Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells

Cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, produce phytochelatins (poly[γ-glutamylcysteinyl]glycines) when exposed to cadmium. The synthesis of these peptides is accompanied by a decline in cellular levels of glutathione. Buthionine sulfoximine, an inhibitor of glutathione synthesis, inhibits the sustained production of phytochelatins. However, phytochelatin synthesis can occur in the presence of buthionine sulfoximine provided that sufficient glutathione is available. These results indicate that glutathione is a substrate for phytochelatin synthesis. The protein synthesis inhibitor cycloheximide does not affect the initial production of phytochelatin.     

Regulation of Glutathione Synthesis by Cadmium in Pisum sativum L

In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCl₂ concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5′-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cd-binding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5′-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis.     

Glutathione Depletion Due to Copper-Induced Phytochelatin Synthesis Causes Oxidative Stress in Silene cucubalus

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 Silene 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 sulfoximine-treated 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.     

Increased Activity of [gamma]-Glutamylcysteine Synthetase in Tomato Cells Selected for Cadmium Tolerance

Two cell lines of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) were systematically compared for their capacity to tolerate cadmium. Unselected CdS cells died in the presence of 0.3 mM CdCl2. CdR6-0 cells, which were selected from CdS, survived and grew in medium supplemented with 0.3 mM CdCl2. Growth of CdR6-0 cells under this condition was accompanied by synthesis of cadmium-binding phytochelatins and maintenance of cellular glutathione (GSH) levels. CdR6-0 cells also exhibited increased tolerance to buthionine sulfoximine, in both the presence and absence of 0.1 mM CdCl2. The specific activity of [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) was approximately 2-fold higher in CdR6-0 cells than in CdS cells, whereas there was no difference between cell lines in specific activity of GSH synthetase (EC 6.3.2.3). Increased activity of the first enzyme of GSH biosynthesis in CdR6-0 cells, presumably a result of selection for increased cadmium tolerance, provides an enhanced capacity to synthesize GSH and to maintain the production of phytochelatins in response to cadmium. This adaptation may contribute to the enhanced cadmium tolerance of CdR6-0 cells.     

Selection and characterization of cadmium tolerant cells in tomato

Tomato (Lycopersicon esculentum cv. VFNT-Cherry)cell lines tolerant of to 5 mM cadmium (Cd) were selected by progressively elevating the level of CdCl₂ in the culture medium (the lethal concentration of Cd for unselected tomato cells is 400 μM). Cd tolerance was not lost during long-term culture (up to 12 months) in the absence of Cd stress. In all the cell lines examined, Cd uptake was rapid and Cd concentration within the cells exceeded that in the culture medium by several fold. While Cd included the synthesis and accumulation of phytochelatins (PCs) [poly[γ-glutamyl-cysteiny)glycine], little change has been observed in protein synthesis during short term Cd stress. PCs formed complexes with Cd. However, uptake and accumulation of Cd was not affected if PC synthesis was inhibited by treatment with buthionine sulfoximine. Selected and unselected cells were compared for their growth characteristics in the presence of various other metal ions. Cd tolerant cells showed a slightly higher tolerance of copper but not of mercury, zinc, lead or silver.     

Effect of cellular glutathione depletion on cadmium-induced cytotoxicity in human lung carcinoma cells

The effect of glutathione depletion on cellular toxicity of cadmium was investigated in a subpopulation (T27) of human lung carcinoma A549 cells with coordinately high glutathione levels and Cd⁺⁺-resistance. Cellular glutathione levels were depleted by exposing the cells to diethyl maleate or buthionine sulfoximine. Depletion was dose-dependent. Exposure of the cells to 0.5 mM diethyl maleate for 4 hours or to 10 mM buthionine sulfoximine for 8 hours eliminated the threshold for Cd⁺⁺ cytotoxic effect and deccreased the LD_50S. Cells that were pretreated with 0.5 mM diethyl maleate or 10 mM buthionine sulfoximine and then exposed to these same concentrations of diethyl maleate or buthionine sulfoximine during the subsequent assay for colony forming efficiency produced no colonies, reflecting an enhanced sensitivity to these agents at low cell density. Diethyl maleate was found to be more cytotoxic than buthionine sulfoximine. Synergistic cytotoxic effects were observed in the response of diethyl maleate pretreated cells exposed to Cd⁺⁺. Thus the results demostrated that depletion of most cellular glutathione in A549-T27 cells prior to Cd⁺⁺ exposure sensitizes them to the agent's cytotoxic effects. Glutathione thus may be involved in modulating the early cellular Cd⁺⁺ cytotoxic response. Comparison of reduced glutathione levels and of Cd⁺⁺ cytotoxic responses in buthionine sulfoximine-treated A549-T27 cells with those levels in other, untreated normal and tumor-derived cells suggests that the higher level of glutathione in A549-T27 is not the sole determinant of its higher level of Cd⁺⁺ resistance.Abbreviations BSO DL-buthionine-(R,S)-sulfoximine - DEM diethyl maleate - DMSO dimethyl sulfoxide - GSH reduced glutathione - MT metallothionein     

Glutathione, a first line of defense against cadmium toxicity

Experimental modulation of cellular glutathione levels has been used to explore the role of glutathione in cadmium toxicity. Mice treated with buthionine sulfoximine [an effective irreversible inhibitor of gamma-glutamylcysteine synthetase (EC 6.3.2.2) that decreases cellular levels of glutathione markedly] were sensitized to the toxic effects of CdCl2. Mice pretreated with a sublethal dose of Cd2+ to induce metallothionein synthesis were not sensitized to Cd2+ by buthionine sulfoximine. Mice sensitized to Cd2+ by buthionine sulfoximine were protected against a lethal dose of Cd2+ by glutathione mono isopropyl ester (L-gamma-glutamyl-L-cysteinylglycylisopropyl ester), but not by glutathione. These results are in accord with studies that showed that glutathione mono esters (in contrast to glutathione) are efficiently transported into cells and converted intracellularly to glutathione. The findings indicate that intracellular glutathione functions in protection against Cd2+ toxicity, and that this tripeptide provides a first line of defense against Cd2+ before induction of metallothionein synthesis occurs. The experimental approach used here in which cellular levels of glutathione are decreased or increased seems applicable to investigation of other types of metal toxicity and of other glutathione-dependent biological phenomena.     

Enhanced cadmium cytotoxicity in A549 cells with reduced glutathione levels is due to neither enhanced cadmium accumulation nor reduced metallothionein synthesis

Glutathione (GSH) depletion sensitizes human lung carcinoma (A549-727) cells to the cytotoxic effects of Cd⁺⁺. The effects of GSH depletion on Cd⁺⁺ accumulation and Cd⁺-induced metallothionein (MT) content were investigated to determine the possible role of these Cd⁺⁺ responses in the sensitization process. Cellular GSH was depleted to 20% to 25% of control levels with buthionine sulfoximine (BSO), or diethyl maleate (DEM), respectively. Neither treatment significantly affected Cd⁺⁺-induced accumulation of exogenous³⁵s-cysteine into intracellular MT in a dose-dependent fashion. The results indicate that neither enhanced Cd⁺⁺ accumulation nor reduced MT synthesis plays a primary role in affecting enhanced Cd⁺⁺ cytotoxicity in A549 cells with reduced GSH levels. Although BSO inhibition of GSH synthesis enhanced MT synthesis, it sensitized the cells to Cd⁺⁺, which suggests an additive effect of GSH and MT in cadmium cytoprotection. This observation also raises the possibility that intracellular cysteine levels limit Cd⁺⁺-induced MT accumulation rates.Abbreviations GSH glutathione - MT metallothionein - BSO DL-buthionine-[S,R]-sulfoximine - DMSO dimethyl sulfoximine - DEM diethyl maleate - NP-40 nonidet-P40 - PBS phosphate buffered saline - HBSS Hank's balanced salt solution - DTT dithiothreitol 3. This work was presented in part at the 72nd Annual Meeting of the Federation of American Societies for Experimental Biology, Las Vegas, Nevada, May 1–5, 1988.     

Glutathione and phytochelatin contents in tomato plants exposed to cadmium

The effect of cadmium on growth and contents of glutathione (GSH) and phytochelatins (PCs) were investigated in roots and leaves of tomato plants (Lycopersicon esculentum Mill. cv. 63/5 F1). The accumulation of Cd increased with external Cd concentrations and was considerably higher in roots than in leaves. Dry mass production decreased under Cd treatment especially in leaves. In both roots and leaves, exposure to Cd caused an appreciable decline in GSH contents and increase in PCs synthesis proportional to Cd concentrations in the growth medium. At the same Cd concentration, PCs production was higher in roots than in leaves. The implication of glutathione in PC synthesis was strongly suggested by the use of buthionine sulfoximine (BSO). The major fraction of Cd accumulated by tomato roots was in the form of a Cd-PCs complex.     

Cadmium-Sensitive Mutants of Arabidopsis thaliana

A screening procedure for identifying Cd-sensitive mutants of Arabidopsis thaliana is described. With this procedure, two Cd-sensitive mutants were isolated. These represent independent mutations in the same locus, referred to as CAD1. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed that the mutation is closely linked to the tt3 locus on chromosome 5. In addition to Cd, the mutants are also significantly more sensitive to mercuric ions and only slightly more sensitive to Cu and Zn, while being no more sensitive than the wild type to Mn, thus indicating a degree of specificity in the mechanism affected by the mutation. Undifferentiated callus tissue is also Cd sensitive, suggesting that the mutant phenotype is expressed at the cellular level. Both wild-type and mutant plants showed increased sensitivity to Cd in the presence of buthionine sulfoximine, an inhibitor of the biosynthesis of the cadmium-binding (γ-glutamylcysteine)_n-glycine peptides, suggesting that the mutant is still able to synthesize these peptides. However, the effects of a cad1 mutation and buthionine sulfoximine together on cadmium sensitivity are essentially nonadditive, indicating that they may affect different aspects of the same detoxification mechanism. Assays of Cd uptake by intact plants indicate that the mutant is deficient in its ability to sequester Cd.     

Nucleotide changes in oxidatively stressed lymphocytes

Similar to HIV-1-induced suppression of thymus-derived lymphocytes (T cells), oxidatively stressed T cells show inhibited DNA synthesis and proliferation. The influence of oxidative stress on nucleotide pools was explored using ³H-uridine addition to OKT3-stimulated peripheral blood lymphocytes. The cells were preincubated and stimulated in the presence of 1 mM buthionine sulfoximine to inhibit GSH synthesis. This treatment gave rise to a significant reduction in dUDP and TTP biosynthesis following 18–32 hours stimulation, indicating possible impairment of ribonucleotide reductase activity.     

Optimization of conditions for cadmium selenide quantum dot biosynthesis in Saccharomyces cerevisiae

The biosynthesis of quantum dots has been explored as an alternative to traditional physicochemical methods; however, relatively few studies have determined optimal synthesis parameters. Saccharomyces cerevisiae sequentially treated with sodium selenite and cadmium chloride synthesized CdSe quantum dots in the cytoplasm. These nanoparticles displayed a prominent yellow fluorescence, with an emission maximum of approximately 540 nm. The requirement for glutathione in the biosynthetic mechanism was explored by depleting its intracellular content through cellular treatments with 1-chloro-2,4-dinitrobenzene and buthionine sulfoximine. Synthesis was significantly inhibited by both of these reagents when they were applied after selenite treatment prior to the addition of cadmium, thereby indicating that glutathione contributes to the biosynthetic process. Determining the optimum conditions for biosynthesis revealed that quantum dots were produced most efficiently at entry into stationary phase followed by direct addition of 1 mM selenite for only 6 h and then immediately incubating these cells in fresh growth medium containing 3 mM Cd (II). Synthesis of quantum dots reached a maximum at 84 h of reaction time. Biosynthesis of 800-μg g⁻¹ fresh weight cells was achieved. For the first time, significant efforts have been undertaken to optimize each aspect of the CdSe biosynthetic procedure in S. cerevisiae, resulting in a 70% increased production.

     

Phytochelatin synthesis and cadmium localization in wild type of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

AlthoughArabidopsis thaliana is known as a model plant, in molecular studies, as well as heavy metal tolerance of higher plants, there have been no detailed studies of its cadmium accumulation, tolerance and cellular distribution in a wild type of this species. In hydroponic experiments the wild type of A. thaliana (L.) Heynh cv. Columbia plants grew at cadmium concentrations varying from 5 to 100 M with phytotoxicity symptoms depending on the concentration and time of application. The concentration of cadmium in roots and shoots increased from 0.28 and 0.08 mg g^–1 d.wt at 5 M Cd treatment after 7 days to 0.82 and 0.85 mg g^–1 d.wt at 100 M Cd treatment after 14 days, respectively. Most of the cadmium (69–88% of its total pool) was found in shoot. Cd application induced the biosynthesis of phytochelatins (PCs) in root and shoot tissues. Studies with buthionine sulfoximine [BSO, specific inhibitor of glutathione (GSH) synthesis] supported the presence of Cd–phytochelatin complexes and their role in Cd detoxification and tolerance in wild type of A. thaliana. Cellular distribution of cadmium was examined using energy-dispersive X-ray micro-analysis. Particularly interesting was the observation of cadmium localized in the root pericycle.     

Involvement of a cadmium-induced low molecular weight protein in regulating cadmium toxicity in the diazotrophic cyanobacterium Anabaena doliolum

This study demonstrated the production of a cadmium-induced low molecular weight (3.5 kDa), buthionine sulfoximine (BSO) sensitive protein in Anabaena doliolum. Production of this protein was accompanied by a decrease in the glutathione level of the cell. Cadmium was found to be differentially toxic to carbon fixation, O₂ evolution, ATP content, nitrate reductase, nitrogenase, alkaline phosphatase and ATPase of control (untreated), BSO, cadmium and (cadmium + BSO) pre-treated A. doliolum. Toxicity was maximum in BSO-grown cells followed be control (untreated), cadmium + BSO and least in cadmium-grown A. doliolum. Cadmium and (cadmium + BSO)-grown cells registered an increased lipid production, reduced metal uptake and low K⁺, Na⁺ loss. In spite of equal cadmium uptake rates, a significant difference in toxicity between cadmium-grown and (cadmium + BSO)-grown cultures was, however, noticed. Better performance of physiological and biochemical variables of cadmium-grown A. doliolum and its tolerance to cadmium could be due to the synthesis of low molecular weight cadmium binding protein (presumably phytochelatin) as well as an increased production of lipid.     

Cadmium is a catalytic inhibitor of DNA topoisomerase II

Cadmium (Cd²⁺) is a highly toxic and carcinogenic metal that is an environmental and occupational hazard. DNA topoisomerase II is an essential nuclear enzyme and its inhibition can result in the formation of genotoxic and recombinogenic DNA double strand breaks. In this study we showed that cadmium chloride strongly inhibited the DNA decatenation activity of human topoisomerase IIα in the low micromolar concentration range and that its inhibitory effects were reduced by glutathione. Because the activity of topoisomerase II is strongly inhibited by thiol-reactive compounds this result suggested that cadmium may be binding to critical topoisomerase II cysteine thiols. Cadmium, however, did not stabilize DNA-topoisomerase II covalent complexes, as measured by the lack of formation of DNA double strand breaks. Hence, it is not likely to be a topoisomerase II poison. Consistent with the idea that cadmium cytotoxicity may be modulated by glutathione levels, buthionine sulfoximine pretreatment to decrease glutathione levels resulted in a greatly increased cadmium-induced cytotoxicity in K562 cells. The results of this study suggest that cadmium may exert some of its cell growth inhibitory, and possibly its toxicity and carcinogenicity, by inhibiting topoisomerase IIα through reaction with critical cysteine thiols.     

Heavy Metal-Activated Synthesis of Peptides in Chlamydomonas reinhardtii

In this study, we have addressed the capacity of the green alga Chlamydomonas reinhardtii to produce metal-binding peptides in response to stress induced by the heavy metals Cd²⁺, Hg²⁺, and Ag⁺. Cells cultured in the presence of sublethal concentrations of Cd²⁺ synthesized and accumulated oligopeptides consisting solely of glutamic acid, cysteine, and glycine in an average ratio of 3:3:1. Cadmium-induced peptides were isolated in their native form as higher molecular weight peptide-metal complexes with an apparent molecular weight of approximately 6.5 × 10³. The isolated complex bound cadmium (as evidenced by absorption spectroscopy) and sequestered (with a stoichiometry of 0.7 moles of cadmium per mole of cysteine) up to 70% of the total cadmium found in extracts of cadmium-treated cells. In Hg²⁺-treated cells, the principal thiol-containing compound induced by Hg²⁺ ions was glutathione. It is possible that glutathione functions in plant cells (as it does in animal cells) to detoxify heavy metals. Cells treated with Ag⁺ ions also synthesized a sulfur-containing component with a charge to mass ratio similar to Cd²⁺-induced peptides. But, in contrast to the results obtained using Cd²⁺ as an inducer, these molecules did not accumulate to significant levels in Ag⁺-treated cells. The presence of physiological concentrations of Cu²⁺ in the growth medium blocked the synthesis of the Ag⁺-inducible component(s) and rendered cells resistant to the toxic effects of Ag⁺, suggesting competition between Cu²⁺ and Ag⁺ ions, possibly at the level of metal uptake.     

Direct one-step labeling of cysteine residues on peptides with [11C]methyl triflate for the synthesis of PET radiopharmaceuticals

Radiolabeled peptides have emerged as an attractive platform for the diagnostic and therapeutic oncology. However, the ¹¹C-radiolabeling of peptides for positron emission tomography (PET) has been poorly explored, owing to the relatively short half-life of carbon-11 (t _1/2 = 20.3 min) and time-consuming multi-step radiochemical reactions. Existing methods have found limited use and are not routinely encountered in the production of radiotracers. Herein, we propose a facile one-step direct ¹¹C-methylation of cysteine residues in peptides using [¹¹C]methyl triflate under ambient temperatures (20 °C) and short reaction times, on the order of seconds. Good regioselectivity of this method was demonstrated by HPLC in a simple peptide (glutathione, GSH) and a more complex test decapeptide (Trp-Tyr-Trp-Ser-Arg-Cys-Lys-Trp-Thr-Gly) bearing multiple nucleophilic sites. In addition, we extend this method towards the synthesis of [¹¹C]Cys(Me)-[Tyr³-octreotate] as a demonstration of applicability for peptides of biological interest. This octreotate derivative was obtained in non-decay-corrected radiochemical yields of 11 ± 2 % (n = 3) with a synthesis time of approx. 30 min.     

Response to copper and cadmium stress in wild-type and copper tolerant strains of the lichen alga <Emphasis Type="Italic">Trebouxia erici</Emphasis>: metal accumulation,toxicity and non-protein thiols

Cysteine, glutathione (GSH) and phytochelatins were determined in the cells of both wild and copper tolerant strains of the lichen alga Trebouxia erici following short-term (24 h) exposure to copper and cadmium and long-term (4 weeks) exposure to copper. Both metals caused concentration dependent synthesis of phytochelatins (PC₂–PC₅), but cadmium was a more potent activator of phytochelatin synthesis, even inducing synthesis of PC₅. The copper-tolerant strain did not reveal a higher degree of phytochelatin synthesis than the wild strain, and at 5 μM Cu production of phytochelatins was in fact significantly lower. Lower levels of phytochelatin correlated with significantly decreased intracellular copper content in the copper-tolerant strain. Both strains maintained high GSH levels even at a high copper concentration of 5 μM, and only the highest copper concentration (10 μM) was toxic for both strains, causing a decrease of GSH and PC content in algal cells. Cadmium had less effect on GSH in the cells of both tested strains. In the long term experiments, only relatively small amounts of PC₂ were detected in both strains, but the copper-tolerant strain retained significantly higher levels of reduced glutathione, probably due to the lesser degree of oxidative stress caused by Cu. The significant increase of cysteine synthesis in the copper-tolerant strain found in the present study may be related to copper tolerance in T. erici, while decreased intracellular Cu uptake, detoxification by PCs and increased free proline levels for protection of chloroplast membranes may also be implicated.     

Plasmodium falciparum: Effect of Solanum nudum steroids on thiol contents and β-hematin formation in parasitized erythrocytes

We studied the effects on total thiols glutathione (GSH) and cysteine contents in Plasmodium falciparum in vitro when treated with four steroid derivatives and a sapogenin (Diosgenone) extracted from Solanum nudum. We also determined their capacity to inhibit β-hematin formation. We showed that SN-1 (16α-acetoxy-26-hydroxycholest-4-ene-3,22-dione) increased total glutathione and cysteine concentrations while SN-4 (26-O-β-d-glucopyranosyloxy-16α-acetoxycholest-4-ene-3,22-dione) decreased the concentration of both thiols. Acetylation in C16 was crucial for the effect of SN-1 while type furostanol and terminal glucosidation were necessary for the inhibitory properties of SN-4. The combination of steroids and buthionine sulfoximine, a specific inhibitor of a step-limiting enzyme in GSH synthesis, did not modify the glutathione contents. Finally, we found that SN-1 inhibited more than 80% of β-hematin formation at 5.0 mM, while the other steroids did not show any effect.     

Glutathione reduces the inhibition of rice seedling root growth caused by cadmium

We investigated the effects of agents known to affect cellular glutathione (reduced form, GSH) levels on the growth of rice seedlings treated with Cd. CdCl₂ was more effective than CdSO₄ in inhibiting root growth. However, CdCl₂ had no effect on shoot growth. GSH, a substrate for phytochelatin synthesis, was effective in counteracting growth inhibition of roots by CdCl₂. Root growth in the CdCl₂ medium was found also to be enhanced by the addition of L-glutamic acid and L-cysteine, both of which are substrates for GSH formation. Buthionine sulfoximine, an inhibitor of GSH synthesis, rendered the roots susceptible to growth inhibition by Cd. Our results suggest that GSH level may play a role in regulating Cd-inhibited growth of rice roots.Abbreviations BSO buthionine sulfoximine - GSH reduced form glutathione