Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1)

The response of tobacco plants genetically engineered with the AtTPS1 gene to stress induced by excess Cu and Cd was evaluated in hydroponic solution (100 and 400 μM Cu and 50 and 200 μM Cd) after a 48 h exposure. Two transgenic lines, transformed with the AtTPS1 (trehalose-6-phosphate synthase) gene from Arabidopsis, with different levels of trehalose-6-phosphate synthase expression (B5H, higher and B1F, lower), and a wild type (WT) were investigated. Protein content, antioxidative enzymes (CAT, POD, SOD, and APX), glucose, fructose, lipid peroxidation, hydrogen peroxide and Cd and Cu contents were determined in leaves. The two transgenic lines were differently influenced by Cd and Cu exposure as they induced a different antioxidant enzymatic defense response. B1F and B5H plants showed a better acclimation to Cd and excess Cu compared to WT. Furthermore B1F was more tolerant than B5H to Cd and excess Cu. B1F accumulated less Cd and Cu in leaves, probably due to a more efficient exclusion mechanism. Catalase was shown to be the most important enzyme in the antioxidative system of these plants.     

Differential response of wheat roots to Cu, Ni and Cd treatment: oxidative stress and defense reactions

Wheat seedlings cv. Zyta were treated with Cu, Ni and Cd at the concentrations causing approximately 50 % root growth inhibition, i.e. 12.5, 50 and 60 μM, respectively. Tissue metal accumulation, membrane permeability, lipid peroxidation, protein oxidation, concentration of thiol compounds as well as protease, glutathione S-transferase (GST) and peroxidase (POD) activities were studied in roots after 7 days of metal exposure. The metals showed different concentrations in root tissues with Cu and Cd being accumulated to the smallest and to the greatest extent, respectively. Membrane permeability was significantly enhanced by Cu and Ni but not by Cd treatment. All metals induced similar increase in protein oxidation, while significant enhancement of lipid peroxidation was observed only in the case of Cu treatment. The detected thiol compounds: cysteine (Cys), homocysteine (Hcy), γ-glutamylcysteine (γ-GluCys) and glutathione (GSH) were differently influenced by the metal treatment. Ni appeared to be the most effective inductor of GSH accumulation while both Cu and Ni similarly increased Cys content in the roots. Accumulation of γ-GluCys was found in response to Cu and Cd applications. Concentration of Hcy was enhanced by Cd treatment but exposure to Ni decreased its content below the level of detection. The activity of GST was considerably elevated by Cd and Ni treatments, while POD activity was increased only in response to Cu application. Our study showed that wheat roots differently responded to treatment with metals used at the concentrations having similar impact on growth.     

The cellular redox state as a modulator in cadmium and copper responses in Arabidopsis thaliana seedlings

The cellular redox state is an important determinant of metal phytotoxicity. In this study we investigated the influence of cadmium (Cd) and copper (Cu) stress on the cellular redox balance in relation to oxidative signalling and damage in Arabidopsis thaliana. Both metals were easily taken up by the roots, but the translocation to the aboveground parts was restricted to Cd stress. In the roots, Cu directly induced an oxidative burst, whereas enzymatic ROS (reactive oxygen species) production via NADPH oxidases seems important in oxidative stress caused by Cd. Furthermore, in the roots, the glutathione metabolism plays a crucial role in controlling the gene regulation of the antioxidative defence mechanism under Cd stress. Metal-specific alterations were also noticed with regard to the microRNA regulation of CuZnSOD gene expression in both roots and leaves. The appearance of lipid peroxidation is dual: it can be an indication of oxidative damage as well as an indication of oxidative signalling as lipoxygenases are induced after metal exposure and are initial enzymes in oxylipin biosynthesis.In conclusion, the metal-induced cellular redox imbalance is strongly dependent on the chemical properties of the metal and the plant organ considered. The stress intensity determines its involvement in downstream responses in relation to oxidative damage or signalling.     

Oxidative enzymes activity during abiotic and biotic stresses in <Emphasis Type="Italic">Zea mays</Emphasis> leaves and roots exposed to Cu,methyl jasmonate and <Emphasis Type="Italic">Trigonotylus caelestialium</Emphasis>

The activities of antioxidative enzymes, i.e. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX), in the leaves and roots of Zea mays L. plants exposed to abiotic (methyl jasmonate, MJ, or/and copper, Cu) and biotic (Trigonotylus caelestialium) factors were examined. The contribution of MJ as a signal molecule in the defense mechanism against abiotic and biotic stresses was studied. All plants were cultivated hydroponically and divided into three groups: not treated by abiotic factors (control), treated by MJ only (MJ) and by MJ and Cu (MJ + Cu) and in each group half of the plants were exposed to T. caelestialium attack. The enzymatic activities of SOD, CAT, APX, and GPX in the leaves were higher in the insect-treated than non-insect-treated control plants, but lower in both MJ + Cu- or MJ- and insect-treated plants. In the roots, the enzyme activities were elevated in all insect-treated plants with the highest rise in MJ + Cu, in comparison with the MJ-treated plants. The results showed that MJ and MJ + Cu were efficient in reducing the activity of the antioxidative enzymes in the leaves under the insect influence by elevating enzyme activity in the roots.     

Root morphology, root-hair development and rhizosheath formation on perennial grass seedlings is influenced by soil acidity

Cadmium (Cd) exerts a detrimental effect on the metabolism of plants, whereas selenium (Se) may protect them against various stressors through its antioxidative activity. In this in vitro study we investigated the impact of Se (2 µM Na₂SeO₄) on the growth, nutrient (P, S, K, Ca, Mg, B, Mn, Fe and Zn) concentrations and cell integrity of rape (Brassica napus oleifera) and two wheat (Triticum aestivum) genotypes subjected to Cd stress (600 µM CdCl₂). Rape accumulated both Cd and Se more than did wheat. In all plants, Cd markedly reduced the biomass, enhanced lipid peroxidation and diminished plasmalemma fluidity. A drop in the K uptake and the reduced plasmalemma permeability diminished the K efflux from the leaf cells. In contrast, Cd elevated S concomitantly with Zn, indicating an activity of detoxifying SH groups and SOD isoenzymes. When added alone, Se promoted the growth of all plants, it enhanced the accumulation of S, but the impact on other nutrients remained minor. In Cd-stressed plants, Se tended to counterbalance the Cd-induced changes in nutrients, it also reduced the lipid peroxidation and exerted positive effects on the cell membrane stability. The Cd stress and the protective role of Se were most evident in rape. The Finnish wheat genotype was less tolerant to Cd than the Polish one.     

Cadmium and lead interactive effects on oxidative stress and antioxidative responses in rice seedlings

Interactive effects of two heavy metal pollutants Cd and Pb in the growth medium were examined on their uptake, production of reactive oxygen species (ROS), induction of oxidative stress and antioxidative defence responses in Indica rice (Oryza sativa L.) seedlings. When rice seedlings in sand culture were exposed to 150 μM Cd (NO₃)₂ or 600 μM Pb (CH₃COO)₂ individually or in combination for 8–16 days, a significant reduction in root/shoot length, fresh weight, relative water content, photosynthetic pigments and increased production of ROS (O₂˙^? and H₂O₂) was observed. Both Cd and Pb were readily taken up by rice roots and localisation of absorbed metals was greater in roots than in shoots. When present together in the growth medium, uptake of both the metals Cd and Pb declined by 25–40 %. Scanning electron microscope (SEM) imaging of leaf stomata revealed that Pb caused more distortion in the shape of guard cells than Cd. Dithizone staining of roots showed localisation of absorbed Cd on root hairs and epidermal cells. Both Cd and Pb caused increased lipid peroxidation, protein carbonylation, decline in protein thiol and increase in non-protein thiol. The level of reduced forms of non-enzymic antioxidants glutathione (GSH) and ascorbate (AsA) and their redox ratios (GSH/AsA) declined, whereas the activities of antioxidative enzymes superoxide dismutase (SOD) and guaiacol peroxidase (GPX) increased in metal treated seedlings compared to controls. In-gel activity staining also revealed increased intensities of SOD and GPX isoforms with metal treatments. Catalase (CAT) activity increased during early days (8 days) of metal exposure and declined by 16 days. Results suggest that oxidative stress is an important component in expression of Cd and Pb toxicities in rice, though uptake of both metals gets reduced considerably when present together in the medium.     

Nitric oxide (NO) counteracts cadmium induced cytotoxic processes mediated by reactive oxygen species (ROS) in Brassica juncea: cross-talk between ROS, NO and antioxidant responses

Research on NO in plants has achieved huge attention in recent years mainly due to its function in plant growth and development under biotic and abiotic stresses. In the present study, we investigated Cd induced NO generation and its relationship to ROS and antioxidant regulation in Brassica juncea. Cd accumulated rapidly in roots and caused oxidative stress as indicated by increased level of lipid peroxidation and H₂O₂ thus, inhibiting the overall plant growth. It significantly decreased the root length, leaf water content and photosynthetic pigments. A rapid induction in intracellular NO was observed at initial exposures and low concentrations of Cd. A 2.74-fold increase in intracellular NO was recorded in roots treated with 25 μM Cd than control. NO effects on Malondialdehyde (MDA) content and on antioxidant system was investigated by using sodium nitroprusside (SNP), a NO donor and a scavenger, [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] (cPTIO). Roots pretreated with 5 mM SNP for 6 h when exposed to 25 μM Cd for 24 h reduced the level of proline, non-protein thiols, SOD, APX and CAT in comparison to only Cd treatments. However, this effect was almost blocked by 100 μM cPTIO pretreatment to roots for 1 h. This ameliorating effect of NO was specific because cPTIO completely reversed the effect in the presence of Cd. Thus, the present study report that NO strongly counteracts Cd induced ROS mediated cytotoxicity in B. juncea by controlling antioxidant metabolism as the related studies are not well reported in this species.     

Glutathione metabolism in Urtica dioica in response to cadmium based oxidative stress

To investigate the antioxidative response of glutathione metabolism in Urtica dioica L. to a cadmium induced oxidative stress, activities of glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GSH-Px), content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation (LPO), and also accumulation of Fe, Zn, Mn, Cu besides Cd were determined in the roots, stems, and leaves of plants exposed to 0 (control), 0.045, and 0.09 mM CdCl₂ for 58 h. Whereas the Cd content continuously increased in all organs, the Fe, Zn, Mn, and Cu content decreased in dependence on the applied Cd concentration and incubation time. The Cd treatment resulted in increased GR and GST activities in all organs, however, GSH-Px activity was dependent on Cd concentration and plant organ. The GSH/GSSG ratio maintained above the control level in the stems at both Cd concentrations. The LPO was generally close to the control values in the roots and stems but it increased in the leaves especially at 0.09 mM Cd.     

Antioxidative response of metal-accumulator and non-accumulator plants under cadmium stress

The present study aims to elucidate the role of antioxidative enzyme in the adaptive responses of metal-accumulators (Thlaspi caerulescens and Brassica juncea) and non-accumulator plant (Nicotiana tabacum) to Cadmium stress. When seedlings of plants were grown in hydroponic condition for a period of 4 days in the presence of 200 or 400 μM CdCl₂, photosynthetic rate, transpiration rate and stomatal conductance in metal-accumulators decreased more slowly than that in tobacco. MDA content and electrolyte leakage increased with elevated Cd concentration and exposure time in all plant species, while the oxidative damage in tobacco was more serious than that in metal-accumulators. The activities of SOD and CAT in metal-accumulators were significantly higher than that in tobacco under normal condition, whereas there was no significant difference in the activity of POD between Indian mustard and tobacco. The activities of antioxidative enzymes increased rapidly in metal-accumulators in response to the Cd treatments, especially SOD and CAT. In tobacco, CAT activity declined rapidly by exposure to the Cd treatment, though the activity of SOD and POD was enhanced, indicating that the antioxidative enzymes in tobacco could not fully scavenge ROS generated by Cd toxicity. These results collectively indicate that the enzymatic antioxidation capacity is one of the important mechanisms responsible for metal tolerance in metal-accumulator plant species.     

Heavy metal-induced oxidative damage is reduced by β-estradiol application in lentil seedlings

The protective effect of β-estradiol (E) application against heavy metal (HM) toxicity in lentil (Lens culinaris) seedlings was investigated. Seeds were treated with distilled water (control) or aqueous solutions of 100 μM CdCl₂, 200 μM CuCl₂ and 1 μM E singly or in combinations (1 μM E+100 μM CdCl₂ and 1 μM E+200 μM CuCl₂). HM treatments resulted in increase in the activities of antioxidative enzymes, including superoxide dismutase (SOD), catalase (CAT), guaicol peroxidase and ascorbate peroxidase. In a similar manner, Cd and Cu affected significantly oxidative injury indicators measured as electrolyte leakage (electrical conductivity of germination medium), lipoxygenase (LOX) activity and contents of malondialdehyde (MDA; lipoperoxidation marker), carbonyl groups (protein oxidation marker) and hydrogen peroxide (a reactive oxygen species). However, E was effective in reducing HM-induced toxicity. The steroid (1) alleviated HM-induced increase in the electrolyte leakage, LOX activity and contents of MDA, carbonyl and H₂O₂ and (2) improved the activities of SOD and CAT, but not the peroxidase ones, as compared to treatments with HM singly. In addition, E application prevented HM-induced decrease in dry weight production, but did not reduce the accumulation of Cd and Cu in tissues. Results of the present study suggest that E is able to protect lentil from HM-induced oxidative damage most likely by avoidance of H₂O₂ generation and improving antioxidative enzyme activities and, thereby, decreasing oxidative stress injury, but not by reducing Cd and Cu uptake.     

Limited sulfur resource forces Arabidopsis thaliana to shift towards non-sulfur tolerance under cadmium stress

Soils in some geographical regions suffer with combined stress of sulfur (S) deficiency and cadmium (Cd) contamination. Although the independent impacts of Cd and S-deficiency on plants are well studied but there are rare reports on synergistic effects of S-deficiency and Cd stress. Thus, this study focuses to investigate the response of Arabidopsis thaliana in terms of defense and growth as influenced by Cd under limited S regime. A. thaliana (Col-0) was grown on S-sufficient MS media for 2 weeks and then subjected to S-deficiency for 15 days. Control (+S/−Cd) and S-starved (−S/−Cd) plants were exposed to Cd (50 μM CdCl₂) for 3–5 days. Results show that S-deficiency (−S/−Cd) induces oxidative stress which was much lesser than Cd (+S/+Cd) but highest in combined stress of S-deficiency along with Cd (−S/+Cd). Interestingly, plant was found to elevate glutathione (GSH) biosynthetic pathway and also improved growth and antioxidative status when sulfur was present during Cd stress (+S/+Cd). Important studies in terms of photosynthetic parameters also support limited loss in +S plants as S-assimilation pathway was up-regulated. Proline accumulation was not influenced much by S-deficiency but stimulated with Cd stress strongly suggesting defense shift towards non-sulfur tolerance mechanism. Levels of glutathione and H₂O₂ removing catalase were also modulated to cope with oxidative stress in a better manner during S-sufficient conditions. Chloroplast ultrastructure showed loss of grana under S-deficiency, however, −S/+Cd resulted in severe disintegration of thylakoids too. Biomass accumulation was also most adversely affected with −S/+Cd followed by Cd stress alone (+S/+Cd) and S-deficiency (−S/−Cd). In conclusion, Arabidopsis maintains equilibrium between defense and growth and thus survive under limited S resource. Also S-assimilation is modulated by Cd stress and Cd-induced stress is prevented by S-nutrition.     

Signal cross talk in Arabidopsis exposed to cadmium, silicon, and Botrytis cinerea

The role of defence gene expression triggered by Cd toxicity in the plant’s response to Botrytis cinerea was investigated in Arabidopsis thaliana Columbia 0. Silicon (0 or 1.5 mM) and Cd (0, 1 or 10 μM) were supplied to 3-month-old solution-cultured plants. After 3 days, half of the plants of each treatment were inoculated with Botrytis. Supplied Cd concentrations were below the toxicity threshold and did not cause shoot growth inhibition or evidence of oxidative stress, while Botrytis infection severely decreased plant growth in all treatments. The expression of marker genes PR1 and BGL2 for the salicylic acid (SA) and the PDF1.2 for the jasmonic acid–ethylene (JA–ET) signalling pathways was enhanced in 10 μM Cd-treated non-infected plants. Twenty hours after inoculation, PDF1.2 expression showed a strong increase in all treatments, while enhanced PR1, BGL2, and CHIB expression was only found 7 days after infection. A great synergistic effect of Cd and Botrytis on PDF1.2 expression was found in 10 μM Cd-treated plants. Silicon decreased PR1, BGL2, and CHIB, while increasing PDF1.2 expression, which indicates its role as a modulator of the signalling pathways involved in the plant’s response to fungal infection. Botrytis growth decreased in 10 μM Cd-treated plants, which could be due to the combined effects of Cd and Botrytis activating the SA and JA–ET-mediated signalling pathways. Taken together, our results provide support for the view that Cd concentrations close to the toxicity threshold induce defence signalling pathways which potentiate the plant’s response against fungal infection.     

Changes of Blue Mussels Mytilus edulis L. Lipid Composition Under Cadmium and Copper Toxic Effect

The lipid and fatty acid composition of the blue mussels Mytilus edulis L. gills and digestive glands was evaluated after 24 and 72 h of cadmium (Cd) and copper (Cu) exposure. Mussels were exposed to different cadmium (10, 100, and 500 μg/L) and copper (5, 50, and 250 μg/L) concentrations. Similar stress response of predominant membrane phospholipids level as well as polyenoic and non-methylene interrupted (NMI) fatty acids content was observed in mussel gills under both cadmium and copper effects. Increased NMI fatty acids level after 24 h, the metal ions treatment suggests that these acids contribute to the protective response to the membrane oxidative stress caused by accumulation of the metals. The content of cholesterol, some minor membrane phospholipids, and storage lipids (triacylglycerols, TAG) in the mussels’ organs alter significantly under the cadmium and copper effect. A two-step response at the digestive glands TAG level depends on the duration of the cadmium and copper treatments (24 and 72 h) on the mussels. The results demonstrate that Cd and Cu impact has adverse effects on gills and digestive glands lipid and fatty acids composition. The type of observed effects varies with the nature and concentration of the metal ions and depends on the role of the metals in the mussels’ life activity.     

Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad).

Pea (Pisum sativum L. cv. Azad) plants exposed to 4 and 40 microM of Cd for 7 d in hydroponic culture were analysed with reference to the distribution of metal, the accumulation of biomass and the metal's effects on antioxidants and antioxidative enzymes in roots and leaves. Cd-induced a decrease in plant biomass. The maximum accumulation of Cd occurred in roots followed by stems and leaves. An enhanced level of lipid peroxidation and an increased tissue concentration of hydrogen peroxide (H2O2) in both roots and leaves indicated that Cd caused oxidative stress in pea plants. Roots and leaves of pea plants responded differently to Cd with reference to the induction of enhanced activities of most of the enzymes monitored in the present study. These differential responses to Cd were further found to be associated with levels of Cd to which the plants were exposed. Cd-induced enhancement in superoxide dismutase (SOD) activity was more at 40 microM than at 4 microM in leaves. While catalase (CAT) prominently increased in leaves both at 4 and 40 microM Cd, ascorbate peroxidase (APX) showed maximum stimulation at 40 microM Cd in roots. Enhancement in glutathione reductase (GR) activity was also more at 40 microM than at 4 microM Cd in roots. While glutathione peroxidase (GPOX) activity decreased in roots and remained almost unmodified in leaves, glutathione S-transferase (GST) showed pronounced stimulation in both roots and leaves of pea plants exposed to 40 microM Cd. Increased activities of antioxidative enzymes in Cd-treated plants suggest that they have some additive function in the mechanism of metal tolerance in pea plants.     

Characterization of Xanthophyll Pigments,Photosynthetic Performance,Photon Energy Dissipation,Reactive Oxygen Species Generation and Carbon Isotope Discrimination during Artemisinin-Induced Stress in Arabidopsis thaliana

Artemisinin, a potent antimalarial drug, is phytotoxic to many crops and weeds. The effects of artemisinin on stress markers, including fluorescence parameters, photosystem II photochemistry, photon energy dissipation, lipid peroxidation, reactive oxygen species generation and carbon isotope discrimination in Arabidopsis thaliana were studied. Arabidopsis ecotype Columbia (Col-0) seedlings were grown in perlite and watered with 50% Hoagland nutrient solution. Adult plants of Arabidopsis were treated with artemisinin at 0, 40, 80, 160 μM for one week. Artemisinin, in the range 40–160 μM, decreased the fresh biomass, chl a, b and leaf mineral contents. Photosynthetic efficiency, yield and electron transport rate in Arabidopsis were also reduced following exposure to 80 and 160 μM artemisinin. The Φ_NPQ and NPQ were less than control. Artemisinin treatment caused an increase in root oxidizability and lipid peroxidation (MDA contents) of Arabidopsis. Calcium and nitrogen contents decreased after 80 and 160 μM artemisinin treatment compared to control. δ¹³C values were less negative following treatment with artemisinin as compared to the control. Artemisinin also decreased leaf protein contents in Arabidopsis. Taken together, these data suggest that artemisinin inhibits many physiological and biochemical processes in Arabidopsis.     

Comparison of cadmium and copper effect on phenolic metabolism, mineral nutrients and stress-related parameters in Matricaria chamomilla plants

Cadmium and copper uptake and its consequence for activity of selected enzymes of phenolic metabolism, phenolic acids accumulation, quantity of mineral nutrients and stress-related parameters in Matricaria chamomilla plants exposed to 60 μM and 120 μM for 7 days has been studied. Cu content in the above-ground biomass was ca. 10-fold lower compared to Cd and amount of Cd in the methanol-soluble fraction was lower than in the water-soluble fraction. “Intra-root” Cd represented 68% and 63% of total Cd content at 60 μM and 120 μM, but no difference was observed in Cu-exposed roots. Cu excess had more pronounced effect on shikimate dehydrogenase, cinnamyl alcohol dehydrogenase, polyphenol oxidase and ascorbate peroxidase activity mainly in the roots. Among eight detected benzoic acid derivatives and four cinnamic acid derivatives, the latter were preferentially accumulated in response to Cd excess. Content of salicylic acid increased in all variants. Amount of superoxide was elevated in both the rosettes (preferentially by Cu) and roots (preferentially by Cd). Accumulation of Ca and Mg was not affected by excess of metals, while potassium decreased in both the rosettes and roots (Cu caused stronger depletion). Amount of Fe increased in the roots in response to both metals (more expressively in Cu-treated ones). Present study using other metabolic parameters (and supplementing our previous studies) has confirmed higher Cu toxicity for chamomile plants, to support its strong pro-oxidant properties. These observations as complex metabolic responses are discussed.     

Increased cold tolerance in Arabidopsis thaliana transformed with Choristoneura fumiferana glutathione S-transferase gene

A glutathione S-transferase (GST) gene cloned from the lepidopteran spruce budworm, Choristoneura fumiferana Clem. was transformed into the model plant Arabidopsis thaliana. The CfGST-transgenic and wild-type A. thaliana were subjected to 4 and 10 °C for 48 h and their cold resistance was studied. The GST activity of the transgenic plants was 46.6 and 35.7 % higher than that of the wild-type plants after 48 h under 4 and 10 °C, respectively. Relative membrane permeability and malondialdehyde content in the transgenic plants were lower while contents of the chlorophyll and proline were higher than those in the wild-type plants under 4 and 10 °C. The survival rate of the transgenic plants was 43.7 % for 24 h under 0 °C, while survival rate of wild-type plants was 28.3 %. The results indicated that the insect GST could enhance cold resistance in the transgenic A. thaliana.