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.     

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.     

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.     

Effect of glutathione on phytochelatin synthesis in tomato cells

Growth of cell suspension cultures of tomato, Lycopersicon esculentum Mill. cv VFNT-Cherry, in the presence of cadmium is inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. Cell growth and phytochelatin synthesis are restored to cells treated with buthionine sulfoximine by the addition of glutathione to the medium. Glutathione stimulates the accumulation of phytochelatins in cadmium treated cells, indicating that availability of glutathione can limit synthesis of these peptides. Exogenous glutathione causes a disproportionate increase in the level of smaller phytochelatins, notably [γ-Glu-Cys]₂-Gly. In the presence of buthionine sulfoximine and glutathione, phytochelatins that are produced upon exposure to cadmium incorporate little [³⁵S]cysteine, indicating that these peptides are probably not synthesized by sequential addition of cysteine and glutamate to glutathione.     

Are Phytochelatins Involved in Differential Metal Tolerance or Do They Merely Reflect Metal-Imposed Strain?

Plants from nontolerant and copper-tolerant populations of Silene vulgaris both produce phytochelatins upon exposure to copper. The threshold copper concentration for induction of phytochelatin and the copper concentration at which maximum phytochelatin contents occurs increase proportionally with the level of tolerance to copper. When exposed to their own highest no-effect concentration or 50%-effect concentration of copper for root growth, tolerant and nontolerant plants exhibit equal phytochelatin contents in the root apex, which is the primary copper target. This also holds for distinctly tolerant nonsegregating F₃ families, derived from a single cross of a nontolerant plant to a tolerant one. Therefore, the phytochelatin content of the root apex can be used as a quantitative tolerance-independent measure of the degree of toxicity experienced by the plant. Differential copper tolerance in S. vulgaris does not appear to rely on differential phytochelatin production.     

Copper tolerance in Silene cucubalus

The uptake, translocation and subcellular distribution of copper as well as its effect on chloroplasts and plastocyanin synthesis were studied in a copper-sensitive and a copper-tolerant population of Silene cucubalus (L.) Wib. As a function of time, the copper concentration in roots of tolerant plants increased more slowly than that in roots of sensitive ones. Translocation to the shoot occurred more rapidly in tolerant plants than in sensitive ones. Although it was accumulated in leaf cells, copper was not accumulated in the chloroplasts of either sensitive or tolerant plants. Chlorophyll content was not affected by copper in tolerant plants, whereas sensitive plants became chlorotic. Plastocyanin synthesis was not enhanced as a result of high copper concentrations and no difference in plastocyanin content between tolerant and sensitive plants was detected. Measurements of copper in purified cell walls revealed that storage of the metal in cell-wall material does not play an important role in tolerance mechanism. Uptake characteristics, distribution and cytoplasmic detoxification of copper are discussed.     

Phytochelatin plays a role in UV-B tolerance in N2-fixing cyanobacterium Anabaena doliolum

To study the role of Cd-induced phytochelatins in UV-B tolerance, lipid peroxidation, antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), glutathione arid phytochelatin contents were measured in buthionine sulphoximine treated and untreated cells of Anabaena doliolum. Cd-pretreatment of the cyanobacterium reduced the lipid peroxidation as well as the antioxidative enzymes in comparison to UV-B treatment alone, whereas the phytochelatin content demonstrated an increase. In contrast to this, buthionine sulphoximine-induced inhibition of phytochelatin synthase, dramatically decreased the Cd-induced co-tolerance against UV-B, hence demonstrating that phytochelatin not only protects the cyanobacterium from heavy metal but participates in UV-B tolerance as well.     

Increased Zinc Tolerance in Silene vulgaris (Moench) Garcke Is Not Due to Increased Production of Phytochelatins

The concentration of acid-soluble thiols other than reduced glutathione (SH - GSH) increases in the roots of zinc-sensitive and zinc-tolerant Silene vulgaris (Moench) Garcke after exposure to zinc for 1 to 3 d. The concentration of SH - GSH in the roots is higher in the sensitive plants than in the tolerant ones, both at equal external zinc concentrations and at zinc concentrations causing the same level of root-length growth inhibition. High performance liquid chromatography analyses show that the increase in the concentration of SH - GSH is not only due to the production of phytochelatins, but is also due to an increase in the concentration of cysteine and the production of nonidentified thiols. The cysteine concentration increases equally in the roots of sensitive and tolerant plants. The accumulation of phytochelatins is higher in the roots of the sensitive plants, whereas the chain length distribution of phytochelatins is the same in sensitive and tolerant plants. It is concluded that increased zinc tolerance in S. vulgaris is not due to increased production of phytochelatins.     

Copper- and arsenate-induced oxidative stress in Holcus lanatus L. clones with differential sensitivity

The biochemical responses of Holcus lanatus L. to copper and arsenate exposure were investigated in arsenate‐tolerant and ‐non‐tolerant plants from uncontaminated and arsenic/copper‐contaminated sites. Increases in lipid peroxidation, superoxide dismutase (SOD) activity and phytochelatin (PC) production were correlated with increasing copper and arsenate exposure. In addition, significant differences in biochemical responses were observed between arsenate‐tolerant and ‐non‐tolerant plants. Copper and arsenate exposure led to the production of reactive oxygen species, resulting in significant lipid peroxidation in non‐tolerant plants. However, SOD activity was suppressed upon metal exposure, possibly due to interference with metallo‐enzymes. It was concluded that in non‐tolerant plants, rapid arsenate influx resulted in PC production, glutathione depletion and lipid peroxidation. This process would also occur in tolerant plants, but by decreasing the rate of influx, they were able to maintain their constitutive functions, detoxify the metals though PC production and quench reactive oxygen species by SOD activity.     

Overexpression of Glutathione Synthetase in Indian Mustard Enhances Cadmium Accumulation and Tolerance

An important pathway by which plants detoxify heavy metals is through sequestration with heavy-metal-binding peptides called phytochelatins or their precursor, glutathione. To identify limiting factors for heavy-metal accumulation and tolerance, and to develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, the Escherichia coli gshII gene encoding glutathione synthetase (GS) was overexpressed in the cytosol of Indian mustard (Brassica juncea). The transgenic GS plants accumulated significantly more Cd than the wild type: shoot Cd concentrations were up to 25% higher and total Cd accumulation per shoot was up to 3-fold higher. Moreover, the GS plants showed enhanced tolerance to Cd at both the seedling and mature-plant stages. Cd accumulation and tolerance were correlated with the gshII expression level. Cd-treated GS plants had higher concentrations of glutathione, phytochelatin, thiol, S, and Ca than wild-type plants. We conclude that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and phytochelatins, and that overexpression of GS offers a promising strategy for the production of plants with superior heavy-metal phytoremediation capacity.     

Effects of Metal Combinations on the Production of Phytochelatins and Glutathione by the Marine Diatom Phaeodactylum tricornutum

Copper, Cd and Zn can be found at elevated concentrations in contaminated estuarine and coastal waters and have potential toxic effects on phytoplankton species. In this study, the effects of these metals on the intracellular production of the polypeptides phytochelatin and glutathione by the marine diatom Phaeodactylum tricornutum were examined in laboratory cultures. Single additions of Cu and Cd (0.4 μM Cu² and 0.45 μM Cd²⁺) to the culture medium induced the production of short-chained phytochelatins ((γ-Glu-Cys)_n-Gly where n = 2–5), whereas a single addition of Zn (2.2 μM Zn²⁺) did not stimulate phytochelatin production. Combination of Zn with Cu resulted in a similar phytochelatin production compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production, which was probably caused by metal competition for cellular binding sites. Glutathione concentrations were affected only upon exposure to Cd (85% increase) or the combination of Cd with Zn (65% decrease), relative to the control experiment. Ratios of phytochelatins to glutathione indicated a pronounced metal stress in response to exposures to Cu or Cd combined with Zn. This study indicates that variabilities in phytochelatin and glutathione production in the field can be explained in part by metal competition for cellular binding sites.     

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant (Silene vulgaris) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal–phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes.     

The possible role of metallothioneins in copper tolerance of Silene cucubalus

Growth and copper-binding of a copper-tolerant and a copper-sensitive population of Silene cucubalus (L.) Wib. have been studied. The copper-tolerant plants showed a much lower uptake and a proportionally higher transport of copper from root to shoot. A copper-binding protein with an apparent M_r of 8500 resembling metallothionein has been isolated from the roots of copper-treated plants of the tolerant population. After 20 d, the protein was observed to be inducible upon copper supply in the copper-tolerant plants, but not yet in the sensitive ones. This could be an indication of a difference in metalregulated synthesis of the protein. Ion-exchange chromatography of the 8500 protein yielded a major copper-containing fraction eluting at high ionic strength. Other characteristics such as UV absorption and amino-acid composition resembled strongly those of metallothioneins. The involvement of metallothioneins in the detoxification of copper within Cu-tolerant plants is discussed in relation to other mechanisms.Abbreviation DEAE diethyloaminoethyl     

A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity

Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.     

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.     

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.     

植物的硫同化及其相关酶活性在镉胁迫下的调节

植物对土壤中硫的利用包括根系对硫酸盐的吸收、转运、同化、分配等过程,也是由一系列酶和蛋白质参与和调节的代谢过程。近年来的研究表明,在植物体内,硫同化与植物对镉等重金属元素的胁迫反应机制有着密切关系。镉胁迫能调节植物对硫酸盐的吸收、转运、同化,以及半胱氨酸、谷胱甘肽（glutathione,GSH）和植物螯合肽（Dhytochelatins,pc）的合成。植物在镉胁迫下通过多种调节机制,增强对硫酸盐的吸收和还原,迅速合成半胱氨酸和谷胱甘肽等代谢物,从而合成足够的PC,以满足植物生理的需要。     

Worms take the 'phyto' out of 'phytochelatins'

Phytochelatin synthase is the enzyme responsible for the synthesis of heavy-metal-binding peptides (phytochelatins) from glutathione and related thiols. It has recently been determined that it is not only restricted to plants and some fungi, as was once thought, but also has an essential role in heavy-metal detoxification in the model nematode Caenorhabditis elegans. These findings and others that demonstrate phytochelatin synthase-coding sequences in the genomes of several other invertebrates, including pathogenic nematodes, schistosomes and roundworms, herald a new era in phytochelatin research, in which these novel post-translationally synthesized peptides will not only be investigated in the context of phytoremediation but also from a clinical parasitological standpoint.     

Accumulation of metal-binding peptides in fission yeast requires hmt2+

The fission yeast Schizosaccharomyces pombe detoxifies cadmium by synthesizing phytochelatins, peptides of the structure (gamma-GluCys)nGly, which bind cadmium and mediate its sequestration into the vacuole. The fission yeast protein HMT2, a mitochondrial enzyme that can oxidize sulphide, appears to be essential for tolerance to multiple forms of stress, including exposure to cadmium. We found that the hmt2- mutant is unable to accumulate normal levels of phytochelatins in response to cadmium, although the cells possess a phytochelatin synthase that is active in vitro. Radioactive pulse-chase experiments demonstrated that the defect lies in two steps: the synthesis of phytochelations and the upregulation of glutathione production. Phytochelatins, once formed, are stable. hmt2- cells accumulate high levels of sulphide and, when exposed to cadmium, display bright fluorescent bodies consistent with cadmium sulphide. We propose that the precipitation of free cadmium blocks phytochelatin synthesis in vivo, by preventing upregulation of glutathione production and formation of the cadmium-glutathione thiolate required as a substrate by phytochelatin synthase. Thus, although sulphide is required for phytochelatin-mediated metal tolerance, aberrantly high sulphide levels can inhibit this pathway. Precise regulation of sulphur metabolism, mediated in part by HMT2, is essential for metal tolerance in fission yeast.     

Induction of heavy-metal binding phytochelatins by inoculation of cell cultures in standard media

A large increase in phytochelatin (PC) synthesis occurred when cell cultures of different plant species were transferred from spent medium to fresh standard media. Phytochelatin accumulation correlated with the initial concentration of zinc ions in the nutrient solution. After reaching stationary growth phase, phytochelatins had almost disappeared from the cells which indicates a high turnover of these molecules under normal conditions. No significant formation of the heavy-metal complexing phytochelatins was observed if the microelement ions zinc and copper were omitted from the nutrient solutions for plant cell cultures. Both the induction and degradation phenomena of these peptides indicate that phytochelatins are involved in metal ion homeostasis in plants.