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.     

Cadmium-responsive thiols in the ectomycorrhizal fungus Paxillus involutus

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (gamma-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and gamma-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.     

Molecular biology of metal tolerances of plants

Abstract. The review discusses some of the important aspects of the molecular biology of metal tolerances in animals, fungi and plants. First, results of classical ecological and genetical studies are briefly outlined. The evidence for the occurrence and properties of metal-binding proteins (metallothioneins) and peptides (phytochelatins) in fungi and plants is described. It is concluded that at present there is no firm evidence to suggest that a protein homologous with the metallothioneins of animals and fungi occurs in plants. The discovery of phytochelatins, γ-glutarnyl peptides, containing only glutamic acid, cysteine and glycine, in plants is described and evidence for their role in heavy metal tolerance is assessed. The difference between sulphur metabolism in animals and plants and its relationship to heavy metal tolerances is discussed in terms of the occurrences of metallothioneins in animals and phytochelalins in plants. Future prospects for research in this area are outlined in terms of identification of plant genes coding for metallothioneins and for the enzymes involved in the synthesis of phytochelatins.     

Cadmium-Responsive Thiols in the Ectomycorrhizal Fungus Paxillus involutus

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (γ-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and γ-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.     

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.     

Interactions between plant hormones and thiol-related heavy metal chelators

Upon toxic metal stress numerous defence mechanisms have been induced, including the synthesis of metal-binding ligands and plant hormones or plant growth regulators in plants. As several elements in the promoter region of the heavy metal-responsive genes can be activated by plant hormones and growth regulators, understanding and revealing possible and special relationships between these regulator compounds and the metal chelator phytochelatins, which are in the first line of heavy metal defence mechanism is of great important. Phytochelatins are synthetized from glutathione and have a structure of [(γ-Glu-Cys)_n]-Gly, where n is the number of repetition of the (γ-Glu-Cys) units. Evidences for the role of PCs in heavy metal tolerance are very strong; however, little information is available on how plant growth regulators influence the phytochelatin synthesis at molecular or even gene expression level. In the present review we provide an overview of the role and synthesis of phytochelatins in metal-tolerance mechanism from a new point of view, i.e. their relation to the plant growth regulator molecules, with special regard also on those cases, when close direct relationship exists because of the partly overlapped synthesis pathways of plant growth regulators and glutathione/phytochelatins.     

Selection <Emphasis Type="Italic">in vitro</Emphasis> and accumulation of phytochelatins in cadmium tolerant cell line of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Cucumber (Cucumis sativus L.) cells from suspension culture were selected for their ability to grow and divide rapidly in toxic concentration of cadmium. As a result of selection a cell suspension tolerant to 100 M cadmium chloride (CdCl₂) was initiated. The selected tolerant line exhibited stable and repeatable increase in fresh and dry weight of cells in the presence of cadmium. The accumulated level of phytochelatins in cadmium sensitive (unselected) and tolerant cell line was measured by high performance liquid chromatography (HPLC) after 3, 24 h and 5 days of cadmium treatment. It was shown that in both cell lines Cd induced accumulation of phytochelatins and simultaneous glutathione depletion occurred. No distinct changes were found after 3 and 24 h of cadmium treatment whereas after 5 days of exposure to the metal, the level of phytochelatins was two times higher in the sensitive cell line as compared to the tolerant one. The accumulation of phytochelatins was correlated with cadmium concentration that increased in both cell lines during the course of cell exposure to metal. However, the level of cadmium was always lower in the tolerant cell line. The results showed no direct correlation between the tolerance of cucumber cells to Cd and the accumulated level of phytochelatins. Other mechanisms responsible for the increased tolerance of cucumber cells exposed to Cd are discussed.     

The role of phytochelates in plant growth and productivity

Plants require minimal amounts of certain metals (Zn,Fe,Cu,etc) for optimal growth and productivity, but excess of these metals leads to cell death. When growth is limited by metal excess or metal deficiency plants respond by synthesizing nonproteinogenic chelating substances. Phytosiderophores are secreted by roots of iron deficient grasses and are important in providing sufficient Fe for normal growth. In response to growth-inhibitory levels of heavy metals plants synthesize metal-binding phytochelatins which detoxify excess metals. Biostimulants such as humic substances and oligomers of lactic acid have properties in common with both phytosiderophores and phytochelatins. The word phytochelates is proposed as a generic term to cover substances that affect plant growth by acting as chelating agents.     

Caenorhabditis elegans expresses a functional phytochelatin synthase.

The formation of phytochelatins, small metal-binding glutathione-derived peptides, is one of the well-studied responses of plants to toxic metal exposure. Phytochelatins have also been detected in some fungi and some marine diatoms. Genes encoding phytochelatin synthases (PCS) have recently been cloned from Arabidopsis, wheat and Schizosaccharomyces pombe. Surprisingly, database searches revealed the presence of a homologous gene in the Caenorhabditis elegans genome, DDBJ/EMBL/GenBank accession no. 266513. Here we show that C. elegans indeed expresses a gene coding for a functional phytochelatin synthase. CePCS complements the Cd2+ sensitivity of a Schizosaccharomyces pombe PCS knock-out strain and confers phytochelatin synthase activity to these cells. Thus, phytochelatins may play a role for metal homeostasis also in certain animals.     

Cd - tolerance of maize, rye and wheat seedlings

The effect of cadmium on growth parameters of seedlings of maize, rye and wheat as well as the role of phytochelatins in Cd detoxication in these species were studied. Cadmium was found to inhibit root growth and decrease fresh weight and water content in roots and shoots of the studied plants. Although a considerably lower Cd accumulation was shown in maize seedlings than in other species, they were characterized by the highest sensitivity to cadmium. Among γ-Glu-Cys peptides synthetized by plant species, phytochelatins — glutathione derivatives predominated. In maize they were synthetized in amounts sufficient for binding the total pool of the metal taken up, and the detoxication mechanism was localized in their roots. Larger amounts of cadmium were accumulated in roots of wheat and rye, but the quantity of the formed γ-Glu-Cys peptides seems insufficient for detoxication of the metal.     

有机酸存在下小麦体内Cd的生物毒性和植物络合素(PCs)合成的关系

采用溶液培养方式 ,研究了有机酸存在下小麦体内 Cd的生物毒性和植物络合素 (PCs)合成的相关关系 ,试图寻求一种与小麦体内 Cd的生物毒性高度相关的评价指标。结果显示 ,Cd胁迫对小麦产生明显的毒害效应并诱导小麦根系内 PCs的大量合成。EDTA、DTPA、柠檬酸、苹果酸和草酸的适量供应可不同程度减轻或消除 Cd的生物毒性 ,其强弱顺序为 EDTA >DTPA 柠檬酸 >苹果酸≈草酸。与此同时 ,小麦根系内 PCs的诱导量也有明显下降 ,与 Cd的生物毒性保持一定的线性关系 ,且在EDTA、DTPA和柠檬酸供应下尤为显著。表明 PCs可以作为一项敏感的生化指标 (biochem ical indicator)用来评价和预测环境中 Cd的污染 ,并有望成为重金属生物有效性评价系统中一种新的补充方法     

Characterization of cadmium- and lead- phytochelatin complexes formed in a marine microalga in response to metal exposure

Phytochelatins (PC_n) are thiol-containing peptides with general structure (-Glu-Cys)_n-Gly enzymatically synthesized by plants and algae in response to metal exposure. They are involved in the cellular detoxification mechanism for their capability to form stable metal-phytochelatin complexes. The speciation of Cd and Pb complexes with phytochelatins has been studied in laboratory cultures of the marine diatom Phaeodactylum tricornutum. An approach based on size-exclusion chromatography (SEC) with off-line detection of phytochelatins, by reverse-phase HPLC, and metal ion, by atomic absorption spectrometry, has been used. The formation of Cd- and Pb-PC_n complexes with n-value from 3 to 6 was demonstrated. The metal-PC_n complexes formed with Cd appear to be different from those formed with Pb for the number of molecules of peptide involved in the complex and for the amount of the metal ion bound. The chromatographic behaviour of metal-PC_n complexes is consistent with Pb-PC_n complexes in which only a molecule of peptide binds the metal ion, and with Cd-PC_n complexes containing two or more molecules of peptide. The metal/peptide molar ratio in Cd-PC_n complexes was higher that in Pb-PC_n complexes. The formation of Cd- or Pb-PC₂ complexes was not demonstrated, probably for a dissociation during the cellular extract preparation. The effectiveness of phytochelatins in the detoxification of these two metal ions in this alga is discussed.     

Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast.

Phytochelatins play major roles in metal detoxification in plants and fungi. However, genes encoding phytochelatin synthases have not yet been identified. By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance. TaPCS1 encodes a protein of approximately 55 kDa with no similarity to proteins of known function. We identified homologs of this new gene family from Arabidopsis thaliana, Schizosaccharomyces pombe, and interestingly also Caenorhabditis elegans. The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast. PCS-expressing cells accumulate more Cd2+ than controls. PCS expression mediates Cd2+ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS-induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd2+ and Cu2+ and are unable to synthesize phytochelatins upon Cd2+ exposure as determined by HPLC analysis. Saccharomyces cerevisiae cells expressing PCS produce phytochelatins. Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity. These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes.     

Metal ion ligands in hyperaccumulating plants

Metal-hyperaccumulating plants have the ability to take up extraordinary quantities of certain metal ions without succumbing to toxic effects. Most hyperaccumulators select for particular metals but the mechanisms of selection are not understood at the molecular level. While there are many metal-binding biomolecules, this review focuses only on ligands that have been reported to play a role in sequestering, transporting or storing the accumulated metal. These include citrate, histidine and the phytosiderophores. The metal detoxification role of metallothioneins and phytochelatins in plants is also discussed.     

Homo-Phytochelatins Are Synthesized in Response to Cadmium in Azuki Beans

In a recent report, it was claimed that azuki beans (Vigna angularis) do not synthesize phytochelatins (PCs) upon exposure to cadmium, although glutathione (GSH), the substrate for PC synthesis, is present in this plant. This legume species thus would be the first exception in the plant kingdom that would fail to complex heavy metals by PCs. Here, we report that not GSH, but only homoglutathione can be detected in this plant and that homo-phytochelatins are formed when azuki beans are challenged with heavy metals such as cadmium. We also show that the 5,5'-dithiobis(2-nitrobenzoic acid)-oxidized GSH reductase recycling assay, used for GSH quantification in the recent study of heavy metal tolerance in azuki beans, reacts both with GSH and homoglutathione and therefore cannot be used when biological samples should be analyzed exclusively for GSH.     

Functional characterization of an unusual phytochelatin synthase, LjPCS3, of Lotus japonicus

In plants and many other organisms, phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins from glutathione in the presence of certain metals and metalloids. We have used budding yeast (Saccharomyces cerevisiae) as a heterologous system to characterize two PCS proteins, LjPCS1 and LjPCS3, of the model legume Lotus japonicus. Initial experiments revealed that the metal tolerance of yeast cells in vivo depends on the concentrations of divalent cations in the growth medium. Detailed in vivo (intact cells) and in vitro (broken cells) assays of PCS activity were performed with yeast expressing the plant enzymes, and values of phytochelatin production for each metal tested were normalized with respect to those of cadmium to correct for the lower expression level of LjPCS3. Our results showed that lead was the best activator of LjPCS1 in the in vitro assay, whereas, for both assays, arsenic, iron, and aluminum were better activators of LjPCS3 and mercury was similarly active with the two enzymes. Most interestingly, zinc was a powerful activator, especially of LjPCS3, when assayed in vivo, whereas copper and silver were the strongest activators in the in vitro assay. We conclude that the in vivo and in vitro assays are useful and complementary to assess the response of LjPCS1 and LjPCS3 to a wide range of metals and that the differences in the C-terminal domains of the two proteins are responsible for their distinct expression levels or stabilities in heterologous systems and patterns of metal activation.     

Phytochelatin synthesis and response of antioxidants during cadmium stress in Bacopa monnieri L.

The phytotoxicity imposed by cadmium (Cd) and its detoxifying responses of Bacopa monnieri L. have been investigated. Effect on biomass, photosynthetic pigments and protein level were evaluated as gross effect, while lipid peroxidation and electrolyte leakage reflected oxidative stress. Induction of phytochelatins and enzymatic and non-enzymatic antioxidants were monitored as plants primary and secondary metal detoxifying responses, respectively. Plants accumulated substantial amount of Cd in different plant parts (root, stem and leaf), the maximum being in roots (9240.11 microg g(-1) dw after 7 d at 100 microM). Cadmium induced oxidative stress, which was indicated by increase in lipid peroxidation and electrical conductivity with increase in metal concentration and exposure duration. Photosynthetic pigments showed progressive decline while protein showed slight increase at lower concentrations. Enzymes viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) showed stimulation except catalase (CAT, EC 1.11.1.6) which showed declining trend. Initially, an enhanced level of cysteine, glutathione and non-protein thiols was observed, which depleted with increase in exposure concentration and duration. Phytochelatins induced significantly at 10 microM Cd in roots and at 50 microM Cd in leaves. The phytochelatins decreased in roots at 50 microM Cd, which may be correlated with reduced level of GSH, probably due to reduced GR activity, which exerted increased oxidative stress as also evident by the phenotypic changes in the plant like browning of roots and slight yellowing of leaves. Thus, besides synthesis of phytochelatins, availability of GSH and concerted activity of GR seem to play a central role for Bacopa plants to combat oxidative stress caused by metal and to detoxify it. Plants ability to accumulate and tolerate high amount of Cd through enhanced level of PCs and various antioxidants suggest it to be a suitable candidate for phytoremediation.     

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.     

Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins

A novel strategy using synthetic phytochelatins is described for the purpose of developing microbial agents for enhanced bioaccumulation of toxic metals. Synthetic genes encoding for several metal-chelating phytochelatin analogs (Glu-Cys)(n)Gly (EC8 (n = 8), EC11 (n = 11), and EC20 (n = 20)) were synthesized, linked to a lpp-ompA fusion gene, and displayed on the surface of E. coli. For comparison, EC20 was also expressed periplasmically as a fusion with the maltose-binding protein (MBP-EC20). Purified MBP-EC20 was shown to accumulate more Cd(2+) per peptide than typical mammalian metallothioneins with a stoichiometry of 10 Cd(2+)/peptide. Cells displaying synthetic phytochelatins exhibited chain-length dependent increase in metal accumulation. For example, 18 nmoles of Cd(2+)/mg dry cells were accumulated by cells displaying EC8, whereas cells exhibiting EC20 accumulated a maximum of 60 nmoles of Cd(2+)/mg dry cells. Moreover, cells with surface-expressed EC20 accumulated twice the amount of Cd(2+) as cells expressing EC20 periplasmically. The ability to genetically engineer ECs with precisely defined chain length could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal.     

Plant responses to metal toxicity

Metal toxicity for living organisms involves oxidative and/or genotoxic mechanisms. Plant protection against metal toxicity occurs, at least in part, through control of root metal uptake and of long distance metal transport. Inside cells, proteins such as ferritins and metallothioneins, and glutathion-derived peptides named phytochelatins, participate in excess metal storage and detoxification. Low molecular weight organic molecules, mainly organic acids and amino acids and their derivatives, also play an important role in plant metal homeostasis. When these systems are overloaded, oxidative stress defense mechanisms are activated. Molecular and cellular knowledge of these processes will be necessary to improve plant metal resistance. Occurrence of naturally tolerant plants which hyperaccumulate metals provides helpful tools for this research.