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.     

植物对重金属镉的耐受机制

镉离子（Cd^2＋）具有强植物毒性,抑制植物生长,甚至使植物死亡。由于长期的环境选择和适应进化,植物发展出耐受机制,可减轻或避免Cd^2＋的毒害。硫转运蛋白、硫还原相关酶类以及半胱氨酸、谷胱甘肽和植物螯合肽合成基因的表达受Cd^2＋调控。同时这些基因的过表达也能提高植物对Cd^2＋的耐性。植物抗氧化系统对Cd^2＋胁迫诱发的活性氧的清除作用,具转运Cd^2＋活性的质膜转运蛋白促进Cd^2＋经共质体途径向木质部运输、装载,而后随蒸腾流向地上部迁移,具转运Cd^2＋活性的液泡膜转运蛋白促进Cd^2＋进入液泡的隔离作用,都在植物对Cd^2＋的耐性中起作用。     

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.     

Accumulation of CdS nanoparticles by yeasts in a fed-batch bioprocess

The yeasts Schizosaccharomyces pombe and Candida glabrata were successfully cultivated in a fed-batch process at cadmium levels up to 100 mg l(-1). S. pombe incorporated 20 mg C dg(-1) dry biomass within 24h. C. glabrata accumulated 8 mg C dg(-1) dry biomass in 24h. The higher Cd uptake from S. pombe cells correlate with the elevated glucose concentrations during and at the end of the cultivation. Analysis of the cells with energy-filtering transmission electron microscopy-element specific imaging (EFTEM-ESI) revealed that cadmium is not precipitated outside the cells or at the cell wall but evenly distributed inside the cell plasma. As Cd is highly toxic this indicates that Cd is immobilized by an intracellular detoxification mechanism. Size exclusion chromatography showed that Cd is associated to a protein fraction between 25 and 67 kDa which corresponds to the theoretical molecular weight of CdS nanoparticles of 35 kDa coated with phytochelatins. This structure has been proposed in literature.     

Comparative proteomic analysis of Cd-responsive proteins in wheat roots

Cadmium (Cd) is a major environmental toxicant to plant cells due to its potential inhibitory effects on many physiological processes. To gain a comprehensive understanding of plant response to Cd, wheat seedlings were exposed to a range of Cd concentrations (10, 100 and 200 μM) for 1 week and a combination of physiological and proteomic approaches were used to evidence Cd effects and to access the plant response to Cd toxicity. Root and shoot elongation was decreased, whereas the H₂O₂ and malondialdehyde content in wheat seedlings was increased significantly at higher Cd concentration. Protein profiles analyzed by two-dimensional electrophoresis revealed that 46 protein spots showed 1.5-fold change in protein abundance following Cd exposure; 31 protein spots were up-regulated and 15 protein spots were down-regulated; 25 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As expected, most of the up-regulated proteins are involved in heavy metal detoxification and antioxidant processes. Enzyme activity analysis revealed that ascorbate peroxidase and glutathione S-transferase activity was stimulated by Cd treatment. Abundance changes of these proteins, together with their putative functions provide us a new insight that can lead to an integrated understanding of the molecular basis of Cd responses in plants.     

Metalloid tolerance based on phytochelatins is not functionally equivalent to the arsenite transporter Acr3p

Active transport of metalloids by Acr3p and Ycf1p in Saccharomyces cerevisiae and chelation by phytochelatins in Schizosaccharomyces pombe, nematodes, and plants represent distinct strategies of metalloid detoxification. In this report, we present results of functional comparison of both resistance mechanisms. The S. pombe and wheat phytochelatin synthase (PCS) genes, when expressed in S. cerevisiae, mediate only modest resistance to arsenite and thus cannot functionally compensate for Acr3p. On the other hand, we show for the first time that phytochelatins also contribute to antimony tolerance as PCS fully complement antimonite sensitivity of ycf1Delta mutant. Remarkably, heterologous expression of PCS sensitizes S. cerevisiae to arsenate, while ACR3 confers much higher arsenic resistance in pcsDelta than in wild-type S. pombe. The analysis of PCS and ACR3 homologues distribution in various organisms and our experimental data suggest that separation of ACR3 and PCS genes may lead to the optimal tolerance status of the cell.     

Cadmium stress tolerance in crop plants: Probing the role of sulfur

Plants can''t move away and are therefore continuously confronted with unfavorable environmental conditions (such as soil salinity, drought, heat, cold, flooding and heavy metal contamination). Among heavy metals, cadmium (Cd) is a non-essential and toxic metal, rapidly taken up by roots and accumulated in various plant tissues which hamper the crop growth and productivity worldwide. Plants employ various strategies to counteract the inhibitory effect of Cd, among which nutrient management is one of a possible way to overcome Cd toxicity. Sulfur (S) uptake and assimilation are crucial for determining crop yield and resistance to Cd stress. Cd affects S assimilation pathway which leads to the activation of pathway responsible for the synthesis of cysteine (Cys), a precursor of glutathione (GSH) biosynthesis. GSH, a non-protein thiol acts as an important antioxidant in mitigating Cd-induced oxidative stress. It also plays an important role in phytochelatins (PCs) synthesis, which has a proven role in Cd detoxification. Therefore, S assimilation is considered a crucial step for plant survival under Cd stress. The aim of this review is to discuss the regulatory mechanism of S uptake and assimilation, GSH and PC synthesis for Cd stress tolerance in crop plants.Key words: cadmium, cysteine, glutathione, phytochelatins, stress tolerance, sulfur     

Proteome analysis of Schizosaccharomyces pombe by two-dimensional gel electrophoresis and mass spectrometry

The fission yeast Schizosaccharomyces pombe (S. pombe) is a unicellular eukaryote and contains many genes and regulatory mechanisms that are close to those of mammals. In this study, we performed a global proteomic analysis of the fission yeast S. pombe wild type h(-S) L 972 proteome. More than 1,500 protein spots were visualized on silver stained 2-D gels in the 3-10 pI range with a high resolution and high reproducibility. Protein identification was carried out by MALDI-TOF-MS and/or nanoLC-MS/MS. Advantage of the complementarity of these two MS approaches was used to enhance the identification quality. So far, 364 proteins (representing 157 different proteins) have been identified. We report here the identification of 117 new proteins on our 2-D reference map of this yeast compared to the first reference map. Of these identified proteins, 40.1% were involved in metabolism. The present work provides a very useful tool for all studies relying on S. pombe as a model organism and is a considerable complement to the first reference map of S. pombe published recently by Sun and coworkers (Sun, N., Jang, J., Lee, S., Kim, S. et al.., Proteomics 2005, 5, 1574-1579).     

Quantitative proteomic analysis of Salmonella enterica serovar Typhimurium under PhoP/PhoQ activation conditions

The PhoP/PhoQ two-component system plays a central regulatory role in the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium), and it can be activated by low Mg(2+) concentrations and sublethal concentrations of cationic antimicrobial peptides (CAMP). Therefore, these two PhoP/PhoQ activation signals are considered as in vivo environmental cues sensed by S. Typhimurium for adaptation and survival. In this work, we conducted a SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative proteomic study to survey the proteomic changes of S. Typhimurium in response to low Mg(2+) concentrations or CAMP. We discovered that CAMP activated a portion of the PhoP/PhoQ regulatory network, whereas low Mg(2+) concentrations upregulated nearly all known members of this network, a number of previously unknown proteins, and some proteins regulated by IHF and RpoS. Systematic analysis following metabolic pathways revealed that low Mg(2+) concentrations selectively influenced proteins of certain metabolic functions while CAMP did not. Our study indicates that the low Mg(2+)-concentration condition may lead S. Typhimurium into a growth-control lifestyle, which provides new perspectives about Salmonella's adaptation to the host environment.     

Cadmium uptake and interaction with phytochelatins in wheat protoplasts.

In order to investigate the role of phytochelatins in short-time uptake of Cd(2+) into the cytosol of wheat protoplasts, a new method was applied, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. The uptake of Cd(2+) into protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum, L. cv. Kadett) was lower in protoplasts from seedlings raised in the presence of 1 microM CdCl(2), than in the absence. Presence of CdCl(2) in the cultivation medium increased the content of phytochelatins (PCs) in the protoplasts. When seedlings were raised in the presence of both Cd(2+) and buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, only little PC was found in the protoplasts. Pre-treatment with BSO alone did not affect the content of PC, but inhibited that of GSH. The inhibition of GSH was independent of pre-treatment with Cd(2+). Unidirectional flux analyses, using (109)Cd(2+), showed approximately the same uptake pattern of Cd(2+) as did the fluorescence experiments showing the cytosolic uptake of Cd(2+). Thus, the diminished uptake of Cd(2+) into protoplasts from cadmium-pre-treated plants was not depending on PCs. Instead, it is likely that pre-treatment with Cd(2+) causes a down-regulation of the short-term Cd(2+) uptake, or an up-regulation of the Cd(2+) extrusion. Moreover, since addition of Cd(2+) to protoplasts from control plants caused a cytosol acidification, it is likely that a Cd(2+/)H(+)-antiport mechanism is involved in the extrusion of Cd(2+) from these protoplasts.     

Phytochelatin synthase catalyzes key step in turnover of glutathione conjugates

Conjugation of xenobiotic compounds and endogenous metabolites to glutathione is an ubiquitous process in eukaryotes. In animals, the first and rate-limiting step of glutathione-S-conjugate metabolism is characterized by the removal of the aminoterminal glutamic acid residue of glutathione. In plants, however, glutathione-S-conjugates are generally metabolized by removal of the carboxylterminal glycine residue of the tripeptide glutathione to give rise to the S-glutamylcysteinyl-derivative. Purification of the glutathione-conjugate catabolizing activity from cell suspension cultures of the plant Silene cucubalus indicated that phytochelatin synthase catalyzes the first step of the pathway. Heterologously expressed phytochelatin synthase from Arabidopsis efficiently converted S-bima ne-glutathione to S-bimane-glutamylcysteine, the formation of which was unequivocally identified by mass spectrometry. No further products, such as S-derivatives of phytochelatins, were observed. Several different glutathione-S-conjugates served as substrates for the enzyme and were processed to the corresponding glutamylcysteinyl-adducts. Affinity-purified phytochelatin synthase preparations required divalent heavy metal ions such as Cd(2+), Zn(2+) or Cu(2+) for detectable turnover of glutathione-S-conjugates. Characterization of the enzymatic properties of phytochelatin synthase argues for both cellular functions of the gamma-glutamylcysteinyl-dipeptidyltransferase: (1) formation of heavy-metal binding peptides and (2) degradation of glutathione-S-conjugates. Mechanistically, the former role is the result of gamma-glutamylcysteinyl transpeptidation onto glutathione or derivatives thereof, while the catabolic function reflects transpeptidation of S-glutamylcysteinyl-adducts onto the acceptor molecule water. Thus, phytochelatin synthase seems to fulfil a second crucial role in glutathione metabolism.     

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.     

CeHMT-1, a putative phytochelatin transporter, is required for cadmium tolerance in Caenorhabditis elegans

Phytochelatins (PCs), (gamma-Glu-Cys)n Gly polymers that were formerly considered to be restricted to plants and some fungal systems, are now known to play a critical role in heavy metal (notably Cd2+) detoxification in Caenorhabditis elegans. In view of the functional equivalence of the gene encoding C. elegans PC synthase 1, ce-pcs-1, to its homologs from plant and fungal sources, we have gone on to explore processes downstream of PC fabrication in this organism. Here we describe the identification of a half-molecule ATP-binding cassette transporter, CeHMT-1, from C. elegans with an equivalent topology to that of the putative PC transporter SpHMT-1 from Schizosaccharomyces pombe. At one level, CeHMT-1 satisfies the requirements of a Cd2+ tolerance factor involved in the sequestration and/or elimination of Cd x PC complexes. Heterologous expression of cehmt-1 in S. pombe alleviates the Cd2+-hypersensitivity of hmt- mutants concomitant with the localization of CeHMT-1 to the vacuolar membrane. Suppression of the expression of ce-hmt-1 in intact worms by RNA interference (RNAi) confers a Cd2+-hypersensitive phenotype similar to but more pronounced than that exhibited by ce-pcs-1 RNAi worms. At another level, it is evident from comparisons of the cell morphology of ce-hmt-1 and cepcs-1 single and double RNAi mutants that CeHMT-1 also contributes to Cd2+ tolerance in other ways. Whereas the intestinal epithelial cells of ce-pcs-1 RNAi worms undergo necrosis upon exposure to toxic levels of Cd2+, the corresponding cells of ce-hmt-1 RNAi worms instead elaborate punctate refractive inclusions within the vicinity of the nucleus. Moreover, a deficiency in CeHMT-1 does not interfere with the phenotype associated with CePCS-1 deficiency and vice versa. Double ce-hmt-1; ce-pcs-1 RNAi mutants exhibit both cell morphologies when exposed to Cd2+. These results and those from our previous investigations of the requirement for PC synthase for heavy metal tolerance in C. elegans demonstrate PC-dependent, HMT-1-mediated heavy metal detoxification not only in S. pombe but also in some invertebrates while at the same time indicating that the action of CeHMT-1 does not depend exclusively on PC synthesis.     

Proteome changes in Arabidopsis thaliana roots upon exposure to Cd2+

Cadmium is a major environmental pollutant that enters human food via accumulation in crop plants. Responses of plants to cadmium exposure--which directly influence accumulation rates--are not well understood. In general, little is known about stress-elicited changes in plants at the proteome level. Alterations in the root proteome of hydroponically grown Arabidopsis thaliana plants treated with 10 microM Cd(2+) for 24 h are reported here. These conditions trigger the synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, shown here as PC2 accumulation. Two-dimensional gel electrophoresis using different pH gradients in the first dimension detected on average approximately 1100 spots per gel type. Forty-one spots indicated significant changes in protein abundance upon Cd(2+) treatment. Seventeen proteins found in 25 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Selected results were independently confirmed by western analysis and selective enrichment of a protein family (glutathione S-transferases) through affinity chromatography. Most of the identified proteins belong to four different classes: metabolic enzymes such as ATP sulphurylase, glycine hydroxymethyltransferase, and trehalose-6-phosphate phosphatase; glutathione S-transferases; latex allergen-like proteins; and unknown proteins. These results represent a basis for reverse genetics studies to better understand plant responses to toxic metal exposure and to the generation of internal sinks for reduced sulphur.     

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.     

Cellular toxicity of cadmium ions and their detoxification by heavy metal-specific plant peptides, phytochelatins, expressed in Mammalian cells

The apoptotic cell death of Jurkat cells due to Cd(2+) toxicity was studied by fluorescence microscopic observation and DNA fragmentation assaying. It was suggested that the apoptotic response to Cd(2+) was less clear than that to a typical apoptosis inducer, ultraviolet light (254 nm). Examination of MAP kinase phosphorylation (p38, JNKs, and c-Jun) due to Cd(2+) toxicity indicated that the phosphorylation was very slowly activated (4 h after stimulation), while UV light could activate the phosphorylation immediately. Therefore, it was suggested that Cd(2+) may not be a typical apoptosis inducer. Antioxidants [glutathione (GSH) and N-acetylcysteine (NAC)] could detoxify Cd(2+), indicating that the toxicity is a kind of oxidative stress. The detoxification effect of antioxidants showed cooperation with Bcl-2, suggesting that Cd(2+)-treatment causes diversified toxic signals including oxidative stress. On the addition of a plant-specific peptide, phytochelatin [PC(7), (gammaGlu-Cys)(7)-Gly], to the medium, the detoxification of Cd(2+) and cooperation with Bcl-2 were more intense than in the cases of GSH and NAC. Using an appropriate vector, a PC synthase gene was transferred from Arabidopsis thaliana to the Jurkat cell. The transfectant exhibited resistance to Cd(2+) and production of plant-specific PC (PC(2-6)).     

Quantitative proteomic analysis of LPS-induced differential immune response associated with TLR4 Polymorphisms by multiplex amino acid coded mass tagging

Polymorphisms at toll-like receptor 4 (TLR4) gene have been found to be associated with immune disorders. A murine macrophage cell line GG2EE derived from C3H/HeJ mice with a polymorphism site at TLR4 is hyposensitive to lipopolysaccharide (LPS). To study the molecular base of diverse TLR4-mediated immune responses, the proteomic changes in both TLR4-deficient and wild-type cell lines in response to the same LPS challenge were quantitatively compared by using multiplex amino acid coded mass tagging (AACT)/SILAC-assisted MS. This strategy allows encoding of two distinct cell populations with different stable isotope-tagged lysine residues as the "in-spectra" quantitative markers. In MS analysis of tryptic peptides derived from the equally mixed three cell populations, the lysine-containing peptides originated from two LPS-stimulated cell populations can be clearly distinguished by their different mass shifts from the unstimulated and unlabeled counterpart. The LPS-induced differential protein expression in TLR4-deficient and wild-type proteomes were obtained by comparing the intensities of isotopically encoded peptides. Among the more than 900 proteins identified, 35 were found to be deregulated at different levels in these two cell lines stimulated by LPS. This multiplex mass-tagging methodology can be readily extended to other comparative proteomic quantitation of different cell populations.     

Regulation of Glutathione Synthesis by Cadmium in Pisum sativum L

In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCl₂ concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5′-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cd-binding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5′-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis.