Expression of phytochelatin synthase from aquatic macrophyte Ceratophyllum demersum L. enhances cadmium and arsenic accumulation in tobacco

Phytochelatin synthase (PCS), the key enzyme involved in heavy metal detoxification and accumulation has been used from various sources to develop transgenic plants for the purpose of phytoremediation. However, some of the earlier studies provided contradictory results. Most of the PCS genes were isolated from plants that are not potential metal accumulators. In this study, we have isolated PCS gene from Ceratophyllum demersum cv. L. (CdPCS1), a submerged rootless aquatic macrophyte, which is considered as potential accumulator of heavy metals. The CdPCS1 cDNA of 1,757?bp encodes a polypeptide of 501 amino acid residues and differs from other known PCS with respect to the presence of a number of cysteine residues known for their interaction with heavy metals. Complementation of cad1-3 mutant of Arabidopsis deficient in PC (phytochelatin) biosynthesis by CdPCS1 suggests its role in the synthesis of PCs. Transgenic tobacco plants expressing CdPCS1 showed several-fold increased PC content and precursor non-protein thiols with enhanced accumulation of cadmium (Cd) and arsenic (As) without significant decrease in plant growth. We conclude that CdPCS1 encodes functional PCS and may be part of metal detoxification mechanism of the heavy metal accumulating plant C. demersum. KEY MESSAGE: Heterologous expression of PCS gene from C. demersum complements Arabidopsis cad1-3 mutant and leads to enhanced accumulation of Cd and As in transgenic tobacco.     

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.     

Expression of Ceratophyllum demersum phytochelatin synthase, CdPCS1, in Escherichia coli and Arabidopsis enhances heavy metal(loid)s accumulation

Phytochelatin synthase (PCS) gene encoding key enzyme for heavy metal detoxification and accumulation has been characterised from different sources and used to develop a technology for bioremediation. Past efforts provided limited success and contradictory results. Therefore, functional characterisation of PCS gene from new sources into different target systems is considered as an important task in the area of bioremediation. Earlier, we isolated and functionally characterised PCS gene from an aquatic macrophyte Ceratophyllum demersum L., a metal accumulator aquatic plant. Expression of this gene, CdPCS1, in tobacco enhanced PC synthesis and metal accumulation of transgenic tobacco plants. In the present study, we have expressed CdPCS1 in more diverse systems, Escherichia coli and Arabidopsis, and studied growth and metal accumulation of transgenic organisms. The expression of CdPCS1 in E. coli offered tolerance against cadmium as well as higher accumulation accompanied with PCS1 activity. The expression of CdPCS1 in Arabidopsis showed a significant enhanced accumulation of heavy metal(loid)s in aerial parts without significant difference in growth parameters in comparison to wild-type Arabidopsis plants. Our study suggests that CdPCS1 can be utilised for enhancing bioremediation potential of different organisms using biotechnological approaches.     

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.     

植物螯合肽合成酶的研究进展

植物螯合肽（phytochelatins,PCs）在植物解除重金属的毒性方面具有重要作用,其结构为（γ-Glu—Cys）n-Gly（n=2—11）,它不是基因的编码产物,而是在植物螯合肽合成酶（phytochelatin synthase,PCS）的催化下以谷胱甘肽（glutathione,GSH）为底物合成的。PCS能够被金属离子激活,高度保守的N-端是催化结构域,而其C-端则是多变的。本文就PCS的结构,功能与催化机制以及PCS的最新研究进行了介绍。     

Heavy metal tolerance of transgenic tobacco plants over-expressing cysteine synthase

Cysteine synthase [O-acetyl-L-serine(thiol)lyase] catalyzes the final step for L-cysteine biosynthesis in plants. The tolerance of transgenic tobacco plants over-expressing cysteine synthase cDNA in cytosol (3F), chloroplasts (4F) and in both organelles (F1) was investigated towards heavy metals such as Cd, Se, Ni, Pb and Cu. The transgenic plants were significantly more tolerant than wild-type plants in agar medium containing Cd, Se and Ni. The F1 transgenic plants had a higher resistance than other transgenic lines towards these metals and could enhance accumulation of Cd in shoot. These results suggest that the transgenic plants over-expressing cysteine synthase both in cytosol and chloroplasts can be applicable to phyto-remediation of Cd from contaminated soils.     

Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast.

Phytochelatins play major roles in metal detoxification in plants and fungi. However, genes encoding phytochelatin synthases have not yet been identified. By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance. TaPCS1 encodes a protein of approximately 55 kDa with no similarity to proteins of known function. We identified homologs of this new gene family from Arabidopsis thaliana, Schizosaccharomyces pombe, and interestingly also Caenorhabditis elegans. The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast. PCS-expressing cells accumulate more Cd2+ than controls. PCS expression mediates Cd2+ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS-induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd2+ and Cu2+ and are unable to synthesize phytochelatins upon Cd2+ exposure as determined by HPLC analysis. Saccharomyces cerevisiae cells expressing PCS produce phytochelatins. Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity. These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes.     

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 phytochelatin synthase in tobacco: distinctive effects of AtPCS1 and CePCS genes on plant response to cadmium

Phytochelatins, heavy-metal-binding polypeptides, are synthesized by phytochelatin synthase (PCS) (EC 2.3.2.15). Previous studies on plants overexpressing PCS genes yielded contrasting phenotypes, ranging from enhanced cadmium tolerance and accumulation to cadmium hypersensitivity. This paper compares the effects of overexpression of AtPCS1 and CePCS in tobacco (Nicotiana tabacum var. Xanthi), and demonstrates how the introduction of single homologous genes affects to a different extent cellular metabolic pathways leading to the opposite of the desired effect. In contrast to WT and CePCS transformants, plants overexpressing AtPCS1 were Cd-hypersensitive although there was no substantial difference in cadmium accumulation between studied lines. Plants exposed to cadmium (5 and 25 muM CdCl2) differed, however, in the concentration of non-protein thiols (NPT). In addition, PCS activity in AtPCS1 transformants was around 5-fold higher than in CePCS and WT plants. AtPCS1 expressing plants displayed a dramatic accumulation of gamma-glutamylcysteine and concomitant strong depletion of glutathione. By contrast, in CePCS transformants, a smaller reduction of the level of glutathione was noticed, and a less pronounced change in gamma-glutamylcysteine concentration. There was only a moderate and temporary increase in phytochelatin levels due to AtPCS1 and CePCS expression. Marked changes in NPT composition due to AtPCS1 expression led to moderately decreased Cd-detoxification capacity reflected by lower SH:Cd ratios, and to higher oxidative stress (assessed by DAB staining), which possibly explains the increase in Cd-sensitivity. The results indicate that contrasting responses to cadmium of plants overexpressing PCS genes might result from species-dependent differences in the activity of phytochelatin synthase produced by the transgenes.     

Genetic improvement of heavy metal tolerance in plants by transfer of the yeast metallothionein gene (CUP1)

Cauliflower (Brassica oleracea var. botrytis) tolerates treatment with 25 µM CdCl2 for eight days, but is killed by that with a 50 µM concentration. However, even 15 µM CdCl2 is toxic in the presence of 1 mM L-buthionine sulfoximine (BSO), suggesting the presence of a Cd-inducible phytochelatin and its involvement in Cd-tolerance in cauliflower. To develop heavy metal-tolerant transgenic plants, we ligated the structural gene of yeast metallothionein gene (CUP1) downstream of CaMV35S promoter and introduced the fused gene into cauliflower. A Cd-tolerant transgenic cauliflower was selected, which grew well in the presence of 400 µM or less Cd, whereas the non-transformed cauliflower tolerated only up to 25 µM Cd. The transgenic cauliflower accumulated more Cd, especially in the upper leaves, than the non-transformed plant.In conclusion, by transfer of the yeast metallothionein gene into cauliflower increased Cd-tolerance and Cd-accumulating ability can be conferred to the plant.     

Phytochelatin synthase (PCS) protein is induced in Brassica juncea leaves after prolonged Cd exposure

Higher plants respond to cadmium exposure with the production of phytochelatins (PCn), small heavy metal binding peptides, which are synthesized from glutathione by phytochelatin synthase (PCS). The isolation of a PCS cDNA clone from Brassica juncea L. cv. Vitasso, a candidate species for phytoremediation, is reported here. CLUSTAL analysis revealed a close relationship of BjPCS1 with PCS proteins from Arabidopsis thaliana and Thlaspi caerulescens. BjPCS1 expressed as recombinant protein in E. coli had PCS activity in vitro that was activated by 50 microM Cu and 200 microM Cd to a similar extent. Immunoblot analysis with an antiserum directed against recombinant BjPCS1 showed constitutive PCS expression during plant development. As a percentage of the total protein, the expression was higher in the roots, internodes and petioles in comparison with the leaf tissue. When B. juncea plants were treated with 25 microM cadmium, PCn accumulated increasingly over a 6 d period. Levels in shoots were about 3-fold higher than in roots. Prolonged cadmium exposure caused a significant increase of PCS protein in leaves, whereas in roots PCS protein levels were not affected.     

Characterization of phytochelatin synthase produced by the primitive red alga Cyanidioschyzon merolae

Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides.     

Enhanced accumulation of Cd2+ by a Mesorhizobium sp. transformed with a gene from Arabidopsis thaliana coding for phytochelatin synthase

We expressed the Arabidopsis thaliana gene for phytochelatin synthase (PCS(At)) in Mesorhizobium huakuii subsp. rengei B3, a microsymbiont of Astragalus sinicus, a legume used as manure. The PCS(At) gene was expressed under the control of the nifH promoter, which regulates the nodule-specific expression of the nifH gene. The expression of the PCS(At) gene was demonstrated in free-living cells under low-oxygen conditions. Phytochelatin synthase (PCS) was expressed and catalyzed the synthesis of phytochelatins [(gamma-Glu-Cys)(n)-Gly; PCs] in strain B3. A range of PCs, with values of n from 2 to 7, was synthesized by cells that expressed the PCS(At) gene, whereas no PCs were found in control cells that harbored the empty plasmid. The presence of CdCl(2) activated PCS and induced the synthesis of substantial amounts of PCs. Cells that contained PCs accumulated 36 nmol of Cd(2+)/mg (dry weight) of cells. The expression of the PCS(At) gene in M. huakuii subsp. rengei B3 increased the ability of cells to bind Cd(2+) approximately 9- to 19-fold. The PCS protein was detected by immunostaining bacteroids of mature nodules of A. sinicus containing the PCS(At) gene. When recombinant M. huakuii subsp. rengei B3 established the symbiotic relationship with A. sinicus, the symbionts increased Cd(2+) accumulation in nodules 1.5-fold.     

Growth and Heavy Metal Binding Properties of Transgenic Chlamydomonas Expressing a Foreign Metallothionein Gene

We have compared the growth rates and cadmium binding capacity of wild-type and transgenic Chlamydomonas reinhardtii cells expressing a foreign class-II metallothionein. We observed that cells expressing metallothionein grew to significantly higher cell densities than wild-type cells in the presence of a toxic cadmium concentration (40 μM). When grown at a low (5 μM) cadmium concentration, cells expressing metallothionein bound twofold more cadmium (0.43 μg Cd)mg Ch1) than wild-type. At cadmium concentrations (40 μM), which induce phytochelatin synthesis in wild-type cells the cadmium binding capacity of both wild-type (79.6 μg Cd)mg Ch1) and transformed cells (86.4 μg Cd)mg Ch1) was similar; however, the transformed cells grew to higher densities than the wild type. These results suggest that under conditions that apparently induce phytochelatin expression, the presence of metallothionein in the cytoplasm reduces heavy metal toxicity. Furthermore, because cells expressing metallothionein grow to higher densities than wild-type cells at a toxic cadmium concentration (40 μM), the transgenic cells sequester more total cadmium (9% of total Cd) from the medium than the wild type (5.5% of total Cd). These results indicate that the trace-metal binding properties of Chlamydomonas can be enhanced through the expression of trace-metal-specific binding proteins.     

Molecular cloning and characterization of a phytochelatin synthase gene, PvPCS1, from Pteris vittata L.

Pteris vittata L. is a staggeringly efficient arsenic hyperaccumulator that has been shown to be capable of accumulating up to 23,000 microg arsenic g(-1), and thus represents a species that may fully exploit the adaptive potential of plants to toxic metals. However, the molecular mechanisms of adaptation to toxic metal tolerance and hyperaccumulation remain unknown, and P. vittata genes related to metal detoxification have not yet been identified. Here, we report the isolation of a full-length cDNA sequence encoding a phytochelatin synthase (PCS) from P. vittata. The cDNA, designated PvPCS1, predicts a protein of 512 amino acids with a molecular weight of 56.9 kDa. Homology analysis of the PvPCS1 nucleotide sequence revealed that it has low identity with most known plant PCS genes except AyPCS1, and the homology is largely confined to two highly conserved regions near the 5'-end, where the similarity is as high as 85-95%. The amino acid sequence of PvPCS1 contains two Cys-Cys motifs and 12 single Cys, only 4 of which (Cys-56, Cys-90/91, and Cys-109) in the N-terminal half of the protein are conserved in other known PCS polypeptides. When expressed in Saccharomyces cerevisae, PvPCS1 mediated increased Cd tolerance. Cloning of the PCS gene from an arsenic hyperaccumulator may provide information that will help further our understanding of the genetic basis underlying toxic metal tolerance and hyperaccumulation.     

苎麻植物螯合肽合成酶(BnPCS1)基因的克隆和表达

根据苎麻转录组测序中的PCS基因片段,利用RT-PCR结合RACE技术从中苎1号中克隆获得了该基因的全长cDNA序列,命名为BnPCS1。该基因的cDNA序列全长为1956 bp,其中开放读码框长1512 bp,编码503个氨基酸,预测其分子量和等电点分别为56.02 kD和7.01。与长喙田菁(ACT87974)、百脉根(Q2TSC7)、狼牙刺(AFM38979)、荷花(BAN08523)和杜梨(AEY68568)的PCS氨基酸序列相似性分别为74%、73%、75%、73%和77%。荧光定量PCR分析表明,BnPCS1在根、茎、茎尖、幼叶、成熟叶中均有表达,其中在成熟叶中的表达量最高,茎中表达量最低,并且该基因受镉和ABA诱导上调表达。BnPCS1基因的克隆将为苎麻抗重金属分子育种和进一步的功能分析奠定基础。     

植物的硫同化及其相关酶活性在镉胁迫下的调节

植物对土壤中硫的利用包括根系对硫酸盐的吸收、转运、同化、分配等过程,也是由一系列酶和蛋白质参与和调节的代谢过程。近年来的研究表明,在植物体内,硫同化与植物对镉等重金属元素的胁迫反应机制有着密切关系。镉胁迫能调节植物对硫酸盐的吸收、转运、同化,以及半胱氨酸、谷胱甘肽（glutathione,GSH）和植物螯合肽（Dhytochelatins,pc）的合成。植物在镉胁迫下通过多种调节机制,增强对硫酸盐的吸收和还原,迅速合成半胱氨酸和谷胱甘肽等代谢物,从而合成足够的PC,以满足植物生理的需要。