Responses of non-protein thiols to Cd exposure in Cd hyperaccumulator Arabis paniculata Franch

We investigated the responses of phytochelatins (PCs), glutathione (GSH) and other non-protein thiols in Cd hyperaccumulator Arabis paniculata after Cd exposure. Applying γ-glutamylcysteine synthetase (γ-ECS) inhibitor, l-buthionine-sulfoximine (BSO), the roles of PCs in Cd tolerance and Cd accumulation in A. paniculata were evaluated. Plants were exposed to four Cd concentrations (0, 50, 100 and 250 μM) for different times (2w or 3w) with and without BSO. Overall, Cd exposure had little impact on plant biomass after 2w or 3w of growth except at the highest Cd level. A. paniculata tolerated ≤100 μM Cd with up to 1127 mg kg^?1 Cd in the shoots and 5624 mg kg^?1 Cd in the roots after 3w of Cd exposure. Cd exposure induced formation of PCs and three unknown thiols in the roots, but none were detected in the shoots. BSO had no significant effect on Cd sensitivity in plants though it reduced Cd accumulation in the roots. In addition, the molar ratio of PCs:Cd, which ranged from 0.7 to 1.3 after exposing to 50–100 μM Cd without BSO in the roots, was close to the value expected for PC-mediated Cd sequestration in plants. Those data indicate that GSH and PCs did not contribute to Cd tolerance in the shoots and Cd transport from the root to shoot in A. paniculata, but they may play an important role in Cd accumulation and Cd complexation in the roots of A. paniculata.     

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.     

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.     

Modulation of Exogenous Glutathione in Ultrastructure and Photosynthetic Performance Against Cd Stress in the Two Barley Genotypes Differing in Cd Tolerance

Greenhouse hydroponic experiments were conducted using Cd-sensitive (Dong 17) and tolerant (Weisuobuzhi) barley genotypes to evaluate genotypic differences in response of photosynthesis and ultrastructure to Cd toxicity in the presence of exogenous glutathione (GSH). Addition of 20 mg L(-1) GSH in 5 μM Cd culture medium (Cd?+?GSH) significantly alleviated Cd-induced growth inhibition and reduced Cd concentration in leaves and roots especially in the sensitive genotype Dong 17. Exogenous GSH greatly ameliorated Cd-induced damages on leaf/root ultrastructure, e.g., compared with Cd alone treatment, chloroplasts in plants treated with Cd?+?GSH become better or in relatively normal shape with well-structured thylakoid membranes and parallel pattern of lamellae and unfolded more starch grains but less osmiophilic plastoglobuli; nuclei of root cells were better formed and chromatin distributed more uniformly in both genotypes, and number of plastids and mitochondria cristae in Dong 17 resumed to control level. The examination of photosynthetic performance revealed GSH dramatically increased net photosynthetic rate (P(n)), stomatal conductance (G(s)), and transpiration rate (T(r)) in the both genotypes and strongly stimulated Cd-induced decrease in the maximal photochemical efficiency (F(v)/F(m)) especially in the sensitive genotype.     

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.     

Characteristics of cadmium accumulation and tolerance in <Emphasis Type="Italic">Rorippa globosa</Emphasis> (Turcz.) Thell., a species with some characteristics of cadmium hyperaccumulation

Characteristics of cadmium (Cd) accumulation and tolerance in Rorippa globosa (Turcz.) Thell., a species with some characteristics of cadmium hyperaccumulation were further investigated and compared with a closely related species, Rorippa islandica. The results showed that there was no phytotoxicity for R. globosa leaves or reduction in biomass when treated with 25 μg Cd g⁻¹, although the concentration of Cd accumulated in the leaves was up to 218.9 μg Cd g⁻¹ dry weight (DW). On the contrary, Cd toxicity was observed in R. islandica leaves by way of determining changes in fresh weight (FW), malondialdehyde (MDA) level and chlorophyll content while treated with 25 μg Cd g⁻¹ DW. R. globosa had stronger self-protection ability than R. islandica to adapt to oxidative stress caused by Cd. Application of Cd significantly increased the activity of superoxide dismutase (SOD) in leaves, the activity of peroxidase (POD) in roots, and the activity of catalase (CAT) in leaves and roots of R. globosa. By contrast, in R. islandica, the activity of antioxidant enzymes was inhibited or unchanged by various Cd treatments. However, R. globosa leaves had higher activity of antioxidant enzymes such as SOD and POD than that of R. islandica. The antioxidative defense systems in R. globosa might play an important role in Cd tolerance. The Cd treatments significantly induced the synthesis of phytochelatins (PCs) in the two species. Leaf PCs and Cd accumulation by R. globosa were much greater than those by R. islandica, but root PCs and Cd accumulation by R. islandica were much greater than those by R. globosa, suggesting that PCs in leaves may be a biomarker of Cd hyperaccumulation, and the synthesis of PCs may be related to an increase in the uptake of Cd ions into the cytoplasm, not the primary mechanism for Cd tolerance.     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.     

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-induced oxidative damage and antioxidative defense mechanisms in <Emphasis Type="Italic">Vigna mungo</Emphasis> L.

Cadmium (Cd)-induced oxidative stress and antioxidant defense mechanisms were analyzed in roots and leaves of Vigna mungo L. Seeds were germinated in perlite-vermiculite and irrigated with Hoagland nutrient solution. At day 6, seedlings were exposed to 40 μM Cd under semi-hydroponic conditions for a period of 12 days. Growth anomalies and abnormal chromatin condensation were observed in Cd-treated plants, in comparison with control ones. Cd accumulation was observed in roots of treated plants. The analyses of antioxidative defense and oxidative parameters in roots, stems and leaves showed different tissue-specific responses. Superoxide dismutase (SOD) and guaiacol peroxidase (GPx) activities and the level of lipid peroxidation (MDA content) decreased in roots. However, they increased in leaves. Catalase activity and chlorophyll content, on the other hand, decreased over exposure to Cd stress. Total glutathione, non-protein thiols, reduced glutathione (GSH) and phytochelatins increased significantly, while oxidized glutathione (GSSG) decreased, as compared with control plants. The present data suggest that the presence of Cd in soil and water can cause oxidative damage that may be detrimental for optimum production of nutritional mung.     

Effects of Interactions Between Cadmium and Lead on Growth, Nitrogen Fixation, Phytochelatin, and Glutathione Production in Mycorrhizal Cajanus cajan (L.) Millsp.

Heavy metals (HM) are a unique class of toxicants because they cannot be broken up into nontoxic forms. Excess HM causes stunted growth, upsets mineral nutrition, and affects membrane structure and permeability. High tolerance to HM toxicity is based on reduced metal uptake or increased internal sequestration in a genotype. Arbuscular mycorrhizal (AM) fungi are important rhizospheric microorganisms that occur in metal-contaminated soils and perhaps detoxify the potential effects of metals. The aim of this work was to study the role of the AM fungus Glomus mosseae in the alleviation of cadmium (Cd) and lead (Pb) toxicities in Cajanus cajan (L.) Millsp. (pigeonpea) genotypes. The effects of interactions between Cd (25 and 50 mg/kg) and Pb (500 and 800 mg/kg) on plant dry mass, nitrogen metabolism, and production of phytochelatins (PCs) and glutathione (GSH) were monitored with and without AM fungus in genotypes Sel-85N (relatively tolerant) and Sel-141-97 (sensitive). Cd treatments were more toxic than Pb, and their combinations led to synergistic inhibitions to growth and nitrogen-fixing potential (acetylene reduction activity [ARA]) in both genotypes. However, the effects were less deleterious in Sel-85N than in Sel-141-97. Exposure to Cd and Pb significantly increased the levels of PCs in a concentration- and genotype-dependent manner, which could be directly correlated with the intensity of mycorrhizal infection (MI). Stimulation of GSH production was observed under Cd treatments, although no obvious effects on GSH levels were observed under Pb treatments. The metal contents (Cd, Pb) were higher in roots and nodules when compared with that in shoots, which was significantly reduced in the presence of AM fungi. The results indicated that PCs and GSH might function as potential biomarkers for metal toxicity, and microbial inoculations showed bioremediation potential by helping pigeonpea plants to grow in multimetal contaminated soils.     

Glutathione in adaptation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> to cadmium stress

The role of glutathione (GSH) in the adaptation of wild type Arabidopsis thaliana plants to Cd stress was investigated. The nutrient solution (control or containing 50 or 100 μM Cd) was supplemented with buthionine sulfoximine (BSO; 50, 100, 500 μM, to decrease the GSH content in plants) or GSH (50, 100, 500 μM, to increase its content in plants) in order to find how GSH content could regulate Cd stress responses. BSO application did not influence plant biomass, while exogenous GSH (especially 500 μM) reduced root biomass. BSO (500μM) in combination with Cd (100 μM) increased Cd toxicity on root growth (by over 50 %), most probably due to reduced GSH content and phytochelatin (PC) accumulation (by over 96 %). On the other hand, combination of exogenous GSH (500 μM) with Cd (100 μM) was also more toxic to plants than Cd alone despite a significant increase in GSH and PC accumulation (up to 2.7 fold in the roots). This fact could indicate that the natural content of endogenous GSH in wild type A. thaliana plants is sufficient for Cd-tolerance. A decrease in this GSH content led to decreased Cd-tolerance of the plants but an increase in GSH content did not enhance Cd-tolerance, and it showed even toxic effect on the plants.     

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.     

Stress Responses of Zea mays to Cadmium and Mercury

A hydroponic experiment was carried out to characterize the oxidative stress responses of maize seedlings (Zea mays L. cv. Dekalb DK604) to cadmium (Cd) and mercury (Hg). Plants were grown hydroponically for 7 days in a nutrient solution supplemented with several concentrations of Cd and Hg: 0.0 (control), 6 or 30 μM. Growth was inhibited by both metals. The effect was more severe in plants exposed to Hg. Oxidative stress was caused by the exposure to the metals, as quantified by malondialdehyde and carbonyl accumulation, by-products of lipid peroxidation and protein oxidation, respectively. The activity of ascorbate peroxidase (APX) and superoxide dismutase (SOD), enzymes involved in the scavenging of reactive oxygen species, were measured upon metal treatment. We found an activation of a cytosolic APX isoform, as identified by using a specific polyclonal antiserum. However, there were negligible changes in SOD activity. Analysis of thiol-peptides revealed that at 6 μM Cd a remarkable increase in root reduced glutathione (GSH) content occurred, and little effect on the relative content of oxidised glutathione (GSSG) was observed. However, at 30 μM Cd and in plants exposed to 6 and 30 μM of Hg, GSH root content either remained stable or decreased significantly, while the proportion of GSSG increased. Moreover, only Cd was able to induce accumulation of phytochelatins at both assayed concentrations. Apparently, Hg was more toxic than Cd, as inferred from the magnitude of the changes found in the physiological parameters tested.     

Contributions of apoplasmic cadmium accumulation,antioxidative enzymes and induction of phytochelatins in cadmium tolerance of the cadmium-accumulating cultivar of black oat (<Emphasis Type="Italic">Avena strigosa</Emphasis> Schreb.)

The contributions of cadmium (Cd) accumulation in cell walls, antioxidative enzymes and induction of phytochelatins (PCs) to Cd tolerance were investigated in two distinctive genotypes of black oat (Avena strigosa Schreb.). One cultivar of black oat ‘New oat’ accumulated Cd in the leaves at the highest concentration compared to another black oat cultivar ‘Soil saver’ and other major graminaceous crops. The shoot:root Cd ratio also demonstrated that ‘New oat’ was the high Cd-accumulating cultivar, whereas ‘Soil saver’ was the low Cd-accumulating cultivar. Varied levels of Cd exposure demonstrated the strong Cd tolerance of ‘New oat’. By contrast, low Cd-accumulating cultivar ‘Soil saver’ suffered Cd toxicity such as growth defects and increased lipid peroxidation, even though it accumulated less Cd in shoots than ‘New oat’. Higher activities of ascorbate peroxidase (EC 1.11.1.11) and superoxide dismutase (EC 1. 15. 1. 1) were observed in the leaves of ‘New oat’ than in ‘Soil saver’. No advantage of ‘New oat’ in PCs induction was observed in comparison to Cd-sensitive cultivar ‘Soil saver’, although Cd exposure increased the concentration of total PCs in both cultivars. Higher and increased Cd accumulation in cell wall fraction was observed in shoots of ‘New oat’. On the other hand, in ‘Soil saver’, apoplasmic Cd accumulation showed saturation under higher Cd exposure. Overall, the present results suggest that cell wall Cd accumulation and antioxidative activities function in the tolerance against Cd stress possibly in combination with vacuolar Cd compartmentation.     

Arsenite treatment induces oxidative stress,upregulates antioxidant system,and causes phytochelatin synthesis in rice seedlings

The effects of arsenite treatment on generation of reactive oxygen species, induction of oxidative stress, response of antioxidative system, and synthesis of phytochelatins were investigated in two indica rice (Oryza sativa L.) cvs. Malviya-36 and Pant-12 grown in sand cultures for a period of 5–20 days. Arsenite (As₂O₃; 25 and 50 μM) treatment resulted in increased formation of superoxide anion (O₂^.−), elevated levels of H₂O₂ and thiobarbituric acid reactive substances, showing enhanced lipid peroxidation. An enhanced level of ascorbate (AA) and glutathione (GSH) was observed irrespective of the variation in the level of dehydroascorbate (DHA) and oxidized glutathione (GSSG) which in turn influenced redox ratios AA/DHA and GSH/GSSG. With progressive arsenite treatment, synthesis of total acid soluble thiols and phytochelatins (PC) increased in the seedlings. Among antioxidative enzymes, the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), total ascorbate peroxidase (APX, EC 1.11.1.11), chloroplastic ascorbate peroxidase, guaiacol peroxidase (EC 1.11.1.7), monodehydroascorbate reductase (EC 1.6.5.4), and glutathione reductase (EC 1.6.4.2) increased in arsenite treated seedlings, while dehyroascorbate reductase (EC 1.8.5.1) activity declined initially during 5–10 days and increased thereafter. Results suggest that arsenite treatment causes oxidative stress in rice seedlings, increases the levels of many enzymatic and non-enzymatic antioxidants, and induces synthesis of thiols and PCs, which may serve as important components in mitigating arsenite-induced oxidative damage.     

Dexamethasone protects cultured rat hepatocytes against cadmium toxicity: involvement of cellular thiols

In the present study, the effects of dexamethasone on cadmium-induced toxicity were evaluated in isolated rat hepatocytes. Hepatocytes were cultured for 24 h in William’s E medium containing fetal calf serum (10%), insulin (0.1 IU/ml), and glucagon (0.01 μM) in the absence or presence of 0.1 μM dexamethasone. Cadmium chloride, 5 or 10 μM, was added to the medium and the toxicity was evaluated for up to 48 h after treatment. Lactate dehydrogenase (LDH) release, the reduced and oxidized glutathione ratio (GSH/GSSG), protein-SH groups, and lipid peroxidation levels were evaluated. Cadmium induced a dose- and time-dependent LDH release in control hepatocytes at 24 h (Cd 10 μM 42%) while hepatocytes pretreated with dexamethasone showed lower necrosis (Cd 10 μM 12% at 24 h). GSH/GSSH ratio and protein-SH groups were higher while lipid peroxidation was lower in dexamethasone-treated hepatocytes as compared with untreated cells. In conclusion, cadmium toxicity was associated with an increase in intracellular oxidative stress responsible for accelerated cell death. The use of dexamethasone prevented cadmium damage, suggesting that the cytoprotective action of this hormone is related to its effect in preventing changes in thiols such as glutathione and protein-SH groups.     

Thiol-peptide level and proteomic changes in response to cadmium toxicity in Oryza sativa L. roots

In the present study, rice seedlings were exposed to a range of Cd concentrations (0.1 μM, 1 μM, 10 μM, 100 μM and 1 mM) for 15 days and a combination of different molecular approaches were used to evidence Cd effects and to assess the plants’ ability to counteract metal toxicity. At a macroscopical level, only the highest Cd concentration (1 mM) caused a complete plant growth inhibition, whereas the lowest concentrations seemed to stimulate growth. At genome level, the amplified fragment length polymorphism (AFLP) technique was applied to detect DNA sequence changes in root cells, showing that all the Cd concentrations induced significant DNA polymorphisms in a dose-dependent manner. Data also evidenced the absence of preferential mutation sites.Plant responses were analysed by measuring the levels of gluthatione (GSH) and phytochelatins (PCs), the thiol-peptides involved in heavy metal tolerance mechanisms. Results showed a progressive increase of GSH up to 10 μM of Cd treatment, whereas a significant induction only of PC₃ was detected in roots of plants exposed to 100 μM of Cd. As suggested by the proteome analysis of root tissues, this last concentration strongly induced the expression of regulatory proteins and some metabolic enzymes. Furthermore, the treatment with 10 μM of Cd induced changes in metabolic enzymes, but it mainly activated defence mechanisms by the induction of transporters and proteins involved in the degradation of oxidatively modified proteins.     

Cadmium Accumulation and Tolerance in <Emphasis Type="Italic">Bradyrhizobium</Emphasis> spp. (Peanut Microsymbionts)

In this study, the effect of cadmium (Cd) on cell viability and its accumulation in Bradyrhizobium spp. (peanut microsymbionts) as well as the role of glutathione (GSH) in the tolerance to this metal were investigated. A reference strain recommended as peanut inoculant (Bradyrhizobium sp. SEMIA6144) grew up to 10 μM Cd meanwhile a GSH-deficient mutant strain (Bradyrhizobium sp. SEMIA6144-S7Z) was unable to grow at this concentration. Two native peanut isolates obtained from Córdoba soils (Bradyrhizobium sp. NLH25 and Bradyrhizobium sp. NOD31) tolerated up to 30 μM Cd. The analysis of Cd content showed that Bradyrhizobium sp. SEMIA6144 accumulated a high amount of this metal, but a considerable inhibition of growth was observed compared to tolerant strains at 10 μM Cd. At this concentration, the intracellular GSH content of all the Bradyrhizobium sp. strains was not modified in comparison to control conditions. However, at 30 μM Cd, the intracellular GSH content significantly increased in Bradyrhizobium sp. strains NLH25 and NOD31. Thus, the distinct response of each Bradyrhizobium sp. strain to Cd reveals that, even in closely related lineages, there are strain-specific variations influencing the levels of tolerance to this metal. Indeed, the native peanut isolates tolerated higher Cd concentration than the reference strain, possibly due to an increase in GSH levels which could act as a detoxifying agent.     

Parietin in the tolerant lichen Xanthoria parietina (L.) Th. Fr. increases protection of Trebouxia photobionts from cadmium excess

Secondary metabolites of lichens can be involved in production of chelates with heavy metals. We hypothesized that parietin plays important role in protection of photobiont cells in Xanthoria parietina from an excess of cadmium ions. Two types of X. parietina lichen thalli, natural with presence of secondary metabolite parietin (p+) as well as without parietin (p−) were exposed to different doses of cadmium (up to 300 μmol g⁻¹ dw). Based on determination of the total and intracellular Cd-accumulation, ergosterol and thiobarbituric acid reactive substances (TBARS) content did not show statistically significant differences in the response of both types of thalli (p+ and p−). However, a stronger toxic effect of the highest Cd-dose on photosynthetic pigment content and chlorophyll a fluorescence was observed in the parietin-depleted thalli. The protective role of parietin against Cd excess was better supported and concluded from the differences observed in the production of non-protein thiol compounds (cysteine, glutathione and phytochelatins) involved in Cd detoxification. In the p+ thalli Cys content was stable but GSH content slightly decreased in the studied Cd range, while in the p− thalli these compounds were completely absent at high Cd doses. At Cd doses higher than 37.5 μmol Cd g⁻¹ dw, toxic to both types of X. parietina thalli, Cys and GSH contents were significantly higher in p+ than in p− thalli. Also, the photobiont partner in the p+ thalli was better protected of the metal exposition, and able to produce phytochelatins (PCs) over the whole range of metal, while in the p− thalli the production was completely inhibited at 75 μmol Cd g⁻¹ dw and higher concentrations, together with the inhibition of cysteine (Cys) and reduced glutathione (GSH) production. The obtained results indicate that the parietin layer is a natural barrier decreasing Cd access to algal cells in X. parietina. Comparison of PCs production appeared to be the most sensitive marker for estimation of Cd availability to photobiont in the symbiotic system.