The relation between loss of glutathione due to metal-induced phytochelatin synthesis and oxidative stress was studied in the roots of copper-sensitive and tolerant SiIene cucubalus (L.) Wib., resistant to 1 and 40 micromolar Cu, respectively. The amount of nonprotein sulfhydryl compounds other than glutathione was taken as a measure of phytochelatins. At a supply of 20 micromolar Cu, which is toxic for sensitive plants only, phytochelatin synthesis and loss of total glutathione were observed only in sensitive plants within 6 h of exposure. When the plants were exposed to a range of copper concentrations for 3 d, a marked production of phytochelatins in sensitive plants was already observed at 0.5 micromolar Cu, whereas the production in tolerant plants was negligible at 40 micromolar or lower. The highest production in tolerant plants was only 40% of that in sensitive plants. In both varieties, the synthesis of phytochelatins was coupled to a loss of glutathione. Copper at toxic concentrations caused oxidative stress, as was evidenced by both the accumulation of lipid peroxidation products and a shift in the glutathione redox couple to a more oxidized state. Depletion of glutathione by pretreatment with buthionine sulfoximine significantly increased the oxidative damage by copper. At a comparably low glutathione level, cadmium had no effect on either lipid peroxidation or the glutathione redox couple in buthionine sulfoximinetreated plants. These results indicate that copper may specifically cause oxidative stress by depletion of the antioxidant glutathione due to phytochelatin synthesis. We conclude that copper tolerance in S. cucubalus does not depend on the production of phytochelatins but is related to the plant's ability to prevent glutathione depletion resulting from copper-induced phytochelatin production, e.g. by restricting its copper uptake.In plants, both essential and nonessential heavy metals induce the formation of thiol-rich peptides, (-y-glutamylcysteinyl),-glycines with n = 2 to 1 1, also known as metal-binding compounds or phytochelatins (8,26). Experiments with BSO,2 an inhibitor of y-glutamylcysteine synthetase, showed that glutathione serves as a precursor in the phytochelatin biosynthesis and that phytochelatins are involved in the detoxification of heavy metals in vivo (17,23,25 2Abbreviations: BSO, buthionine sulfoximine; TBA-rm: 2-thiobarbituric acid-reactive material; SH, sulfhydryl.lyzed by a specific y-glutamylcysteine dipeptidyl transpeptidase, called phytochelatin synthase, which is activated in the presence of metal ions and uses GSH as a substrate (9). Phytochelatins are the major if not the only thiol-rich compounds induced in metal-exposed plants (8,26), although it has been reported that copper induces metallothionein-like compounds as well (27). Phytochelatins probably play a central role in the homeostatic control of metal ions in plants (26). They may also be involved in the physiological mechanism of metal tolerance of selected cell lines and intact plants (4,12,22...