Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2+ tolerance and accumulation but not translocation to the shoot

Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd²⁺ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd²⁺ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd²⁺ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd²⁺ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd²⁺ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd²⁺ transport.     

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.     

Effect of heavy metals on the glutathione status in different ectomycorrhizal <Emphasis Type="Italic">Paxillus involutus</Emphasis> strains

The glutathione (GSH) status and heavy metal tolerance were investigated in four Paxillus involutus strains isolated from different heavy-metal-polluted and non-polluted regions of Europe. The heavy metal burden in the habitats did not affect significantly either the heavy metal (Cr₂O₇²⁻, Cd²⁺, Hg²⁺, Pb²⁺, Zn²⁺, Cu²⁺) tolerance and accumulation or the GSH production of the strains tested. Exposures to heavy metals increased the intracellular GSH concentrations in 12 from 24 experimental arrangements (four strains exposed to six heavy metals) independently of the habitats of the strains. The importance of GSH in heavy metal tolerance (high MIC values, ability to accumulate heavy metals and to grow in the presence of heavy metals) was thus demonstrated in this ectomycorrhizal fungus.     

Non-Ionic Contrast Media Induces Oxidative Stress and Apoptosis Through Ca2+ Influx in Human Neutrophils

Non-ionic contrast media (CM) can induce tissue kidney injury via activation of phagocytosis and oxidative stress, although the mechanisms of injury via neutrophils are not clear. We investigated the effects of CM on oxidative stress and Ca²⁺ concentrations in serum and neutrophils of humans. Ten migraine patients were used in the study. Serum and neutrophil samples from patients?? peripheral blood were obtained before (control) and 30?min after non-ionic (iopromide) CM injection. The neutrophils were incubated with non specific transient receptor potential 2 (TRPM2) channel blocker, 2-aminoethoxydiphenyl borate (2-APB), and voltage gated Ca²⁺ channel blockers, verapamil plus diltiazem. Serum and neutrophil lipid peroxidation, apoptosis and intracellular Ca²⁺ concentrations levels were higher in the CM group than in controls. The neutrophilic reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) levels as well as serum vitamin E and ??-carotene concentrations were lower in the CM group than in controls. Neutrophil lipid peroxidation levels were lower in the CM+2-APB and CM+verapamil-diltiazem groups than in the CM group, although GSH, GSH-Px and intracellular Ca²⁺ values increased in the CM+2-APB and CM+verapamil-diltiazem groups. However, caspase-3, caspase-9, vitamin A and vitamin C values were unaltered by CM treatment. In conclusion, we observed that CM induced oxidative stress and Ca²⁺ influx by decreasing vitamin E, ??-carotene and Ca²⁺ release levels in human serum and neutrophils. However, we observed protective effects of Ca²⁺ channel blockers on Ca²⁺ influx in neutrophils.     

Heavy metal detoxification in higher plants - a review

A set of heavy-metal-complexing peptides was isolated from plants and plant suspension cultures. The structure of these peptides was established as (γ-glutamic acid-cysteine)_n-glycine (n=2–11) [(γ-Glu-Cys)_n-Gly]. These peptides appear upon induction of plants with metals of the transition and main groups (Ib-Va, Z=29−83) of the periodic table of elements. These peptides, called phytochelatins (PC), are induced in all autotrophic plants so far analyzed, as well as in select fungi. Some species of the order Fabales and the family Poaceae synthesize aberrant PC that contain, at their C-terminal end, either β-alanine, serine or glutamic acid. For this group of peptides the name iso-PC is proposed. The biosynthesis of PC proceeds by metal activation of a constitutive enzyme that uses glutathione (GSH) as a substrate; this enzyme is a γ-glutamylcysteine dipeptidyl transpeptidase which was given the trivial name PC synthase. It catalyzes the following reaction: γ-Glu-Cys-Gly+(γ-Glu-Cys)_n-Gly→(γ-Glu-Cys)_n+1-Gly+Gly. The plant vacuole is the transient storage compartment for these peptides. They probably dissociate, and the metal-free peptide is subsequently degraded. Sequestration of heavy metals by PC confers protection for heavy-metal-sensitive enzymes. The isolation of a Cd²⁺-sensitive cadl mutant of Arabidopsis thaliana, that is deficient in PC synthase, demonstrates conclusively the importance of PC for heavy metal tolerance. In spite of the fact that nucleic acid sequences and proteins are found in higher plants that have distant homology to animal metallothioneins, there is absolutely no experimental evidence that these ‘plant metallothioneins’ are involved in the detoxification of heavy metals. PC synthase will be an interesting target for biotechnological modification of heavy metal tolerance in higher plants.     

Does Cd2+ use Ca2+ channels to penetrate into chloroplasts? — a preliminary study

Effects of Cd²⁺ toxicity on the photochemistry of primary leaves at two different growth stages of runner bean plants were taken into consideration to study whether Cd²⁺ can use Ca²⁺ channels to get into chloroplasts. Different concentrations of Cd²⁺, ionophore A 23187 and Ca²⁺ were vacuum infiltrated into leaf discs. Toxicity of Cd²⁺ at the donor side of PSII depending on the metal concentration and age of the plants was confirmed. Application of ionophore caused an increase in the sensitivity of the PSII donor side to low Cd²⁺ concentrations. Additional supply of Ca²⁺ in the infiltration medium abolished toxic effect of Cd²⁺ on photochemical activity, except for older plants, where it was not observed for the highest Cd²⁺ concentration. In our opinion it is possible that Cd²⁺ penetrates into chloroplasts via Ca²⁺ channels. Age-dependent Ca²⁺ content in the primary leaves seems to be a very important factor protecting photochemical activity from the toxic action of Cd²⁺.     

Stress response in two strains of the aquatic hyphomycete <Emphasis Type="BoldItalic">Heliscus lugdunensis</Emphasis> after exposure to cadmium and copper ions

Biochemical responses to cadmium (Cd²⁺) and copper (Cu²⁺) exposure were compared in two strains of the aquatic hyphomycete (AQH) Heliscus lugdunensis. One strain (H4-2-4) had been isolated from a heavy metal polluted site, the other (H8-2-1) from a moderately polluted habitat. Conidia of the two strains differed in shape and size. Intracellular accumulation of Cd²⁺ and Cu²⁺ was lower in H4-2-4 than in H8-2-1. Both␣strains synthesized significantly more glutathione (GSH), cysteine (Cys) and γ-glutamylcysteine (γ-EC) in the presence of 25 and 50 μM Cd²⁺, but quantities and rates of synthesis were different. In H4-2-4, exposure to 50 μM Cd²⁺ increased GSH levels to 262% of the control; in H8-2-1 it increased to 156%. Mycelia of the two strains were analysed for peroxidase, dehydroascorbate reductase, glutathione reductase and glucose-6-phosphate dehydrogenase. With Cd²⁺ exposure, peroxidase activity increased in both strains. Cu²⁺ stress increased dehydroascorbate reductase activity in H4-2-4 but not in H8-2-1. Dehydroascorbate reductase and glucose-6-phosphate dehydrogenase activities progressively declined in the presence of Cd²⁺, indicating a correlation with Cd²⁺ accumulation in both strains. Cd²⁺ and Cu²⁺ exposure decreased glutathione reductase activity.     

Effects of cadmium on gas exchange and phytohormone contents in citrus

The effect of increased Cd²⁺ concentrations in the watering solution on citrus physiology was studied by using two citrus genotypes, Cleopatra mandarin and Carrizo citrange. Cadmium content in roots and leaves was tested together with measurements of leaf damage, gas exchange parameters, and hormonal contents. Citrus roots efficiently retained Cd²⁺ avoiding its translocation to the shoots and Cleopatra mandarin translocated less Cd²⁺ than Carrizo. With increasing Cd²⁺ concentration all gas exchange parameters were decreased more in Carrizo than in Cleopatra mandarin. Cd-induced increases in abscisic acid and salicylic acid contents were observed in leaves but not in roots of both genotypes.     

Localization and functional characterization of metal-binding sites in phytochelatin synthases

Metal-binding domains consisting of short, contiguous stretches of amino acids are found in many proteins mediating the transport, buffering, trafficking or detoxification of metal ions. Phytochelatin synthases are metal-activated enzymes that function in the detoxification of Cd²⁺ and other toxic metal and metalloid ions. In order to localize Cd²⁺-binding sites, peptide libraries of two diverse phytochelatin synthases were synthesized and incubated with ¹⁰⁹Cd²⁺. Distinct binding sites and binding motifs could be localized based on the patterns of Cd²⁺-binding. The number of binding sites was consistent with previous findings for recombinant protein. Positions of binding sites appeared to be conserved even among diverse phytochelatin synthases. Mutant peptide analysis was used to assess the contribution of exemplary amino acids to binding. Several binding motifs contain cysteines or glutamates. For cysteines a strong correlation was found between binding activity and degree of conservation among known phytochelatin synthases. These findings indicate the suitability of peptide scanning for the identification of metal-binding sites. The functional role of several cysteines was investigated by expression of hemagglutinin-tagged phytochelatin synthases in phytochelatin synthase-deficient, Cd²⁺-hypersensitive Schizosaccharomyces pombe cells. The data are consistent with a model suggesting functionally essential metal-binding activation sites in the N-terminal catalytic part of phytochelatin synthases and additional binding sites at the C-terminus not essential for activity.Abbreviations EMM Edinburgh's minimal medium - GSH glutathione - HA hemagglutinin - PC phytochelatin - PCS phytochelatin synthase     

重金属镉(Cd)在植物体内的转运途径及其调控机制

重金属镉(Cd)的毒害效应与其由土壤向植物地上部分运输有关,揭示Cd~(2+)转运途径及其调控机制可为提高植物抗镉性以及镉污染的植物修复提供依据。对Cd~(2+)在植物体内的转运途径,特别是限制Cd~(2+)移动的细胞结构和分子调控机制研究进展进行了回顾。Cd~(2+)通过共质体和质外体途径穿过根部皮层进入木质部的过程中,大部分在皮层细胞间沉积,少部分抵达中柱后转移到地上部分。为了免受Cd~(2+)的危害,植物体产生了多种限制Cd~(2+)吸收和转移的生理生化机制:1)环绕在内皮层径向壁和横向壁上的凯氏带阻止Cd~(2+)以质外体途径进入木质部;2)螯合剂与进入根的Cd~(2+)螯合形成稳定化合物并区隔在液泡中;3)通过H+/Cd~(2+)离子通道等将Cd~(2+)逆向转运出根部。植物共质体和质外体途径转运重金属镉的能力以及两条途径的串扰尚待进一步明晰和阐明。     

Effects of cadmium and lead on the accumulation of Ca2+ and K+ and on the influx and translocation of K+ in wheat of low and high K+ status

The effects of cadmium and lead on the internal concentrations of Ca²⁺ and K⁺, as well as on the uptake and translocation of K(⁸⁶Rb⁺) were studied in winter wheat (Triticum aestivum L. a. MV-8) grown hydroponically at 2 levels of K⁺ (100 uM and 10 mM). Cd²⁺ and Pb²⁺ were applied in the nutrient solution in the range of 0.3 to 1000 u.M. Growth was more severely inhibited by Cd²⁺ and in the high-K⁺ plants as compared to Pb^z+ and low-K⁺ plants. Ions of both heavy metals accumulated in the roots and shoots, but the K⁺ status influenced their levels. Ca²⁺ accumulation was increased by low concentrations of Cd²⁺ mainly in low-K⁺ shoots, whereas it was less influenced by Pb²⁺. The distribution of Cd²⁺ and Ca²⁺ in the plant and in the growth media indicated high selectivity for Cd²⁺ in the root uptake, while Ca²⁺ was preferred in the radial and/or xylem transport. Cd²⁺ strongly inhibited net K⁺ accumulation in high-K⁺ plants but caused stimulation at low K⁺ supply. In contrast, the metabolis-dependent influx of K⁺(⁸⁶Rb⁺) was inhibited in low-K⁺ plants, while the passive influx in high-K⁺ plants was stimulated. Translocation of K⁺ from the roots to the shoots was inhibited by Cd²⁺ but less influenced in Pb²⁺-treated plants. It is concluded that the effects of heavy metals depend upon the K⁺-status of the plants.     

Polyamines and heavy metal stress: the antioxidant behavior of spermine in cadmium- and copper-treated wheat leaves

Polyamine metabolism, as well as spermine (Spm) antioxidant properties, were studied in wheat leaves under Cd²⁺ or Cu²⁺ stress. The oxidative damage produced by both metals was evidenced by an increased of thiobarbituric acid reactive substances (TBARS) and a significant decrease in glutathione under both metal treatments. Ascorbate peroxidase (APOX) and glutathione reductase (GR) activities were reduced by both metals to values ranging from 30% to 64% of the control values. Conversely, copper produced a raise in superoxide dismutase activity. The high putrescine (Put) content detected under Cd²⁺ stress (282% over the control) was induced by the increased activity of both enzymes involved in Put biosynthesis, arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). However, only ODC activity was increased in wheat leaves subjected to Cu²⁺ stress, leading to a lower Put rise (89% over the controls). Spermidine (Spd) content was not affected by metal treatments, while Spm was significantly reduced. Pretreatment with Spm completely reverted the metals-induced TBARS increase whereas metals-dependent H₂O₂ deposition on leaf segments (revealed using diaminobenzidine), was considerably reduced in Spm pretreated leaf segments. This polyamine failed to reverse the depletion in APOX activity and glutathione (GSH) content produced by Cd²⁺ and Cu²⁺, although it showed an efficient antioxidant behavior in the restoration of GR activity to control values. These results suggest that Spm could be exerting a certain antioxidant function by protecting the tissues from the metals-induced oxidative damage, though this effect was not enough to completely avoid Cd²⁺ and Cu²⁺ effect on certain antioxidant enzymes, though the precise mechanism of protection still needs to be elucidated.     

Effect of cadmium on antioxidative enzymes,glutathione content,and glutathionylation in tall fescue

The aim of this work was to assess the effect of different Cd2+concentrations on some antioxidant enzymes in Festuca arundinacea. Increased activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione S-transferase, and glutathione reductase were ascertained in response to low Cd²⁺ concentrations (0–20 μM), whereas the enzyme activities were less increased or decreased at a higher Cd²⁺ dosage (50 μM) and a longer exposure. The content of reduced glutathione (GSH) decreased significantly with increasing Cd²⁺ concentrations, whereas the content of oxidized glutathione (GSSG) increased proportionally to the amount of Cd²⁺ applied. Further experiments, performed by incubating the enzyme extracts with oxidized glutathione, evidenced that the addition of GSSG to the incubation mixtures caused significant decreases of some enzymatic activities. Finally, the effect of glutathione S-transferase, FaGST I, extracted from fescue seedlings and purified till homogeneity, on these enzyme activities was investigated. It was found that FaGST I enhanced the decreased enzymatic activities caused by GSSG.     

Catch me if you can! Novel aspects of cadmium transport in mammalian cells

Cadmium (Cd²⁺) is a nonessential divalent metal ion that causes toxicity in multiple organs in humans. In order for toxicity to occur Cd²⁺ must first enter cells by utilizing transport pathways for essential metals. This review focuses on studies in which Cd²⁺ transport was directly demonstrated by electrophysiological, radiotracer or Cd²⁺-sensitive fluorescent dye techniques. The chemistry of Cd²⁺ and metal ions in general is addressed in the context of properties relevant for transport through membrane proteins, such as hydration energy. Apart from transport by the ZIP transporters SLC39A8 and SLC39A14, which is not topic of the review, uptake of free Cd²⁺ has been demonstrated for the Fe²⁺/H⁺ cotransporter divalent metal transporter 1. Moreover, the multiligand endocytic receptors megalin and cubilin take up cadmium-metallothionein complexes via receptor-mediated endocytosis. The role of ATP binding cassette transporters in Cd²⁺ efflux from cells is also discussed. Both the multidrug resistance-associated protein 1 and cystic fibrosis transmembrane conductance regulator are likely to transport cadmium–glutathione complexes out of cells, whereas transport of free Cd²⁺ by the multidrug resistance P-glycoprotein remains controversial. Finally, arguments for and against Cd²⁺ transport by Ca²⁺ channels are presented. Most N- and L-type Ca²⁺ channels are closed at resting membrane potential (with the exception of CaV1.3 channels) and therefore unlikely to allow significant Cd²⁺ influx under physiological conditions. CaV3.1 and CaV3.2 T-type calcium channels are permeated by divalent metal ions, such as Fe²⁺ and Mn²⁺ because of considerable “window” currents close to resting membrane potential and could be responsible for tonic Cd²⁺ entry. TRPM7 and the mitochondrial Ca²⁺ uniporter are other likely candidates for Cd²⁺ transporters, whereas the role of Orai proteins, the store-operated calcium channels carrying Ca²⁺ release-activated Ca²⁺ current, in Cd²⁺ influx remains to be investigated.     

Cadmium impairs ion homeostasis by altering K+ and Ca2+ channel activities in rice root hair cells

Cadmium (Cd²⁺) interferes with the uptake, transport and utilization of several macro‐ and micronutrients, which accounts, at least in part, for Cd²⁺ toxicity in plants. However, the mechanisms underlying Cd²⁺ interference of ionic homeostasis is not understood. Using biophysical techniques including membrane potential measurements, scanning ion‐selective electrode technique for non‐invasive ion flux assays and patch clamp, we monitored the effect of Cd²⁺ on calcium (Ca²⁺) and potassium (K⁺) transport in root hair cells of rice. Our results showed that K⁺ and Ca²⁺ contents in both roots and shoots were significantly reduced when treated with exogenous Cd²⁺. Further studies revealed that three cellular processes may be affected by Cd²⁺, leading to changes in ionic homeostasis. First, Cd²⁺‐induced depolarization of the membrane potential was observed in root hair cells, attenuating the driving force for cation uptake. Second, the inward conductance of Ca²⁺ and K⁺ was partially blocked by Cd²⁺, decreasing uptake of K⁺ and Ca²⁺. Third, the outward K⁺ conductance was Cd²⁺‐inducible, decreasing the net content of K⁺ in roots. These results provide direct evidence that Cd²⁺ impairs uptake of Ca²⁺ and K⁺, thereby disturbing ion homeostasis in plants.     

Antioxidant response to cadmium in Phragmites australis plants

Phragmites (Phragmites australis Cav. (Trin.) ex Steud) plants exposed to a high concentration of CdSO₄ (50 μM) for 21 d were analysed with respect to the distribution of metal, its effects on antioxidants, the antioxidant enzymes and the redox status in leaves, roots and stolons. The highest accumulation of Cd²⁺ occurred in roots followed by leaves, and it was not significant in the stolons when compared with the control plants. In particular, in roots from Cd-treated plants, both the high amount of GSH and the parallel increase of glutathione-S-transferase (EC 2.5.1.18; GST) activity seemed to be associated with an induction of the detoxification processes in response to the high cadmium concentration. Superoxide dismutase (EC 1.15.1.1; SOD), ascorbate peroxidase (EC 1.11.1.11; APX), glutathione reductase (EC 1.6.4.2; GR) and catalase (EC 1.11.1.6; CAT) activities as well as reduced and oxidised glutathione contents in all samples of leaves, roots and stolons were increased in the presence of Cd²⁺ when compared to control plants. Despite the fact that Cd²⁺ has a redox characteristic not compatible with the Fenton-type chemistry that produces active oxygen species, the antioxidant response is widespread and generic. Increased activities of antioxidant enzymes in Cd-treated plants suggest that metal tolerance in Phragmites plants might be associated to the efficiency of these mechanisms.     

The role of glutathione in cadmium ion detoxification: coordination modes and binding properties--a density functional study

In this investigation the reduced form of glutathione molecule (GSH) was considered as a model in the coordination chemistry of Cd²⁺ and group thiol-containing peptides. Three different forms of GSH, corresponding to the prevalent ones in gas-phase and in aqueous solution, were taken into account in the metallation process. The obtained complexes were characterized at the density functional B3LYP level with the purpose to give better insight in the chelation mechanism of GSH with heavy metal ions in living organisms. Solvent effects, whether with explicit water molecules or with polarizable continuum model (PCM), were considered on the most stable cadmium-complexes obtained by every GSH charged species examined.     

Possible role of ca2+ in heavy metal cytotoxicity

• 1.1. Organic xenobiotic metabolism often results in oxidative stress, involving GSH depletion, alteration of thiol/disulphide balance and peroxidation of membrane lipids. These events can lead to the disruption of Ca²⁺ homeostasis, through impairment of the Ca²⁺ translocases present in cellular membranes. Inhibition of the activity of Ca,Mg-ATPases due to oxidation of their SH groups would lead to uncontrolled rises in cytosolic Ca²⁺ levels resulting in loss of cell viability.
• 2.2. These observations seem to be of interest when interpreting the biochemical mechanisms of heavy metal cytotoxicity. Since these cations (such as Hg²⁺, Cu²⁺, Cd²⁺ and Zn) have an extremely high affinity for SH groups, they may affect the function of SH containing proteins, such as the Ca,Mg-ATPases, as in the case of oxidative stress.
• 3.3. Results are reported indicating that Hg²⁺ may stimulate Ca²⁺ influx through voltage-dependent channels in different experimental systems. Moreover, evidence is presented that heavy metals can inhibit Ca,Mg-ATPase activity and affect mitochondrial functions in the cells of different organisms.
• 4.4. The possibility that heavy metal cytotoxicity is mediated through disruption of Ca²⁺ homeostasis is discussed.