TY - JOUR
AU - Meagher, Richard B.
AB - Abstract 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 Arabidopsisthaliana 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 µM 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, γ-glutamyl cysteine (γ-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. (Received June 25, 2004; Accepted September 15, 2004) Introduction Phytoremediation relies on plants to extract, sequester and/or detoxify pollutants, and it is widely regarded as a less expensive, more effective and an environmentally friendly alternative to physical remediation methods such as excavation and reburial (Salt et al. 1995, Meagher 2000). The plants used to phytoremediate toxic elemental pollutants must meet at least two major requirements: they must be able to resist the toxicity of these elements and they must be able to hyperaccumulate them above ground (Salt et al. 1995, Meagher 2000). Genetic engineering can enhance the natural abilities of plants to uptake, accumulate and detoxify toxicants by overexpressing the critical enzymes limiting these abilities (Cobbett and Meagher 2002). Phytochelatins (PCs) are a family of cysteine-rich, thiol-reactive peptides that bind many toxic metals and metalloids, making them good candidates for genetically enhanced phytoremediation strategies (Cobbett and Meagher 2002). It seems logical to propose that overexpression of thiol-rich peptides could help phytoremediating plants meet both requirements for resistance and increased accumulation for thiol-reactive metals and metalloids. Several of the most hazardous elemental pollutants, such as the heavy metal cadmium and the metalloid arsenic, have chemical species that are thiol reactive. Two common forms of arsenic in the environment are the oxyanions arsenate AsO4III– and arsenite AsO3III–. Arsenate is the form of arsenic most readily taken up by cells, because it is a phosphate analog. In contrast, arsenite is the thio-reactive form that is expected to bind γ-glutamylcysteine (γ-EC) and the downstream peptides glutathione (GSH) and the PCs (Shi et al. 1996). Animals, plants, yeast and bacteria appear to efficiently and electrochemically reduce most of the arsenate in cells to arsenite (Pickering et al. 2000, Dhankher et al. 2002, Liu et al. 2002). The arsenite is then specifically pumped out of bacteria (Dey et al. 1994), yeast (Rosen 1999) and animal cells (Liu et al. 2002), conferring resistance. However, this efflux mechanism has not yet been demonstrated in plants. In the case of cadmium, the divalent cation Cd(II) is extremely thiol reactive and the role of the PC pathway, shown in Fig. 1A, in Cd(II) resistance has been partially explored. Schizosaccharomyces pombe PC synthase- (PCS-)deficient mutants are hypersensitive to cadmium (Ha et al. 1999) as are yeast mutants blocked in the synthesis of the upstream enzyme, γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase (GS) (Mutoh and Hayashi 1988). Arabidopsis mutants cad1-3 and cad2-1 with PCS and γ-ECS deficiencies, respectively, are hypersensitive to cadmium (Howden et al. 1995, Cobbett et al. 1998), suggesting key roles of this pathway in cadmium resistance and detoxification. Thus, while electrochemical reduction and efflux are all parts of cellular arsenic resistance, previous work on PC pathway mutants suggests that these thiol-rich peptides may have roles in toxic cadmium processing at both the cellular and organism levels (Cobbett and Meagher 2002). The goal of our research was to explore arsenic and cadmium resistance and accumulation in plants that overexpress Arabidopsis PCS (AtPCS1). Therefore, we generated a set of transgenic Arabidopsis lines overexpressing AtPCS1 from strong constitutive actin regulatory sequences (Dhankher et al. 2002). Overexpression of AtPCS1 resulted in high levels of PC synthesis, significant levels of arsenic tolerance and in contrast, cadmium hypersensitivity relative to wild type (WT). Results Expression of a modified AtPCS1 gene in Escherichia coli The WT Arabidopsis thalianaAtPCS1 sequence was modified to contain a 10 amino acid hemagglutinin (HA) epitope tag immediately after the start codon. This facilitated detection of the recombinant protein using a commercial monoclonal antibody (see Materials and Methods). The modified AtPCS1 was cloned into E. coli under control of the bacterial lacZ promoter (pAtPCS1/BSKS). WT E. coli expresses endogenous γ-ECS and GS, the first two enzymes in the pathway for PC biosynthesis (Fig. 1A), but it does not express PCS. Thus, expression of PCS was expected to complement these existing enzymes, result in PC synthesis, and enhance the metal ion sequestration and resistance of the transformed bacteria. We used an E. coli cadmium-hypersensitive strain RW3110, which lacks a zinc and cadmium metal ion export pump (Rensing et al. 1997), to assay AtPCS1 activity. A filter disk containing Cd(II) was applied to a lawn of RW3110 bacteria containing either pAtPCS1/BSKS plasmid or the KS empty plasmid vector, as shown in Fig. 2. RW3110 containing pAtPCS1/BSKS was more resistant to Cd(II) and grew significantly closer to filter disks than did the control strain containing the KS empty vector. These data suggest that the HA-modified AtPCS1 cDNA encoded a functional PCS in E. coli. Expression of the modified AtPCS1 gene in transgenic Arabidopsis Arabidopsis plants were engineered to overexpress the HA-containing version of AtPCS1 under control of a strong constitutive Arabidopsis actin-2 expression cassette (A2). A physical map of the A2::AtPCS1 gene is shown in Fig. 1B. Transgenic plant lines containing the construct were identified by screening the transgenic seedlings for a linked kanamycin resistance marker. The AtPCS1 protein expression levels were tested with Western immunoblot assays using an anti-HA monoclonal antibody that reacts with the epitope tag. Five transgenic lines (A5, A25, A27, A31, A35) were found that expressed detectable HA-tagged AtPCS1 protein among the 38 T2 generation plant lines tested. These five positive AtPCS1 transgenic plant lines showed no noticeable phenotypic differences relative to WT under normal growth conditions on soil or in aseptic culture on half-strength Murashige and Skoog (MS) medium. Western assays for two representative lines, A5 and A35, are shown in Fig. 3. PCS protein was observed in both leaves and roots. Transgenic PCS expression was stable in these plant lines at least through the T5 generation. Preliminary studies suggested that these five lines all behaved similarly when exposed to the toxicants arsenic, cadmium and mercury. Therefore, the A35 line was selected for further quantitative analysis. Levels of thiol-peptides in WT and A2::AtPCS1-overexpressing plants Transgenic line A35 and WT plants were analyzed for levels of PCs and their metabolic precursors γ-EC and GSH. Transgenic and WT plants were harvested after a long (48 h) exposure to arsenate or cadmium, or a shorter 1 h exposure to cadmium or a 4 h exposure to arsenate. Using monobromobimane (mBBr)-derivatized cysteine, γ-EC, GSH, PC2, PC3 and PC4 standards, fluorescent HPLC analysis clearly identified the mBBr-labeled peptides in the leaves and roots of A35 plants, shown in Fig. 4. In general, the levels of the products of AtPCS1 were dramatically increased in A35 transgenic plants relative to WT in response to arsenic or cadmium. Even in plants grown in half-strength MS medium without exposure to toxic ions, higher levels of PC2 and PC3 were detected in the A35 plants relative to WT (compare Fig. 4A with B). The levels of PC2, PC3 and PC4 were significantly increased in both the A35 and WT plants compared with normal media controls when plants were exposed to Cd(II) for 48 h (Fig. 4A, B and E, F). The levels of these peptides were much higher in roots than in leaves. Grown under cadmium stress, the roots of the A35 transgenic plants contained at least 5- to 10-fold more PC2 and PC3 relative to WT plants, as summarized in Fig. 5. In response to arsenate, the levels of thiol-reactive products were also significantly altered in both WT and the A35 plants compared with unchallenged plants, and the increases were more extreme than after treatment with cadmium (Fig. 4, 5). In response to arsenate or cadmium, PC4 was easily detected in the A35 plants, while it was not detected in WT plants (Fig. 4C, D, 5F). In addition, in response to arsenate, at least three unknown peaks (labeled a, b and c in Fig. 4) were detected in the roots of the A35 plants, which were at least 6- to 16-fold greater than in WT plants exposed to arsenate (Fig. 5G). Among the unknown fluorescent peaks, peak a was adjacent to GSH, and both b and c were adjacent to PC2. Surprisingly, after exposure to arsenate, no significant difference was detected in PC2 levels between the A35 and WT plants, and PC3 levels of the A35 plants were slightly lower than those of WT (P < 0.05) (Fig. 4C, D, 5D, E). Enhanced PC biosynthesis should have drained off the substrates, γ-EC and GSH, in the A35 plants relative to WT (Fig. 1A). As expected, compared with unchallenged growth or following cadmium treatment, GSH levels were found to decrease in roots of both WT and the A35 transgenic plants when exposed to arsenate for 48 h (P < 0.00001) (Fig. 4C, D, 5C). Paradoxically, the levels of γ-EC in roots of A35 transgenic plants were 7- to 10-fold greater relative to WT in response to a 48 h exposure to arsenate (P = 0.00378) or cadmium (P = 0.0042) (Fig. 5B). The level of γ-EC in WT plants also significantly increased in response to arsenate, but just not as dramatically as in the A35 plants. A short exposure to arsenate or cadmium also produced significant increases in thiol-peptides as summarized in Fig. 6. In response to a 4 h exposure to arsenate, PC2 levels in the A35 leaf tissues were 3- to 4-fold higher than in WT (P < 0.05) (Fig. 6B). None of the other fluorescent PCs (PC3, PC4) were detected in leaf tissue. However, in A35 root tissue, the level of PC4 was 2-fold greater than in WT (P < 0.05), and the levels of both PC2 and PC3 were higher than in WT (Fig. 6B). After exposure to cadmium for 1 h, the γ-EC level in the roots of the A35 plants was not substantially altered, but the level of root PC2 was found to be 7-fold greater than in WT (P < 0.05) (Fig. 6D). However, there was no significant difference in the levels of these peptides in leaf tissue of A35 plants relative to WT (Fig. 6C). Increased levels of PC2 and PC3 after 1 and 4 h exposure to Cd(II) and arsenate, respectively, suggested that rates for both synthesis and turnover of PC peptides were very rapid. Arsenate resistance of AtPCS-expressing plants Arsenic resistance was assayed in the transgenic lines overexpressing AtPCS1 and compared with WT. Arsenate is readily taken up by plants and converted to arsenite, the thiol-reactive form, by endogenous plant enzymes (Pickering et al. 2000, Dhankher et al. 2002). T3 generation A35 seeds and WT seeds were plated onto solid half-strength MS medium containing 200–300 µM arsenate. At these arsenic concentrations, the WT seeds germinated, but the seedlings bleached white and died after 2 weeks. However, the transgenic lines grew vigorously, as shown for A35 plants in Fig. 7B. On normal half-strength MS media, there were no significant growth differences between WT and the transgenic lines (Fig. 7A). To quantify arsenic tolerance, the fresh shoot weight (FSW) was measured. The FSW of the A35 line was 20–100 times higher than that of WT under these arsenic stress conditions (P < 0.0001) (Fig. 7C). Thus, it appeared that the higher levels of thiol-peptides in the A35 plant lines and other transgenic plant lines (not shown) provided high-level resistance to arsenic, presumably by sequestering reduced arsenite in plant tissues. Cadmium hypersensitivity of AtPCS1-expressing plants Cadmium resistance was assayed and compared among the WT and the transgenic plant lines that overexpressed AtPCS1. The seeds were plated on half-strength MS medium containing various concentrations of cadmium chloride (50–100 µM) (Fig. 7D, F). Contrary to our expectations, the representative A35 plants overexpressing AtPCS1 were significantly more sensitive to cadmium than WT. For example, A35 plants accumulated several times less fresh weight after 3 weeks than WT on medium with 50 (P < 0.05), 75 (P < 0.01) and 100 µM Cd(II) (P < 0.01) (Fig. 7F). The increased Cd(II) sensitivity in an AtPCS1-overexpressing line was surprising considering that Cd(II) is a highly thiol-reactive metal that should bind the product of the PCS-catalyzed reactions and considering that AtPCS1 expression conferred Cd(II) resistance on E. coli. Similar cadmium sensitivity was observed for the other AtPCS1-overexpressing lines A5, A25, A27 and A31 (data not shown). Arsenic and cadmium accumulation It seemed logical that the higher thiol-peptide levels in PCS transgenic plant leaves might act as a sink for thiol-reactive metalloids and metals and thus these plants should be able to sequester higher levels of arsenic and cadmium. Therefore, we examined arsenic and cadmium levels in the leaf tissues of these plants. Plants were grown in solid, half-strength MS medium containing 150 µM arsenate or 30 µM cadmium chloride for 3 weeks and shoot tissues were collected. At these low levels of arsenate and cadmium, WT plants grow at only slightly different rates than A2::AtPCS1 transgenic plant lines. The arsenic and cadmium concentrations were quantified using inductively coupled plasma optical emission spectroscopy (ICP-OES) as described in Materials and Methods. As shown in Fig. 8, there were no significant differences in the concentrations of arsenic or cadmium in shoot tissues of the transgenic line A35 and WT. Discussion Three successive reactions catalyzed by γ-ECS, GS and PCS (Fig. 1) are responsible for biosynthesis of PCs from common amino acids. Previous efforts examined the potential of γ-ECS and GS overexpression to enhance tolerance to cadmium in plants (Goldsbrough 1998, Zhu et al. 1999a, Zhu et al. 1999b, Xiang et al. 2001). Our goal was to determine whether overexpression of PCS would confer on plants high-level resistance to arsenic, cadmium and mercury. We achieved significant resistance to arsenic and weak resistance to mercury (data not shown), while the transgenic plants were hypersensitive to Cd(II) compared with WT plants. Considering that PCS is constitutively expressed in some plants (Steffens 1990) and that PCS is energetically activated by thiol-reactive metals or metalloids (Vatamaniuk et al. 2000, Vatamaniuk et al. 2004), it was not clear that overexpression of PCS in plants would substantially increase production of PCs and therefore would enhance metal or metalloid resistance any further compared with WT. However, the A2::AtPCS1 transgenic lines showed significant increases in the levels of PC and PC-related peptides over the increase observed in WT in response to short or long exposure to cadmium or arsenic. Response to arsenic In response to 48 h exposure to arsenate, the levels of the thiol-peptides were increased dramatically in the AtPCS1 plants relative to WT. However, the greatest increases were observed in the three unknown thiol-rich peptide peaks (a, b, c in Fig. 4). These peaks were most significantly enhanced in root tissue of the A35 plants relative to WT. The identity of these peaks and their exact roles in arsenate resistance are unknown. Peaks b and c might be closely related to the significant decrease in levels of PC2 and PC3 as well as significant increase of γ-EC in A35 plants. One possibility is that arsenic alters the substrate specificity of AtPCS1, which allows substrates other than GSH and γ-EC to be incorporated into PC assembly. For example, PCs with alanine and serine replacing the C-terminal glycine have been reported in yeast and plants (Cavener and Ray 1991). Peaks a, b and c may represent PC-related derivatives with alternative C-terminal amino acids. It has been suggested that several endogenous mechanisms confer arsenic resistance on prokaryotes and higher plants (Xu et al. 1998, Salerno et al. 2002). Arsenate (AsO43–) is an analog of phosphate (PO43–) and probably competes with phosphate for uptake by high-affinity phosphate transporters in higher plants, as it does in yeast and the aquatic monocot Lemna gibba (Ullrich-Eberius et al. 1989, Bun-ya et al. 1996). Plants grown in arsenate medium convert more than 90% of arsenate to arsenite in plant tissues via arsenate reductase (Pickering et al. 2000, Dhankher et al. 2002). The reduced arsenite is more toxic than arsenate, but may be eliminated from cells through arsenite efflux mechanisms in bacteria, fungi and plants (Rosen 1999, Sharples et al. 2000, Hartley-Whitaker et al. 2002). Reduced arsenite that is not effluxed is thought to be chelated by PCs or related thiol-peptides like γ-EC and GSH in plants and filamentous fungi (Grill 1987, Scott et al. 1993, Ha et al. 1999, Pickering et al. 2000, Schmoger et al. 2000, Sharples et al. 2000, Dhankher et al. 2002). Our previous study demonstrated that at least some arsenate remained to be reduced by an exogenous E. coli reductase expressed in shoots and be trapped in thiol-peptide complexes (Dhankher et al. 2002). Our data herein on AtPCS1 overexpression suggest that AtPCS1 partially complements other existing systems for arsenic resistance in plants by increasing the levels of PCs. It seemed surprising that the order of magnitude increases in thiol-peptide levels in the leaves of AtPCS plants did not result in increases in above-ground accumulation of arsenic (Fig. 8). PCS overexpression in E. coli resulted in an order of magnitude increase in cellular arsenic accumulation (Sauge-Merle et al. 2003). Clearly significant amounts of arsenic were transported to leaves of both WT and AtPCS plants, and increased concentrations of thiol-peptides might have acted as an enlarged sink to draw more arsenic up into the leaves. Furthermore, a previous study shows that electrochemical reduction of arsenate to arsenite in leaves coupled to increased thiol-peptide levels does result in significant increases in leaf arsenic, demonstrating that such a gradient for arsenic movement exists in plants (Dhankher et al. 2002). An alternative explanation for the lack of greater metal ion concentrations in leaves shown herein is first, that the higher levels of thiol-peptides in roots might have resulted from the presence of appropriate cofactor metal ions enhancing the enzymic activity of PCS being present in roots. Second, increased root PC levels might in turn support the enhanced efflux of arsenite from root cells, parallel to the efflux mechanisms in yeast and bacteria. Cellular efflux could then support short- and long-distance transport of arsenic and arsenic elimination from the entire plant. Enhanced efflux of arsenic would explain the lack of increased arsenic concentrations in AtPCS plants. One paradoxical observation was that in roots exposed to arsenate the level of γ-EC increased 12-fold in the transgenic plants relative to WT plants. This did not happen after exposure to cadmium. To explain these phenomena, it is necessary to consider that the stability, processing and storage of As–PC complexes is probably distinct from that of Cd–PC complexes. For example, it has been suggested that natural plant resistance to arsenic results from prolonged increases in PC biosynthesis rates (Schmoger et al. 2000, Hartley-Whitaker et al. 2001, Hartley-Whitaker et al. 2002). Cd–PC complexes are transported across the tonoplast into root vacuoles, where they eventually dissociate due to the acidic vacuolar pH (Vogeli-Lange and Wagner 1990, Salt and Rauser 1995). Furthermore, Cd–thiol-peptide complexes may block the ability of free Cd(II) to activate PCS (Vatamaniuk et al. 2000, Vatamaniuk et al. 2004). It has been suggested that the PCs released from Cd–PC complexes are either degraded or shuttled back into the cytoplasm (Hartley-Whitaker et al. 2001). Parallel fates for the As–PC complexes have not been described. However, if As–PC complexes are also transported into the root vacuoles, they might be dissociated into PCs and metalloid ions, and the PCs degraded into their precursors (e.g. γ-EC and GSH). The γ-EC could then be shuttled back into the cytoplasm. This would explain the 12-fold higher levels of γ-EC in A2::AtPCS1 plants compared with WT plants exposed to arsenate for 48 h. Response to cadmium There is strong evidence to support a connection between the synthesis of PCs in plants and cadmium resistance. Perhaps most significantly, the first two Arabidopsis cadmium-sensitive mutants isolated, cad1-3 and cad2-1, were blocked in the biosynthesis of PCs and γ-EC, respectively (Howden et al. 1995, Cobbett et al. 1998). In addition, both AtPCS1 (herein) and S. pombe SpPCS conferred resistance to Cd(II) in a sensitive bacterial strain (RW3110) (Li et al. 2001; Y. Li and R. Meagher, unpublished) and resulted in significant increases in bacterial cadmium accumulation (Sauge-Merle et al. 2003). Thus, it was logical to expect that overexpression of PCS enzyme would lead to cadmium resistance and accumulation in plants. It is striking that the A35 transgenic Arabidopsis plants, reported herein with 2- to 6-fold higher levels of PCs than WT in response to cadmium, were even more sensitive to toxic levels of cadmium ion than WT. Similar results were recently reported for transgenic Arabidopsis plants overexpressing native AtPCS1 in roots (Lee et al. 2003). While these plants accumulated 1.3- to 2.5-fold higher levels of PCs in roots in response to cadmium compared with WT, they were also more sensitive to cadmium than WT. However, transgenic tobacco Nicotiana glauca expressing Triticum aestivum PCS (TaPCS1) show a slight increase in tolerance to Cd(II) (Gisbert et al. 2003). Two distinct interpretations seem plausible. First, there could be a functional difference between AtPCS1 and TaPCS1 enzymes. Second, and more likely, tobacco and Arabidopsis differ in the downstream processing of Cd–PC complexes. For example, Cd–PC complexes or Cd(II) itself might poison required steps in metal-ion processing in Arabidopsis, such as transport into vacuoles. The discrepancy between AtPCS1 overexpression, the high levels of thiol-peptides achieved and hypersensitive response to cadmium suggests that much is still to be learned about the processing of cadmium in Cd–PC peptide complexes. In conclusion, the expression of an epitope-tagged Arabidopsis AtPCS1 protein from a strong plant actin regulatory cassette resulted in high levels of enzyme expression in transgenic Arabidopsis plants. After exposure to arsenic or cadmium, the A2::AtPCS1 plants showed order of magnitude increases in the production of PC-related thiol-peptide products relative to WT. These increases are substantially higher than those reported in earlier studies. Furthermore, the AtPCS1 plants were highly resistant to concentrations of arsenic that killed WT plants. Thus, PCS overexpression may become part of various strategies for arsenic phytoremediation by providing high levels of arsenic resistance. However, these plants did not accumulate more arsenic above ground, and thus, genetic amendments or alternative approaches will be needed to develop arsenic hyperaccumulation. The strong cadmium hypersensitivity of the AtPCS1 plants was surprising, and suggests unknown cadmium-sensitive step(s) for processing Cd–PC complexes. Materials and Methods Plant growth during heavy metal and metalloid treatments A.thaliana (ecotype Columbia) WT and transgenic plant lines were grown with a photoperiod of 16 h light and 8 h darkness at 23°C constant temperature. Seeds were sterilized as described previously (Li et al. 2001) or by treatment with Cl2 gas, as follows. Commercial bleach (100 ml) was mixed with 1 ml of concentrated hydrochloride acid in a 200 ml beaker inside a 2,000 cm3 desiccator. Seeds layered no more than 1/8 inch in a 1.5 ml microfuge tube were incubated for 5 h in the Cl2-filled chamber. These sterile seeds were then plated onto the half-strength MS medium solidified with phytagar (0.8 g liter–1) supplied with various levels of arsenate or cadmium. After the seeds germinated, the plates were positioned vertically for 3 weeks before the fresh-shoot weight was quantified. For resistance assays the medium was supplemented with sodium arsenate (200–300 µM) or cadmium chloride (50, 75 and 100 µM). Plants were grown in hydroponics medium to avoid agar contamination in element accumulation assays. The sterilized seeds were germinated for 4 d on nylon mesh (52 × 52) in Petri dishes with half-strength MS medium. Then the meshes with seedlings were transferred to Majenta boxes containing 100 ml of half-strength MS supplied with 35 µg ml–1 kanamycin. Half-strength MS medium was supplemented with 150 µM sodium arsenate (Na3AsO4) or 30 µM CdCl2 for 3 weeks and the shoot tissues were collected. DNA manipulation for bacterial and plant expression The AtPCS1 cDNA sequence (Vatamaniuk et al. 1999) (accession #AF085230) was PCR modified and cloned into a vector containing a constitutive Arabidopsisactin ACT2 promoter region and terminator (termed collectively A2). Two oligonucleotide primers added synthetic flanking sequences necessary for cloning and bacterial expression. The sense primer, AtPCS1-5′S, consisted of the 86-nt sequence: 5′-CACAGCCTCGAGTAAGGAGGATCATGAGTGGATACCCATACGATGTTCCAGATTACGCTGCTATGGCGAGTTTATATCGGCGATCT. It also contained cloning site XhoI, a TAA stop codon, bacterial translational signals (SD) (Rugh et al. 1996), and encoded a HA tag amino acid sequence GYPYDVPDYA (Green et al. 1982) located immediately after the start codon, which recognizes the influenza HA epitope. The antisense primer, AtPCS-3′A, had the 46 nt sequence 5′-CACAGCGGATCCAAGCTTTTAAGTGTAGAGAACGTGGGATTCAAAT, with cloning sites BamHI and HindIII. The PCR product was first cloned into XhoI/BamHI sites in pBluescript KS(II) to make the plasmid AtPCS1/KS for sequencing. The PCR-modified AtPCS1 coding sequence was cloned as a NcoI–BamHI fragment under control of the actin promoter and teminator to make A2::AtPCS1, which was then subcloned into the plant binary vector pBin19. Agrobacterium tumefaciens strain C58C1 (pMP90) (Koncz and Schell 1986) carrying the binary plasmid with A2::AtPCS1 was used to transform A. thaliana Columbia by vacuum infiltration as described previously (Chan et al. 1999). Metal-ion sensitivity disk assay in E. coli Because a 10 amino acid HA tag was added to the native PCS cDNA sequence, it was necessary to determine whether the modified AtPCS1 sequence was still functional. The plasmid AtPCS1/BSKS was transformed into the host strain RW3110, an E. coli mutant lacking a zinc/cadmium export pump (ZntA). The RW3110 strain is more sensitive to Cd(II) compared with WT. RW3110 containing the parent plasmid pBluescript KS(II) was used in these experiments as the negative control bacteria. All metal-ion sensitivity disk assays were performed in the presence of IPTG (100 µM ml–1) to induce expression of AtPCS1, and ampicillin (100 µg ml–1) to maintain the AtPCS1/BSKS plasmid. Four microliters of a 100 µM CdCl2 stock solution was loaded onto each filter paper disk, and there were two disks in each plate. The diameter of the inhibition zone was measured. The data reported herein are the average of several replicates and the standard error is given. Assays of AtPCS1 protein levels The crude protein levels were quantified according to the Bradford assay (Bradford 1976) and confirmed by Coomassie blue staining of a gel run in parallel. Equal amounts of crude protein extract from shoots or roots were separated by SDS–PAGE. Proteins were electrotransferred to Immobilon-P membrane (Millipore Corp., Bedford, MA, U.S.A.) and reacted with commercial monoclonal antibody that reacts with the HA epitope (Covance, Princeton, NJ, U.S.A.). Goat anti-mouse antibody conjugated to horseradish perioxidase (Amersham, Piscataway, NJ, U.S.A.) was used as the secondary antibody. Signals were amplified with the ECL System (Amersham) and recorded on X-ray film. HPLC analysis of thiol-peptide levels Cysteine- and thiol-containing peptides γ-EC, GSH, PC2, PC3 and PC4 were analyzed using fluorescence detection HPLC as described (Fahey and Newton 1987). Peptides were extracted and derivatized with mono-bromobimane (mBBr) as described perviously (Sneller et al. 2000, Cazale and Clemens 2001, Sauge-Merle et al. 2003), but with some modifications of these methods. Fresh tissues were ground in liquid nitrogen and peptides were extracted with extraction buffer (6.3 mM diethylenetrianmine pentacetic acid in 0.1% trifluoroacetic acid) at 1 ml (g fresh weight)–1 for leaf tissue and 2 ml (g fresh root tissue)–1. The homogenate was centrifuged at 10,000×g for 10 min at 4°C and the supernatant was filtered (0.22 µm filter; Millipore). The peptides were separated on a reverse-phase Nova-Pak C18 column (pore size, 60 Å; particle size, 4 µm; dimensions, 3.9 × 300 mm; Waters) at 27°C and fluorescence was monitored on a Thermo Finnagan Fluoromonitor S1100 Series fluorescence detector (Agilent Technologies, Milford, MA, U.S.A.) with λexcitation = 395 nm/λemission = 485 nm. The standards for cysteine, γ-EC and GSH, PC2, PC3, and PC4 are commercially available peptides from Sigma-Aldrich (St. Louis, MO, U.S.A.) and PC2, PC3 and PC4 standards were synthesized in the Molecular Genetics Instrument Facility, University of Georgia, U.S.A. The levels of the peptides were quantified from the intensity of the fluorescence in chromatographs and normalized to the fresh weight of plant tissues. Quantification of arsenic and cadmium concentrations in leaves Arsenic-treated or cadmium treated shoots were collected and dried as described by Dhankher et al. (2002). To extract total arsenic and cadmium, 20–100 mg freeze-dried powdered plant tissue was mixed with 2 ml of 1 : 7 (v/v), HClO4:HNO3 at room temperature for more than 48 h, following the protocol of Suszcynsky and Shann (1995). The sample volume was adjusted to 4 ml with deionized water. Total arsenic and mercury were determined using ICP-OES at the University of Georgia’s Chemical Analysis Laboratory using a Thermo Jarrell-Ash SH1000. Concentrations were normalized to dried plant tissue weight. Acknowledgments We would like to thank Gay Gragson, Dr. Arron Smith and Dr. Andrew C.P. Heaton for their critical comments on the manuscript and A.C.P.H. for his help in the quantification of arsenic and cadmium levels. Dr. Barry Rosen at Wayne State University kindly provided the bacterial strain RW3110. We also thank Dr. William Randle and his lab members (Horticulture Department, University of Georgia) for generous use of their freeze-drying equipment. This research was supported by grants from the Department of Energy Environmental Management Sciences (DEG0796ER20257) and National Institutes of Health (GM 36397–14) to R.B.M., and National Institutes of Environmental Health Sciences (1P42ES10337) to J.I.S. and EPA START fellowship U-91582701-1 to D.L. 4 Corresponding author: E-mail, meagher@uga.edu; Fax, +1-706-542-1387. View largeDownload slide Fig. 1 PC synthesis pathway and map of the A2::AtPCS1 gene. (A) Three steps for phytochelatins biosynthesis catalyzed by enzymes, γ-ECS, GS and PCS, respectively. (B) Physical map of A2::AtPCS1: The AtPCS1 gene under control of the ArabidopsisACT2 actin promoter, 5′UTR, leader intron and 3′UTR with polyadenylation sequences. Abbreviations: TATA box, the characterized sequence specifying the start of transcription; ts, start of transcription; PA, characterized poly(A) addition sites; ATG and TAA, initiation and termination codons, respectively. View largeDownload slide Fig. 1 PC synthesis pathway and map of the A2::AtPCS1 gene. (A) Three steps for phytochelatins biosynthesis catalyzed by enzymes, γ-ECS, GS and PCS, respectively. (B) Physical map of A2::AtPCS1: The AtPCS1 gene under control of the ArabidopsisACT2 actin promoter, 5′UTR, leader intron and 3′UTR with polyadenylation sequences. Abbreviations: TATA box, the characterized sequence specifying the start of transcription; ts, start of transcription; PA, characterized poly(A) addition sites; ATG and TAA, initiation and termination codons, respectively. View largeDownload slide Fig. 2 Filter disk inhibition assay showing Cd(II) resistance conferred by AtPCS expression in E. coli. (A) The growth of E. coli cadmium mutant strain RW3110 containing either the AtPCS1/BSKS plasmid or the pBluescript KS(II) parent control plasmid (BSKS) were compared when exposed to a filter disk with Cd(II). (B) Difference in the size of the zone of inhibition (diameter in mm) for the two strains examined in (A). Standard errors are indicated. View largeDownload slide Fig. 2 Filter disk inhibition assay showing Cd(II) resistance conferred by AtPCS expression in E. coli. (A) The growth of E. coli cadmium mutant strain RW3110 containing either the AtPCS1/BSKS plasmid or the pBluescript KS(II) parent control plasmid (BSKS) were compared when exposed to a filter disk with Cd(II). (B) Difference in the size of the zone of inhibition (diameter in mm) for the two strains examined in (A). Standard errors are indicated. View largeDownload slide Fig. 3 Analysis of AtPCS1 protein expression levels in leaves and roots of plant lines overexpressing A2::AtPCS1. (A) Western analysis of AtPCS1 expression in protein extracts from the leaves and roots of transgenic plant lines A5 and A35 as compared with WT after resolution on an SDS–PAGE gel. The filter imprint was probed with anti-HA antibody. The 56 kDa AtPCS protein band is indicated (PCS with arrow). (B) Coomasine blue-stained gel of identical samples to those in (A) showing equal loading. View largeDownload slide Fig. 3 Analysis of AtPCS1 protein expression levels in leaves and roots of plant lines overexpressing A2::AtPCS1. (A) Western analysis of AtPCS1 expression in protein extracts from the leaves and roots of transgenic plant lines A5 and A35 as compared with WT after resolution on an SDS–PAGE gel. The filter imprint was probed with anti-HA antibody. The 56 kDa AtPCS protein band is indicated (PCS with arrow). (B) Coomasine blue-stained gel of identical samples to those in (A) showing equal loading. View largeDownload slide Fig. 4 Fluorescence HPLC chromatograms of the mBBr-labeled plant extracts. (A, B) Roots of WT A. thaliana (var. Columbia) plants and transgenic A35 plants without toxicants; (C, D) WT and A35 plants exposed to 100 µM arsenate for 48 h; (E, F) WT and A35 plants exposed to 25 µM CdCl2 for 48 h. Peaks corresponding to cysteine, γ-EC, GSH, PC2, PC3 and PC4 are indicated. View largeDownload slide Fig. 4 Fluorescence HPLC chromatograms of the mBBr-labeled plant extracts. (A, B) Roots of WT A. thaliana (var. Columbia) plants and transgenic A35 plants without toxicants; (C, D) WT and A35 plants exposed to 100 µM arsenate for 48 h; (E, F) WT and A35 plants exposed to 25 µM CdCl2 for 48 h. Peaks corresponding to cysteine, γ-EC, GSH, PC2, PC3 and PC4 are indicated. View largeDownload slide Fig. 5 Comparison of cysteine, γ-EC, GSH and PC levels in leaves and roots of WT and transgenic plants that express Arabidopsis AtPCS1. The transgenic line (A35) shows increased levels of γ-EC and PC peptides compared with WT plants, when exposed to 25 µM CdCl2 (Cd), 25 µM HgCl2 (Hg) and 100 µM arsenate (As) for 48 h. (A to F) Levels of cysteine, γ-EC, GSH, PC2, PC3 and PC4 normalized to fresh weight of roots and leaves of WT and A35 transgenic plants. (G) Relative levels of unknown thiol-peptides a, b and c assayed as peak area normalized to equal amounts of fresh tissue weight. Symbols: 0, heavy metal free control; As, arsenate (100 µM) and Cd, CdCl2 (25 µM). The data shown are the average ± the standard error of the mean (SEM, error bar) of at least three independent experiments, each experiment consisting of 25 individual plants. Significant differences from WT are indicated with one asterisk (P < 0.05) or two (P < 0.01). View largeDownload slide Fig. 5 Comparison of cysteine, γ-EC, GSH and PC levels in leaves and roots of WT and transgenic plants that express Arabidopsis AtPCS1. The transgenic line (A35) shows increased levels of γ-EC and PC peptides compared with WT plants, when exposed to 25 µM CdCl2 (Cd), 25 µM HgCl2 (Hg) and 100 µM arsenate (As) for 48 h. (A to F) Levels of cysteine, γ-EC, GSH, PC2, PC3 and PC4 normalized to fresh weight of roots and leaves of WT and A35 transgenic plants. (G) Relative levels of unknown thiol-peptides a, b and c assayed as peak area normalized to equal amounts of fresh tissue weight. Symbols: 0, heavy metal free control; As, arsenate (100 µM) and Cd, CdCl2 (25 µM). The data shown are the average ± the standard error of the mean (SEM, error bar) of at least three independent experiments, each experiment consisting of 25 individual plants. Significant differences from WT are indicated with one asterisk (P < 0.05) or two (P < 0.01). View largeDownload slide Fig. 6 Analysis of γ-EC, GSH and PC2, PC3 and PC4 levels in leaves and roots of WT and A35 plants exposed to 25 µM Cd(II) for 1 h or 100 µM arsenate for 4 h. (A, B) Levels of GSH and PC2 were assayed in shoot tissues in response to 100 µM arsenate for 4 h. (C) Levels of PC2, PC3 and PC4 in root tissues that were exposed to 100 µM arsenate for 4 h. Levels of γ-EC, GSH, PC2, PC3 and PC4 were normalized to the dry weight of plant material and were higher in roots of the A35 line than in WT plants. (D, E) A35 plants showed higher levels of GSH and PC2 peptides than WT plants when exposed to 25 µM cadmium for 1 h. The data shown are the average ± SEM of at least two independent experiments, each experiment consisting of two individual plants, with significant differences from WT indicated with one asterisk (P < 0.05) or two (P < 0.01). View largeDownload slide Fig. 6 Analysis of γ-EC, GSH and PC2, PC3 and PC4 levels in leaves and roots of WT and A35 plants exposed to 25 µM Cd(II) for 1 h or 100 µM arsenate for 4 h. (A, B) Levels of GSH and PC2 were assayed in shoot tissues in response to 100 µM arsenate for 4 h. (C) Levels of PC2, PC3 and PC4 in root tissues that were exposed to 100 µM arsenate for 4 h. Levels of γ-EC, GSH, PC2, PC3 and PC4 were normalized to the dry weight of plant material and were higher in roots of the A35 line than in WT plants. (D, E) A35 plants showed higher levels of GSH and PC2 peptides than WT plants when exposed to 25 µM cadmium for 1 h. The data shown are the average ± SEM of at least two independent experiments, each experiment consisting of two individual plants, with significant differences from WT indicated with one asterisk (P < 0.05) or two (P < 0.01). View largeDownload slide Fig. 7 Arsenate resistance and cadmium sensitivity of the transgenic plants expressing the A2::AtPCS1 construct was compared with WT. Sterilized seeds were plated onto half-strength MS phytagar as a control (A) or on half-strength MS supplemented with 200 µM arsenate (Na3AsO4) (B). After germination, the plates were incubated in a vertical orientation for 3 weeks. The fresh weight of plants was quantified after 3 weeks growth under various conditions. The data shown are the average ± SEM of three independent experiments, with each experiment consisting of weighing 10 individual plants. (D, F) Sterilized seeds were plated onto half-strength MS phytagar as a control (D) or on half-strength MS supplemented with 75 µM of cadmium (CdCl2) (E). After germination, the plates were incubated in a vertical orientation for 3 weeks. Quantification of the fresh weight of plants grown under the various conditions (F) after 3 weeks’ growth. (C, F) Significant differences from WT are indicated by one asterisk (P < 0.05) or two (P < 0.01) (see legends to Fig. 4, 5). View largeDownload slide Fig. 7 Arsenate resistance and cadmium sensitivity of the transgenic plants expressing the A2::AtPCS1 construct was compared with WT. Sterilized seeds were plated onto half-strength MS phytagar as a control (A) or on half-strength MS supplemented with 200 µM arsenate (Na3AsO4) (B). After germination, the plates were incubated in a vertical orientation for 3 weeks. The fresh weight of plants was quantified after 3 weeks growth under various conditions. The data shown are the average ± SEM of three independent experiments, with each experiment consisting of weighing 10 individual plants. (D, F) Sterilized seeds were plated onto half-strength MS phytagar as a control (D) or on half-strength MS supplemented with 75 µM of cadmium (CdCl2) (E). After germination, the plates were incubated in a vertical orientation for 3 weeks. Quantification of the fresh weight of plants grown under the various conditions (F) after 3 weeks’ growth. (C, F) Significant differences from WT are indicated by one asterisk (P < 0.05) or two (P < 0.01) (see legends to Fig. 4, 5). View largeDownload slide Fig. 8 Arsenic and cadmium accumulation in tissues of A2::AtPCS1 and WT plants. The shoot tissues were collected from the plants grown in half-strength MS medium supplemented with (A) 150 µM arsenate or (B) 30 µM Cd(II) and grown for 3 weeks. The plant samples were lyophylized, dried, hydrolyzed and subjected to ICP-OES to determine metalloid or metal concentration (see Materials and Methods). Comparing each experimental sample with WT, the data shown are the average ± SEM of three independent experiments, normalized to micrograms of metal or metalloid per gram dry plant tissue. View largeDownload slide Fig. 8 Arsenic and cadmium accumulation in tissues of A2::AtPCS1 and WT plants. The shoot tissues were collected from the plants grown in half-strength MS medium supplemented with (A) 150 µM arsenate or (B) 30 µM Cd(II) and grown for 3 weeks. The plant samples were lyophylized, dried, hydrolyzed and subjected to ICP-OES to determine metalloid or metal concentration (see Materials and Methods). 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TI - Overexpression of Phytochelatin Synthase in Arabidopsis Leads to Enhanced Arsenic Tolerance and Cadmium Hypersensitivity
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pch202
DA - 2004-12-15
UR - https://www.deepdyve.com/lp/oxford-university-press/overexpression-of-phytochelatin-synthase-in-arabidopsis-leads-to-Z97g7lVCpi
SP - 1787
EP - 1797
VL - 45
IS - 12
DP - DeepDyve
ER -