Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced by Overexpressing γ-Glutamylcysteine Synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding γ-glutamylcysteine synthetase (γ-ECS), targeted to the plastids. The γ-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, γ-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, γ-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the γ-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of γ-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of γ-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.     

Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress

Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 microM CdCl(2) stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 microM CdCl(2). Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels.     

Enhanced drought resistance in fructan-producing sugar beet

Fructans are soluble polymers of fructose that are produced by approximately 15 % of the flowering plant species. Production of bacterial fructans in tobacco has been shown previously to lead to improved biomass production under polyethylene glycol-mediated drought stress. Here, we used the same SacB gene from Bacillus subtilis to produce bacterial fructans in sugar beet (Beta vulgaris L.). The transgenic sugar beets accumulated fructans to low levels (max. 0.5 % of dry weight) in both roots and shoots. Two independent transgenic lines of fructan-producing sugar beets showed significantly better growth under drought stress than untransformed beets. Drought stressed fructan-producing plants attained higher total dry weights (+25–35 %) than wildtype sugar beet, due to higher biomass production of leaves (+30–33 %), storage roots (+16–33 %) and fibrous roots (+37–60 %). Under well-watered conditions, no significant differences were observed between the transgenic and wildtype beets. In conclusion, the introduction of fructan biosynthesis in transgenic plants is a promising approach to improve crop productivity under drought stress.     

Effects of Municipal Sewage Sludge Doses on the Chlorophyll Contents and Heavy Metal Concentration of Sugar Beet (Beta vulgaris var. saccharifera)

The present study was conducted to assess the suitability of sewage sludge amendment (SSA) in soil for Beta vulgaris var. saccharifera (sugar beet) by evaluating the heavy metal accumulation and physiological responses of plants grown at a 10%, 25%, and 50% sewage sludge amendment rate. The sewage sludge amendment was modified by the physicochemical properties of soil, thus increasing the availability of heavy metals in the soil and consequently increasing accumulation in plant parts. Cd, Pb, Ni, and Cu concentrations in roots were significantly higher in plants grown at 25% as compared to 50% SSA; however, Cr and Zn concentration was higher at 50% than 25% SSA. The concentrations of heavy metal showed a trend of Zn > Ni > Cu > Cr > Pb > Cd in roots and Zn > Cu > Ni > Cr > Pb > Cd in leaves. The only instance in which the chlorophyll content did not increase after the sewage sludge treatments was 50%. There were approximately 1.12-fold differences between the control and 50% sewage sludge application for chlorophyll content. The sewage sludge amendment led to a significant increase in Pb, Cr, Cd, Cu, Zn, and Ni concentrations of the soil. The heavy metal accumulation in the soil after the treatments did not exceed the limits for the land application of sewage sludge recommended by the US Environmental Protection Agency (US EPA). The increased concentration of heavy metals in the soil due to the sewage sludge amendment led to increases in heavy metal uptake and the leaf and root concentrations of Ni, Zn, Cd, Cu, Cr, Pb, and Zn in plants as compared to those grown on unamended soil. More accumulation occurred in roots and leaves than in shoots for most of the heavy metals. The concentrations of Cd, Cr, and Pb were more than the permissible limits of national standards in the edible portion of sugar beet grown on different sewage sludge amendment ratios. The study concludes that the sewage sludge amendment in the soil for growing sugar beet may not be a good option due to risk of contamination of Cr, Pb, and Cd.     

Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco

Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl₂ over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants.     

Significantly improved Escherichia coli tolerance and accumulation of Cd2+, Zn2+ and Cu2+ expressing Streptococcus thermophilus StGCS-GS with high glutathione content

Streptococcus thermophilus γ-glutamylcysteine synthetase-glutathione synthetase (StGCS-GS) which synthesized glutathione (GSH) without limit feedback inhibition was over-expressed as a fusion protein of TrxA-StGCS-GS to analyze its possibly functional role in heavy metal tolerance of Escherichia coli (BL21). For comparative analyses, Arabidopsis γ-glutamylcysteine synthetase (AtGCS) and glutathione synthetase (AtGS) were introduced into Escherichia coli (E. coli) in the same manner, respectively. The results showed that the growth and survivability of E. coli over-expressing TrxA-StGCS-GS were slightly influenced by 1 mM Cd²⁺, Zn²⁺ and Cu²⁺ toxicity, and it could withstand duration of these heavy metal stresses competently. In contrast, the two strains over-expressing TrxA-AtGCS and TrxA-AtGS were impacted apparently; the BL21 empty strain was even almost suppressed. Meanwhile, a much higher bioaccumulation of Cd²⁺, Zn²⁺, Cu²⁺ ions and glutathione content were observed in the strain over-expressing TrxA-StGCS-GS than in the other comparison strains. It could be concluded that over-expression of StGCS-GS offered a more significant enhancement of heavy metal tolerance to E. coli with superior GSH content to accumulate considerable heavy metal.     

Phytochelatin is not a primary factor in determining copper tolerance

Phytochelatin (PC) is involved in the detoxification of harmful, non-essential heavy metals and the homeostasis of essential heavy metals in plants. Its synthesis can be induced by either cadmium (Cd) or copper (Cu), and can form stable complexes with either element. This might suggest that PC has an important role in determining plant tolerance to both. However, this is not clearly apparent, as evidenced by a PC-deficient and Cd-sensitiveArabidopsis mutant (cad1-3) that shows no significant increase in its sensitivity to copper. Therefore, we investigated whether the mechanism for Cu tolerance differed from that for Cd by analyzing copper sensitivity in Cd-tolerant transgenics and Cd-sensitive mutants ofArabidopsis. Cadmium-tolerant transgenic plants that over-expressedA. thaliana phytochelatin synthase 1 (AtPCS1) were not tolerant of copper stress, thereby supporting the hypothesis that PC is not primarily involved in this tolerance mechanism. We also investigated Cu tolerance incad2-1, a Cd-sensitive and glutathione (GSH)-deficientArabidopsis mutant. Paradoxically,cad2-1 was more resistant to copper stress than were wild-type plants. This was likely due to the high level of cysteine present in that mutant. However, when the growth medium was supplemented with cysteine, the wild types also exhibited copper tolerance. Moreover,Saccharomyces cerevisiae that expressedAtPCS1 showed tolerance to Cd but hypersensitivity to Cu. All these results indicate that PC is not a major factor in determining copper tolerance in plants.     

Heavy Metal Tolerance and Accumulation in Indian Mustard (Brassica Juncea L.) Expressing Bacterial γ-Glutamylcysteine Synthetase or Glutathione Synthetase

The overexpression of either γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase (GS) in Brassica juncea transgenics was shown previously to result in higher accumulation of glutathione (GSH) and phytochelatins (PCs), as well as enhanced Cd tolerance and accumulation. The present study was aimed at analyzing the effects of γ-ECS or GS overexpression on tolerance to and accumulation of other metal/loids supplied individually in agar medium (seedlings) or in hydroponics (mature plants). Also, as pollution in nature generally consists of mixtures of metals, glutamylcysteine synthetase (ECS) and GS seedlings were tested on combinations of metals. Compared to wild-type plants, ECS and GS transgenics exhibited a significantly higher capacity to tolerate and accumulate a variety of metal/loids (particularly As, Cd, and Cr) as well as mixed-metal combinations (As, Cd, Zn/As, Pb, and Zn). This enhanced metal tolerance and accumulation of the ECS and GS transgenics may be attributable to enhanced production of PCs, sustained by a greater availability of GSH as substrate, as suggested by their higher concentrations of GSH, PC2, PC3, and PC4 as compared to wild-type plants. Overexpression of GS and γ-ECS may represent a promising strategy for the development of plants with an enhanced phytoremediation capacity for mixtures of metals.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Investigation of the Effects of Phosphate Fertilizer Application on the Heavy Metal Content in Agricultural Soils with Different Cultivation Patterns

The use of phosphate fertilizers is essential in agriculture, because they supply farmland with nutrients for growing plants. However, heavy metals might be included as impurities in natural materials and minerals, so heavy metals can also be present in phosphate fertilizers or other chemical fertilizers. The aim of this work was to assess the heavy metal content and contamination status of agricultural soils in the Hamadan province of Iran used for the cultivation of different crops, including cucumber, potatoes, and sugar beet. Surface soil samples were collected and analyzed to determine the total concentration of specific elements (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn), before the pollution index was calculated for each element. Soils used for the cultivation of the three types of crop were not contaminated with As, Cr, Cu, Pb, or Zn. However, the pollution indices for Cd were 1.1, 4.4, and 3.8 in cucumber, potato, and sugar beet fields, respectively, which indicated moderate, high, and high levels of contamination, respectively. Soils from potato and sugar beet fields were heavily contaminated with Cd, which may have resulted from long-term overuse of phosphate fertilizers.     

Influence of exogenous application of glutathione on rubisco and rubisco activase in heavy metal-stressed tobacco plant grown in vitro

The effect of glutathione on the influences of heavy metals affecting rubisco and rubisco activase was studied in tobacco plants grown in vitro where the shoot explants of the tobacco plant cultured on MS medium under aseptic conditions and two explants were placed in the control, 0.1 mM GSH, 1 mM GSH, 0.2 mM Cd, 0.2 mM Cu, 0.2 mM Zn, and a mixture of Cd and GSH, Cu and GSH, Zn and GSH, respectively. The effect of GSH on the growth of the tobacco plant was minimal, but the heavy metals clearly retarded its growth. GSH recovered the growth retarded by heavy metals, and the concentration of GSH required to recover the growth differed depending on the heavy metals. The content of chlorophyll in the plant increased through GSH and Zn, and decreased through Cd and Cu. The chlorophyll content which decreased due to Cd and Cu was recovered by GSH, and the content which increased due to Zn was decreased by 1 mM GSH. The content of rubisco decreased due to GSH and heavy metals, and the content which decreased due to heavy metals was recovered by GSH, and when GSH was treated with Zn, the increased rate was maximum compared to other heavy metals. The activity of rubisco was increased due to GSH and heavy metals, and the activity increased by Cd and Zn decreased through GSH. In the case of Cu, the activity of GSH increased even more. There was no effect of GSH on the influences of heavy metals on the content and activity of rubisco activase. The activity of rubisco decreased by thiourea among six denaturing agents, and increased by l-cysteine, and in most cases the activity level was recorded as high. The activity of rubisco activase all decreased as a result of six denaturing agents, and the effect caused by EDTA and guanidine-HCl was the greatest, while the effect caused by l-cysteine and urea was minimal.     

Possible roles of phytochelatins and glutathione metabolism in cadmium tolerance in chickpea roots

The possible roles of phytochelatin (PC) and glutathione (GSH) in the heavy metal detoxification in plants were examined using two varieties (CSG-8962 and C-235) of chickpea (Cicer arietinum L.). The seedlings were grown for 5 days and the roots were treated with 0–20 μM CdSO₄ for 3 days. The CSG-8962 seedlings exhibited more Cd-tolerant characteristics than did the C-235, where the roots, rather than shoots, suffered from more toxic effects by Cd. Both the seedlings synthesized the large amounts of PCs and homo-phytochelatins (hPCs) in roots, but only a little in shoots in response to Cd. The Cd treatments also caused a marked increase in the levels of GSH and cysteine in both the root and shoot tissues, suggesting that Cd may activate the GSH biosynthesis and, hence, enhance PC synthesis in the plants. Such a Cd-sensitive PC synthesis in chickpea plants does not explain the difference in Cd sensitivity in the varieties, but can be used as a biochemical indicator for Cd contamination in various environments. In the chickpea plants, possible PC-dependent and independent mechanisms for Cd tolerance are discussed. Electronic Publication     

Glutathione and transition-metal homeostasis in Escherichia coli

Glutathione (GSH) and its derivative phytochelatin are important binding factors in transition-metal homeostasis in many eukaryotes. Here, we demonstrate that GSH is also involved in chromate, Zn(II), Cd(II), and Cu(II) homeostasis and resistance in Escherichia coli. While the loss of the ability to synthesize GSH influenced metal tolerance in wild-type cells only slightly, GSH was important for residual metal resistance in cells without metal efflux systems. In mutant cells without the P-type ATPase ZntA, the additional deletion of the GSH biosynthesis system led to a strong decrease in resistance to Cd(II) and Zn(II). Likewise, in mutant cells without the P-type ATPase CopA, the removal of GSH led to a strong decrease of Cu(II) resistance. The precursor of GSH, gamma-glutamylcysteine (gammaEC), was not able to compensate for a lack of GSH. On the contrary, gammaEC-containing cells were less copper and cadmium tolerant than cells that contained neither gammaEC nor GSH. Thus, GSH may play an important role in trace-element metabolism not only in higher organisms but also in bacteria.     

Heavy metal stress and sulfate uptake in maize roots

ZmST1;1, a putative high-affinity sulfate transporter gene expressed in maize (Zea mays) roots, was functionally characterized and its expression patterns were analyzed in roots of plants exposed to different heavy metals (Cd, Zn, and Cu) interfering with thiol metabolism. The ZmST1;1 cDNA was expressed in the yeast (Saccharomyces cerevisiae) sulfate transporter mutant CP154-7A. Kinetic analysis of sulfate uptake isotherm, determined on complemented yeast cells, revealed that ZmST1;1 has a high affinity for sulfate (Km value of 14.6 +/- 0.4 microm). Cd, Zn, and Cu exposure increased both ZmST1;1 expression and root sulfate uptake capacity. The metal-induced sulfate uptakes were accompanied by deep alterations in both thiol metabolism and levels of compounds such as reduced glutathione (GSH), probably involved as signals in sulfate uptake modulation. Cd and Zn exposure strongly increased the level of nonprotein thiols of the roots, indicating the induction of additional sinks for reduced sulfur, but differently affected root GSH contents that decreased or increased following Cd or Zn stress, respectively. Moreover, during Cd stress a clear relation between the ZmST1;1 mRNA abundance increment and the entity of the GSH decrement was impossible to evince. Conversely, Cu stress did not affect nonprotein thiol levels, but resulted in a deep contraction of GSH pools. Our data suggest that during heavy metal stress sulfate uptake by roots may be controlled by both GSH-dependent or -independent signaling pathways. Finally, some evidence suggesting that root sulfate availability in Cd-stressed plants may limit GSH biosynthesis and thus Cd tolerance are discussed.     

Analysis of gene inheritance and expression in hybrids between transgenic sugar beet and wild beets

Reciprocal gene exchange between cultivated sugar beet and wild beets in seed production areas is probably the reason for the occurence of weed beets in sugar beet production fields. Therefore, when releasing transgenic sugar beet plants into the environment, gene transfer to wild beets ( Beta vulgaris ssp. maritima ) has to be considered. In this study the transfer of BNYVV- (beet necrotic yellow vein virus) resistance and herbicide-tolerance genes from two transgenic sugar beet lines that were released in field experiments in 1993 and 1994 in Germany to different wild beet accessions was investigated. In order to evaluate the consequences of outcrossing, manual pollinations of emasculated wild beet plants with homozygous transgenic sugar beet plants were performed. In the resulting hybrids the transgenes were stably inherited according to Mendelian law. Gene expression in leaves and roots of the hybrids was in the same range as in the original transgenic sugar beet plants. Moreover, it was found that in one of the wild beet accessions, transfer and expression of the BNYVV resistance gene did considerably increase the level of virus resistance.     

Phytochelatins as biomarkers for heavy metal stress in maize (Zea mays L.) and wheat (Triticum aestivum L.): combined effects of copper and cadmium

Heavy metal contaminated soils often show increased levels of more than one metal, e.g. copper (Cu), cadmium (Cd), zinc (Zn), lead (Pb) or nickel (Ni). In case such soils are used for crop production, prediction of yield reduction or quality decline due to heavy metals in the soil is inadequate when based only on chemical soil analysis. The use of biomarkers such as phytochelatins (PC), non-protein thiols specifically induced in plants upon exposure to heavy metals, may be an additional tool or diagnostic criterion in heavy metal research and in practice. In the present work, Cu and Cd uptake and induction of PC synthesis are studied with hydroponically grown maize and wheat plants exposed to mixtures of the two metals. We observed a close positive relationship between the concentrations of Cd and PC in the plant shoot material. A decreased shoot concentration of Cd after addition of Cu, due to metal competition at common root absorption sites, coincided with lower shoot PC levels. Also differences in metal uptake and xylary metal transport among the two plant species were reflected in corresponding differences in PC concentration. The observed direct relationship between shoot PC concentration and the degree of metal-induced growth inhibition makes the use of PC promising for the purpose tested for.     

Overexpression of ZmIRT1 and ZmZIP3 Enhances Iron and Zinc Accumulation in Transgenic Arabidopsis

Iron and zinc are important micronutrients for both the growth and nutrient availability of crop plants, and their absorption is tightly controlled by a metal uptake system. Zinc-regulated transporters, iron-regulated transporter-like proteins (ZIP), is considered an essential metal transporter for the acquisition of Fe and Zn in graminaceous plants. Several ZIPs have been identified in maize, although their physiological function remains unclear. In this report, ZmIRT1 was shown to be specifically expressed in silk and embryo, whereas ZmZIP3 was a leaf-specific gene. Both ZmIRT1 and ZmZIP3 were shown to be localized to the plasma membrane and endoplasmic reticulum. In addition, transgenic Arabidopsis plants overexpressing ZmIRT1 or ZmZIP3 were generated, and the metal contents in various tissues of transgenic and wild-type plants were examined based on ICP-OES and Zinpyr-1 staining. The Fe and Zn concentration increased in roots and seeds of ZmIRT1-overexpressing plants, while the Fe content in shoots decreased. Overexpressing ZmZIP3 enhanced Zn accumulation in the roots of transgenic plants, while that in shoots was repressed. In addition, the transgenic plants showed altered tolerance to various Fe and Zn conditions compared with wild-type plants. Furthermore, the genes associated with metal uptake were stimulated in ZmIRT1 transgenic plants, while those involved in intra- and inter- cellular translocation were suppressed. In conclusion, ZmIRT1 and ZmZIP3 are functional metal transporters with different ion selectivities. Ectopic overexpression of ZmIRT1 may stimulate endogenous Fe uptake mechanisms, which may facilitate metal uptake and homeostasis. Our results increase our understanding of the functions of ZIP family transporters in maize.     

Metal(loid)s and radionuclides cytotoxicity in Saccharomyces cerevisiae. Role of YCF1, glutathione and effect of buthionine sulfoximine

The presence of heavy metal(loid)s in soils and waters is an important issue with regards to human health. Taking into account speciation problems, in the first part of this report, we investigated under identical growth conditions, yeast tolerance to a set of 15 cytotoxic metal(loid)s and radionuclides. The yeast cadmium factor 1 (YCF1) is an ATP-Binding Cassette transporter mediating the glutathione detoxification of heavy metals. In the second part, metal(loid)s that could be handled by YCF1 and a possible re-localisation of the transporter after heavy metal exposure were evaluated. YCF1 and a C-terminal GFP fusion, YCF1-GFP, were overexpressed in wild-type and Deltaycf1 strains. Both forms were functional, conferring a tolerance to Cd, Sb, As, Pb, Hg but not to Ni, Zn, Cu, Ag, Se, Te, Cr, Sr, Tc, U. Confocal experiments demonstrated that during exposure to cytotoxic metals, the localisation of YCF1-GFP was restricted to the yeast vacuolar membrane. In the last part, the role of glutathione in this resistance mechanism to metal(loid)s was studied. In the presence of heavy metals, application of buthionine sulfoximine (BSO), a well-known inhibitor of gamma-glutamylcysteine synthetase, led to a decrease in the cytosolic pool of GSH and to a limitation of yeast growth. Surprisingly, BSO was able to phenocopy the deletion of gamma-glutamylcysteine synthetase after exposure to Cd but not to Sb or As. In the genetic context of gsh1 and gsh2 yeast mutants, the critical role of GSH for Cd, As, Sb and Hg tolerance was compared to that of wild-type and Deltaycf1.