Heavy Metal Detoxification In Plants Research Articles

An Insight into Microbes Mediated Heavy Metal Detoxification in Plants: a Review

An Insight into Microbes Mediated Heavy Metal Detoxification in Plants: a Review

Transcriptome Response to Cadmium Exposure in Barley (Hordeum vulgare L.).

Cadmium is an environmental pollutant with high toxicity that negatively affects plant growth and development. To understand the molecular mechanisms of plant response to cadmium stress, we have performed a genome-wide transcriptome analysis on barley plants treated with an increased concentration of cadmium. Differential gene expression analysis revealed 10,282 deregulated transcripts present in the roots and 7,104 in the shoots. Among them, we identified genes related to reactive oxygen species metabolism, cell wall formation and maintenance, ion membrane transport and stress response. One of the most upregulated genes was PLANT CADMIUM RESISTACE 2 (HvPCR2) known to be responsible for heavy metal detoxification in plants. Surprisingly, in the transcriptomic data we identified four different copies of the HvPCR2 gene with a specific pattern of upregulation in individual tissues. Heterologous expression of all five barley copies in a Cd-sensitive yeast mutant restored cadmium resistance. In addition, four HvPCR2 were located in tandem arrangement in a single genomic region of the barley 5H chromosome. To our knowledge, this is the first example showing multiplication of the PCR2 gene in plants.

The Role of Phytochelatin Synthase in Phytoremediation Agent: Structural Conservation of Phytochelatin (PC) Synthase to Maintain Its Activity as Heavy Metal Detoxification in Plant

Phytochelatin (PC) Enzyme has crucial role in heavy metal detoxification and homeostasis in plants. This study aimed to evaluate the genetic variation of PC synthase related to its activity based on structural comparison. We evaluated PC genes and protein sequences from 6 plants namely, Brassicasp., Amaranthussp., Noccaeasp., Arabidopsissp., Nicotianasp., and Pterissp. All sequences were aligned based on CLUSTALW matrix for DN sequences and MUSCLE algorithm for protein sequences. Data were clustered using MEGA Software for similarity clustering. Selected data were further modeled using SWISSMODEL to evaluate the 3D-structural analysis based on homology modeling. Thus, all protein models were superimposed and evaluated the structure comparison based on RMSD data. The result showed that genetic variation of PC gene is high among species. But it is clustered for the same species has similar sequence. In addition, protein sequences also showed the high diversity among species and it is still clustered basedon their genus. RMSD data showed that PC synthase from 6 plant has similar structure and tend to conserved even there is genetic variation or amino acid modification. We concluded that structural of PC gene is more conserved than its sequences. It is important to keep its function among species.

Moonlighting Function of Phytochelatin Synthase1 in Extracellular Defense against Fungal Pathogens.

Phytochelatin synthase (PCS) is a key component of heavy metal detoxification in plants. PCS catalyzes both the synthesis of the peptide phytochelatin from glutathione and the degradation of glutathione conjugates via peptidase activity. Here, we describe a role for PCS in disease resistance against plant pathogenic fungi. The pen4 mutant, which is allelic to cadmium insensitive1 (cad1/pcs1) mutants, was recovered from a screen for Arabidopsis mutants with reduced resistance to the nonadapted barley fungal pathogen Blumeria graminis f. sp. hordei PCS1, which is found in the cytoplasm of cells of healthy plants, translocates upon pathogen attack and colocalizes with the PEN2 myrosinase on the surface of immobilized mitochondria. pcs1 and pen2 mutant plants exhibit similar metabolic defects in the accumulation of pathogen-inducible indole glucosinolate-derived compounds, suggesting that PEN2 and PCS1 act in the same metabolic pathway. The function of PCS1 in this pathway is independent of phytochelatin synthesis and deglycination of glutathione conjugates, as catalytic-site mutants of PCS1 are still functional in indole glucosinolate metabolism. In uncovering a peptidase-independent function for PCS1, we reveal this enzyme to be a moonlighting protein important for plant responses to both biotic and abiotic stresses.

Cd2+ and Pb2+ complexation by glutathione and the phytochelatins

Phytochelatins or PCn, (γGlu-Cys)n-Gly, and their glutathione (GSH) precursor are thiol-rich peptides that play an important role in heavy metal detoxification in plants and microorganisms. Complex formation between Cd2+ and Pb2+ and GSH or PCn (n = 2, 4 and 6) are investigated by microcalorimetry, absorption spectrophotometry and T-jump kinetics. Complex formation with Pb2+ or Cd2+ is exothermic, and induces ligand metal charge transfer bands in UV absorption spectral range, which implies the formation of a coordination bond between the metal and the thiol groups of the phytochelatins. Absorption spectra and microcalorimetry experiments allow the determination of the affinity constants and the stoichiometry of the complexes. We show that the three PCn interact with Pb2+ to form the 1:1 and 2:1 M:L complexes, with similar affinity constants (log K11Pb∼4.6, log K21Pb∼11.4). These affinities are independent of the number of thiols and are, moreover, lower than those determined for complex formation with Cd2+. On the other hand, with Cd2+, PC2-Cd, PC2-Cd2, (PC2)3-Cd2, PC4-Cd, PC4-Cd2, PC6-Cd, (PC6)2-Cd3 and PC6-Cd3 complexes are detected. Furthermore, for PC4-Cd, the 1:1 complex is the most stable: affinity constant (log K11Cd∼7.5). Kinetic studies indicate that complex formation between Cd2+ and GSH occurs in the ms range; direct rate constant kobs = (6.8 ± 0.3) 106 M−1 s−1 and reverse rate constant k−obs = 340 ± 210 s−1. Thus, when encapsulated in a silica matrix, PCn can be good candidates for heavy metal detection.

Vacuolar compartmentalization as indispensable component of heavy metal detoxification in plants.

Plant cells orchestrate an array of molecular mechanisms for maintaining plasmatic concentrations of essential heavy metal (HM) ions, for example, iron, zinc and copper, within the optimal functional range. In parallel, concentrations of non-essential HMs and metalloids, for example, cadmium, mercury and arsenic, should be kept below their toxicity threshold levels. Vacuolar compartmentalization is central to HM homeostasis. It depends on two vacuolar pumps (V-ATPase and V-PPase) and a set of tonoplast transporters, which are directly driven by proton motive force, and primary ATP-dependent pumps. While HM non-hyperaccumulator plants largely sequester toxic HMs in root vacuoles, HM hyperaccumulators usually sequester them in leaf cell vacuoles following efficient long-distance translocation. The distinct strategies evolved as a consequence of organ-specific differences particularly in vacuolar transporters and in addition to distinct features in long-distance transport. Recent molecular and functional characterization of tonoplast HM transporters has advanced our understanding of their contribution to HM homeostasis, tolerance and hyperaccumulation. Another important part of the dynamic vacuolar sequestration syndrome involves enhanced vacuolation. It involves vesicular trafficking in HM detoxification. The present review provides an updated account of molecular aspects that contribute to the vacuolar compartmentalization of HMs.

Tobacco phytochelatin synthase (NtPCS1) plays important roles in cadmium and arsenic tolerance and in early plant development in tobacco

Phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins, which are involved in heavy metal detoxification in plants and other living organisms. Previously, we cloned a PCS1 gene from tobacco (Nicotiana tabacum) and showed that its expression in yeast (Saccharomyces cerevisiae) resulted in increased cadmium (Cd) tolerance and Cd accumulation (Kim et al., J Plant Biol 48:440–447, 2005). To examine the role of NtPCS1 in tobacco, we generated transgenic tobacco lines over-expressing NtPCS1 in the sense or antisense direction. Compared with other PCS1-expressing plants, NtPCS1-expressing tobacco exhibited a unique phenotype: increased tolerance to cadmium and arsenite, but no change in cadmium and arsenic accumulation. In addition, the antisense-NtPCS1 tobacco lines showed growth retardation in the early stage, suggesting that phytochelatin also plays a role in plant development. These results demonstrate that NtPCS1 plays important roles in metal(loid) tolerance as well as in growth and development in tobacco.

Heavy-metal-induced reactive oxygen species: phytotoxicity and physicochemical changes in plants.

As a result of the industrial revolution, anthropogenic activities have enhanced there distribution of many toxic heavy metals from the earth's crust to different environmental compartments. Environmental pollution by toxic heavy metals is increasing worldwide, and poses a rising threat to both the environment and to human health.Plants are exposed to heavy metals from various sources: mining and refining of ores, fertilizer and pesticide applications, battery chemicals, disposal of solid wastes(including sewage sludge), irrigation with wastewater, vehicular exhaust emissions and adjacent industrial activity.Heavy metals induce various morphological, physiological, and biochemical dysfunctions in plants, either directly or indirectly, and cause various damaging effects. The most frequently documented and earliest consequence of heavy metal toxicity in plants cells is the overproduction of ROS. Unlike redox-active metals such as iron and copper, heavy metals (e.g, Pb, Cd, Ni, AI, Mn and Zn) cannot generate ROS directly by participating in biological redox reactions such as Haber Weiss/Fenton reactions. However, these metals induce ROS generation via different indirect mechanisms, such as stimulating the activity of NADPH oxidases, displacing essential cations from specific binding sites of enzymes and inhibiting enzymatic activities from their affinity for -SH groups on the enzyme.Under normal conditions, ROS play several essential roles in regulating the expression of different genes. Reactive oxygen species control numerous processes like the cell cycle, plant growth, abiotic stress responses, systemic signalling, programmed cell death, pathogen defence and development. Enhanced generation of these species from heavy metal toxicity deteriorates the intrinsic antioxidant defense system of cells, and causes oxidative stress. Cells with oxidative stress display various chemical,biological and physiological toxic symptoms as a result of the interaction between ROS and biomolecules. Heavy-metal-induced ROS cause lipid peroxidation, membrane dismantling and damage to DNA, protein and carbohydrates. Plants have very well-organized defense systems, consisting of enzymatic and non-enzymatic antioxidation processes. The primary defense mechanism for heavy metal detoxification is the reduced absorption of these metals into plants or their sequestration in root cells.Secondary heavy metal tolerance mechanisms include activation of antioxidant enzymes and the binding of heavy metals by phytochelatins, glutathione and amino acids. These defense systems work in combination to manage the cascades of oxidative stress and to defend plant cells from the toxic effects of ROS.In this review, we summarized the biochemiCal processes involved in the over production of ROS as an aftermath to heavy metal exposure. We also described the ROS scavenging process that is associated with the antioxidant defense machinery.Despite considerable progress in understanding the biochemistry of ROS overproduction and scavenging, we still lack in-depth studies on the parameters associated with heavy metal exclusion and tolerance capacity of plants. For example, data about the role of glutathione-glutaredoxin-thioredoxin system in ROS detoxification in plant cells are scarce. Moreover, how ROS mediate glutathionylation (redox signalling)is still not completely understood. Similarly, induction of glutathione and phytochelatins under oxidative stress is very well reported, but it is still unexplained that some studied compounds are not involved in the detoxification mechanisms. Moreover,although the role of metal transporters and gene expression is well established for a few metals and plants, much more research is needed. Eventually, when results for more metals and plants are available, the mechanism of the biochemical and genetic basis of heavy metal detoxification in plants will be better understood. Moreover, by using recently developed genetic and biotechnological tools it may be possible to produce plants that have traits desirable for imparting heavy metal tolerance.

Regulation of the Phytochelatin Synthase Gene <italic>AtPCS2 </italic>in <italic>Arabidopsis thaliana</italic>

Phytochelatin synthase (PCS) plays an essential role in heavy metal detoxification in plants and fungi, via the catalyzation of phytochelatins (PCs) biosynthesis from the substrate of glutathione (GSH). Two PCS genes <italic>AtPCS1</italic> and <italic>AtPCS2</italic> are present in <italic>Arabidopsis</italic> genome. However, loss of function of only the <italic>AtPCS1</italic> gene resulted in undetectable PCs and the consequent metal hypersensitivity in the corresponding mutant <italic>cad1</italic>-<italic>3</italic>,while <italic>in vitro</italic> assay indicated that AtPCS2 is capable to efficiently catalyze PCs biosynthesis. These results suggest that repression mechanisms exist in <italic>Arabidopsis</italic> to tightly control <italic>AtPCS2</italic> function <italic>in vivo</italic>. To test and decipher this hypothesis, we generated the construct 35S/AtPCS2::cMyc and transformed it into the PCs-deficient mutant <italic>cad1</italic>-<italic>3</italic>. Our results showed that both the <italic>AtPCS2</italic> mRNA and protein levels were steadily high in the transgenic plants. Consistently, PCs biosynthesis and metal tolerance were also observed when <italic>AtPCS2</italic> was introduced into <italic>cad1</italic>-<italic>3</italic>. Furthermore, in addition to the cytosol, AtPCS2 was also localized to the nuclei. Taken together, all these results indicate that <italic>AtPCS2</italic> is under tight control mainly at the transcription level in <italic>Arabidopsis</italic>, and it may function beyond catalyzing PCs biosynthesis.

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

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

A papain-like enzyme at work: native and acyl-enzyme intermediate structures in phytochelatin synthesis.

Phytochelatin synthase (PCS) is a key enzyme for heavy-metal detoxification in plants. PCS catalyzes the production of glutathione (GSH)-derived peptides (called phytochelatins or PCs) that bind heavy-metal ions before vacuolar sequestration. The enzyme can also hydrolyze GSH and GS-conjugated xenobiotics. In the cyanobacterium Nostoc, the enzyme (NsPCS) contains only the catalytic domain of the eukaryotic synthase and can act as a GSH hydrolase and weakly as a peptide ligase. The crystal structure of NsPCS in its native form solved at a 2.0-A resolution shows that NsPCS is a dimer that belongs to the papain superfamily of cysteine proteases, with a conserved catalytic machinery. Moreover, the structure of the protein solved as a complex with GSH at a 1.4-A resolution reveals a gamma-glutamyl cysteine acyl-enzyme intermediate stabilized in a cavity of the protein adjacent to a second putative GSH binding site. GSH hydrolase and PCS activities of the enzyme are discussed in the light of both structures.

Simultaneous femtomole determination of cysteine, reduced and oxidized glutathione, and phytochelatin in maize ( Zea mays L.) kernels using high-performance liquid chromatography with electrochemical detection

Thiol compounds such as cysteine (Cys), reduced (GSH) and oxidized (GSSG) gluathione, and phytochelatins (PCs) play an important role in heavy metal detoxification in plants. These thiols are biological active compounds whose function is elimination of oxidative stress in plant cells. The aim of our work was to optimise sensitive and rapid method of high-performance liquid chromatography coupled with electrochemical detector (HPLC-ED) for determination of the abovementioned thiol compounds in maize ( Zea mays L.) kernels. New approach for evaluation of HPLC-ED parameters is described. The most suitable isocratic mobile phase for the separation and detection of Cys, GSH, GSSG and PC 2 consisted of methanol (MeOH) and trifluoroacetic acid (TFA). In addition, the influence of concentrations of TFA and ratio of MeOH:TFA on chromatographic separation and detection of the thiol compounds were studied. The mobile phase consisting from methanol and 0.05% (v/v) TFA in ratio 97:3 (%; v/v) was found the most suitable for the thiol compounds determination. Optimal flow rate of the mobile phase was 0.18 ml min −1 and the column and detector temperature 35 °C. Hydrodynamic voltammograms of all studied compounds was obtained due to the selection of the most effective working electrodes potentials. Two most effective detection potentials were selected: 780 mV for the GSSG and PC 2 and 680 mV for determination of Cys and GSH. The optimised HPLC-ED method was capable to determine femtomole levels of studied compounds. The detection limits (3 S/N) of the studied thiol compounds were for cysteine 112.8 fmol, GSH 63.5 fmol, GSSG 112.2 fmol and PC 2 2.53 pmol per injection (5 μl). The optimised HPLC-ED method was applied to study of the influence of different cadmium concentrations (0, 10 and 100 μM Cd) on content of Cys, GSH, GSSG and PC 2 in maize kernels. According to the increasing time of Cd treatment, content of GSH, GSSG and PC 2 in maize kernels increased but content of Cys decreased. Decreasing Cys concentration probably relates with the increasing GSH and phytochelatins synthesis.

Functional analysis of phytochelatin synthase from Arabidopsis thaliana and its expression in Escherichia coli and Saccharomyces cerevisiae

Plants, like other organisms, have adaptive mechanisms whereby they are able to respond to both nutrient deficiencies and toxicities. Phytochelatins (PCs) play an essential role in heavy-metal detoxification in plants, fungi and worms. PCs chelate heavy metals and then PCmetal complexes are translocated across the tonoplast and sequestered in vacuoles. PCs are synthesized from glutathione by the enzyme PC synthase (PCS). Comparison of the deduced amino acid sequences of PCS suggests that the C-terminal domain may be important for activation of the enzyme. We established the method for purification of PCS from Arabidopsis thaliana to perform enzymatic characterization. Moreover, the PCS gene was expressed in E. coli and S. cerevisiae to enhance tolerance to toxicity of cadmium ion. The obtained results implied that the some regions of the PCS may serve as regulatory region through interaction with cadmium ion and/or oxygen related compounds. Moreover, PCS expression dramatically enhanced cadmium ion tolerance of different organisms. Based on our findings, functional mechanism for PCS activation was hypothesized.

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

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

Possible roles of phytochelatins and glutathione metabolism in cadmium tolerance in chickpea roots.

The possible roles of phytochelatin (PC) and glutathione (GSH) in the heavy metal detoxification in plants were examined using two varieties (CSG-8962 and C-235) of chickpea ( Cicer arietinumL.). The seedlings were grown for 5 days and the roots were treated with 0-20 micro M CdSO(4) for 3 days. The CSG-8962 seedlings exhibited more Cd-tolerant characteristics than did the C-235, where the roots, rather than shoots, suffered from more toxic effects by Cd. Both the seedlings synthesized the large amounts of PCs and homo-phytochelatins (hPCs) in roots, but only a little in shoots in response to Cd. The Cd treatments also caused a marked increase in the levels of GSH and cysteine in both the root and shoot tissues, suggesting that Cd may activate the GSH biosynthesis and, hence, enhance PC synthesis in the plants. Such a Cd-sensitive PC synthesis in chickpea plants does not explain the difference in Cd sensitivity in the varieties, but can be used as a biochemical indicator for Cd contamination in various environments. In the chickpea plants, possible PC-dependent and independent mechanisms for Cd tolerance are discussed.

Heavy Metal Detoxification in Plants: Phytochelatin Biosynthesis and Function

AbstractPhytochelatins (PCs) are a family of peptides important in the detoxification of heavy metals such as cadmium in plants and some microorganisms. PCs are synthesised enzymatically from glutathione. Molecular genetic studies, particularly in the yeast Schizosaccharomyces pombe and in the plant Arabidopsis thaliana, have identified a number of genes important in the biosynthesis or function of PCs. PC‐deficient mutants of Arabidopsis have confirmed both the role of glutathione as the substrate for PC biosynthesis and the role of PCs themselves in heavy metal detoxification in plants. PC synthase genes have been identified in Arabidopsis and other plant species as well as in a number of animal species, suggesting PCs play a wider role in metal detoxification than previously anticipated. PC synthesis is regulated at a number of levels, most importantly through the activation of PC synthase by metal ions. This article reviews recent advances in our understanding of the biosynthesis and function of PCs in plants and other organisms.

Cadmium lets increase the glutathione pool in bryophytes

Summary Glutathione (GSH) plays an important role in protecting plants from environmental stresses like oxidative stress and xenobiotics. Glutathione-derived peptides are involved in heavy metal detoxification in plants and fungi. Terrestrial and aquatic bryophytes were investigated for their biochemical response to heavy metals. The GSH pool increased significantly in the first two days after supply of 100 μmol/L Cd(II). PCs were not detected. Cd(II) also induced the enhancement of the GSH pool in the water moss Fontinalis antipyretica. Cysteine and γ-glutamyl-cysteine also increased during Cd(II) treatment, but remained on a lower level. Uptake experiments with Cd(II) showed a fast regulation of equilibrium between the Cd(II) content of the medium and the plant surface, followed by a slow migration of Cd(II) to intracellular sites. The main storage compartment of heavy metals in Fontinalis are the vacuoles, where they are precipitated as phosphates. In the cytoplasm, the S-content increased during Cd(II) exposition. EEL-spectra indicate that in the cytoplasm, Cd(II) is chelated by SH-groups. All findings support the idea that in the investigated moss species, GSH plays an essential role in heavy metal detoxification during the transport of the metals through the cytoplasm.

Application of high performance liquid chromatography - inductively coupled plasma mass spectrometry to the investigation of phytochelatin complexes and their role in heavy metal detoxification in plants

Application of high performance liquid chromatography - inductively coupled plasma mass spectrometry to the investigation of phytochelatin complexes and their role in heavy metal detoxification in plants

Application of high performance liquid chromatography - inductively coupled plasma mass spectrometry to the investigation of phytochelatin complexes and their role in heavy metal detoxification in plants

Application of high performance liquid chromatography - inductively coupled plasma mass spectrometry to the investigation of phytochelatin complexes and their role in heavy metal detoxification in plants