Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity

Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC2, PC3, PC4), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.     

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.     

Enhanced arsenic accumulation by engineered yeast cells expressing Arabidopsis thaliana phytochelatin synthase

Phytochelatins (PCs) are naturally occurring peptides with high-binding capabilities for a wide range of heavy metals including arsenic (As). PCs are enzymatically synthesized by phytochelatin synthases and contain a (gamma-Glu-Cys)(n) moiety terminated by a Gly residue that makes them relatively proteolysis resistant. In this study, PCs were introduced by expressing Arabidopsis thaliana Phytochelatin Synthase (AtPCS) in the yeast Saccharomyces cerevisiae for enhanced As accumulation and removal. PCs production in yeast resulted in six times higher As accumulation as compared to the control strain under a wide range of As concentrations. For the high-arsenic concentration, PCs production led to a substantial decrease in levels of PC precursors such as glutathione (GSH) and gamma-glutamyl cysteine (gamma-EC). The levels of As(III) accumulation were found to be similar between AtPCS-expressing wild type strain and AtPCS-expressing acr3Delta strain lacking the arsenic efflux system, suggesting that the arsenic uptake may become limiting. This is further supported by the roughly 1:3 stoichiometric ratio between arsenic and PC2 (n = 2) level (comparing with a theoretical value of 1:2), indicating an excess availability of PCs inside the cells. However, at lower As(III) concentration, PC production became limiting and an additive effect on arsenic accumulation was observed for strain lacking the efflux system. More importantly, even resting cells expressing AtPCS pre-cultured in Zn(2+) enriched media showed PCs production and two times higher arsenic removal than the control strain. These results open up the possibility of using cells expressing AtPCS as an inexpensive sorbent for the removal of toxic arsenic.     

Enhancing the tolerance of zebrafish (Danio rerio) to heavy metal toxicity by the expression of plant phytochelatin synthase

Phytochelatin synthase (PC synthase) catalyzes a biosynthesis of phytochelatins (PCs), which are small molecules and glutathione (GSH)-derived metal-binding peptides that are essential for the detoxification of heavy metal ions in plants, fungi and worms. In order to enhance tolerance to heavy metal cytotoxicity, mRNA coding for PC synthase from Arabidopsis thaliana (AtPCS1) was introduced into the early embryos of zebrafish. As a result, the heterogeneous expression of PC synthase and the synthesis of PCs from GSH in embryos could be detected. The developing embryos expressing PC synthase (PC-embryos) became more tolerant to Cd toxicity (500 microM exposure). PC-embryos had significantly longer apparent lethal times for 50% of the population (LT50) of 8.17+/-1.08 days, although control embryos had apparent LT50 of 5.43+/-0.66 days. These data suggest that PC synthase can function in developmental zebrafish, and that PCs are highly effective in detoxifying Cd toxicity even in the whole body of a vertebrate species.     

Glutathione and phytochelatin contents in tomato plants exposed to cadmium

The effect of cadmium on growth and contents of glutathione (GSH) and phytochelatins (PCs) were investigated in roots and leaves of tomato plants (Lycopersicon esculentum Mill. cv. 63/5 F1). The accumulation of Cd increased with external Cd concentrations and was considerably higher in roots than in leaves. Dry mass production decreased under Cd treatment especially in leaves. In both roots and leaves, exposure to Cd caused an appreciable decline in GSH contents and increase in PCs synthesis proportional to Cd concentrations in the growth medium. At the same Cd concentration, PCs production was higher in roots than in leaves. The implication of glutathione in PC synthesis was strongly suggested by the use of buthionine sulfoximine (BSO). The major fraction of Cd accumulated by tomato roots was in the form of a Cd-PCs complex.     

Enhanced toxic metal accumulation in engineered bacterial cells expressing Arabidopsis thaliana phytochelatin synthase

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes.     

Metal ion resistance in fungi: molecular mechanisms and their regulated expression

One stress response in cells is the ability to survive in an environment containing excessive concentrations of metal ions. This paper reviews current knowledge about cellular and molecular mechanisms involved in the response and adaptation of various fungal species to metal stress. Most cells contain a repertoire of mechanisms to maintain metal homeostasis and prevent metal toxicity. Roles played by glutathione, related (gamma-EC)nG peptides, metallothionein-like polypeptides, and sulfide ions are discussed. In response to cellular metal stress, the biosynthesis of some of these molecules are metalloregulated via intracellular metal sensors. The identify of the metal sensors and the role of metal ions in the regulation of biosynthesis of metallothionein and (gamma-EC)nG peptides are subjects of much current attention and are discussed herein.     

Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient.

An allelic series of cad1, cadmium-sensitive mutants of Arabidopsis thaliana, was isolated. These mutants were sensitive to cadmium to different extents and were deficient in their ability to form cadmium-peptide complexes as detected by gel-filtration chromatography. Each mutant was deficient in its ability to accumulate phytochelatins (PCs) as detected by high-performance liquid chromatography and the amount of PCs accumulated by each mutant correlated with its degree of sensitivity to cadmium. The mutants had wild-type levels of glutathione, the substrate for PC biosynthesis, and in vitro assays demonstrated that each of the mutants was deficient in PC synthase activity. These results demonstrate conclusively the importance of PCs for cadmium tolerance in plants.     

Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding gamma-glutamylcysteine synthetase (gamma-ECS), targeted to the plastids. The gamma-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, gamma-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, gamma-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the gamma-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of gamma-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of gamma-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.     

Oxidative stress and phytochelatin characterisation in bread wheat exposed to cadmium excess.

In this work, we first investigated if the bread wheat (Triticum aestivum L.) cv. Albimonte can be defined as "shoot cadmium excluder"--by comparing the cadmium (Cd) content in leaves and roots and by calculating the shoot-to-root Cd concentration ratio. Furthermore, we evaluated if the exposure to Cd excess could generate oxidative stress in leaves and roots of this cv., in terms of hydrogen peroxide (H(2)O(2)) accumulation, NAD(P)H oxidation rate, and variations in reduced glutathione (GSH) content and peroxidase (POD, EC 1.11.1.7) activity. Finally, we surveyed possible quali- quantitative differences in thiol-peptide compound pattern between roots and leaves, in order to verify whether phytochelatins (PCs) and related thiol-peptides could contribute in limiting the Cd-induced oxidative stress. Unambiguous characterisation of PCs and related forms present in the root samples was obtained by electrospray ionisation mass spectrometry (ESI-MS) and ESI-tandem MS (ESI-MS/MS). Our results indicate that in leaves the stress generated by the low accumulation of Cd (due to a moderate translocation in planta) seems to be counteracted by the antioxidant response and by the PC biosynthesis. On the contrary, in roots, in spite of the elevated presence of PCs and related thiol-peptide-compounds, the excess of Cd causes a decline in the antioxidant protection of the organ, with the consequent generation of considerable amounts of H(2)O(2), a direct agent of oxidative stress.     

Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress

Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12- to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3- to 2.1-fold under 85 microM CdCl(2) stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 microM CdCl(2). Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels.     

Relief of Arsenate Toxicity by Cd-Stimulated Phytochelatin Synthesis in the Green Alga Chlamydomonas reinhardtii

In most photosynthetic organisms, inorganic arsenic taken up into the cells inhibits photosynthesis and cellular growth. In a green alga, Chlamydomonas reinhardtii, 0.5 mM arsenate inhibited photosynthesis almost completely within 30 min. However, in cells acclimated with a sublethal concentration (0.05 to 0.1 mM) of Cd, the inhibition of photosynthesis at 30 min after the addition of arsenate was relieved by more than 50%. The concentrations of arsenic incorporated into the cells were not significantly different between the Cd-acclimated and the non-acclimated cells. The Cd-acclimated cells accumulated Cd and synthesized phytochelatin (PC) peptides, which are known to play an important role in detoxification of heavy metals in plants. By the addition of an inhibitor of glutathione (an intermediate in the PC biosynthetic pathway) biosynthesis, buthionine sulfoximine, cells lost not only Cd tolerance but also arsenate tolerance. These results suggest that glutathione and/or PCs synthesized in Cd-acclimated cells are involved in mechanisms of arsenate tolerance. The authors contributed equally to this work.     

Role of Arbuscular Mycorrhizal (AM) Fungi on Growth, Cadmium Uptake, Osmolyte, and Phytochelatin Synthesis in Cajanus cajan (L.) Millsp. Under NaCl and Cd Stresses

Application of phosphatic (P) fertilizers and biosolids is known to enhance cadmium (Cd) contamination in saline soils. Increased concentration of dissolved chloride (Cl^?) in soil solution significantly influences Cd bioavailability in P fertilizer- or biosolid-amended soils. Arbuscular mycorrhizal (AM) fungi have an ability to protect plants against salinity and heavy metals by mediating interactions between toxic ions and plant roots. The effects of Glomus mosseae (AM) and NaCl and Cd stresses on Cd uptake and osmolyte and phytochelatin (PCs) synthesis in Cajanus cajan (L.) Millsp. (pigeonpea) were studied under greenhouse conditions. Two genotypes [Sel 85?N (tolerant) and ICP 13997 (sensitive)] were subjected to NaCl (4 and 6?dS?m^?1) and Cd (CdCl₂, 25 and 50?mg?kg^?1 dry soil) treatments. NaCl and Cd applied individually as well as in combination caused dramatic reductions in plant biomass and induced membrane peroxidation, ionic perturbations, and metabolite synthesis in both genotypes, although Sel 85?N was less affected than ICP 13997. Cadmium uptake was enhanced when NaCl was added along with Cd. The protection of growth in Sel 85?N was associated with restricted accumulation of Na⁺, Cl^?, and Cd²⁺ and higher concentrations of stress metabolites (sugars, proteins, free amino acids, proline, glycine betaine). Cd led to a significant increase in biothiols (NP-SH) and glutathione (GSH), with a larger pool of NP-SH which strongly induced accumulation of phytochelatins, whereas no significant effects in their concentrations were detectable under NaCl stress. The interactive effects of NaCl and Cd on all parameters were larger than those of individual treatments. Fungal inoculations improved plant growth and reduced accumulation of toxic ions. Higher stress metabolite synthesis and PCs observed in AM plants of Sel 85?N indicated the role of an efficient AM symbiosis capable of attenuating NaCl and Cd stresses.     

Exogenous Application of Selenium Mitigates Cadmium Toxicity in <Emphasis Type="Italic">Brassica juncea</Emphasis> L. (Czern &amp; Cross) by Up-Regulating Antioxidative System and Secondary Metabolites

The main aim of the present study was to examine the role of selenium (Se) in ameliorating the toxic effect of cadmium (Cd) in mustard (Brassica juncea) plants. The plants exposed to elevated levels of Cd exhibited reduced biomass, pigment content, and relative water content (RWC). However, supplementation of Se restores the negative effect of Cd and increases biomass, pigment content, and RWC. Osmolyte (proline and glycine betaine) and sugar content were increased under Cd stress and further increase was observed with addition of Se. Cd decreased protein content and supplementation of Se increases it to appreciable levels. Cd also increased production of H₂O₂ and lipid peroxidation, electrolyte leakage, and the activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, and glutathione reductase. Supplementation of Se decreased accumulation of H₂O₂ and lipid peroxidation, increased the activities of antioxidant enzymes to greater levels, and regulates Cd accumulation in roots and shoots. Ascorbic acid (AsA) and flavonoids decreased with elevated concentrations of Cd; however, tocopherol and total phenols were increased with the same concentrations of Cd. Se application maintains AsA and flavonoid content, and further increase in tocopherol and total phenols were observed with Se in the present study. Overall the results confirm that exogenous application of Se mitigates the negative effects of Cd stress in mustard plants through the regulation of osmoprotectants, antioxidant enzymes, and secondary metabolites.     

Characterization of phytochelatin synthase-like protein encoded by alr0975 from a prokaryote, Nostoc sp. PCC 7120

Phytochelatins (PCs) are well known as the heavy metal-detoxifying peptides in higher plants, eukaryotic algae, fungi, and nematode. In contrast, neither PCs nor PC synthase genes have ever been identified in any prokaryotes. The genome sequences for the cyanobacterium Nostoc sp. PCC 7120 were recently completed and allowed us to identify a gene encoding a PC synthase-like protein, termed alr0975. The predicted product of alr0975 contains the conserved N-terminal domain but not the variable C-terminal domain found in eukaryotic PC synthases. The recombinant alr0975 protein strongly catalyzed the first step of PC synthesis, in which glutathione (GSH) is converted to gamma-glutamylcysteine (gamma-EC), although the protein only weakly catalyzed the second step of PC synthesis, namely the transfer of gamma-EC moiety to an acceptor GSH molecule to form PC(2). These results suggest alr0975 protein may be a more primitive form of the PC synthases found in eukaryotes.     

Hydroxymethyl-phytochelatins [(gamma-glutamylcysteine)n-serine] are metal-induced peptides of the Poaceae.

Exposure of several species of the family Poaceae to cadmium results in the formation of metal-induced peptides of the general structure (gamma-Glu-Cys)n-Ser (n=2-4). They are assumed to be formed from hydroxymethyl-glutathione (gamma-Glu-Cys-Ser) and are termed hydroxymethyl-phytochelatins (hm-PCs) in analogy to the homo-phytochelatins [(gamma-Glu-Cys)n-beta-Ala], discovered in legumes, and the phytochelatins [PCs, (gamma-Glu-Cys)n-Gly] found in most other plants and many fungi. The hm-PCs were isolated from the roots of cadmium-exposed rice (Oryza sativa L. cv Strella), and their structure was confirmed by amino acid analysis after total and enzymic hydrolysis and by tandem mass spectrometry. The hm-PCs probably play a significant role in heavy metal detoxication in rice. In addition to this new form of gamma-Glu-Cys (gamma EC) peptide, PCs and gamma EC peptides without C-terminal Ser or Gly are found. All gamma EC peptides are synthesized without delay after incubation of rice plants in 100 microM CdCl2 in the roots as well as in the shoots. Incubation times exceeding 24 h or higher concentrations of cadmium result in a selective enrichment of gamma EC peptides with higher chain length and an increased ratio of PCs to hm-PCs. gamma EC peptide synthesis is accompanied by a decrease of the glutathione content and an increase of the hydroxymethyl-glutathione content in roots and shoots of rice plants.     

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

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

Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation

Phytochelatins (PCs) are glutathione-derived peptides that function in heavy metal detoxification in plants and certain fungi. Recent research in Arabidopsis has shown that PCs undergo long-distance transport between roots and shoots. However, it remains unknown which tissues or vascular systems, xylem or phloem, mediate PC translocation and whether PC transport contributes to physiologically relevant long-distance transport of cadmium (Cd) between shoots and roots. To address these questions, xylem and phloem sap were obtained from Brassica napus to quantitatively analyze which thiol species are present in response to Cd exposure. High levels of PCs were identified in the phloem sap within 24 h of Cd exposure using combined mass spectrometry and fluorescence HPLC analyses. Unexpectedly, the concentration of Cd was more than four-fold higher in phloem sap compared to xylem sap. Cadmium exposure dramatically decreased iron levels in xylem and phloem sap whereas other essential heavy metals such as zinc and manganese remained unchanged. Data suggest that Cd inhibits vascular loading of iron but not nicotianamine. The high ratios [PCs]/[Cd] and [glutathione]/[Cd] in the phloem sap suggest that PCs and glutathione (GSH) can function as long-distance carriers of Cd. In contrast, only traces of PCs were detected in xylem sap. Our results suggest that, in addition to directional xylem Cd transport, the phloem is a major vascular system for long-distance source to sink transport of Cd as PC–Cd and glutathione–Cd complexes.     

Selection and characterization of cadmium tolerant cells in tomato

Tomato (Lycopersicon esculentum cv. VFNT-Cherry)cell lines tolerant of to 5 mM cadmium (Cd) were selected by progressively elevating the level of CdCl₂ in the culture medium (the lethal concentration of Cd for unselected tomato cells is 400 μM). Cd tolerance was not lost during long-term culture (up to 12 months) in the absence of Cd stress. In all the cell lines examined, Cd uptake was rapid and Cd concentration within the cells exceeded that in the culture medium by several fold. While Cd included the synthesis and accumulation of phytochelatins (PCs) [poly[γ-glutamyl-cysteiny)glycine], little change has been observed in protein synthesis during short term Cd stress. PCs formed complexes with Cd. However, uptake and accumulation of Cd was not affected if PC synthesis was inhibited by treatment with buthionine sulfoximine. Selected and unselected cells were compared for their growth characteristics in the presence of various other metal ions. Cd tolerant cells showed a slightly higher tolerance of copper but not of mercury, zinc, lead or silver.