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.

Overexpression of a Functional Vicia sativa PCS1 Homolog Increases Cadmium Tolerance and Phytochelatins Synthesis in Arabidopsis.

Phytochelatins (PCs) catalyzed by phytochelatin synthases (PCS) are important for the detoxification of metals in plants and other living organisms. In this study, we isolated a PCS gene (VsPCS1) from Vicia sativa and investigated its role in regulating cadmium (Cd) tolerance. Expression of VsPCS1 was induced in roots of V. sativa under Cd stress. Analysis of subcellular localization showed that VsPCS1 was localized in the cytoplasm of mesophyll protoplasts of V. sativa. Overexpression of VsPCS1 (35S::VsPCS1, in wild-type background) in Arabidopsis thaliana could complement the defects of Cd tolerance of AtPCS1-deficent mutant (atpcs1). Compared with atpcs1 mutants, 35S::VsPCS1/atpcs1 (in AtPCS1-deficent mutant background) transgenic plants significantly lowered Cd-fluorescence intensity in mesophyll cytoplasm, accompanied with enhanced Cd-fluorescence intensity in the vacuoles, demonstrating that the increased Cd tolerance may be attributed to the increased PC-based sequestration of Cd into the vacuole. Furthermore, overexpressing VsPCS1 could enhance the Cd tolerance in 35S::VsPCS1, but have no effect on Cd accumulation and distribution, showing the same level of Cd-fluorescence intensity between 35S::VsPCS1 and wild-type (WT) plants. Further analysis indicated this increased tolerance in 35S::VsPCS1 was possibly due to the increased PCs-chelated Cd in cytosol. Taken together, a functional PCS1 homolog from V. sativa was identified, which hold a strong catalyzed property for the synthesis of high-order PCs that retained Cd in the cytosol rather the vacuole. These findings enrich the original model of Cd detoxification mediated by PCS in higher plants.

Metal accumulation and detoxification mechanisms in mycorrhizal Betula pubescens

Metal detoxification in plants is a complex process that involves different mechanisms, such as the retention of metals to the cell wall and their chelation and subsequent compartmentalization in plant vacuoles. In order to identify the mechanisms involved in metal accumulation and tolerance in Betula pubescens, as well as the role of mycorrhization in these processes, mycorrhizal and non-mycorrhizal plants were grown in two industrial soils with contrasting concentrations of heavy metals.Mycorrhization increased metal uptake at low metal concentrations in the soil and reduced it at high metal concentrations, which led to an enhanced growth and biomass production of the host when growing in the most polluted soil. Our results suggest that the sequestration on the cell wall is the main detoxification mechanism in white birch exposed to acute chronic metal-stress, while phytochelatins play a role mitigating metal toxicity inside the cells. Given its high Mn and Zn root-to-shoot translocation rate, Betula pubescens is a very promising species for the phytoremediation of soils polluted with these metals.

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.

Arabidopsis thaliana phytochelatin synthase 2 is constitutively active in vivo and can rescue the growth defect of the PCS1-deficient cad1-3 mutant on Cd-contaminated soil.

Phytochelatins play a key role in the detoxification of metals in plants and many other eukaryotes. Their formation is catalysed by phytochelatin synthases (PCS) in the presence of metal excess. It appears to be common among higher plants to possess two PCS genes, even though in Arabidopsis thaliana only AtPCS1 has been demonstrated to confer metal tolerance. Employing a highly sensitive quantification method based on ultraperformance electrospray ionization quadrupole time-of-flight mass spectrometry, we detected AtPCS2-dependent phytochelatin formation. Overexpression of AtPCS2 resulted in constitutive phytochelatin accumulation, i.e. in the absence of metal excess, both in planta and in a heterologous system. This indicates distinct enzymatic differences between AtPCS1 and AtPCS2. Furthermore, AtPCS2 was able to partially rescue the Cd hypersensitivity of the AtPCS1-deficient cad1-3 mutant in a liquid seedling assay, and, more importantly, when plants were grown on soil spiked with Cd to a level that is close to what can be found in agricultural soils. No rescue was found in vertical-plate assays, the most commonly used method to assess metal tolerance. Constitutive AtPCS2-dependent phytochelatin synthesis suggests a physiological role of AtPCS2 other than metal detoxification. The differences observed between wild-type plants and cad1-3 on Cd soil demonstrated: (i) the essentiality of phytochelatin synthesis for tolerating levels of Cd contamination that can naturally be encountered by plants outside of metal-rich habitats, and (ii) a contribution to Cd accumulation under these conditions.

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.

Natural Abiotic Formation of Oxalic Acid in Soils: Results from Aromatic Model Compounds and Soil Samples

Oxalic acid is the smallest dicarboxylic acid and plays an important role in soil processes (e.g., mineral weathering and metal detoxification in plants). We have first proven its abiotic formation in soils and investigated natural abiotic degradation processes based on the oxidation of soil organic matter, enhanced by Fe(3+) and H(2)O(2) as hydroxyl radical suppliers. Experiments with the model compound catechol and further hydroxylated benzenes were performed to examine a common degradation pathway and to presume a general formation mechanism of oxalic acid. Two soil samples were tested for the release of oxalic acid and the potential effects of various soil parameters on oxalic acid formation. Additionally, the soil samples were treated with different soil sterilization methods to prove the oxalic acid formation under abiotic soil conditions. Different series of model experiments were conducted to determine a range of factors including Fe(3+), H(2)O(2), reaction time, pH, and chloride concentration on oxalic acid formation. Under certain conditions, catechol is degraded up to 65.6% to oxalic acid referring to carbon. In serial experiments with two soil samples, oxalic acid was produced, and the obtained results are suggestive of an abiotic degradation process. In conclusion, Fenton-like conditions with low Fe(3+) concentrations and an excess of H(2)O(2) as well as acidic conditions were required for an optimal oxalic acid formation. The presence of chloride reduced oxalic acid formation.

Characterization and Expression of a Phytochelatin Synthase Gene in Birch-leaf Pear (Pyrus betulaefolia Bunge)

Phytochelatins play a major role in metal detoxification in plants. However, the molecular mechanisms of heavy metal detoxification remain unknown, and birch-leaf pear genes related to metal detoxification have not yet been identified. Here, we report the isolation of cDNA and genomic DNA sequences encoding a phytochelatin synthase (PCS) from birch-leaf pear (Pyrus betulaefolia Bunge). The PbPCS1 cDNA sequence contained 1,965 nucleotides of a 1,494 bp open reading frame (ORF), which encodes a 497-amino-acid protein with a molecular weight of 55.00 kDa. Its corresponding genomic DNA sequence consists of 3,820 nucleotides and eight exons separated by seven introns. The deduced amino acid sequence of PbPCS1 contains five Cys residues (56, 90, 91, 109 and 113 amino acids) that are highly conserved in the plant PCS1 family, and 12 cysteine residues putatively involved in heavy metal binding sites, which included three adjacent Cys–Cys components (331–332, 351–352 and 369–370 amino acids) in the C-terminal variable domain. Homology analysis of the deduced PbPCS1 amino acid sequence revealed that it shares a high sequence similarity amongst N-terminal amino acids and low similarity with C-terminal amino acids with plant PCS1 proteins deposited with NCBI. PbPCS1 was most related to PCS1 from legume plants Lotus japonicus (GenBank accession no. AAT80342) and soybean (Glycine max L.; AAL78384) as they were clustered into the same clade in a neighbor-joining phylogenetic tree. Quantitative real-time PCR (qPCR) expression analysis revealed that PbPCS1 had a very low basal expression level in untreated whole seedlings, and levels were higher in roots than in leaves and stems. After 24 h of exposure to 20 μM CdSO4, CuSO4 or ZnSO4, PbPCS1 expression increased significantly in different organs. In addition, L-buthionine-sulfo-ximine (BSO) can inhibit PbPCS1 expression in roots, stems and leaves, while L-glutathionereduced (GSH) stimulates PbPCS1 expression in different organs of birch-leaf pear.

Implications of metal accumulation mechanisms to phytoremediation

Trace elements (heavy metals and metalloids) are important environmental pollutants, and many of them are toxic even at very low concentrations. Pollution of the biosphere with trace elements has accelerated dramatically since the Industrial Revolution. Primary sources are the burning of fossil fuels, mining and smelting of metalliferous ores, municipal wastes, agrochemicals, and sewage. In addition, natural mineral deposits containing particularly large quantities of heavy metals are found in many regions. These areas often support characteristic plant species thriving in metal-enriched environments. Whereas many species avoid the uptake of heavy metals from these soils, some of them can accumulate significantly high concentrations of toxic metals, to levels which by far exceed the soil levels. The natural phenomenon of heavy metal tolerance has enhanced the interest of plant ecologists, plant physiologists, and plant biologists to investigate the physiology and genetics of metal tolerance in specialized hyperaccumulator plants such as Arabidopsis halleri and Thlaspi caerulescens. In this review, we describe recent advances in understanding the genetic and molecular basis of metal tolerance in plants with special reference to transcriptomics of heavy metal accumulator plants and the identification of functional genes implied in tolerance and detoxification. Plants are susceptible to heavy metal toxicity and respond to avoid detrimental effects in a variety of different ways. The toxic dose depends on the type of ion, ion concentration, plant species, and stage of plant growth. Tolerance to metals is based on multiple mechanisms such as cell wall binding, active transport of ions into the vacuole, and formation of complexes with organic acids or peptides. One of the most important mechanisms for metal detoxification in plants appears to be chelation of metals by low-molecular-weight proteins such as metallothioneins and peptide ligands, the phytochelatins. For example, glutathione (GSH), a precursor of phytochelatin synthesis, plays a key role not only in metal detoxification but also in protecting plant cells from other environmental stresses including intrinsic oxidative stress reactions. In the last decade, tremendous developments in molecular biology and success of genomics have highly encouraged studies in molecular genetics, mainly transcriptomics, to identify functional genes implied in metal tolerance in plants, largely belonging to the metal homeostasis network. Analyzing the genetics of metal accumulation in these accumulator plants has been greatly enhanced through the wealth of tools and the resources developed for the study of the model plant Arabidopsis thaliana such as transcript profiling platforms, protein and metabolite profiling, tools depending on RNA interference (RNAi), and collections of insertion line mutants. To understand the genetics of metal accumulation and adaptation, the vast arsenal of resources developed in A. thaliana could be extended to one of its closest relatives that display the highest level of adaptation to high metal environments such as A. halleri and T. caerulescens. This review paper deals with the mechanisms of heavy metal accumulation and tolerance in plants. Detailed information has been provided for metal transporters, metal chelation, and oxidative stress in metal-tolerant plants. Advances in phytoremediation technologies and the importance of metal accumulator plants and strategies for exploring these immense and valuable genetic and biological resources for phytoremediation are discussed. A number of species within the Brassicaceae family have been identified as metal accumulators. To understand fully the genetics of metal accumulation, the vast genetic resources developed in A. thaliana must be extended to other metal accumulator species that display traits absent in this model species. A. thaliana microarray chips could be used to identify differentially expressed genes in metal accumulator plants in Brassicaceae. The integration of resources obtained from model and wild species of the Brassicaceae family will be of utmost importance, bringing most of the diverse fields of plant biology together such as functional genomics, population genetics, phylogenetics, and ecology. Further development of phytoremediation requires an integrated multidisciplinary research effort that combines plant biology, genetic engineering, soil chemistry, soil microbiology, as well as agricultural and environmental engineering.

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.

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.

Different effects of calcium and lanthanum on the expression of phytochelatin synthase gene and cadmium absorption in Lactuca sativa

Phytochelatins (PCs) have an important role in metal detoxification in plants, however the relationship of the expression of Lactuca sativa phytochelatin synthase gene, its catalyzing products—PCs and the absorption of cadmium (Cd) was still not clear. To investigate this relationship, a full-length cDNA of a phytochelatin synthase gene ( LsPCS1) was cloned from L. sativa and the physiological changes were observed. When L. sativa (cabbage lettuce) plants were exposed to toxic cadmium, growth was retarded. Though L. sativa plants cadmium tolerance was enhanced when synchronously treated with lanthanum (La 3+) or calcium (Ca 2+), the effect on the expression of phytochelatin synthase gene and cadmium absorption was different. The transcript level of LsPCS1 and the PCs content in the plants grown under normal conditions were very low, and the Cd absorption was minimal. In comparison with these plants, the expression level of LsPCS1 and the absorption of Cd were enhanced both in leaf and root under Cd 2+, Cd 2+ + Ca 2+ and Cd 2+ + La 3+ treatments. In comparison with Cd 2+ treatment, the accumulated mRNA level of LsPCS1, PCs content and the absorption of Cd were enhanced with Cd 2+ + Ca 2+ treatment. Though Cd 2+ + La 3+ treatment showed a similar expression profile with Cd 2+ + Ca 2+ treatment, the absorption of Cd of Cd 2+ + La 3+ treatment was less than that of Cd 2+ treatment. Ca 2+ alone has little effect on the expression of LsPCS1, whereas La 3+ itself enhances the mRNA and PCs accumulation. It is suggested that Ca 2+ increases the expression of PC synthase gene under Cd 2+ stress, which in turn enhances the plant tolerance and Cd accumulation. Thus, fertilization with Ca 2+ appears to be a promising strategy for increasing phytoremediation capacity. While La 3+ could decrease the accumulation of Cd, La 3+ may play an important role on reducing the accumulation of Cd of plants grown in contaminated soil.

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.