Effects of cadmium and copper on peroxidase, NADH oxidase and IAA oxidase activities in cell wall, soluble and microsomal membrane fractions of pea roots

Twelve-day-old seedlings of pea (Pisum sativum L.) that were treated for 4 days by 20 and 100 micromol/l Cd(NO3)2 or CuSO4 showed a growth reduction in all organs. From root protein extracts, the activities of guaiacol peroxidase (GPX; EC 1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11), coniferyl alcohol peroxidase (CAPX), NADH oxidase, and indole-3-acetic acid (IAA) oxidase were measured in covalently--and ionically--[symbol: see text] bound cell wall, soluble, and microsomal membrane fractions. With the exception of 20 micromol/l Cu, metal treatments enhanced GPX activity in all fractions. Only IAA oxidase activity was metal-elevated in the covalently bound cell wall fraction, while the ionic one showed Cd stimulation for all assayed enzymic activities. These effects were not entirely observed in Cu-treated plants, since APX and IAA oxidase activities were only enhanced in this fraction. However, soluble extract showed stimulation of APX activity, while in the microsomal fraction metal exposure also increased the activities of CAPX and NADH oxidase. Differential responses of root cell fractions to the presence of cadmium and copper ions are discussed in regard to the contribution of their enzymic capacities in antioxidant, lignification, and auxin degradation pathways. Comparisons between metals and dose effects are also underlined.     

In situ Inactivation of the Oxidase Activity of Xylem Peroxidases by H2O2 in the H2O2-Producing Xylem of Zinnia elegans

Zinnia elegans stems with 3,3′, 5, 5′-tetramethylbenzidine (TMB) in the presence and in the absence of catalase reveals the presence of xylem oxidase activities in the H₂O₂-producing lignifying xylem cells. This staining of lignifying xylem cells with TMB is the result of two independent mechanisms: one is the catalase-sensitive (H₂O₂-dependent) peroxidase-mediated oxidation of TMB, and the other the catalase-insensitive (H₂O₂-independent) oxidation of TMB, probably due to the oxidase activity of xylem peroxidases. The response of this TMB-oxidase activity of xylem peroxidases to different exogenous H₂O₂ concentrations was studied, and the results showed that H₂O₂ at high concentrations (100–1,000 mM) clearly acted as an inactivator of this xylem TMB-oxidase activity, although some inhibitory effect could still be appreciated at 10 mM H₂O₂. This xylem TMB-oxidase activity resided in a strongly basic cell wall-bound peroxidase (pl about 10.5). Given such a scenario, it may be concluded that this TMB-oxidase activity of peroxidase is located in tissues capable of sustaining H₂O₂ production, and that the in situ oxidase activity shown by this enzyme is inactivated by high H₂O₂ concentrations. Received 20 April 1999/ Accepted in revised form 16 August 1999     

Copper-induced oxidative stress and antioxidant defence in Arabidopsis thaliana

Content of reactive oxygen species (ROS): O2*-, H2O2 and OH* as well as activities of antioxidant enzymes: superoxide dismutase (SOD), guaiacol peroxidase (POX) and catalase (CAT) were studied in leaves of Arabidopsis thaliana ecotype Columbia, treated with Cu excess (0, 5, 25, 30, 50, 75, 100, 150 and 300 microM). After 7 days of Cu action ROS content and the activity of SOD and POX increased, while CAT activity decreased in comparison with control. Activities of SOD, POX and CAT were correlated both with Cu concentration (0-75 microM) in the growth medium and with OH* content in leaves. Close correlation was also found between OH* content and Cu concentration. Oxidative stress in A. thaliana under Cu treatment expressed in elevated content of O2*-, H2O2 and OH* in leaves. To overcome it very active the dismutase- and peroxidase-related (and not catalase-related, as in other plants) ROS scavenging system operated in A. thaliana. Visual symptoms of phytotoxicity: chlorosis, necrosis and violet colouring of leaves as well as a reduction of shoot biomass occurred in plants.     

The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H₂O₂ in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H₂O₂ production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H₂O₂ and to sustain H₂O₂ production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H₂O₂ production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H₂O₂ production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H₂O₂ production in lignifying xylem was obtained from the observation that areas of H₂O₂ production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H₂O₂ which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H₂O₂-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H₂O₂ to peroxidase in the lignifying xylem of Z. elegans. Received: 6 February 1998 / Accepted: 14 August 1998     

The apoplastic antioxidant enzymatic system in the wood-forming tissues of trees

The complete apoplastic enzymatic antioxidant system, composed by class I ascorbate peroxidases (class I APXs), class III ascorbate peroxidases (class III APXs), ascorbate oxidases (AAOs), and other class III peroxidases (PRX), of wood-forming tissues has been studied in Populus alba, Citrus aurantium, and Eucalyptus camaldulensis. The aim was to ascertain whether these enzymatic systems may regulate directly (in the case of APXs), or indirectly (in the case of AAOs), apoplastic H₂O₂ levels in lignifying tissues, whose capacity to produce and to accumulate H₂O₂ is demonstrated here. Although class I APXs are particularly found in the apoplastic fraction of P. alba (poplar), and class III APXs are particularly found in the apoplastic fraction of C. aurantium (bitter orange tree), the results showed that the universal presence of AAO in the extracellular cell wall matrix of these woody species provokes the partial or total dysfunction of apoplastic class I and class III APXs, and of the whole plethora of non-enzymatic redox shuttles in which ascorbic acid (ASC) is involved, by the competitive and effective removal of ASC. In fact, the redox state (ASC/ASC+DHA) in intercellular wash fluids (IWFs) of these woody species was zero, and thus strongly shifted towards DHA (dehydroascorbate), the oxidized product of ASC. This imbalance of the apoplastic antioxidant enzymatic system apparently results in the accumulation of H₂O₂ in the apoplast of secondary wood-forming tissues, as can be experimentally observed. Furthermore, it is hypothesized that since AAO uses O₂ to remove ASC, it could regulate O₂ availability in the lignifying xylem and, thorough this mechanism, AAO could also control the activity of NADPH oxidase (the enzyme responsible for H₂O₂ production in lignifying tissues) at substrate level, by controlling the tension of O₂. That is, the presence of AAO in the extracellular cell wall matrix appears to be essential for finely tuning the oxidative performance of secondary wood-forming tissues.     

Effects of nitrogen on the activity of antioxidant enzymes and gene expression in leaves of Populus plants subjected to cadmium stress

The research aimed to verify the important physiological effect of nitrogen (N) on plants exposed to cadmium (Cd). The poplar plants were grown in a Hoagland nutrient solution and treated with extra N, Cd, and N + Cd. After treatment, plant growth and chlorophyll content were recorded. The oxidative stress, the activity of antioxidant enzymes, and the expression of related genes were also examined. The results indicated the plants treated with sole Cd presented obvious toxicity symptoms, i.e. growth inhibition, reactive oxygen species accumulation, and chlorophyll content decrement. However, when N was added to the plants under Cd stress, plant growth was enhanced, chlorophyll synthesis was promoted, and the oxidative stress was alleviated. Further, the expression of antioxidant enzymes genes was upregulated by N. The results indicated that N partially reversed the toxic effect of Cd on poplar plants, which can provide new methodology to enhance the phytoremediation technology for heavy metal pollution soil.     

The activity of lipoxygenase in Arabidopsis thaliana (L.) Heynh -- a preliminary study

The activity of lipoxygenase (EC 1.13.11.12) in Arabidopsis thaliana (L.) Heynh seedlings and mature plants was estimated spectrophotometrically at 234 nm. Linoleic acid was used as a substrate. Lipoxygenase activity showed two pH optima: at 7.0 and 10.0 in seedlings, and at pH 8.0 and 10.0 in leaves of mature plants. Seven-week-old plants were transferred to a hydroponic system and treated with different concentrations of Cd(2+) or Cu(2+) [in microM]: 0, 5, 25, 50, 100 for 7 days. The lipoxygenase activities at pH 8.0 and 10.0 depended on the metal that was added to the nutrient solution. The main change in lipoxygenase activity was under Cd(2+)stress at pH 8.0 and under Cu(2+)excess at pH 10.0.     

Lipid peroxidation and antioxidative response in Arabidopsis thaliana exposed to cadmium and copper

Formation of lipid hydroperoxides, malondialdehyde (MDA) and hydroxyalkenals (HAEs), membrane damages and antioxidative response of plants expressed as changes in glutathione S-transferase activity (GST) and anthocyanin accumulation were studied in Arabidopsis thaliana (L.) Heynh cv. Columbia plants treated for 7 days with various concentrations: 5, 25, 50, 100 μM Cd and Cu. Increased lipid hydroperoxide content was metal concentration-dependent. The level of MDA + HAE was elevated in Cd- and Cu- treated plants, but it was metal concentration-dependent under Cu stress. Electrolyte leakage measurements showed a larger membrane damage under Cu- than Cd-treatment. In Cu-stressed plants, GST activity was always enhanced in comparison with control, while in plants exposed to Cd it dropped slightly at lower metal concentrations; but at 100 μM Cd it was even higher than in plants treated with the same Cu concentration. Anthocyanin accumulation was considerably higher under Cu than Cd stress. Both lipid peroxidation and antioxidative response was stronger in Cu- than Cd-treated Arabidopsis thaliana plants. Various mechanisms of defense against the lipid peroxidation products, depending on the metal type, are discussed.     

Effect of copper excess on superoxide dismutase,catalase, and peroxidase activities in sunflower seedlings (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

The changes in lipid peroxidation, antioxidative and lignifying enzyme activities were studied in leaves and stems of Cu-stressed sunflower seedlings. In both organs, membrane lipid peroxidation was enhanced by copper treatment. Additionally, catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) activities were modulated: The activity of superoxide dismutase was enhanced in both plant organs. Differently, catalase activity was not affected in leaves but significantly reduced in stems. Peroxidase (EC 1.11.1.7) activities were also changed. Guaiacol peroxidase activity was increased in leaves and stems. In the same way, electrophoretic analysis of the anionic isoperoxidases involved in lignification (syringaldazine peroxidase) revealed qualitative and quantitative changes on the isoenzyme patterns. These modifications were accompanied by the increase of the NADH-oxidase activity in ionically cell wall bound fraction. It appeared that the growth delay caused by copper excess could be related to the activation of lignifying peroxidases.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Cadmium-induced changes in the growth and oxidative metabolism of pea plants

The effect of growing pea (Pisum sativum L.) plants with CdCl(2) (0-50 microM) on different plant physiological parameters and antioxidative enzymes of leaves was studied in order to know the possible involvement of this metal in the generation of oxidative stress. In roots and leaves of pea plants Cd produced a significant inhibition of growth as well as a reduction in the transpiration and photosynthesis rate, chlorophyll content of leaves, and an alteration in the nutrient status in both roots and leaves. The ultrastructural analysis of leaves from plants grown with 50 microM CdCl(2), showed cell disturbances characterized by an increase of mesophyll cell size, and a reduction of intercellular spaces, as well as severe disturbances in chloroplast structure. Alterations in the activated oxygen metabolism of pea plants were also detected, as evidenced by an increase in lipid peroxidation and carbonyl-groups content, as well as a decrease in catalase, SOD and, to a lesser extent, guaiacol peroxidase activities. Glutathione reductase activity did not show significant changes as a result of Cd treatment. A strong reduction of chloroplastic and cytosolic Cu,Zn-SODs by Cd was found, and to a lesser extent of Fe-SOD, while Mn-SOD was only affected by the highest Cd concentrations. Catalase isoenzymes responded differentially, the most acidic isoforms being the most sensitive to Cd treatment. Results obtained suggest that growth of pea plants with CdCl(2) can induce a concentration-dependent oxidative stress situation in leaves, characterized by an accumulation of lipid peroxides and oxidized proteins as a result of the inhibition of the antioxidant systems. These results, together with the ultrastructural data, point to a possible induction of leaf senescence by cadmium.     

Pre-treatment of seeds with static magnetic field ameliorates soil water stress in seedlings of maize (Zea mays L.)

The effect of magnetic field (MF) treatments of maize (Zea mays L.) var. Ganga Safed 2 seeds on the growth, leaf water status, photosynthesis and antioxidant enzyme system under soil water stress was investigated under greenhouse conditions. The seeds were exposed to static MFs of 100 and 200 mT for 2 and 1 h, respectively. The treated seeds were sown in sand beds for seven days and transplanted in pots that were maintained at -0.03, -0.2 and -0.4 MPa soil water potentials under greenhouse conditions. MF exposure of seeds significantly enhanced all growth parameters, compared to the control seedlings. The significant increase in root parameters in seedlings from magnetically-exposed seeds resulted in maintenance of better leaf water status in terms of increase in leaf water potential, turgor potential and relative water content. Photosynthesis, stomatal conductance and chlorophyll content increased in plants from treated seeds, compared to control under irrigated and mild stress condition. Leaves from plants of magnetically-treated seeds showed decreased levels of hydrogen peroxide and antioxidant defense system enzymes (peroxidases, catalase and superoxide dismutase) under moisture stress conditions, when compared with untreated controls. Mild stress of -0.2 MPa induced a stimulating effect on functional root parameters, especially in 200 mT treated seedlings which can be exploited profitably for rain fed conditions. Our results suggested that MF treatment (100 mT for 2 h and 200 for 1 h) of maize seeds enhanced the seedling growth, leaf water status, photosynthesis rate and lowered the antioxidant defense system of seedlings under soil water stress. Thus, pre sowing static magnetic field treatment of seeds can be effectively used for improving growth under water stress.     

The beneficial role of potassium in Cd-induced stress alleviation and growth improvement in Gladiolus grandiflora L.

Heavy metal contaminated agricultural soils are one of the most important constraints for successful cultivation of crops. The current research was conducted to evaluate the role of potassium (K) on plant growth and amelioration of cadmium (Cd) stress in Gladiolus grandiflora under greenhouse conditions. G. grandiflora corms were sown in media contaminated with 0 (C), 50 (Cd50) and 100 (Cd100) mg Cd kg^?1 soil. The plants growing in Cd-contaminated media exhibited reduced gas exchange attributes, chlorophyll (Chl) contents, vegetative and reproductive growth as compared to control. The plants raised in Cd contaminated media showed reduced nutrition yet higher Cd contents. However, supplementation of 60 mg Kg^?1 K in treated plants (C+K, Cd50+K and Cd100+K) improved quantity of total soluble protein and proline (Pro) along with activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) under Cd stress. Similarly, K supplementation reduced the level of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) in treated plants. Potassium supplemented plants exhibited better vegetative and reproductive growth. The improved stress tolerance in K supplemented plants was attributed to the reduced quantity of MDA and H₂O₂, enhanced synthesis of protein, proline, phenols, flavonides and improved activity of antioxidant enzymes. The present research supports the application of K for alleviation of Cd stress in G. grandiflora.     

Changes in Growth,Photosynthetic Pigments,Cell Viability,Lipid Peroxidation and Antioxidant Defense System in Two Varieties of Chickpea (<i>Cicer arietinum</i> L.) Subjected to Salinity Stress

Salinity is one of the most severe abiotic stresses for crop production. The present study investigates the salinity-induced modulation in growth indicators, morphology and movement of stomata, photosynthetic pigments, activity of carbonic anhydrase as well as nitrate reductase, and antioxidant systems in two varieties of chickpea (Pusa-BG5023, and Pusa-BGD72). On 20^th day of sowing, plants were treated with varying levels of NaCl (0, 50, 100, 150 and 200 mM) followed by sampling on 45 days of sowing. Recorded observations on both the varieties reveal that salt stress leads to a significant decline in growth, dry biomass, leaf area, photosynthetic pigments, protein content, stomatal behavior, cell viability, activity of nitrate reductase and carbonic anhydrase with the rise in the concentration of salt. However, quantitatively these changes were less in Pusa-BG5023 as compared to Pusa-BGD72. Furthermore, salinity-induced oxidative stress enhanced malondialdehyde content, superoxide radicals, foliar proline content, and the enzymatic activities of superoxide dismutase, catalase, and peroxidase. The variety Pusa-BGD72 was found more sensitive than Pusa-BG5023 to salt stress. Out of different graded concentrations (50, 100, 150 and 200 mM) of sodium chloride, 50 mM was least toxic, and 200 mM was most damaging. The differential behavior of these two varieties measured in terms of stomatal behavior, cell viability, photosynthetic pigments, and antioxidant defense system can be used as prospective indicators for selection of chickpea plants for salt tolerance and sensitivity.     

Identification and partial purification of an oxidase from lignifying xylem of Leyland cypress (X Cupressocyparis leylandii)

 Oxidase activity was exclusively present in lignifying cells of developing xylem of Leyland cypress. The oxidase was enriched in 200 mM CaCl₂ extracts of crude cell walls and seems to be ionically associated with the cell walls. Oxidase activity was selected and concentrated using affinity chromatography on Concanavalin-A Sepharose which suggests that it is a high-mannose type glycoprotein. A subsequent purification step using gel permeation chromatography on Sephadex GF-150 partially separated the oxidase activity from peroxidase activity. An oxidase band of apparent Mr 92 kD capable of oxidising N, N, N′, N′ - tetramethyl phenylene diamine/α-naphthol was identified after non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 92 kD oxidase band was enriched in the oxidase-rich fraction and absent from the peroxidase-rich fraction from the gel permeation step. In addition, the 92 kD oxidase band could be differentiated from peroxidase bands because it was not intensified by the addition of hydrogen peroxide. The partially purified oxidase effectively oxidised and polymerised coniferyl alcohol to form insoluble material that yielded a Fourier transform infra-red spectrum similar to dehydrogenation polymers of coniferyl alcohol. This coniferyl alcohol oxidase appears to be specific to lignifying xylem cells and may participate in lignin deposition but further studies are required to fully define this oxidase and its possible homology with other oxidases identified in the lignifying xylem of different species of trees. Received: 20 May 1997 / Accepted: 7 August 1997     

Enzymatic responses to metal exposures in a freshwater fish Oreochromis niloticus

Freshwater fish Oreochromis niloticus were exposed to 5, 10 and 20 microM concentrations of Cd, Cu, Zn and Pb for 14 days and responses of several enzymes were determined subsequently. Liver catalase (CAT) activity was influenced by Cd and Pb exposures, while it was inhibited by Zn exposure. Copper, on the other hand, did not cause significant changes in CAT activity. Liver alkaline phosphatase (AP) activity was first stimulated at lowest (5 microM) exposure concentration, while there were significant inhibitions at higher (10 microM) exposure concentration. At the highest (20 microM) exposure concentration, AP activity was compensated coming to the control level, except Pb exposure. Intestine and serum AP activities were stimulated by all Zn exposures and 10 microM Cu exposure, while other exposures did not cause significant changes on AP activity. Na,K-ATPase activity in the gill and intestine was inhibited by all the metal exposures, except 20 microM Pb exposure that resulted in an increase in the activity in the gill. Similarly, muscle Ca-ATPase activity was inhibited by all the metal exposures, except Cu exposures. This study indicated that enzymatic systems may be used as a sensitive bioindicator of metal contamination in aquatic systems. Nevertheless, the results also stress the importance of addressing the sensitivity of these enzymes when they are used as a bioindicator for metal contamination, because the responses of the enzymes varied considerably according to metal types and concentrations.     

Copper-stress induced alterations in protein profile and antioxidant enzymes activities in the in vitro grown Withania somnifera L.

Withania somnifera L. seedlings were grown in half-strength MS (Murashige and Skoog) basal medium for 4 weeks and then transferred to full-strength MS liquid medium for 3 weeks. The sustainable plants were subcultured in the same medium but with different concentrations (0, 25, 50, 100 and 200 μM) of Cu for 7 and 14 days. The growth parameters (root length, shoot length, leaf length and total number of leaves per plant) showed a declining trend in the treated plants in a concentration dependant manner. Roots and leaves were analyzed for protein profiling and antioxidant enzymes [catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (GPX, EC 1.11.1.7)]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude protein extracts showed the appearance of some new proteins due to Cu treatment. In plant samples grown with 25 and 50 μM of Cu, a rapid increase in antioxidant activities were noticed but at higher concentration (100 and 200 μM) the activities declined. Isoforms of CAT, SOD and GPX were separated using non-denaturing polyacrylamide gel electrophoresis and concentration specific new isoforms were noticed during the study. Isoforms of the antioxidant enzymes synthesized due to Cu stress may be used as biomarkers for other species grown under metal stress.     

Metal accumulation and response of antioxidant enzymes in seedlings and adult sunflower mutants with improved metal removal traits on a metal-contaminated soil

Sunflower mutant lines with an enhanced tolerance and metal accumulation capacity obtained by mutation breeding have been proposed for Zn, Cd and Cu removal from metal-contaminated soils in previous studies. However, soils contaminated with trace elements induce various biochemical alterations in plants leading to oxidative stress. There is a lack of knowledge concerning the metal accumulation and antioxidant responses during the growth and development of sunflowers. This study, therefore, aimed to characterise metal accumulation and possible metal detoxification mechanisms in young seedlings and adult sunflowers. Beside the inbred line, two mutant lines with an improved growth and enhanced metal uptake capacity on a metal contaminated soil were investigated in more detail.Sunflowers cultivated on a metal-contaminated soil in the greenhouse showed a decrease in shoot biomass and chlorophyll concentration in two different developmental stages. Adult sunflowers showed a lower sensitivity to metal toxicity than young seedlings, whereas mutant lines were more tolerant to metal stress than the control. Mutant lines also produced a higher amount of carotenoids on a metal-contaminated soil than on the control soil, indicating a possible protective mechanism of sunflower mutants against oxidative stress caused by Cd and excess Zn.Heavy metals primarily increased the activity of antioxidant enzymes involved in the ascorbate–glutathione cycle in sunflower leaves. Activity of dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) was strongly increased in young seedlings exposed to heavy metals. The enzyme activities were even more pronounced in mutant lines. A significantly increased ascorbate peroxidase (APOX) activity in adult sunflowers exposed to heavy metals indicated an elevated use of ascorbate after a longer exposure to metal stress.An increased antioxidant level corresponded to a high Cd and Zn accumulation in young and adult sunflowers. Metal distribution, zinc translocation in particular, from the root into the shoot tissue obviously increased during sunflower growth and ripening. Altogether, these results suggest that sunflower plants, primarily the mutant lines, possess an efficient defence mechanism against oxidative stress caused by metal toxicity. A good tolerance of sunflowers toward heavy metals coupled with an increased metal accumulation capacity might contribute to an efficient removal of heavy metals from a polluted area.