Glutathione and phytochelatin contents in tomato plants exposed to cadmium

The effect of cadmium on growth and contents of glutathione (GSH) and phytochelatins (PCs) were investigated in roots and leaves of tomato plants (Lycopersicon esculentum Mill. cv. 63/5 F1). The accumulation of Cd increased with external Cd concentrations and was considerably higher in roots than in leaves. Dry mass production decreased under Cd treatment especially in leaves. In both roots and leaves, exposure to Cd caused an appreciable decline in GSH contents and increase in PCs synthesis proportional to Cd concentrations in the growth medium. At the same Cd concentration, PCs production was higher in roots than in leaves. The implication of glutathione in PC synthesis was strongly suggested by the use of buthionine sulfoximine (BSO). The major fraction of Cd accumulated by tomato roots was in the form of a Cd-PCs complex.     

Effect of spermine on the phytochelatin concentration and composition in cadmium-treated roots ofCanavalia lineata seedlings

Effect of spermine on the phytochelatins (PCs) in cadmium-treated roots ofCanavalia lineata seedlings was studied. With the treatment of spermine, total nonprotein thiol (SH) contents decreased by 55% in roots of Cd-treated plants. Glutathione (GSH) synthetase activity was inhibited by 36.8% in roots and cysteine synthase was also inhibited by 9.5% while y-GluCys synthetase activity was not affected. From the PC-Cd complex analyses by gel column chromatography, it was found that Cd+spermine-treated roots contain an additional PC that has low affinity for Cd, in addition to Cd-induced PC whose SH:Cd ratio is 1:1. Spermine affected the PC concentration and composition in the Cd-treated roots ofC. lineata seedlings.     

Changes in phytochelatins and their biosynthetic intermediates in red spruce (<Emphasis Type="Italic">Picea rubens </Emphasis>Sarg.) cell suspension cultures under cadmium and zinc stress

Cell suspension cultures of red spruce (Picea rubens Sarg.) were selected to study the effects of cadmium (Cd) and zinc (Zn) on phytochelatins (PCs) and related metabolites after 24 h exposure. The PC₂ and its precursor, γ-glutamylcysteine (γ-EC) increased two to fourfold with Cd concentrations ranging from 12.5 to 200 μM as compared to the control. However, Zn-treated cells showed a less than twofold increase in γ-EC and PC₂ levels as compared to the control even at the highest concentration of 800 μM. In addition, unidentified higher chain PCs were also found in both the Cd and Zn treated cells and they increased significantly with increasing concentrations of Cd and Zn. The cellular ratio of PC₂ : Cd or Zn content clearly indicated that Cd (with ratios ranging from 0.131 to 0.546) is a more effective inducer of PC₂ synthesis/accumulation than Zn (with ratios ranging from 0.032 to 0.102) in red spruce cells. A marginal decrease in glutathione (GSH) was observed in both Cd and Zn treated cells. However, the GSH precursor, cysteine, declined twofold with all Cd concentrations while the decrease with Zn was 1.5–2-fold only at the higher treatment concentrations of Zn as compared to control. In addition, changes in other free amino acids, polyamines, and inorganic ions were also studied. These results suggest that PCs and their biosynthetic intermediates play a significant role in red spruce cells protecting against Cd and Zn toxicity.     

Separation and Determination of Heavy Metals Associated with Low Molecular Weight Chelators in Xylem Saps of Indian Mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) by Size Exclusion Chromatography and Atomic Absorption Spectrometry

To elucidate the role of low molecular weight chelators in long-distance root-to-shoot transport of heavy metals in Indian mustard, an “off-line” size exclusion high-performance liquid chromatography–graphite furnace atomic absorption spectrometry was developed to investigate heavy metals associated with low molecular weight chelators in xylem saps of Indian mustard (Brassica juncea). The size exclusion chromatogram presented only the peaks with molecular weight for all xylem saps and directly indicated the long-distance transport of phytochelatins (PCs) of Indian mustard under Cd stress. In the absence of Cd stress, only organic acids and inorganic anions participated in the long-distance transport of Cd, but organic acids, inorganic anions, glutathione (GSH), and cysteine might relate to the long-distance transport of Cu or Zn. In the presence of Cd stress, PCs were induced, and Cd ions in xylem saps were associated with the induced PCs. As the Cd levels in nutrient solution increased, more Cd in xylem saps adopted the form of PC–Cd. Although PCs might participate in the long-distance transport of Cd under Cd stress, the majority of Cd was still transported by organic acids and inorganic anions in xylem vessels. Moreover, results indicated the existence of complexation competition for GSH and cysteine between Cd and Cu (or Zn) and complexation competition for Cd between PCs and GSH (or cysteine) in xylem vessels. Our work might be very useful for understanding the mechanism of long-distance transport of heavy metals in hyperaccumulator.     

Enhancement of tolerance to heavy metals and oxidative stress in Dunaliella tertiolecta by Zn-induced phytochelatin synthesis

The synthesis of phytochelatins (PCs) in a marine alga, Dunalliela tertiolecta, is strongly induced by Zn. Pretreatment of the cells with Zn enhances the tolerance toward toxic heavy metals such as Cd, Hg, Cu, Pb, and arsenate. Moreover, the pretreatment also increases the tolerance toward oxidative stress caused by hydrogen peroxide or paraquat. In vitro analysis shows that PC is a stronger scavenger of hydrogen peroxide and superoxide radical than glutathione. These results suggest that PCs inducibly synthesized by Zn treatment could play a role not only in detoxification of heavy metals but also in mitigation of oxidative stress.     

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 arietinum L.). The seedlings were grown for 5 days and the roots were treated with 0–20 μM CdSO₄ 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. Electronic Publication     

Oxidative stress and phytochelatin characterisation in bread wheat exposed to cadmium excess.

In this work, we first investigated if the bread wheat (Triticum aestivum L.) cv. Albimonte can be defined as "shoot cadmium excluder"--by comparing the cadmium (Cd) content in leaves and roots and by calculating the shoot-to-root Cd concentration ratio. Furthermore, we evaluated if the exposure to Cd excess could generate oxidative stress in leaves and roots of this cv., in terms of hydrogen peroxide (H(2)O(2)) accumulation, NAD(P)H oxidation rate, and variations in reduced glutathione (GSH) content and peroxidase (POD, EC 1.11.1.7) activity. Finally, we surveyed possible quali- quantitative differences in thiol-peptide compound pattern between roots and leaves, in order to verify whether phytochelatins (PCs) and related thiol-peptides could contribute in limiting the Cd-induced oxidative stress. Unambiguous characterisation of PCs and related forms present in the root samples was obtained by electrospray ionisation mass spectrometry (ESI-MS) and ESI-tandem MS (ESI-MS/MS). Our results indicate that in leaves the stress generated by the low accumulation of Cd (due to a moderate translocation in planta) seems to be counteracted by the antioxidant response and by the PC biosynthesis. On the contrary, in roots, in spite of the elevated presence of PCs and related thiol-peptide-compounds, the excess of Cd causes a decline in the antioxidant protection of the organ, with the consequent generation of considerable amounts of H(2)O(2), a direct agent of oxidative stress.     

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.     

Accumulation, detoxification, and genotoxicity of heavy metals in Indian mustard (Brassica juncea L.)

Plants of Indian mustard (Brassica juncea L.) were exposed to different concentrations (15, 30, 60, 120 microM) of (Cd, Cr, Cu, Pb) for 28 and 56 d for accumulation and detoxification studies. Metal accumulation in roots and shoots were analyzed and it was observed that roots accumulated a significant amount of Cd (1980 microg g(-1) dry weight), Cr (1540 microg g(-1) dry weight), Cu (1995 microg g(-1) dry weight), and Pb (2040 microg g(-1) dry weight) after 56 d of exposure, though in shoot this was 1110, 618, 795, and 409 microg g(-1) dry weight of Cd, Cr, Cu, and Pb, respectively. In order to assess detoxification mechanisms, non-protein thiols (NP-SH), glutathione (GSH) and phytochelatins (PCs) were analyzed in plants. An increase in the quantity of NP-SH (9.55), GSH (8.30), and PCs (1.25) micromol g(-1) FW were found at 15 microM of Cd, however, a gradual decline in quantity was observed from 15 microM of Cd onwards, after 56 d of exposure. For genotoxicity in plants, cytogenetic end-points such as mitotic index (MI), micronucleus formation (MN), mitotic aberrations (MA) and chromosome aberrations (CA) were examined in root meristem cells of B. juncea. Exposure of Cd revealed a significant (P < 0.05) inhibition of MI, induction of MA, CA, and MN in the root tips for 24 h. However, cells examined at 24 h post-exposure showed concentration-wise recovery in all the endpoints. The data revealed that Indian mustard could be used as a potential accumulator of Cd, Cr, Cu, and Pb due to a good tolerance mechanisms provided by combined/concerted action of NP-SH, GSH, and PCs. Also, exposure of Cd can cause genotoxic effects in B. juncea L. through chromosomal mutations, MA, and MN formation.     

Thiols in cadmium- and copper-treated maize (Zea mays L.)

Maize plants (Zea mays L. cv. Honeycomb F-1) were grown on quartz sand containing amounts of Cd or Cu which resulted in comparable internal contents in the roots. Fresh and dry weights and the content of Cd or Cu were measured in roots and shoots after eight weeks. In addition, cysteine, γ-glutamylcysteine (γEC), glutathione (GSH) and the thiols in heavy-metal-binding peptides (HMBPs) were determined in the roots. At low internal contents, Cd and Cu inhibited root growth to the same extent. Inhibition by Cu was enhanced, however, at high internal contents, indicating that Cu was more toxic than Cd. Separation of extracts from roots of Cd- and Cutreated plants on a Sephadex G-50 column resulted in HMBP complexes with relative molecular masses (M_rs) of 6200 and 7300, respectively. Separation of these HMBP-complexes using HPLC resulted in a distinct pattern of thiol compounds for each heavy metal. The accumulation of HMBPs was linearly dependent on the content of Cd at all values examined. In Cu-treated roots, HMBP accumulation was linearly dependent on the internal Cu content only up to 7.1 μmol·g^?1 dry weight. At internal contents which caused an enhanced inhibition of root growth, no further significant increase in the HMBP content was detected. At these internal Cu contents an increased transport of Cu to the shoot was measured. This result indicates that HMBPs are involved in reducing heavy-metal transport from roots to shoots.     

Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii

Sedum alfredii has been reported to be a cadmium (Cd) hyperaccumulator. Phytochelatins (PCs) and other thiol (SH)-containing compounds have been proposed to play an important role in the detoxification and tolerance of some heavy metals, but it is not clear whether PCs are responsible for Cd hyperaccumulation and tolerance in S. alfredii. In this study, two geographically isolated populations of S. alfredii were studied: one population grew on an old Pb/Zn mine site, while the other on a non-mine site. The mine population of this species exhibited a stronger heavy metal tolerance than in the other population. Root-to-shoot transport of Cd was higher in population located at the mine site than at the non-mine site. Considerable amounts of Cd were accumulated in leaves and stems of mine plants, while most Cd was distributed in roots of non-mine plants. Non-protein SH in plant tissues of two populations were further investigated by a HPLC pre-column derivatization system. Upon exposure to Cd, no PCs were detected in all tissues of mine population, while an appreciable amount of glutathione (GSH) was observed in the descending order of stem>root>leaf. The concentrations of GSH consistently increased with the increase of exogenous Cd concentrations and time. On the contrary, Cd exposure strongly induced the production of PCs (mainly PC(2) and PC(3)) and GSH in plant tissues of non-mine population, and the concentrations of GSH showed an initial drop over the duration of 7-d exposure. The present results provided strong evidence that PCs are not involved in Cd transport, hyperaccumulation and tolerance in mine population of S. alfredii.     

PRODUCTION OF PHYTOCHELATINS AND GLUTATHIONE BY MARINE PHYTOPLANKTON IN RESPONSE TO METAL STRESS1

Phytoplankton deal with metal toxicity using a variety of biochemical strategies. One of the strategies involves glutathione (GSH) and phytochelatins (PCs), which are metal‐binding thiol peptides produced by eukaryotes and these compounds have been related to several intracellular functions, including metal detoxification, homeostasis, metal resistance and protection against oxidative stress. This paper assesses our state of knowledge on the production of PCs and GSH by marine phytoplankton in laboratory and field conditions and the possible applications of PCs for environmental purposes. Good relationships have been observed between metal exposure and PC production in phytoplankton in the laboratory with Cd, Pb, and Zn showing the greatest efficacy, thereby indicating that PCs have a potential for application as a biomarker. Fewer studies on PC distributions in particulate material have been undertaken in the field. These studies show that free Cu has a strong relationship with the levels of PC in the particulate material. The reason for this could be because Cu is a common contaminant in coastal waters. However it could also be due to the lack of measurements of other metals and their speciation. GSH shows a more complex relationship to metal levels both in the laboratory and in the field. This is most likely due to its multifunctionality. However, there is evidence that phytoplankton act as an important source of dissolved GSH in marine waters, which may form part of the strong organic ligands that control metal speciation, and hence metal toxicity.     

Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis

Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.     

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.     

Phytochelatin synthesis plays a similar role in shoots of the cadmium hyperaccumulator Sedum alfredii as in non‐resistant plants

Phytochelatin (PC) synthesis is considered necessary for Cd tolerance in non‐resistant plants, but roles for PCs in hyper‐accumulating species are currently unknown. In the present study, the relationship between PC synthesis and Cd accumulation was investigated in the Cd hyperaccumulator Sedum alfredii Hance. PCs were most abundant in leaves followed by stems, but hardly detected by the reversed‐phase high‐performance liquid chromatography (HPLC) in roots. Both PC synthesis and Cd accumulation were time‐dependent and a linear correlation between the two was established with about 1:15 PCs : Cd stoichiometry in leaves. PCs were found in the elution fractions, which were responsible for Cd peaks in the anion exchange chromatograph assay. About 5% of the total Cd was detected in these elution fractions as PCs were found. Most Cd was observed in the cell wall and intercellular space of leaf vascular cells. These results suggest that PCs do not detoxify Cd in roots of S. alfredii. However, like in non‐resistant plants, PCs might act as the major intracellular Cd detoxification mechanism in shoots of S. alfredii.     

Cadmium stress tolerance in crop plants: Probing the role of sulfur

Plants can''t move away and are therefore continuously confronted with unfavorable environmental conditions (such as soil salinity, drought, heat, cold, flooding and heavy metal contamination). Among heavy metals, cadmium (Cd) is a non-essential and toxic metal, rapidly taken up by roots and accumulated in various plant tissues which hamper the crop growth and productivity worldwide. Plants employ various strategies to counteract the inhibitory effect of Cd, among which nutrient management is one of a possible way to overcome Cd toxicity. Sulfur (S) uptake and assimilation are crucial for determining crop yield and resistance to Cd stress. Cd affects S assimilation pathway which leads to the activation of pathway responsible for the synthesis of cysteine (Cys), a precursor of glutathione (GSH) biosynthesis. GSH, a non-protein thiol acts as an important antioxidant in mitigating Cd-induced oxidative stress. It also plays an important role in phytochelatins (PCs) synthesis, which has a proven role in Cd detoxification. Therefore, S assimilation is considered a crucial step for plant survival under Cd stress. The aim of this review is to discuss the regulatory mechanism of S uptake and assimilation, GSH and PC synthesis for Cd stress tolerance in crop plants.Key words: cadmium, cysteine, glutathione, phytochelatins, stress tolerance, sulfur     

Induction of non-protein thiols and phytochelatins by cadmium in Eichhornia crassipes

Abstract

Impact of root Cd concentration on production of cysteine, non-protein thiols (NP-SH), glutathione (GSH), reduced glutathione (GSSG), and phytochelatins (PCs) in Eichhornia crassipes exposed to different dilutions of brass and electroplating industry effluent (25%, 50%, and 75%), and synthetic metal solutions of Cd alone (1, 2.5, and 3.5?ppm) and with Cr (1?ppm Cd + 1?ppm Cr, 2.5?ppm Cd + 3?ppm Cr, and 3.5?ppm Cd + 4?ppm Cr) was assessed in a 45?days study. Different treatments were used to understand and compare differential antioxidant defense response of plant under practical drainage (effluent) and experimental synthetic solutions. The production of NP-SH and cysteine was maximum under 2.5?ppm Cd + 3?ppm Cr treatments i.e., 1.78?µmol/g fw and 288?nmol/g fw, respectively. The content of GSH declined whereas that of GSSG increased progressively with exposure duration in all treatments. HPLC chromatograms revealed that the concentrations of PC2, PC3, and PC4 (248, 250, and 288?nmol-SH equiv.g^?1 fw, respectively) were maximum under 1?ppm Cd, 1?ppm Cd + 1?ppm Cr, and 2.5?ppm Cd + 3?ppm Cr treatments, respectively. PC2, PC3, and PC4 concentrations increased with Cd accumulation in the range 812–1354?µg/g dry wt, 1354–2032?µg/g dry wt and 2032–3200?µg/g dry wt, respectively. Thus, the study establishes a direct proportionality relationship between concentration/length of phytochelatins and root Cd concentrations, upto threshold limits, in E. crassipes.     

Regulation of Glutathione Synthesis by Cadmium in Pisum sativum L

In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCl₂ concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5′-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cd-binding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5′-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis.