properties of zinc uptake in yeast expressing the ZIP1, ZIP2, or ZIP3 genes. zrt1 zrt2 (ZHY3) transformants expressing ZIP1, ZIP2, or ZIP3 were grown to exponential phase (≈5 × 10⁶ cells/ml) in synthetic-defined medium and assayed for zinc uptake with ⁶⁵Zn. Because of their different pH optima for uptake activity, the ZIP1 and ZIP3 activities were assayed at pH 4.7, and ZIP2 activity was assayed at pH 6.0. (A–C) Time and temperature dependence of zinc accumulation assayed with 10 μM (ZIP1, ZIP3) or 1 μM ⁶⁵Zn (ZIP2) at 0°C (open symbols) and at 30°C (closed symbols). (D–F) Concentration dependence of zinc uptake was determined by measuring zinc accumulation for 5 min over a range of substrate concentrations. Each point in A–F represents the mean of two experiments each performed in duplicate, and the error bars represent ±1 SD. (G–I) Inhibition of ZIP-dependent uptake in yeast by other metals. Cells were assayed for zinc uptake rate with 10 μM (ZIP1, ZIP3) or 1 μM (ZIP2) ⁶⁵Zn in the absence (−) or presence of 10-fold excess of the chloride salts of the indicated metal ions. Each value represents the mean of four replicates. The asterisks indicate significant differences from control values (P < 0.05) as determined by one-way ANOVA followed by Scheffe’s test.

Figure 3

Regulation of ZIP1, ZIP3, and ZIP4 mRNA levels by zinc availability. Gene probes were hybridized to a Northern blot containing 1 μg of poly(A) mRNA prepared from the roots or shoots of hydroponically grown plants. The same blot was used sequentially (after stripping) for hybridization to each of the gene probes.

Figure 4

Dendrogram showing amino acid sequence relationships among the ZIP family members. GenBank accession numbers for sequences designated by name are as follows: IRT1, U27590; IRT2, T04324; ZIP4, U95973; IRT3, M35868; ZRT1, P32804; and ZRT2, X91258. The amino acid sequence of human 998569 is only available in the Entrez database. The Oryza sativa D49213, IRT2, IRT3, and Homo sapiens H20615 clones have not been sequenced completely.