Effects of Buthionine Sulfoximine on Cd-Binding Peptide Levels in Suspension-Cultured Tobacco Cells Treated with Cd, Zn, or Cu

Abstract

ABSTRACThaving small size and similar amino acid composition in fission yeast and showed that they contained 2 or 3 pairs of Glu-Cys groups attached to a single Gly residue. In addition, they demonstrated that the Gly-Cys pairs were not bound through standard peptide linkages, but were attached through the -y-carboxyl group of Glu residues, in a manner analogous to glutathione. Recent work by Grill et al. (6) using different methods for primary sequence determination showed that peptides (termed phytochelatins) formed by Rauvolfia cultured cells in the presence of Cd are structurally identical to those of fission yeast. Direct comparison of the properties of plant Cd-peptide and rat liver Cd, Zn-thionein show that these ligands differ greatly in composition, metal affinity, and metal selectivity (16).The repeating y-glutamylcysteine groups in Cd-binding peptides of higher plants and fission yeast suggest that their synthesis may involve enzymes ofglutathione metabolism. The occurrence of this pathway has been documented in whole plants and cultured cells (see Rennenberg [17] for review) as have been numerous related pathways (10). BSO3 is a specific inhibitor of y-glutamylcysteine synthetase (EC 6.3.2.2) in animal cells (3) and has been shown to inhibit glutathione synthesis in cultured tomato cells (2). Since this enzyme may be involved in the synthesis of plant Cd-binding peptide, we examined the effects of BSO on the synthesis of this peptide in suspension-cultured tobacco cells exposed to Cd. We also examined the effects of Zn and Cu on formation of ligand of Cd-binding peptide, as these metals have been reported to induce the ligand (albeit very low levels) in fission yeast (5)

Relationships between Cadmium, Zinc, Cd-Peptide, and Organic Acid in Tobacco Suspension Cells

“…(Table III) (12,23 We were interested to determine if the absence of Cdpeptide would affect the capacity of the cell to accumulate Cd. Cells were exposed to 250 Mm Cd in the presence and absence of 0.2 mM BSO which was shown to inhibit synthesis by >85% (not shown) (18). As shown in Figure 5, …”

“…The above discussion raises the question of the form of vacuolar Cd in cells containing, and also those not containing, ligand of Cd-peptide. The latter condition occurs in tobacco cultured cells exposed to relatively low levels (<1 uM) of Cd, cells exposed to < 1000 uM Zn (18), immediately after exposure of nonacclimated cells to high levels of Cd (this study), and to cells grown in the presence of BSO,' an inhibitor of peptide formation (18). It is noteworthy that most plants in nature and agricultural crops are probably exposed to very low levels (50.1 uM) of this metal (31).…”

“…Positive correlations between occurrence of Cd peptide and tolerance (lack of growth inhibition) have been documented for two plant tissue-culture systems, suggesting a role for peptide in tolerance to high level Cd exposure (7,18). It is not known if Cd-peptide is formed in plants exposed to low levels of metal as occur in agriculture (31).…”

“…Therefore, the question arises as to the mechanism of Cd (and Zn) accommodation and accumulation in the absence of Cd-peptide. Zn binding to this peptide has not been demonstrated (19), though peptide induction by very high levels of Zn (possibly secondary response) has been reported (3,18).…”

“…Ernst was first to postulate vacuolar accumulation of Zn organic acid complexes as a mechanism for Zn tolerance in naturally tolerant ecotypes (32). Considerable, but not entirely consistent, evidence exists for a correlation between Zn content and organic acid content in various plants and cultured cells (2,8,13,15,25,26,32 (18). Homogenates were centrifuged (4°C) at 16,000g for 5 min and the resulting supernatants were centrifuged at 100,000g, 30 min.…”