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We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter)Several heavy metals are essential during plant growth and development, but their excess can easily lead to toxic effects. Contamination of soils with heavy metals, either by natural causes or due to pollution, often has pronounced effects on the vegetation, resulting in the appearance of metallophytes, heavy-metal-tolerant plants. The precise mechanisms of uptake, transport, and accumulation of heavy metals in plants are poorly understood, but several genes likely to be involved in these processes have been described. Recently, a family of ZIP genes that are expressed in roots upon Zn deficiency was isolated from Arabidopsis (Grotz et al., 1998). The proteins encoded by the ZIP genes have eight predicted TM regions and a high degree of similarity to the ZRT genes from yeast that are involved in Zn uptake. Expression of the ZIP genes in yeast conferred Zn-uptake activities to these cells, demonstrating that they are probably functional homologs of the yeast ZRT genes (Grotz et al., 1998). The only other metaltransporting protein recently identified in plants belongs to the large family of cation-transporting P-type ATPases (Tabata et al., 1997), but these proteins are structurally very different from the metal-transporting proteins mentioned above.Recent data have provided more insight into the mechanisms of heavy-metal tolerance. Metallophytes often exhibit tolerance to several different heavy metals, but all of these metals need not be present at toxic levels in their habitat (Schat and ten Bookum, 1992a; Schat and Vooijs, 1997, and refs. therein;Schat and Verkleij, 1998). Although such a feature is suggestive of a general mechanism of heavy-metal tolerance, recent genetic evidence has shown that a number of different mechanisms must exist, each with its own metal specificity (Schat and Vooijs, 1997). In Arabidopsis, a plant species with a typical level of tolerance to heavy metals, it has been demonstrated that the Cd-sensitive mutants cad1 and cad2 are defective in the synthesis of the metal-binding compound phytochelatin (Howden et al., 1995). cad1 plants were only slightly more sensitive to Cu and Zn, indicating that phytochelatinmediated detoxification is not sufficient for Cu and Zn detoxification (Howden et al., 1995b). Metallothioneins appear to be of major importance for constitutive Cu tolerance in Arabidopsis (Zhou and Goldsbrough, 1994).Aside from complexation of heavy metals by heavymetal-binding proteins, there is evidence that transportmediated sequestration can contribute to heavy-metal tolerance. In the Zn-tolerant plant Silene vulgaris it was shown that Zn transport across the tonoplast was about 2.5 times higher than in Zn-sensitive plants of the same species (Verkleij et al

The results presented in this paper describe the short-and long-term toxicity of arsenate in Silene vulgaris. Shortterm toxicity, measured as inhibition of root elongation, depended on phosphate nutrition, arsenate being much less toxic at high phosphate supply. At low phosphate levels more arsenic was taken up by the plants. Under chronic exposure, toxicity (measured as inhibition of biomass production) did not increase with time. In addition, the accumulation of phytochelatins (PCs) as a function of toxicity and duration of exposure was studied. Shortterm PC accumulation (over a 3 d period) was positively correlated with exposure. Isolation of peptide complexes from prolongedly exposed plants showed that PC # , PC $ and PC % were present, although the latter not until at least 3 d exposure. Arsenic co-eluted mainly with PC # and PC $ . Fractions containing PC % were devoid of As, probably due to dissociation of the complexes during extraction or elution. The breakdown of PCs after arresting As exposure was very slow. This could explain the continuous accumulation of PCs throughout longer periods of As exposure.Key words : Silene vulgaris, arsenic toxicity, arsenic detoxification, phytochelatins. Arsenic is taken up mainly by plant roots as arsenate (AsO % $−) (Macnair & Cumbes, 1987) through the phosphate-uptake system (Asher & Reay, 1979). Once the arsenate, As(V), is taken up it is reduced to arsenite, As(III), by glutathione (GSH) (Thompson, 1993). Only in phosphate-deficient conditions is arsenate subsequently methylated in plants (Nissen & Benson, 1982). Between the successive methylation steps, GSH serves to reduce the intermediate products (Scott et al., 1993 ;Thompson, 1993). Arsoniumphospholipids in freshwater plants (Nissen & Benson, 1982) and arsenic sugars in marine brown algae (Edmonds & Francesconi, 1981) have also been identified.Mostly there is little transport of As to the aboveground parts of the plants. Dicotyledonous plants appear to transport more As to the shoots than monocotyledonous plants (Otte, 1991). The form in *Author for correspondence (fax j31 20 444 7123 ; e-mail elsesnel!bio.vu.nl). which the As is transported is unknown. There is some indication that dimethylarsenic acid is transported to the shoots (Marin et al., 1993).Increased As levels may cause toxic symptoms in plants, such as a decrease in plant growth and fruit yield (Carbonell-Barrachina et al., 1995), root discoloration and root plasmolysis, wilting and necrosis of leaf tips and leaf margins (Machlis, 1941), and a decrease in photosynthetic capacity (Marin et al., 1993).Some authors have reported the accumulation of heavy metal-binding, thiol-rich phytochelatins (PCs) on exposure to As (Grill et al., 1986a(Grill et al., ,b, 1987Maitani et al., 1996). Phytochelatins have the structure (γ-glu-cys) n -gly, where n l 2-11 (Grill et al., 1985), and are produced in plants on exposure to a variety of heavy metals and metalloids (Gekeler et al., 1989). Phytochelatins are synthesized from GSH (Hayashi et ...

Phytochelatins (PCs) are a family of thiol-rich peptides, with the general structure (gamma-Glu-Cys)(n)()-Gly, with n = 2-11, induced in plants upon exposure to excessive amounts of heavy metals and some metalloids, such as arsenic. Two types of PC analyses are currently used, i.e., acid extraction and separation on HPLC with either precolumn derivatization (pH 8.2) with monobromobimane (mBBr) or postcolumn derivatization (pH 7.8) with Ellman's reagent [5, 5'-dithiobis(2-nitrobenzoic acid), DTNB]. Although both methods were satisfactory for analysis of Cd-induced PCs, formation of (RS)(3)-As complexes during extraction of As-induced PCs rendered the DTNB method useless. This paper shows that precolumn derivatization with mBBr, during which the (RS)(3)-As complexes are disrupted, provides a qualitative and quantitative analysis of both Cd- and As-induced PCs. In addition, derivatization efficiencies of both methods for the oligomers with n = 2-4 (PC(2)(-)(4)) are compared. Derivatization efficiency decreased from 71.8% and 81.4% for mBBr and DTNB derivatization, respectively, for PC(2) to 27.4% and 50.2% for PC(4). This decrease is most likely due to steric hindrance. Correction of measured thiol concentration is therefore advised for better quantification of PC concentrations in plant material.

The metabolism of sulphur in angiosperms is reviewed under the aspect of exposure to ecologically relevant concentrations of sulphur, heavy metals and metalloids. Because of the inconsistent use of the term 'metal tolerance', in this review the degree of tolerance to arsenic and heavy metals is divided into three categories: hypotolerance, basal tolerance and hypertolerance.The composition of nutrient solutions applied to physiological experiments let see that the well-known interactions of calcium, sulphate and zinc supply with uptake of heavy metals, especially cadmium are insufficiently considered. Expression of genes involved in reductive sulphate assimilation pathway and enzyme activities are stimulated by cadmium and partially by copper, but nearly not by other heavy metals. The synthesis of the sulphur-rich compounds glucosinolates, metallothioneins and phytochelatins is affected in a metal-specific way. Phytochelatin levels are low in all metal(loid)-hypertolerant plant species growing in the natural environment on metal(loid)-enriched soils.If laboratory experiments mimic the natural environments, especially high Zn/Cd ratios and good sulphur supply, and chemical analyses are extended to more mineral elements than the single metal(loid) under investigation, a better understanding of the impact of metal(loid)s on the sulphur metabolism can be achieved.Key-words: glucosinolates; glutathione; hypertolerance; hypotolerance; metallothionein; phytochelatin; zinc/ cadmium ratio.Abbreviations: EC50, effect concentration of a metal(loid) which diminishes plant performance by 50%, often measured as root elongation and/or plant biomass.