Responses of tobacco (Nicotiana tabacum) suspension cells to Cd and Zn were studied in the presence and absence of ligand of Cd-peptide in order to understand the role of this peptide versus other mechanisms in Cd and Zn accumulation and accommodation in plants. With 45 micromolar Cd and 300 micromolar Zn (non-growth-inhibiting levels), metals appeared rapidly within cells, and intracellular Cd and Zn reached medium concentrations after 6 to 10 hours. Cd-peptide was observed in response to Cd after 2 hours, but this form only accounted for -30% of soluble Cd after 24 hours. Peptide was not observed in cells exposed to 300 micromolar Zn for up to 7 days. Organic acid-to-metal stoichiometry indicated that endogenous organic acid content of cells was more than sufficient to complex absorbed metals and no evidence was found for stimulation of organic acid biosynthesis by Cd or Zn. Metal-complexing potential of organic acids for Cd and Zn versus endogenous cations is discussed as is vacuolarextravacuolar distribution of metals. The absence of Cd-peptide does not limit Cd-accumulation in the system studied. Results suggest that tobacco suspension cells accommodate the presence of non-growth-inhibiting and growth-inhibiting levels of Cd and Zn by sequestration in the vacuole as complexes with endogenous organic acids and that this may be a principal means for accommodation of Cd as well as Zn in the presence and absence of Cd-peptide.Recent interest in unique (-y-Glu-Cys), Gly Cd-binding peptides, also called cadystins, phytochelatins, etc., centers on their structure, biosynthesis, and possible role in tolerance of plants and plant cultured cells to challenge with high levels of this metal (4,6,9,16,20,21,24,31 Prior to the demonstration that plants exposed to high levels of Cd form Cd-peptide, models to explain the mechanisms of heavy metal tolerance in plants focused on Zn and Cu and possible roles of the cell wall and vacuolar organic acids in metal sequestration (32). 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 Other extraction media and methods were used as noted. A previously described procedure was used to determine Cdpeptide content (18). Homogenates were centrifuged (4°C) at 16,000g for 5 min and the resulting supernatants were centrifuged at 100,000g, 30 min. Pellets were combined and washed with growth media less metal; insoluble fractions were dried and digested with 9:1 (v/v) HNO3:HClO4 and the digest was evaporated and analyzed for Cd and Zn content in 1 % HCI using flame atomic absorption spectroscopy (with background correction for Zn). For determination of K+, Mg2+, Ca2+, NO3-, C1-, S042-, PO34, and H+, water washed cells were homogenized in freshly boiled, distilled, deionized H20. The K+, Mg2+, and Ca2+ contents ...

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Various mechanisms have been suggested for the quenching of Cd ion activity in plant vacuoles. These include solution complexation with organic acids and sulfhydryl-containing peptides and precipitation as sulfides. Because direct experimental support for these mechanisms is lacking and difficult to obtain, we have used a computer model to evaluate the quenching role of possible organic and inorganic ligands of tobacco cultured cells exposed to Cd. Results of this thermodynamic evaluation, which assumes that a chemical equilibrium state is met in the vacuole, support the conclusion that sulfhydryl-containing peptides and certain organic acids may form soluble Cd complexes. Although complexation of malate and oxalate with Cd is predicted to be less significant, citrate in the concentration range encountered in the tobacco cultured cell vacuoles has high potential for forming soluble complexes with Cd over the entire possible vacuolar pH range, especially 4.3 to 7.0, even in the presence of low levels of Cd-binding peptides. In addition, results show that inorganic chloride, sulfide (if present), and phosphate may also act to sequester Cd ion activity in the vacuole by forming soluble Cd-Cl and insoluble CdS and Cd-phosphate.Understanding the fate ofCd in plants is of interest because of concerns of Cd transfer from plants to animals and man. Recent studies suggest that heavy metals accumulated in the higher plant are mainly compartmentalized in the vacuole (5, 7, 9, 27). Various mechanisms have been proposed to account for the accumulation of these potentially toxic heavy metal ions in the plant vacuole (18,30). In general, these mechanisms include formation of soluble metal-organic acid complexes (4, 9, 14) or metal-phytate (25, 26), formation ofmetalpeptide or metal-peptide-sulfide complexes (9, 18, 27), or precipitation of metal-sulfides (2,21,25).Support for a particular mechanism of accumulation/sequestration of an ion is gained if the compartment of accumulation/sequestration is verified and if speciation of the ion in that compartment is determined. Both direct and indirect approaches (10) used to determine compartmentation ofions, including heavy metals, can only provide qualitative and quantitative estimates of vacuole contents (28). They cannot identify the species of ion complexes occurring in vacuoles. Therefore, mechanisms proposed on the basis of compartmentation analysis alone are not sufficient to argue the validity of a mechanism of accumulation/sequestration. However, it is possible with computer assistance to simulate the ion species distribution in the plant vacuole and thus to evaluate a proposed mechanism.Computer calculations have been used to model the chemistry of xylem sap of soybean and tomato (29). Here, we use data obtained previously regarding sap composition of vacuoles from Cd-treated tobacco (Nicotiana tabacum) cultured cells and the GEOCHEM-PC computer model (16,23) to predict ion species of these vacuoles in vivo. The prediction of ion speciation in vacuoles of Zn-treate...

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Surface chemical characteristics of root cell walls extracted from two tobacco genotypes exhibiting differential tolerance to Mn toxicity were studied using potentiometric pH titration and Fourier transform infrared spectroscopy. The Mn-sensitive genotype KY 14 showed a stronger interaction of its cell wall surface with metal ions than did the Mn-tolerant genotype Tobacco Introduction (T.l.) 1112. This observation may be attributed to the relatively higher ratio of COO to COOH in KY 14 cell walls than that found in the cell walls of T.I. 1 1 12 in the pH range of 4 to 10. For both genotypes, the strength of binding between metal ions and cell wall surface was in the order of Cu > Ca > Mn > Mg > Na. However, a slightly higher preference of Ca over Mn was observed with the T.l. 1112 cell wall. This may explain the high accumulation of Mn in the leaves of Mn-tolerant genotype T.l. 1112 rather than the high accumulation of Mn in roots, as occurred in Mn-sensitive KY 14. It is concluded that surface chemical characteristics of cell walls may play an important role in plant metal ion uptake and tolerance.Recently, more evidence has suggested that cell wall exchange properties may well affect ion availability for uptake, diffusion rates in the apoplast, the chemical and electrical environment of the membrane and its transporters, growth of the cell wall, and function of cell wall enzymes (1). Therefore, an evaluation of the role of the root cell wall in the processes of metal ion uptake and tolerance is indeed necessary.In this study, purified cell wall materials from two tobacco (Nicotiana tabacum L.) genotypes demonstrating different tolerance to Mn toxicity were investigated. The study examined the intrinsic surface chemical characteristics of root cell walls and related these characteristics to genotypic differences in Mn uptake and tolerance. MATERIALS AND METHODSMn toxicity has been a significant growth-limiting factor for many crops in various regions, especially where crops are grown in relatively low pH soils (e.g. pH < 5.5 (14,19,20) and trichomes (3). The Mn trapped in plant vacuoles has been speculated to be complexed with organic acids (14). This hypothesis is supported by thermodynamic predictions using computer simulation models (24,25 Plant Physiol. Vol. 100, 1992 equilibrated for a relatively long period (e.g. 20 h). The titration curves shown in Figure 2 obtained with a 30-min equilibration period imply that the neutralization reaction between OH-and the H-form of the cell wall of KY 14 genotype in the Na solution background may be faster than that of T.I. 1112. This result indicates that the architecture and/or composition of the cell walls of the two genotypes are different.The shape of the titration curve of KY 14 in Figure 2 suggests that there are at least two apparent pKas in the cell wall material. However, the pKas obtained from such a titration plot cannot be considered as intrinsic pKas because the latter require additional information on ionic strength effect and residue ...

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Genotypic differences in tolerating toxic levels of heavy metals have been observed in various plant species. This research was conducted to study the effect of manganese (Mn), iron (Fe), calcium (Ca), and magnesium (Mg) accumulation on two tobacco genotypes, Tobacco Introduction (T.I.) 1112 and KY 14, that have exhibited different sensitivity to toxic levels of Mn. The investigation was carried out employing a solution culture technique in which combinations of three levels of Mn exposure at varying strengths and pH of an Hoagland solution were made. Increasing the strength of the Hoagland solution suppressed Mn uptake and the occurrence of Mn toxicity symptoms in both genotypes, suggesting the potential role of other ions on reducing Mn toxicity. A change in solution pH from 4.5 to 6.5 had no significant effect on the accumulation of Mn, Fe, Ca, and Mg in tobacco leaves as well as the occurrence of Mn toxicity symptoms in the tobacco plant. Accumulations of Mn, Fe, Ca and Mg were significantly higher in leaves of the Mn-tolerant genotype (T.I. 1112) than in those of the Mn-sensitive genotype (KY 14), but ratios of Mn/cation (cation denotes Fe or Ca or Mg) were almost the same in both genotypes. These data suggest that the difference between the Mn-sensitive and Mn-tolerant tobacco genotypes were not due to the simple imbalance of Mn in relation to Ca, Mg and Fe in the plant. Plant physiological studies are currently under way to elucidate this difference in Mn tolerance between the two genotypes.