Metal(loid)s and radionuclides cytotoxicity in Saccharomyces cerevisiae. Role of YCF1, glutathione and effect of buthionine sulfoximine

The presence of heavy metal(loid)s in soils and waters is an important issue with regards to human health. Taking into account speciation problems, in the first part of this report, we investigated under identical growth conditions, yeast tolerance to a set of 15 cytotoxic metal(loid)s and radionuclides. The yeast cadmium factor 1 (YCF1) is an ATP-Binding Cassette transporter mediating the glutathione detoxification of heavy metals. In the second part, metal(loid)s that could be handled by YCF1 and a possible re-localisation of the transporter after heavy metal exposure were evaluated. YCF1 and a C-terminal GFP fusion, YCF1-GFP, were overexpressed in wild-type and Deltaycf1 strains. Both forms were functional, conferring a tolerance to Cd, Sb, As, Pb, Hg but not to Ni, Zn, Cu, Ag, Se, Te, Cr, Sr, Tc, U. Confocal experiments demonstrated that during exposure to cytotoxic metals, the localisation of YCF1-GFP was restricted to the yeast vacuolar membrane. In the last part, the role of glutathione in this resistance mechanism to metal(loid)s was studied. In the presence of heavy metals, application of buthionine sulfoximine (BSO), a well-known inhibitor of gamma-glutamylcysteine synthetase, led to a decrease in the cytosolic pool of GSH and to a limitation of yeast growth. Surprisingly, BSO was able to phenocopy the deletion of gamma-glutamylcysteine synthetase after exposure to Cd but not to Sb or As. In the genetic context of gsh1 and gsh2 yeast mutants, the critical role of GSH for Cd, As, Sb and Hg tolerance was compared to that of wild-type and Deltaycf1.     

Sources and Ecological Risk Assessment of Heavy Metal(loid)s in Agricultural Soils of Huzhou,China

To identify sources of heavy metal(loid) (HM) contamination in agricultural soils of Huzhou, surface soil samples were sampled from 89 different agricultural regions in 2012. Concentrations of heavy metal(loid)s, along with pH, total phosphorus (TP), total nitrogen (TN), and soil organic matter (SOM), were determined. Ecological risk was then assessed using a modified Hakanson ecological risk index, and the sources of contamination were determined using principal component analysis (PCA). Mean concentrations of heavy metal(loid)s were 10.26, 23.21, 83.75, 22.81, 0.25, 61.86, 33.03, and 0.15 mg kg^?1 for As, Cu, Zn, Ni, Cd, Cr, Pb, and Hg, respectively. Cu, Zn, Ni, Cr, Cd, Hg, and Pb were correlated positively with TP and there were obvious positive correlations among Cu, Zn, Ni, Cr, and Cd. Risk index (RI) values varied from 39 to 1246 with a mean value of 137. Enrichment of Pb, Zn, Cu, and especially Cd can be attributed to excessive use of nitrogen and phosphorus fertilizers containing heavy metals, as well to surface irrigation and natural soil formation. While the ecological risk of most agricultural soils in Huzhou is low, it is recommended that the use of phosphate and nitrogen fertilizers be restricted and production technology be improved to reduce the heavy metal(loid) concentrations. Results suggest that the Chinese environmental quality standard for soil should be revised to better address heavy metal(loid) contamination.     

A novel heavy metal ATPase peptide from <Emphasis Type="Italic">Prosopis juliflora</Emphasis> is involved in metal uptake in yeast and tobacco

Heavy metal pollution of agricultural soils is one of the most severe ecological problems in the world. Prosopis juliflora, a phreatophytic tree species, grows well in heavy metal laden industrial sites and is known to accumulate heavy metals. Heavy Metal ATPases (HMAs) are ATP driven heavy metal pumps that translocate heavy metals across biological membranes thus helping the plant in heavy metal tolerance and phytoremediation. In the present study we have isolated and characterized a novel 28.9 kDa heavy metal ATPase peptide (PjHMT) from P. juliflora which shows high similarity to the C-terminal region of P_1B ATPase HMA1. It also shows the absence of the invariant signature sequence DKTGT, and the metal binding CPX motif but the presence of conserved regions like MVGEGINDAPAL (ATP binding consensus sequence), HEGGTLLVCLNS (metal binding domain) and MLTGD, GEGIND and HEGG motifs which play important roles in metal transport or ATP binding. PjHMT, was found to be upregulated under cadmium and zinc stress. Heterologous expression of PjHMT in yeast showed a higher accumulation and tolerance of heavy metals in yeast. Further, transgenic tobacco plants constitutively expressing PjHMT also showed increased accumulation and tolerance to cadmium. Thus, this study suggests that the transport peptide from P. juliflora may have an important role in Cd uptake and thus in phytoremediation.     

Engineering polyphosphate metabolism in Escherichia coli: implications for bioremediation of inorganic contaminants

Polyphosphate metabolism plays an important role in the bioremediation of phosphate contamination in municipal wastewater, and may play a key role in heavy metal tolerance and bioremediation. However, little is known about the regulation of polyphosphate metabolism in microorganisms and its role in heavy metal toxicity. We have manipulated polyphosphate metabolism in Escherichia coli by overexpressing the genes for polyphosphate kinase (ppk) and for polyphosphatase (ppx) under control of their native promoters and inducible promoters. Overexpression of ppk results in high levels of intracellular polyphosphate, improved phosphate uptake, but no increase in tolerance to heavy metals. Overexpression of both ppk and ppx results in lower levels of intracellular polyphosphate, secretion of phosphate from the cell, and increased tolerance to heavy metals. Metabolic flux analysis indicates that the cell responds to increased flux through the PPK-PPX pathway by altering flux through the TCA cycle.     

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.     

Metals and seeds: Biochemical and molecular implications and their significance for seed germination

Seeds contain the embryo as a new plant in miniature and have two major functions, reproduction and dispersal. Seed formation completes the process of plant reproduction and, with seed germination, the next plant generation starts. Given the ever-increasing environmental pollution with metal(loid)s, it is perhaps surprising that relatively few reports detail the impacts of metals on seed metabolism, viability and germination in comparison to the numerous publications on the effects of metals in vegetative tissues, particularly roots and shoots. This review provides information on metal(loid) homeostasis, detoxification and tolerance in relation to seed metabolism and performance. The delivery of metals from the mother plant into seeds and their implications on seed development are discussed, as are their uptake upon seed imbibition and subsequent effects on seed germination. Implications for seeds and seedlings on the biochemical and molecular level are discussed and finally, applied aspects are considered regarding the use of seeds for soil and water purification, and in phytoremediation programmes. We conclude with a perspective on future metal research in relation to seed biology.     

镉污染条件下水稻对假单胞菌TCd-1微生物修复的生理响应

微生物在修复重金属污染土壤中发挥重要作用。为阐明假单胞菌TCd-1降低水稻镉吸收的机理,通过加镉土培盆栽试验,在10 mg/kg镉处理下,对比研究了接种菌株对2种镉耐性不同水稻品种(特优671、百香139)镉含量、根系活力、光合作用、抗氧化酶活性及抗氧化物质含量的影响。结果表明：与10 mg/kg镉处理相比,接种假单胞菌TCd-1后水稻“特优671”和“百香139”的根、茎、叶、糙米的镉含量分别依次降低了30.4%、39.1%、40.7%、29.2%和52.2%、51.7%、18.4%、38.8%;提高了2种水稻的根系活力、光合作用,促进了水稻的生长;“特优671”、“百香139”叶片的超氧化物歧化酶(Superoxide dismutase, SOD)活性分别提高了7.3%、138.5%,过氧化物酶(Peroxidase, POD)活性提高了82.0%、106.2%,过氧化氢酶(Catalase, CAT)活性提高了58.8%、172.7%;叶片的类黄酮含量提高了139.4%、73.6%,总酚含量提高了27.2%、23.1%;超氧阴离子含量降低了44.2%、29.0%,丙二醛(Mal...     

单一与复合污染条件下两种敏感性植物对Cd、Zn、Pb的吸收效应

为了进一步研究镉、锌、铅 3种重金属元素间的相互作用以及对植物吸收重金属能力的影响 ,在模拟单一重金属污染试验研究的基础上 ,采用正交回归设计方案 ,研究了 Cd、Zn、Pb复合污染情况下紫花苜蓿和披碱草两种敏感性植物对 3种重金属的吸收效应。结果表明 ,在单一污染条件下 ,镉元素对紫花苜蓿生长的影响大于锌、铅 ,铅元素对披碱草生长的影响大于锌、镉 ;紫花苜蓿对于镉的吸收累积显著高于披碱草 ,植物内镉元素浓度最高达到 1 0 88.5 mg/kg,而披碱草对于铅元素的吸收则高于紫花苜蓿 ,植物内铅元素浓度最高达到 1 3 4 5 .5 mg/kg。在复合污染条件下 ,两种植物对铅、锌和铅、镉的吸收在不同浓度范围内分别存在存在着协同效应和拮抗效应 ;同时两种植物对锌、镉元素在实验涉及浓度范围内都存在着拮抗效应。这对于深入研究复合污染条件下重金属的土壤环境化学行为 ,对植物的综合毒性以及不同植物对重金属的吸收累积效应等 ,具有一定的参考意义     

Heavy metals in rat liver cadmium binding protein.

The simultaneous determination of heavy metals in microsamples of chromatographically isolated cadmium-binding protein (Cd-BP) from rat liver was performed by neutron activation analysis. The results suggested that metals other than those already reported (Cd, Zn, Cu, and Hg) can bind the protein. These observations were confirmed by in vivo radiotracer experiments by injecting i.p. 21 labelled metal ions in cadmium-treated rats. Of the metals tested, 109Cd, 65Zn, 64Cu, 203Hg, 106Ag and 113Sn were found incorporated in the Cd-BP. The incorporation of 35S-cysteine, used as an indicator of Cd-BP biosynthesis, was increased in rats exposed to cadmium as compared to untreated animals. In order to establish the influence of other metal ions on the biosynthesis of Cd-BP and the incorporation of cadmium in the protein, in vivo experiments were carried out by i.p. injection of 109Cd and 35S-cysteine. In the presence of 42 metal ions no influence was observed on the incorporation of the two radioisotopes in the Cd-BP. These observations tend to support the hypothesis that cadmium can act as a highly specific inducer of Cd-BP and that this protein might be involved in the metabolism of several heavy metals.     

Toxicity, accumulation, and removal of heavy metals by three aquatic macrophytes

A comprehensive understanding of the uptake, tolerance, and transport of heavy metals by plants will be essential for the development of phytoremediation technologies. In the present paper, we investigated accumulation, tissue and intracellular localization, and toxic effects of cadmium (Cd), lead (Pb), zinc (Zn), and copper (Cu) in three aquatic macrophytes (the angiosperms Lemna minor and Elodea canadensis, and the moss Leptodictyum riparium). We also tested and compared their capacity to absorb heavy metal from water under laboratory conditions. Our data showed that all the three species examined could be considered good bioaccumulators for the heavy metals tested. L. riparium was the most resistant species and the most effective in accumulating Cu, Zn, and Pb, whereas L. minor was the most effective in accumulating Cd. Cd was the most toxic metal, followed by Pb, Cu, and Zn. At the ultrastructural level, sublethal concentrations of the heavy metals tested caused induced cell plasmolysis and alterations of the chloroplast arrangement. Heavy metal removal experiments revealed that the three macrophytes showed excellent performance in removing the selected metals from the solutions in which they are maintained, thus suggesting that they could be considered good candidates for wastewaters remediation purpose.     

Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization

The aim of this review is to assess the mode of action and role of antioxidants as protection from heavy metal stress in roots, mycorrhizal fungi and mycorrhizae. Based on their chemical and physical properties three different molecular mechanisms of heavy metal toxicity can be distinguished: (a) production of reactive oxygen species by autoxidation and Fenton reaction; this reaction is typical for transition metals such as iron or copper, (b) blocking of essential functional groups in biomolecules, this reaction has mainly been reported for non-redox-reactive heavy metals such as cadmium and mercury, (c) displacement of essential metal ions from biomolecules; the latter reaction occurs with different kinds of heavy metals. Transition metals cause oxidative injury in plant tissue, but a literature survey did not provide evidence that this stress could be alleviated by increased levels of antioxidative systems. The reason may be that transition metals initiate hydroxyl radical production, which can not be controlled by antioxidants. Exposure of plants to non-redox reactive metals also resulted in oxidative stress as indicated by lipid peroxidation, H(2)O(2) accumulation, and an oxidative burst. Cadmium and some other metals caused a transient depletion of GSH and an inhibition of antioxidative enzymes, especially of glutathione reductase. Assessment of antioxidative capacities by metabolic modelling suggested that the reported diminution of antioxidants was sufficient to cause H(2)O(2) accumulation. The depletion of GSH is apparently a critical step in cadmium sensitivity since plants with improved capacities for GSH synthesis displayed higher Cd tolerance. Available data suggest that cadmium, when not detoxified rapidly enough, may trigger, via the disturbance of the redox control of the cell, a sequence of reactions leading to growth inhibition, stimulation of secondary metabolism, lignification, and finally cell death. This view is in contrast to the idea that cadmium results in unspecific necrosis. Plants in certain mycorrhizal associations are less sensitive to cadmium stress than non-mycorrhizal plants. Data about antioxidative systems in mycorrhizal fungi in pure culture and in symbiosis are scarce. The present results indicate that mycorrhization stimulated the phenolic defence system in the Paxillus-Pinus mycorrhizal symbiosis. Cadmium-induced changes in mycorrhizal roots were absent or smaller than those in non-mycorrhizal roots. These observations suggest that although changes in rhizospheric conditions were perceived by the root part of the symbiosis, the typical Cd-induced stress responses of phenolics were buffered. It is not known whether mycorrhization protected roots from Cd-induced injury by preventing access of cadmium to sensitive extra- or intracellular sites, or by excreted or intrinsic metal-chelators, or by other defence systems. It is possible that mycorrhizal fungi provide protection via GSH since higher concentrations of this thiol were found in pure cultures of the fungi than in bare roots. The development of stress-tolerant plant-mycorrhizal associations may be a promising new strategy for phytoremediation and soil amelioration measures.     

Arabidopsis arenosa (Brassicaceae) from a lead–zinc waste heap in southern Poland – a plant with high tolerance to heavy metals

The morphological traits and tolerance to heavy metals (zinc, cadmium and lead) of two populations of Arabidopsis arenosa (Brassicaceae) were compared. One population was from a zinc–lead waste heap in Bolesław near Olkusz (southern Poland), the other one from the Kampinoski National Park (central Poland). Biometric measurements were done in the field and repeated after cultivation under controlled conditions (garden soil, phytotron chamber). Significant heritable morphological differences between the two populations were found. The plants from the waste-heap are smaller in comparison with the reference population, and their leaves are narrower, thicker with fewer trichomes, indicating. genetic adaptation to xerothermic conditions. The level of tolerance to heavy metals (zinc, cadmium, and lead) was compared by the root test. Very high tolerance to the three metals tested was found in the waste-heap population. Its tolerance exceeded that of four other predominant plant species populations growing on the same waste heap that had previously been tested in our laboratory. We consider the waste-heap population of A. arenosa to be a very suitable ecotype for the study of heavy metal tolerance mechanisms in plants. Responsible Editor: SE: Henk Schat.     

Local adaptation of microbial communities to heavy metal stress in polluted sediments of Lake Erie

Microbial communities must balance the assimilation of biologically necessary metals with resistance to toxic metal concentrations. To investigate the impact of heavy metal contaminants on microbial communities, we performed two experiments measuring extracellular enzyme activities (EEA) in polluted and unpolluted sediments of Lake Erie. In the first experiment, inoculations with moderate concentrations of copper and zinc appreciably diminished EEA from uncontaminated sites, whereas EEA from contaminated sediments increased or were only negligibly affected. In the second experiment, we compared the effects of three separate metals (i.e. copper, arsenic, and cadmium) on microbial community metabolism in polluted and unpolluted locations. Although copper and arsenic elicited differential effects by inhibiting EEA only in unpolluted sediments, cadmium inhibited EEA in both polluted and unpolluted sediments. Multivariate analyses of EEA from polluted sediments revealed direct associations among hydrolytic enzymes and inverse or absent associations between hydrolases and oxidases; these associations demonstrated resilience to heavy metal stress. In contrast, addition of heavy metals to unpolluted sediments appeared to have a higher impact on the multivariate pattern of EEA associations as revealed by an increase in the number of associations, more inverse relationships, and potential enzymatic trade-offs. The results of this study suggest community-level adaptations through the development of resistance mechanisms to the types and local levels of heavy metals in the environment.     

Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis

The genome of Pseudomonas putida KT2440 encodes an unexpected capacity to tolerate heavy metals and metalloids. The availability of the complete chromosomal sequence allowed the categorization of 61 open reading frames likely to be involved in metal tolerance or homeostasis, plus seven more possibly involved in metal resistance mechanisms. Some systems appeared to be duplicated. These might perform redundant functions or be involved in tolerance to different metals. In total, P. putida was found to bear two systems for arsenic (arsRBCH), one for chromate (chrA), four to six systems for divalent cations (two cadA and two to four czc chemiosmotic antiporters), two systems for monovalent cations: pacS, cusCBA (plus one cryptic silP gene containing a frameshift mutation), two operons for Cu chelation (copAB), one metallothionein for metal(loid) binding, one system for Te/Se methylation (tpmT) and four ABC transporters for the uptake of essential Zn, Mn, Mo and Ni (one nikABCDE, two znuACB and one mobABC). Some of the metal-related clusters are located in gene islands with atypical genome signatures. The predicted capacity of P. putida to endure exposure to heavy metals is discussed from an evolutionary perspective.     

Comparative analysis of the contribution of phytochelatins to cadmium and arsenic tolerance in soybean and white lupin

The biosynthesis of phytochelatins (PCs) plays a crucial role in the detoxification and homeostasis of heavy metals and metalloids in plants. However, in an increasing number of plant species metal(loid) tolerance is not well correlated with the accumulation of PCs: tolerant ecotypes frequently contain lower levels of PCs than non-tolerant ecotypes. In this study we have compared the responses of soybean (Glycine max L. cv. Resnik) and white lupin (Lupinus albus L. cv. Marta) to cadmium and arsenate in order to assess the role of homophytochelatins (hPCs) in the tolerance of soybean to these toxic elements. Soybean plants treated with Cd and As showed a high contribution of homo-glutathione (hGSH) to the pool of thiols in shoots in comparison to white lupin. Higher levels of hPCs in Cd-treated soybeans compared to PCs in lupins did not prevent growth inhibition. In contrast, the role of hPCs in the detoxification mechanism to arsenate in soybean seems to be clearer, showing higher thiol concentrations and lower growth reductions than those present in lupin plants.     

Environmental relevance of heavy metal-substituted chlorophylls using the example of water plants

Following experiments which studied the substitution of thecentral ion of isolated chlorophylls by heavy metal ions invitro, in vivo experiments with submersed water plants werecarried out. It was discovered that the substitution of thecentral atom of chlorophyll, magnesium, by heavy metals (mercury,copper, cadmium, nickel, zinc, lead) in vivo is an importantdamage mechanism in stressed plants. This substitution preventsphotosynthetic light-harvesting in the affected chlorophyllmolecules, resulting in a breakdown of photosynthesis. The reactionvaries with light intensity. In low light irradiance all thecentral atoms of the chlorophylls are accessible to heavy metals,with heavy metal chlorophylls being formed, some of which aremuch more stable towards irradiance than Mg-chlorophyll. Consequently,plants remain green even when they are dead. In high light,however, almost all chlorophyll decays, showing that under suchconditions most of the chlorophylls are inaccessible to heavymetal ions. Key words: Heavy metal chlorophylls, submersed water plants, antenna pigments, copper, zinc     

大蒜植物络合素合酶基因转化对酵母重金属抗性的提高

重金属污染是全球面临的亟待解决的生态问题。利用植物对重金属的富集作用来清除环境重金属污染即植物修复已成为重要的环境生物技术之一。这一技术的长远发展有赖于在重金属富集或耐受中起关键作用的基因的克隆和应用。植物络合素是植物体内一类重要的对重金属起螯合作用的多肽, 其合成受植物络合素合酶的催化。该文取得了如下研究结果:1)通过原子吸收测定表明,在大蒜(Allium sativum)的根部可以积累3 000 mg·kg^-1的重金属镉;2)将克隆的大蒜植物络合素合酶基因(AsPCS)置于酵母表达启动子之下,构建酵母表达载体,并将其分别转入了因CUP1和acr3基因缺失而对重金属镉和砷敏感的酵母突变体菌株后,发现来自大蒜的AsPCS基因的表达使酵母CUP1缺失菌株对镉的耐受性提高了4倍, acr3缺失菌株对砷的耐受性提高了两倍;3)表达AsPCS基因酵母的生长模式证实了AsPCS基因的表达是酵母对重金属耐受性提高的原因。这些结果暗示, 大蒜植物络合素合酶基因在大蒜对重金属的抗性及大蒜根部对镉的积累中起关键作用,可作为重要的基因元件应用到修复污染的植物基因工程中。