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An allelic series of cadl, cadmium-sensitive mutants of Arabidopsis fhaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants.

Phytochelatins (PCs), a family of heavy metal-inducible peptides important in the detoxification of heavy metals, have been identified in plants and some microorganisms, including Schizosaccharomyces pombe, but not in animals. PCs are synthesized enzymatically from glutathione (GSH) by PC synthase in the presence of heavy metal ions. In Arabidopsis, the CAD1 gene, identified by using Cd-sensitive, PC-deficient cad1 mutants, has been proposed to encode PC synthase. Using a positional cloning strategy, we have isolated the CAD1 gene. Database searches identified a homologous gene in S. pombe, and a mutant with a targeted deletion of this gene was also Cd sensitive and PC deficient. Extracts of Escherichia coli cells expressing a CAD1 cDNA or the S. pombe gene catalyzing GSH-dependent, heavy metal-activated synthesis of PCs in vitro demonstrated that both genes encode PC synthase activity. Both enzymes were activated by a range of metal ions. In contrast, reverse transcription-polymerase chain reaction experiments showed that expression of the CAD1 mRNA is not influenced by the presence of Cd. A comparison of the two predicted amino acid sequences revealed a highly conserved N-terminal region, which is presumed to be the catalytic domain, and a variable C-terminal region containing multiple Cys residues, which is proposed to be involved in activation of the enzyme by metal ions. Interestingly, a similar gene was identified in the nematode, Caenorhabditis elegans, suggesting that PCs may also be expressed in some animal species.

The roots of the cadmium-sensitive mutant of Arabidopsis thaliana, cad7-7, become brown in the presence of cadmium. A new cadmium-sensitive mutant affected at a second locus, cad2, has been identified using this phenotype. Cenetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Assays of cadmium accumulation by intact plants indicated that the mutant is deficient in its ability to sequester cadmium. Undifferentiated callus tissue was also cadmium sensitive, suggesting that the mutant phenotype is expressed at the cellular level. The level of cadmium-binding complexes formed in vivo was decreased compared with the wild type and accumulation of phytochelatins was about 10% of that in the wild type. The level of glutathione, the substrate for phytochelatin biosynthesis, in tissues of the mutant was decreased to about 15 to 30% of that in the wild type. Thus, the deficiency in phytochelatin biosynthesis can be explained by a deficiency in glutathione.In plants and in some yeast species a class of inducible peptides, sometimes termed PCs, with the molecular structures (y-Glu-Cys),-Gly is known to play an important role in heavy-metal detoxification processes. The biosynthesis, structure, and function of PCs have been extensively reviewed (Rauser, 1990;Steffens, 1990). Their biosynthesis in vivo is rapidly induced in the presence of heavy metals (Grill et al., 1987) and appears to result from the activation by heavy metals of an enzyme, PC synthase, that synthesizes PCs from the substrate GSH (Grill et al., 1989). Use of BSO, an inhibitor of the enzyme y-Glu-Cys synthetase and thus of GSH biosynthesis, has provided strong evidence that this biosynthetic pathway is necessary for heavy-metal detoxification. The addition of BSO to cell cultures inhibits the biosynthesis of both GSH and PCs and increases the sensitivity of cells to added heavy metals. Furthermore, this effect can be reversed by the addition of GSH (Scheller et al., 1987;Mendum et al., 1990).

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