Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation

The enzymatically synthesized thiol peptide phytochelatin (PC) plays a central role in heavy metal tolerance and detoxification in plants. In response to heavy metal exposure, the constitutively expressed phytochelatin synthase enzyme (PCS) is activated leading to synthesis of PCs in the cytosol. Recent attempts to increase plant metal accumulation and tolerance reported that PCS over-expression in transgenic plants paradoxically induced cadmium hypersensitivity. In the present paper, we investigate the possibility of synthesizing PCs in plastids by over-expressing a plastid targeted phytochelatin synthase (PCS). Plastids represent a relatively important cellular volume and offer the advantage of containing glutathione, the precursor of PC synthesis. Using a constitutive CaMV 35S promoter and a RbcS transit peptide, we successfully addressed AtPCS1 to chloroplasts, significant PCS activity being measured in this compartment in two independent transgenic lines. A substantial increase in the PC content and a decrease in the glutathione pool were observed in response to cadmium exposure, when compared to wild-type plants. While over-expressing AtPCS1 in the cytosol importantly decreased cadmium tolerance, both cadmium tolerance and accumulation of plants expressing plastidial AtPCS1 were not significantly affected compared to wild-type. Interestingly, targeting AtPCS1 to chloroplasts induced a marked sensitivity to arsenic while plants over-expressing AtPCS1 in the cytoplasm were more tolerant to this metalloid. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.     

Leaf-targeted phytochelatin synthase in Arabidopsis thaliana

One of the key steps in developing transgenic plants for the phytoremediation of metal containing soils is to develop plants that accumulate metals in the aerial tissues. With the goal of changing the distribution of phytochelatin (PC)-dependent cadmium accumulation from roots to the leaves, the phytochelatin synthase (PCS) deficient cad1-3 mutant and wild type (Col-0) Arabidopsis plants were transformed with an Arabidopsis phytochelatin synthase (AtPCS1) under the control of a leaf-specific promoter. Three independent transformant lines from each genetic background were chosen for further analysis and designated cad-PCS and WT-PCS. PCS activity in the cadPCS lines was restored in the leaves, but not in the roots. Additionally, when whole plants were treated with cadmium, PCs were found only in the leaves of cad-PCS plants. Although the inserted AtPCS1 gene was leaf-specific, cad-PCS lines showed an overall decrease in cadmium toxicity evidenced by a partial amelioration of the "brown-root" phenotype and root growth was restored to wild type levels when treated with cadmium and arsenate. WT-PCS lines showed an increase in leaf PCS activity but had only wild type PC levels. In addition, cadmium uptake studies indicated that there was no difference in cadmium accumulation among all types tested. So, while we were able to protect the plants against cadmium by expressing PC synthase only in the leaves, we were not able to limit cadmium accumulation to aerial tissues.     

Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2+ tolerance and accumulation but not translocation to the shoot

Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd²⁺ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd²⁺ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd²⁺ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd²⁺ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd²⁺ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd²⁺ transport.     

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.     

Expression of Caenorhabditis elegans PCS in the AtPCS1‐deficient Arabidopsis thaliana cad1‐3 mutant separates the metal tolerance and non‐host resistance functions of phytochelatin synthases

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.     

Arabidopsis thaliana expresses a second functional phytochelatin synthase.

Phytochelatins represent a major detoxifying pathway for heavy metals in plants and many other organisms. The Arabidopsis thaliana CAD1 (=AtPCS1) gene encodes a phytochelatin synthase and cad1 mutants are phytochelatin deficient and cadmium hypersensitive. The Arabidopsis genome contains a highly homologous gene, AtPCS2, of which expression and function were studied in order to understand the apparent non-redundancy of the two genes. Low constitutive AtPCS2 expression is detected in all plant organs analyzed. The AtPCS2 gene encodes a functional phytochelatin synthase as shown by expression in Saccharomyces cerevisiae and the complementation of a Schizosaccharomyces pombe phytochelatin synthase knockout strain.     

Mechanism of heavy metal ion activation of phytochelatin (PC) synthase: blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides

The dependence of phytochelatin synthase (gamma-glutamylcysteine dipeptidyltranspeptidase (PCS), EC ) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana (AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd(2+)- and Zn(2+)-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g. Cd.GS(2) or Zn.GS(2)) and free glutathione act as gamma-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both gamma-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity.     

High concentration of cadmium induces AtPCS2 gene expression in Arabidopsis thaliana (L.) Heynh ecotype Wassilewskija seedlings

In Arabidopsis thaliana, two genes encoding phytochelatin synthase (PCS; EC 2.3.2.15), AtPCS1 and AtPCS2, have been identified. Until now, only AtPCS1 was shown to play a role in response to Cd. To gain insight into the putative role of AtPCS2, three Cd concentrations (50, 100 and 200 μM) and long-term exposure (7 days) were tested on 1-week-old A. thaliana ecotype Wassilewskija (Ws) seedlings. Since 100 μM Cd did not alter seedling metabolism, as shown by unchanged total soluble protein and free proline contents, we investigated plantlet response to this concentration in addition to Cd accumulation. Seedlings accumulated Cd in roots and shoots. As phytochelatins and glutathione (GSH) contents increased in treated seedlings, we suggested that Cd might be translocated via the phytochelatin pathway. Specific enzymatic activities of γ-glutamylcysteine synthetase (GCS; EC 6.3.2.2), glutathione synthetase (GS; EC 6.3.2.3) and PCS were twice much more stimulated in shoots and roots after Cd exposure except GS that remained constant in shoots. As expression of genes encoding GCS and GS was unchanged in response to Cd, we suggested a regulation at translational or post-translational level. Surprisingly, AtPCS1 and AtPCS2 were differentially up-regulated after Cd treatment: AtPCS1 in shoots and AtPCS2 in whole plantlets. This last result suggests that PCS2 could be involved in plant response to high concentration of Cd in Ws ecotype and supports a putative role of PCS2, not redundant with PCS1, in a long-term response to Cd.     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">AtPCS1</Emphasis>) exhibit enhanced As and Cd tolerance

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.     

Overexpression of genes involved in phytochelatin biosynthesis in Escherichia coli: effects on growth, cadmium accumulation and thiol level

In Escherichia coli, heterologous production of Schizosaccharomyces pombe phytochelatin synthase (PCS) along with overproduction of E. coli serine acetyltransferase (SAT) and gamma-glutamylcysteine synthase (gammaECS) was achieved and resulted in the accumulation of phytochelatins in bacterial cells. Overproduction of either gammaECS alone or simultaneous production of all three proteins in bacterial cells were accompanied by reduced growth rate in liquid cultures. Interestingly, bacteria overproducing either gammaECS or both SAT and gammaECS (with elevated level of gamma-glutamylcysteine but not of phytochelatins) were able to accumulate more cadmium per dry weight than the control. However, the most efficient cadmium accumulation was observed in bacteria with elevated levels of all three proteins: SAT, gammaECS and PCS. Therefore, "pushing" the entire pathway might be the most promising approach in modification of bacteria for potential bioremediation purposes because the level of intermediates, cysteine and glutathione, can limit the rate of production of phytochelatins. However, in such bacteria other metabolic process might become limiting for efficient growth.     

A reassessment of substrate specificity and activation of phytochelatin synthases from model plants by physiologically relevant metals

Phytochelatin synthases (PCS) catalyze phytochelatin (PC) synthesis from glutathione (GSH) in the presence of certain metals. The resulting PC-metal complexes are transported into the vacuole, avoiding toxic effects on metabolism. Legumes have the unique capacity to partially or completely replace GSH by homoglutathione (hGSH) and PCs by homophytochelatins (hPCs). However, the synthesis of hPCs has received little attention. A search for PCS genes in the model legume Lotus (Lotus japonicus) resulted in the isolation of a cDNA clone encoding a protein (LjPCS1) highly homologous to a previously reported homophytochelatin synthase (hPCS) of Glycine max (GmhPCS1). Recombinant LjPCS1 and Arabidopsis (Arabidopsis thaliana) PCS1 (AtPCS1) were affinity purified and their polyhistidine-tags removed. AtPCS1 catalyzed hPC synthesis from hGSH alone at even higher rates than did LjPCS1, indicating that GmhPCS1 is not a genuine hPCS and that a low ratio of hPC to PC synthesis is an inherent feature of PCS1 enzymes. For both enzymes, hGSH is a good acceptor, but a poor donor, of gamma-glutamylcysteine units. Purified AtPCS1 and LjPCS1 were activated (in decreasing order) by Cd2+, Zn2+, Cu2+, and Fe3+, but not by Co2+ or Ni2+, in the presence of 5 mm GSH and 50 microm metal ions. Activation of both enzymes by Fe3+ was proven by the complete inhibition of PC synthesis by the iron-specific chelator desferrioxamine. Plants of Arabidopsis and Lotus accumulated (h)PCs only in response to a large excess of Cu2+ and Zn2+, but to a much lower extent than did with Cd2+, indicating that (h)PC synthesis does not significantly contribute in vivo to copper, zinc, and iron detoxification.     

Domain organization of phytochelatin synthase: functional properties of truncated enzyme species identified by limited proteolysis

Phytochelatin synthase (PCS) is a major determinant of heavy metal tolerance in plants and other organisms. No structural information on this enzyme is as yet available. It is generally believed, however, that the active site region is located in the more conserved N-terminal portion of PCS, whereas various, as yet unidentified (but supposedly less critical) roles have been proposed for the C-terminal region. To gain insight into the structural/functional organization of PCS, we have conducted a limited proteolysis analysis of the enzyme from Arabidopsis (AtPCS1), followed by functional characterization of the resulting polypeptide fragments. Two N-terminal fragments ending at positions 372 (PCS_Nt1) and 283 (PCS_Nt2) were produced sequentially upon V8 protease digestion, without any detectable accumulation of the corresponding C-terminal fragments. As revealed by the results of in vivo and in vitro functional assays, the core PCS_Nt2 fragment is biosynthetically active in the presence of cadmium ions and supports phytochelatin formation at a rate that is only approximately 5-fold lower than that of full-length AtPCS1. The loss of the C-terminal region, however, substantially decreases the thermal stability of the enzyme and impairs phytochelatin formation in the presence of certain heavy metals (e.g. mercury and zinc, but not cadmium or copper). The latter phenotype was shared by PCS_Nt2 and by its precursor fragment PCS_Nt1, which, on the other hand, was almost as stable and biosynthetically active (in the presence of cadmium) as the full-length enzyme. AtPCS1 thus appears to be composed of a protease-resistant (and hence presumably highly structured) N-terminal domain, flanked by an intrinsically unstable C-terminal region. The most upstream part of such a region (positions 284-372) is important for enzyme stabilization, whereas its most terminal part (positions 373-485) appears to be required to determine enzyme responsiveness to a broader range of heavy metals.     

Adaptive Engineering of Phytochelatin-based Heavy Metal Tolerance

Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation.     

Expression of Arabidopsis phytochelatin synthase 2 is too low to complement an AtPCS1-defective Cad1-3 mutant

Phytochelatins play an important role in heavy metal detoxification in plants as well as in other organisms. The Arabidopsis thaliana mutant cad1-3 does not produce detectable levels of phytochelatins in response to cadmium stress. The hypersensitivity of cad1-3 to cadmium stress is attributed to a mutation in the phytochelatin synthase 1 (AtPCS1) gene. However, A. thaliana also contains a functional phytochelatin synthase 2 (AtPCS2). In this study, we investigated why the cad1-3 mutant is hypersensitive to cadmium stress despite the presence of AtPCS2. Northern and Western blot analyses showed that expression of AtPCS2 is weak compared to AtPCS1 in both roots and shoots of transgenic Arabidopsis. The lower level of AtPCS2 expression was confirmed by RT-PCR analysis of wild type Arabidopsis. Moreover, no tissue-specific expression of AtPCS2 was observed. Even when AtPCS2 was under the control of the AtPCS1 promoter or of the cauliflower mosaic virus 35S promoter (CaMV 35S) it was not capable of fully complementing the cad1-3 mutant for cadmium resistance.     

Function of phytochelatin synthase in catabolism of glutathione-conjugates

Detoxification of xenobiotic compounds and heavy metals is a pivotal capacity of organisms, in which glutathione (GSH) plays an important role. In plants, electrophilic herbicides are conjugated to the thiol group of GSH, and heavy metal ions form complexes as thiolates with GSH-derived phytochelatins (PCs). In both detoxification processes of plants, phytochelatin synthase (PCS) emerges as a key player. The enzyme is activated by heavy metal ions and catalyzes PC formation from GSH by transferring glutamylcysteinyl residues (gamma-EC) onto GSH. In this study with Arabidopsis, we show that PCS plays a role in the plant-specific catabolism of glutathione conjugates (GS-conjugates). In contrast to animals, breakdown of GS-conjugates in plants can be initiated by cleavage of the carboxyterminal glycine residue that leads to the generation of the corresponding gamma-EC-conjugate. We used the xenobiotic bimane in order to follow GS-conjugate turnover. Functional knockout of the two PCS of Arabidopsis, AtPCS1 and AtPCS2, revealed that AtPCS1 provides a major activity responsible for conversion of the fluorescent bimane-GS-conjugate (GS-bimane) into gamma-EC-bimane. AtPCS1 deficiency resulted in a gamma-EC-bimane deficiency. Transfection of PCS-deficient cells with AtPCS1 recovered gamma-EC-bimane levels. The level of the gamma-EC-bimane conjugate was enhanced several-fold in the presence of Cd2+ ions in the wild type, but not in the PCS-deficient double mutant, consistent with a PCS-catalyzed GS-conjugate turnover. Thus AtPCS1 has two cellular functions: mediating both heavy metal tolerance and GS-conjugate degradation.     

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.     

Effects of simultaneous expression of heterologous genes involved in phytochelatin biosynthesis on thiol content and cadmium accumulation in tobacco plants

Transgenic tobacco (Nicotiana tabacum cv. LA Burley 21) lines expressing three genes encoding enzymes thought to be critical for the efficient production of phytochelatins, (i) serine acetyltransferase (EC 2.3.1.30) involved in the production of O-acetylserine, the cysteine precursor, (ii) gamma-glutamylcysteine synthetase (EC 6.3.2.2) involved in the production of gamma-glutamylcysteine, the precursor of glutathione, and (iii) phytochelatin synthase (EC 2.3.2.15), were obtained and analysed for non-protein thiol content and cadmium accumulation. After a 3 week exposure to 15 microM CdCl2, plants expressing transgenes (either separately or in combination) had increased cadmium concentration in roots but not in shoots compared with the wild type. Nearly all transgenic lines analysed had more non-protein thiols than the wild type. The greatest effects (about 8-fold elevation of thiols) were found in one of the lines simultaneously expressing the three transgenes. Despite the fact that a multi-transgene strategy described in this work resulted in a strong increase in the levels of several classes of non-protein thiols in transgenic plants, other factors appeared to restrict cadmium accumulation in shoots.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Nonspecific binding of monoclonal anti-FLAG M2 antibody in Indian mustard (Brassica juncea)

Chimeric constructs with the hydrophilic octapeptide FLAG epitope (DYKDDDDK) have been widely used as multipurpose tags for identification, detection, and purification of FLAG fusion proteins. Constructs consisting of C-terminal FLAG-tagged genomic and cDNA clones of anArabidopsis phytochelatin synthase gene,AtPCS1, were used in developing transgenic lines of Indian mustard. Presence and expression ofAtPCS1 in transgenic lines were confirmed by using PCR and Northern blot analyses. However, immunoblot analysis revealed strong nonspecific binding of a monoclonal anti-FLAG M2 antibody to an endogenous protein in both shoot and leaf tissues of wild-type Indian mustard (85-kDa) that masked presence of the phytochelatin synthase (PCS) protein of interest (55-kDa). Further analysis revealed absence of a nonspecific protein in root tissues of transgenic plants, thus allowing detection of the FLAG-tagged PCS protein.