Changes in the levels of phytochelatins and related metal-binding peptides in chickpea seedlings exposed to arsenic and different heavy metal ions

Phytochelatin-related peptides were analyzed in chickpea plants exposed to six different heavy-metal ions. Cadmium and arsenic stimulated phytochelatin and homophytochelatin synthesis in roots but other metals did not. These metals, however, caused an overall increase in the precursors, glutathione, homoglutathione and cysteine. These changes may be different biochemical indexes for heavy-metal contamination.     

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.     

Comparison of the toxicity and distribution of cadmium and lead in plant cells

Summary. The toxicity of heavy metals (Cd, Zn, and Pb) was assessed by in vivo observations of their effect on cytoplasmic streaming in Allium cepa L. bulb scale epidermal cells. On the basis of our results, the order of toxicity of the studied cations is Zn < Pb ≪ Cd. The difference in toxicity between cadmium and lead was found to be very large. When cytoplasmic streaming was assessed, this difference was threefold. When the total content of cadmium and lead (determined by inductively coupled plasma mass spectrometry) was the criterion, the difference in toxicity was 15-fold. Fractionation of the tissue and enzymatic digestion of the cells revealed that the largest proportion of cadmium was located in the cell walls (56%), whereas almost all of the lead (97.6%) was accumulated in an insoluble form. The speciation of water-soluble Pb and Cd fractions is discussed on the basis of analysis by capillary zone electrophoresis interfaced with inductively coupled plasma mass spectrometry of water extracts from epidermal cells. Lead and cadmium appeared to be bound mainly to salts, which explains their toxicity. Cadmium was complexed (detoxified) by organic acids, while thiols were the metal-complexing species for lead. Histidine formed complexes with both cadmium and lead. Ultrastructural analyses showed that lead was encapsulated in small vesicles in the cytoplasm. Fluorescence studies of the endoplasmic reticulum (ER) revealed that it underwent extensive fragmentation under the influence of lead, with numerous ER vesicles appearing in the cells. In other words, the lead deposits in the cytoplasm were contained in vesicles arising from fragmentation of the ER. These observations indicate that epidermal cells have a rapid and effective mechanism for detoxifying lead involving the ER, and this may be one of the mechanisms accounting for the lower toxicity of lead in comparison with cadmium. The suitability of Allium cepa bulb scale epidermal cells for use in ecotoxicological studies is also discussed. Step-by-step directions for this test are given. Correspondence and reprints: Department of Ecotoxicology, Institute of Experimental Plant Biology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.     

Induction of non-protein thiols and phytochelatins by cadmium in Eichhornia crassipes

Abstract

Impact of root Cd concentration on production of cysteine, non-protein thiols (NP-SH), glutathione (GSH), reduced glutathione (GSSG), and phytochelatins (PCs) in Eichhornia crassipes exposed to different dilutions of brass and electroplating industry effluent (25%, 50%, and 75%), and synthetic metal solutions of Cd alone (1, 2.5, and 3.5?ppm) and with Cr (1?ppm Cd + 1?ppm Cr, 2.5?ppm Cd + 3?ppm Cr, and 3.5?ppm Cd + 4?ppm Cr) was assessed in a 45?days study. Different treatments were used to understand and compare differential antioxidant defense response of plant under practical drainage (effluent) and experimental synthetic solutions. The production of NP-SH and cysteine was maximum under 2.5?ppm Cd + 3?ppm Cr treatments i.e., 1.78?µmol/g fw and 288?nmol/g fw, respectively. The content of GSH declined whereas that of GSSG increased progressively with exposure duration in all treatments. HPLC chromatograms revealed that the concentrations of PC2, PC3, and PC4 (248, 250, and 288?nmol-SH equiv.g^?1 fw, respectively) were maximum under 1?ppm Cd, 1?ppm Cd + 1?ppm Cr, and 2.5?ppm Cd + 3?ppm Cr treatments, respectively. PC2, PC3, and PC4 concentrations increased with Cd accumulation in the range 812–1354?µg/g dry wt, 1354–2032?µg/g dry wt and 2032–3200?µg/g dry wt, respectively. Thus, the study establishes a direct proportionality relationship between concentration/length of phytochelatins and root Cd concentrations, upto threshold limits, in E. crassipes.     

Cellular and subcellular effects of very heavy ions

The biological effects of irradiation with ions of masses larger than 40 and energies up to 20 MeV per atomic mass unit are reviewed. The objects are viruses, bacterial spores, yeast and mammalian cells. Experimental parameters include loss of colony forming ability, induction of mutants, chromosomal aberrations, cell cycle progression, inhibition of biochemical activities and the formation of strand breaks. Some of the pertinent physical questions--e.g. track structure--are also discussed. It is shown that with very heavy ions the biological effectiveness is no longer unambiguously related to a single parameter like l.e.t. or Z*2/beta 2 but depends strongly on ion energy. This points to the importance of far-reaching delta-electrons. The analysis indicates also that even with very high l.e.t., cells are not killed by the passage of a single particle through their nucleus. Possible implications of the findings for fundamental radiation biology are outlined.     

From cysteine to longer chain thiols: thermodynamic analysis of cadmium binding by phytochelatins and their fragments

Isothermal Titration Calorimetry (ITC) was used to study the binding of Cd(2+) by phytochelatins ((γGlu-Cys)(n)-Gly, PC(n); n = 1-5) and their selected fragments (Cys, Cys-Gly and γGlu-Cys) in order to understand the influence of the chain length on the complex stabilities and the origin of the enhanced affinities in Tris buffer at pH 7.5 and 8.5 and at 25 °C. Different complexes are formed with glutathione (GSH) and its fragments, Cys, Cys-Gly and γGlu-Cys, and their stabilities depend on the corresponding pK(a) value of the thiol group in the ligands. The stability of Cd-PC(n) complexes increases moving towards higher PC(2-5), as well as the complexing capacity expressed as the number of metal ions that can be bound by one ligand molecule. The affinity of Cd(2+) for the PC(n) can be described by the following GSH < PC(2) < PC(3)≤ PC(4)≤ PC(5) sequence. On the basis of these thermodynamic data it is possible to explain the abundance of certain Cd-PC(n) complexes found in nature. The comprehension of the thermodynamic rules that govern the interactions of Cd(2+) with PC(n) and their constituents is of great service in the research with real plant samples subjected to metal stress and in the development of new strategies of bio/phytoremediation.     

Comparison of plant uptake and plant toxicity of various ions in wheat

The effects of varying solution concentrations of manganese (Mn), zinc (Zn), copper (Cu), boron (B), iron (Fe), gallium (Ga) and lanthanum (La) on plant chemical concentrations, plant uptake and plant toxicity were determined in wheat (Triticum aestivum L.) grown in a low ionic strength (2.7×10^–3 M solution culture). Increasing the solution concentration of Mn, Zn, Cu, B, Fe, Ga and La increased plant concentrations of that ion. Asymptotic maximum plant concentrations were reached for Zn (10 mg kg DM^–1 in the roots), Ga (2 mg kg DM^–1 in the tops and 18 mg kg DM^–1 in the roots) and La (0.4 mg kg DM^–1 in the tops and 4 mg kg DM^–1 in the roots). Plant ion concentrations were, on average, 3 times higher in the roots than the tops for Mn and Zn, 7 times for Cu, 9 times for Fe, 12 times for Ga and 15 times for La. In contrast, B concentrations were higher in the tops than the roots by, on average, 2 times. The estimated toxicity threshold (plant concentration at which a rapid decrease in yield occurred) in the tops was 0.4 mg g DM^–1 for B, 2 for Zn, 0.075 for Cu and 0.09 for La and in the roots 0.2 mg g DM^–1 for B, 5 for Zn, 0.3 for Cu and 3 for La. Plant uptake rates of the ions (as estimated by the slope of the relationship between solution ion concentrations and plant ion concentrations) was in the order B 250 mg kg DM^–1 M ^–1). Plant toxicity was estimated as the reciprocal of the plant concentration that reduced yield by 50% (change in relative yield per mg ion kg DM^–1). The plant toxicity of the ions tested was in the order Mn相似文献   

Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides

Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane of E. coli. Isolated cell envelopes of E. coli bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity. The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd2+ from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+. However, Cu2+ ceased contribution of HP for Cd2+ accumulation, probably due to the strong binding of Cu2+ to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.     

Electrochemical survey of the chain length influence in phytochelatins competitive binding by cadmium

Multivariate curve resolution with alternating least squares (MCR-ALS) was applied to voltammetric data obtained in the analysis of the competitive binding of glutathione (GSH) and phytochelatins [(γGlu-Cys)_n-Gly, PC_n, n = 2-5] by Cd²⁺. The displacements between ligands and chain length influence on the competitive binding of PC_n toward Cd²⁺ were investigated. The analysis of the resulting pure voltammograms and concentration profiles of the resolved components suggests that ligands containing more thiol groups are able to displace the shortest chain ligands from their metal complexes, whereas the opposite does not happen. However, when the length of the chain surpasses that of PC₃, the binding capacity of the molecule still increases (i.e., it can bind more metal ions), but the position and shape of the voltammetric signals practically rest unchanged. This suggests that at this level, the stability of metal binding could depend more on the nature of the binding sites separately than on the quantity of the sites (i.e., the chain length).     

Cellular sequestration of cadmium in the hyperaccumulator plant species Sedum alfredii

Spatial imaging of cadmium (Cd) in the hyperaccumulator Sedum alfredii was investigated in vivo by laser ablation inductively coupled plasma mass spectrometry and x-ray microfluorescence imaging. Preferential Cd accumulation in the pith and cortex was observed in stems of the Cd hyperaccumulating ecotype (HE), whereas Cd was restricted to the vascular bundles in its contrasting nonhyperaccumulating ecotype. Cd concentrations of up to 15,000 μg g(-1) were measured in the pith cells, which was many fold higher than the concentrations in the stem epidermis and vascular bundles in the HE plants. In the leaves of the HE, Cd was mainly localized to the mesophyll and vascular cells rather than the epidermis. The distribution pattern of Cd in both stems and leaves of the HE was very similar to calcium but not zinc, irrespective of Cd exposure levels. Extended x-ray absorption fine structure spectroscopy analysis showed that Cd in the stems and leaves of the HE was mainly associated with oxygen ligands, and a larger proportion (about 70% in leaves and 47% in stems) of Cd was bound with malic acid, which was the major organic acid in the shoots of the plants. These results indicate that a majority of Cd in HE accumulates in the parenchyma cells, especially in stems, and is likely associated with calcium pathways and bound with organic acid (malate), which is indicative of a critical role of vacuolar sequestration of Cd in the HE S. alfredii.     

Bioremediation of cadmium induced renal toxicity in Rattus norvegicus by medicinal plant Catharanthus roseus

Cadmium is the second most hazardous metals with bio-concentration factor (BCF)?>?100 Although WHO permitted cadmium concentration in drinking water is 0.005?mg/L, yet the reality is far above to this limit because of industrial utility of this metal. Oral exposure of cadmium to human results in dreadful symptoms of metabolic disorders especially in liver and kidneys. Endogenous protection could be supported by some exogenous herbal supplement (viz., Catharanthus roseus in this case) to overcome the toxic effects. Present Study has been designed to find out the functional renal changes under the effect of cadmium and Catharanthus roseus in the model organism albino rats. Cadmium significantly (p?>?0.01) increases the level of nitrogenous waste (Urea, BUN, Uric Acid and Creatinin), while decreases the serum protein profile in acute and sub-acute sets. Urea concentration of control ranged from 16.56 to 17.72?mg/dl while that of Group-B and D were 19.84 to 20.87?mg/dl and 17.56 to 17.59?mg/dl respectively. Similarly uric acid concentration ranged in control form 6.98 to 8.01?mg/dl in group-B from 7.58 to 10.25?mg/dl, in Group-D 8.02 to 8.59?mg/dl respectively. Creatinin concentration ranged in control 0.57 to 0.65?mg/dl, in group-B 0.97 to 1.02?mg/dl, in group-D – 0.95 to 0.98?mg/dl respectively.These results might be due to altered filtration rate of kidney because of protein disruption. The studies conclude the efficient nephro-protection offered by Catharanthus roseus extract against Cadmium toxicity.     

Induction of proline prototrophs in CHO-K1 cells by heavy ions

Using an established mammalian cell line, Chinese hamster ovary cells (CHO-K1), we have observed the induction of prototrophs by various heavy ions. This cell line requires proline for normal growth in medium with low serum concentration. X-rays, three types of heavy particles (600 MeV/u iron, 670 MeV/u neon, and 320 MeV/u silicon ions), ethylmethane sulphonate and 5-azacytidine were used to induce revertants which were proline independent. Log-phase cells treated with 5-azacytidine showed a very high reversion frequency. The induction frequency per viable cell appears to be dose dependent for these four types of radiation, and the dose-response curves are approximately linear. Our results also indicate that the effectiveness of high-LET particles in inducing proline prototrophs is much greater than that of low-LET radiation. The RBE value for the induction of prototrophs was calculated for neon, silicon, and iron particles and found to be about 1.3, 1.7 and 4.5, respectively. At equal survival level, the reversion frequency for X-rays and EMS was about the same.     

食品中重金属的存在形态及其与毒性的关系

简要介绍了近年来有关食品中重金属形态及其毒性关系的研究进展,归纳了食品中重金属的化学形态特征、不同类型食品中重金属的存在形态、食品中重金属形态与生物有效性和毒性的关系,以及该领域中急需研究解决的问题．     

Poly(γ-glutamylcysteinyl)glycines or phytochelatins and their role in cadmium tolerance of Silene vulgaris

Abstract. The effect of cadmium on growth of Cd-tolerant and -sensitive plants of Silene vulgaris and on the production of metal-binding compounds in both types of plants was studied. The Cd-content of the roots and the Cd-root/shoot ratio was higher in Cd-tolerant plants. A Cd-binding compound (Cd-BC) with an apparent molecular mass of 14.5 kD was isolated from the roots of Cd-tolerant and -sensitive plants, grown in 40 mmol m⁻³ Cd for 21 d. More than 60% of the total Cd in the roots was associated with this compound. Determination of the amino-acid content of the purified Cd-containing compound from both types of plants showed that they possessed a similar amino-acid composition to that of phytochelatins. Only the bis- and tris-forms were present. The amount of Cd and sulphide associated with phytochelatin was greater in tolerant plants than in sensitive ones suggesting that an increased sulphide content of complexes containing peptide, sulphide and Cd may form the basis of evolved Cd-tolerance in Silence vulgaris.     

Fixation and imaging of biological elements: heavy metals, diffusible substances, ions, peptides, and lipids

We tested various fixation and analysis methods to demonstrate by electron microscopy elemental imaging in tissues and cells, i.e., soluble substances such as many kinds of ionic elements, water soluble low molecular peptides, and even organic solvent soluble substances such as lipids. For the ionic elements, we tested frozen dried or freeze-substituted methods and organic or inorganic special chemical precipitation methods combined with microwaved fixation methods. The data were analyzed with electron beam X-ray microanalysis, electron energy filtered imaging analysis, and electron microscope autoradiography. The data were demonstrated as elemental distribution images and were calculated quantitatively. For the soluble low molecular peptides, we developed a tannic acid and aldehyde method combined with microwaved fixation. We discuss the theoretical background of the tannic acid fixation and microwaved fixation methods. For the organic solvent soluble substances, i.e., lipids including steroids, we successfully tested the use of a mixed fixative of aldehyde and osmium, digitonization, and osmification with the use of p-phenylendiamine or imidazole. We also proposed some new ideal biotracers for electron beam X-ray microanalysis and electron energy filtered imaging analysis.     

Changes in glutathione and phytochelatins in roots of maize seedlings exposed to cadmium

The effects of excess Cd on the contents of free cysteine, total glutathione and phytochelatin (PC) were measured in roots of intact maize seedlings. Exposure to 3 /tmM Cd for 15 min caused PCs to appe substrates for formation of longer PCs. Total glutathione levels declined with PC synthesis, free cysteine contents changed little. The reactions to excess Cd differed along the length of roots. In the 1 cm apical region a high production of PCs occurred with a moderate loss of total glutathione. In the mature region, PC content was 2.5-fold less than in apices, several unidentified thiols accumulated, and total glutathione levels declined drastically. Exposure to 0.05 μM Cd for 24 h induced PCs, the contents rose as Cd concentrations were increased. The roots produced PCs in excess of that required to chelate the Cd present, as if some PCs were compartmentalized or had not yet formed Cd-PC complexes. Phytochelatin formation was stimulated most effectively by Cd, less by Zn and Cu and negligibly by Ni. Total glutathione declined with Cd and Zn exposure, however, with excess Cu the roots contained 45% more total glutathione than did the controls.     

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

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

Detection of phytochelatins in the hyperaccumulator Sedum alfredii exposed to cadmium and lead

Phytochelatins (PCs) are known to play an essential role in the heavy metal detoxification of some higher plants and fungi by chelating heavy metals. However, three recent papers reported that no PCs could be detected in the hyperaccumulator Sedum alfredii Hance upon cadmium, lead or zinc treatment, respectively. In this paper, PC synthesis was assayed again in the mine population of S. alfredii with the help of reversed phase high-performance liquid chromatography (HPLC), HPLC-mass spectrometry, and HPLC-tandem mass spectrometry. Our data showed that PC formation could be induced in the leaf, stem and root tissues of S. alfredii upon exposure to 400 microM cadmium, and only in the stem and root when exposed to 700 microM lead. However, no PCs were found in any part of S. alfredii when it was subjected to exposure to 1600 microM zinc.     

Heavy metals toxicity in plants: An overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants

Plants experience oxidative stress upon exposure to heavy metals that leads to cellular damage. In addition, plants accumulate metal ions that disturb cellular ionic homeostasis. To minimize the detrimental effects of heavy metal exposure and their accumulation, plants have evolved detoxification mechanisms. Such mechanisms are mainly based on chelation and subcellular compartmentalization. Chelation of heavy metals is a ubiquitous detoxification strategy described in wide variety of plants. A principal class of heavy metal chelator known in plants is phytochelatins (PCs), a family of Cys-rich peptides. PCs are synthesized non-translationally from reduced glutathione (GSH) in a transpeptidation reaction catalyzed by the enzyme phytochelatin synthase (PCS). Therefore, availability of glutathione is very essential for PCs synthesis in plants at least during their exposure to heavy metals. Here, I reviewed on effect of heavy metals exposure to plants and role of GSH and PCs in heavy metal stress tolerance. Further, genetic manipulations of GSH and PCs levels that help plants to ameliorate toxic effects of heavy metals have been presented.