Changes in glutathione and phytochelatins in roots of maize seedlings exposed to cadmium

The effects of excess Cd on the contents of free cysteine, total glutathione and phytochelatin (PC) were measured in roots of intact maize seedlings. Exposure to 3 /tmM Cd for 15 min caused PCs to appe substrates for formation of longer PCs. Total glutathione levels declined with PC synthesis, free cysteine contents changed little. The reactions to excess Cd differed along the length of roots. In the 1 cm apical region a high production of PCs occurred with a moderate loss of total glutathione. In the mature region, PC content was 2.5-fold less than in apices, several unidentified thiols accumulated, and total glutathione levels declined drastically. Exposure to 0.05 μM Cd for 24 h induced PCs, the contents rose as Cd concentrations were increased. The roots produced PCs in excess of that required to chelate the Cd present, as if some PCs were compartmentalized or had not yet formed Cd-PC complexes. Phytochelatin formation was stimulated most effectively by Cd, less by Zn and Cu and negligibly by Ni. Total glutathione declined with Cd and Zn exposure, however, with excess Cu the roots contained 45% more total glutathione than did the controls.     

Effects of Metal Combinations on the Production of Phytochelatins and Glutathione by the Marine Diatom Phaeodactylum tricornutum

Copper, Cd and Zn can be found at elevated concentrations in contaminated estuarine and coastal waters and have potential toxic effects on phytoplankton species. In this study, the effects of these metals on the intracellular production of the polypeptides phytochelatin and glutathione by the marine diatom Phaeodactylum tricornutum were examined in laboratory cultures. Single additions of Cu and Cd (0.4 μM Cu² and 0.45 μM Cd²⁺) to the culture medium induced the production of short-chained phytochelatins ((γ-Glu-Cys)_n-Gly where n = 2–5), whereas a single addition of Zn (2.2 μM Zn²⁺) did not stimulate phytochelatin production. Combination of Zn with Cu resulted in a similar phytochelatin production compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production, which was probably caused by metal competition for cellular binding sites. Glutathione concentrations were affected only upon exposure to Cd (85% increase) or the combination of Cd with Zn (65% decrease), relative to the control experiment. Ratios of phytochelatins to glutathione indicated a pronounced metal stress in response to exposures to Cu or Cd combined with Zn. This study indicates that variabilities in phytochelatin and glutathione production in the field can be explained in part by metal competition for cellular binding sites.     

Glutathione-mediated Antioxidative Mechanisms in Sunflower (<Emphasis Type="Italic">Helianthus Annuus</Emphasis> L.) Cells in Response to Cadmium Stress

Cadmium (Cd) homeostasis and detoxification in sunflower (Helianthus annuus L.) cells differing in Cd sensitivity/tolerance were studied by analyzing the glutathione-mediated antioxidant mechanism vis-à-vis phytochelatin biosynthesis in vitro. Calluses exposed to Cd-shock/-acclimatization (150μM) were assayed for oxidative stress, reduced glutathione (GSH), glutathione disulfide (GSSG), phytochelatins (PCs) and reactive oxygen species (ROS). Although Cd did not induce any oxidative stress in Cd-tolerant callus (TCd), it generated oxidative stress in Cd-shock callus (SCd) both in terms of lipid peroxidation and protein oxidation. GSH/GSSG ratio remained similar to control values in the cadmium-acclimatized calluses. However, after acute treatment, there was a decline in both GSH and GSSG levels in SCd with concomitant reduction in the GSH/GSSG ratio. Analysis of PCs was performed using HPLC and mass spectrometry methods. PC concentration in TCd were approximately twice those that in SCd, showing in both cases a 1:2:1 relative proportion for PC n = 2 (PC2): PC n = 3 (PC3): PC n = 4 (PC4). Calluses growing in the presence of Cd developed an increased resistance to paraquat oxidative stress generation. These results indicated that PCs synthesis was an important mechanism for Cd detoxification in sunflower calluses, but the capacity to grow in the presence of Cd is related to the tissues ability to maintain high intracellular levels of GSH.     

Organic Acids Accumulation and Antioxidant Enzyme Activities in Thlaspi caerulescens under Zn and Cd Stress

Growth, organic acid and phytochelatin accumulation, as well as the activity of several antioxidative enzymes, i.e. superoxide dismutase (SOD), ascorbate peroxidase (APX) guaiacol peroxidase (POX) and catalase (CAT) were investigated under Zn and Cd stress in hydroponically growing plants of Thlaspi caerulescens population from Plombières, Belgium. Tissue Zn and Cd concentration increased (the highest concentration of both was in roots) as the concentration of these metals increased in the nutrient solution. Increasing Zn concentration enhanced plant growth, while with Cd it declined compared to the control. Both metals stimulated malate accumulation in shoots, Zn also caused citrate to increase. Zn did not induce phytochelatin (PC) accumulation. In plants exposed to Cd, PC concentration increased with increasing Cd concentration, but decreased with time of exposure. Under Zn stress SOD activity increased, but APX activity was higher at 500 and 1000 μM Zn and CAT activity only at 500 μM Zn in comparison with the control. CAT activity decreased in Cd- and Zn-stressed plants. The results suggest that relative to other populations, a T. caerulescens population from Plombières, when grown in hydroponics, was characterized by low Zn and Cd uptake and their translocation to shoots and tolerance to both metals. The accumulation of malate and citrate, but not PC accumulation was responsible for Zn tolerance. Cd tolerance seems to be due to neither PC production nor accumulation of organic acids.     

Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding gamma-glutamylcysteine synthetase (gamma-ECS), targeted to the plastids. The gamma-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, gamma-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, gamma-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the gamma-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of gamma-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of gamma-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.     

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     

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。     

植物对重金属镉的耐受机制

镉离子（Cd^2＋）具有强植物毒性,抑制植物生长,甚至使植物死亡。由于长期的环境选择和适应进化,植物发展出耐受机制,可减轻或避免Cd^2＋的毒害。硫转运蛋白、硫还原相关酶类以及半胱氨酸、谷胱甘肽和植物螯合肽合成基因的表达受Cd^2＋调控。同时这些基因的过表达也能提高植物对Cd^2＋的耐性。植物抗氧化系统对Cd^2＋胁迫诱发的活性氧的清除作用,具转运Cd^2＋活性的质膜转运蛋白促进Cd^2＋经共质体途径向木质部运输、装载,而后随蒸腾流向地上部迁移,具转运Cd^2＋活性的液泡膜转运蛋白促进Cd^2＋进入液泡的隔离作用,都在植物对Cd^2＋的耐性中起作用。     

Physiological Responses and Tolerance Mechanisms to Cadmium in Conyza canadensis

Experiments were conducted to examine the effects of different concentrations of Cd on the performance of the Cd accumulator Conyza canadensis. Cd accumulation in roots and leaves (roots > leaves) increased with increasing Cd concentration in soil. High Cd concentration inhibited plant growth, increased the membrane permeability of leaves, and caused a significant decline in plant height and chlorophyll [chlorophyll (Chl) a, Chl b, and total Chl] content. Leaf ultrastructural analysis of spongy mesophyllic cells revealed that excessive Cd concentrations cause adverse effects on the chloroplast and mitochondrion ultrastructures of C. canadensis. However, the activities of antioxidant enzymes, such as superoxide dismutase, catalase, peroxidase, total non-protein SH compounds, glutathione, and phytochelatin (PC) concentrations, showed an overall increase. Specifically, the increase in enzyme activities demonstrated that the antioxidant system may play an important role in eliminating or alleviating the toxicity of Cd in C. canadensis. Furthermore, results demonstrate that PC synthesis in plant cells is related to Cd concentration and that PC production levels in plants are related to the toxic effects caused by soil Cd level. These findings demonstrate the roles played by these compounds in supporting Cd tolerance in C. canadensis.     

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

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

The protective role of selenium in rape seedlings subjected to cadmium stress

The effect of selenium (Se) on rape (Brassica napus) seedlings subjected to cadmium (Cd) stress was studied in vitro by investigating plant growth and changes in fatty acid composition, activity of antioxidative enzymes and DNA methylation pattern. Physiological experiments were carried out on seedlings cultured for 2 weeks on Murashige-Scoog (MS) media with Cd concentrations of 0, 400 and 600 μM, and on corresponding media supplied with Se (2 μM). Exposure to increasing Cd concentrations reduced the fresh weight of the upper part (hypocotyls+cotyledons) of the seedlings more strongly than that of the root system, which was accompanied by higher Cd accumulation in these tissues. In the upper part, Cd exposure led to significant changes in the biochemical parameters: fatty acid unsaturation of plasmalemma decreased, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPOX) diminished and that of ascorbate peroxidase (APX) increased. In contrast, the roots showed an increase in fatty acid unsaturation and in the activity of antioxidative enzymes. In both parts of rape seedlings H₂O₂ level and lipid peroxidation increased. Se addition to medium considerably reversed the Cd-induced decrease in fresh mass as well as the changes in lipid unsaturation and peroxidation. Se applied separately or in combination with Cd did not significantly affect the activity of antioxidative enzymes in the roots, but diminished it in the upper part. Moreover, the presence of Se in medium prevented changes in the DNA methylation pattern triggered in rape seedlings by high Cd concentrations. Two possible mechanisms for the action of Se were considered: (1) removal of Cd from metabolically active cellular sites, and (2) reduction of oxygen radicals.     

A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity

Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.     

Response to copper and cadmium stress in wild-type and copper tolerant strains of the lichen alga <Emphasis Type="Italic">Trebouxia erici</Emphasis>: metal accumulation,toxicity and non-protein thiols

Cysteine, glutathione (GSH) and phytochelatins were determined in the cells of both wild and copper tolerant strains of the lichen alga Trebouxia erici following short-term (24 h) exposure to copper and cadmium and long-term (4 weeks) exposure to copper. Both metals caused concentration dependent synthesis of phytochelatins (PC₂–PC₅), but cadmium was a more potent activator of phytochelatin synthesis, even inducing synthesis of PC₅. The copper-tolerant strain did not reveal a higher degree of phytochelatin synthesis than the wild strain, and at 5 μM Cu production of phytochelatins was in fact significantly lower. Lower levels of phytochelatin correlated with significantly decreased intracellular copper content in the copper-tolerant strain. Both strains maintained high GSH levels even at a high copper concentration of 5 μM, and only the highest copper concentration (10 μM) was toxic for both strains, causing a decrease of GSH and PC content in algal cells. Cadmium had less effect on GSH in the cells of both tested strains. In the long term experiments, only relatively small amounts of PC₂ were detected in both strains, but the copper-tolerant strain retained significantly higher levels of reduced glutathione, probably due to the lesser degree of oxidative stress caused by Cu. The significant increase of cysteine synthesis in the copper-tolerant strain found in the present study may be related to copper tolerance in T. erici, while decreased intracellular Cu uptake, detoxification by PCs and increased free proline levels for protection of chloroplast membranes may also be implicated.     

铜、镉胁迫下外源NO介导的番茄解毒途径

一氧化氮(NO)作为信号分子,在抵御重金属胁迫中起重要作用,但对不同离子胁迫下的解毒机制尚缺乏研究.本研究采用营养液培养法,研究了铜(Cu)、镉(Cd)单一或复合胁迫下,番茄幼苗对Cu、Cd的吸收转运特性及对外源NO的响应机制.结果表明: 50 μmol·L^-1的Cu²⁺、Cd²⁺均显著抑制番茄植株的生长,其中Cd胁迫对生长的抑制效应远高于Cu胁迫.Cu、Cd单一或复合胁迫均使番茄根系Cu、Cd含量显著升高,但根系对Cu、Cd吸收存在严格选择性.根细胞对必需元素Cu表现出“奢侈吸收”的现象,而对毒性较强的Cd则吸收相对较少,胞内Cd浓度仅为Cu的1/10左右.外源NO处理可不同程度地缓解Cu、Cd胁迫,其中缓解Cd胁迫的效能更强.番茄对被动进入细胞的Cu、Cd具有相似的解毒机制:一方面,Cu、Cd胁迫诱导细胞质中产生谷胱甘肽(GSH)、植物螯合肽(PCs)和金属硫蛋白(MTs),络合过多的Cu、Cd离子,降低其生物毒性;另一方面,过多的Cu、Cd离子或螯合物被转运至液泡区隔化.外源NO通过调控GSH-GSSG(氧化型谷胱甘肽)氧化还原状态及GSH-PCs代谢方向的改变,促进Cu、Cd离子转运至液泡区隔化来缓解胁迫抑制;NO还可诱导植株叶片或根系表达更多的金属硫蛋白、GSH和PCs,而且上述响应普遍存在叠加效应.这可能是NO介导番茄对Cu、Cd胁迫的另一主要解毒途径.     

Selenium alleviates cadmium toxicity by preventing oxidative stress in sunflower (Helianthus annuus) seedlings

The present study investigated the possible mediatory role of selenium (Se) in protecting plants from cadmium (Cd) toxicity. The exposure of sunflower seedlings to 20 μM Cd inhibited biomass production, decreased chlorophyll and carotenoid concentrations and strongly increased accumulation of Cd in both roots and shoots. Similarly, Cd enhanced hydrogen peroxides content and lipid peroxidation as indicated by malondialdehyde accumulation. Pre-soaking seeds with Se (5, 10 and 20 μM) alleviated the negative effect of Cd on growth and led to a decrease in oxidative injuries caused by Cd. Furthermore, Se enhanced the activities of catalase, ascorbate peroxidase and glutathione reductase, but lowered that of superoxide dismutase and guaiacol peroxidase. As important antioxidants, ascorbate and glutathione contents in sunflower leaves exposed to Cd were significantly decreased by Se treatment. The data suggest that the beneficial effect of Se during an earlier growth period could be related to avoidance of cumulative damage upon exposure to Cd, thus reducing the negative consequences of oxidative stress caused by heavy metal toxicity.     

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.     

Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1

Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length.     

Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced by Overexpressing γ-Glutamylcysteine Synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding γ-glutamylcysteine synthetase (γ-ECS), targeted to the plastids. The γ-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, γ-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, γ-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the γ-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of γ-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of γ-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.