Recipient Business Office and phone number: DOE Contract Administrator: Heavy metal toxicity poses major environmental and health problems, and heavy metals are more difficult to remediate than chemical contaminants, which can be degraded by microorganisms. Cadmium and arsenic, for example, are non-essential heavy metals which are toxic to living cells at very low concentrations. Cd 2+ ions displace Ca 2+ or Zn 2+ in proteins and can cause oxidative stress, while arsenic also causes oxidative

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... ress damage and is a well known carcinogen. Schizosaccharomyces pombe and other fungi as well as plants synthesize phytochelatins which chelate Cd 2+ , Cu 2+ and other heavy metal ions. Phytochelatins are thiolate peptides with the primary structure (-Glu-Cys) n -Gly, which are nontranslationally synthesized from glutathione. Phytochelatins play major roles in metal detoxification in plants and fungi and have been proposed to be central to phytoremediation of heavy metal contaminated soils and waters. We were interested in exploring this hypothesis, however, genes encoding phytochelatin synthases had not been identified until DOE-supported results in the P.I.'s lab and parallel research elsewhere (Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 1999) . By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression results in a dramatic increase in cadmium tolerance (Clemens et al, 1999) . TaPCS1 encodes a protein of ~55 kDa with no similarity to proteins of known function. We identified homologues of this new gene family from Arabidopsis and fungi. The Arabidopsis and fungal genes were also demonstrated to confer substantial increases in metal tolerance upon expression. PCS-expressing cells accumulate more Cd 2+ than controls. PCS expression mediates Cd 2+ tolerance even in mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS-induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Disruption in a fungal PCS gene results in hypersensitivity to Cd 2+ and Cu 2+ and inability to synthesize phytochelatins upon Cd 2+ exposure. Moreover, cells overexpressing PCS produce phytochelatins. These data demonstrate a central role for the PCS gene family in phytochelatin synthesis and metal detoxification in eukaryotes and suggests that PCS genes could be of central importance for engineering plants or other organisms for remediation of metal contaminated soils and waters. We are continuing to experiment with using PCS to increase the metal tolerance of plants. Another focus of this grant was to identify plant membrane proteins that can facilitate uptake of the toxic metal cadmium. We identified a first plant cDNA , named LCT1 (Schachtman et al., 1997) , that mediates uptake of the toxic metal cadmium (Clemens et al., 1998) . LCT1 was further shown to mediate uptake of calcium into yeast (Clemens et al., 1998) . A protocol was further prepared describing newly developed methods for identifying new genes that control plant metal sensitivity (Lee and Schroeder, in press). In further research we identified a different class of plant proteins that mediates uptake of toxic metals, the Arabidopsis Nramp proteins (Thomine et al., 2000) . Metal