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EffectofCopperExcessonH2O2AccumulationandPeroxidaseActivitiesinBeanRoots

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We studied oxidative stress and peroxidase activity resulting from application of excess copper in the

nutrient medium on the roots of young bean seedlings. The change in H2O2 content, lipid peroxidation

and oxidative enzymes activities were quantified and located. Excess of copper caused a loss of mem-

brane integrity and the formation of hydrogen peroxide (H2O2) as visualized in the transmission electron

microscopy and measured using spectrophotometry. H2O2 accumulated in the intercellular spaces and in

the cell wall. The production of H2O2 was accompanied by an increase in the activity of soluble and ionic

GPX (Guaicol peroxidase, EC 1.11.17), CAPX (Coniferyl alcohol peroxidase) and NADH oxidase.

Keywords: Bean – peroxidase – H2O2 – cell wall – copper

INTRODUCTION

In plant cells, an excess of heavy metal ions shift the balance of free radicals metab-

olism towards an accumulation of hydrogen peroxide (H2O2). In the presence of

redox-active transition metals such as Cu2+ and Fe2+, H2O2 can be converted to the

highly reactive molecules like .OH [13, 14, 31, 38].

These reactive molecules induce the activation of chain reactions leading to oxida-

tive stress.

Cu (copper) toxicity was previously believed to be mainly due to the loss of mem-

brane integrity and the formation of free radicals [36].

Transition metals like copper and iron catalyze the formation of .OH and H2O2

radicals from the non enzymatic chemical reaction via the Haber-Weiss and Fenton

reaction [14, 15].

Acta Biologica Hungarica 59 (2), pp. 233–245 (2008)

DOI: 10.1556/ABiol.59.2008.2.9

0236-5383/$ 20.00 © 2008 Akadémiai Kiadó, Budapest

EFFECT OF COPPER EXCESS

ON H2O2 ACCUMULATION

AND PEROXIDASE ACTIVITIES IN BEAN ROOTS

HOUDA BOUAZIZI,1 H. JOUILI,1 ANJA GEITMANN2 and E. EL FERJANI1*

1 Laboratoire de Bio-Physiologie Cellulaires, Faculté des Sciences de Bizerte, 7021 Zarzouna, Tunisia2 Laboratoire de Microscopie, Institut de recherche en biologie végétale 4101 rue Sherbrooke,

Montréal, Québec H1X 2B2, Canada

(Received: May 3, 2006; accepted: April 13, 2007)

* Corresponding author; e-mail: ezzferjani2002@yahoo.fr

234 H. BOUAZIZI et al.

Acta Biologica Hungarica 59, 2008

H2O2 is generated via superoxide, during electron transport processes such as pho-

tosynthesis and mitochondrial respiration.

The enzymatic sources of H2O2 generated in response to specific stimuli are still

under discussion; various potential sources have been described in plants. These

include NADPH oxidase [16], cell wall peroxidases, oxalate oxidase, plast amine,

diamine and polyamine oxidase-type enzyme and flavin-containing oxidases [5,

6, 21].

Hydrogen peroxide generated by abiotic and biotic stress can act as a signalling

molecule that regulates plant development, stress adaptation and programmed cell

death [1].

In the cell, the over-production of H2O2 activates peroxide-detoxifying mecha-

nisms; these include scavenging the reactive oxygen by non enzymatic systems such

as ascorbate and α-tocopherol and the use of an enzymatic antioxidant system that

includes glutathion reductase (GR), superoxide dismutase (SOD), catalase (CAT),

guaiacol peroxidase (GPX) and ascorbate peroxidase (APX).

In this work, we tried to highlight the involvement of copper excess in the stimu-

lation of the production of hydrogen peroxide and we located the molecule cyto-

chemically using transmission electron microscopy. Also, the effect of copper excess

on growth, metal accumulation and peroxidase activities in bean roots were studied.

MATERIALS AND METHODS

Plant material and growth conditions

Bean seeds (Phaseolus vulgaris) were germinated on glass plates covered with moist

filter paper in darkness at 25 °C for 3 days. The seedlings were grown in a controlled

chamber at 25 °C ± 2 °C and 65% RH, with a photoperiod of 16 h and a light inten-

sity of 150 µmol/m2/s. Fourteen-day old seedlings were treated for 3 days by addi-

tion of 50 and 75 µM of CuSO4 to the nutrient medium, as previously described by

Mazhoudi et al. [20].

Total copper content and cell wall fractions content

Total copper content

Dried root material was ground to powder and wet-digested in 65% nitric acid (1 ml

per 0.1 g of dry matter). The digested material was resuspended in distilled water.

The Cu content of the roots was determined using an atomic absorption spectropho-

tometer (Perkin Elmer-model 2380, C.R.G.R.).

Copper content in the cell wall fractions

Excised roots were incubated in 1% (v/v) Triton X-100. This solution was periodi-

cally renewed during 3 weeks, which was sufficient for the lysis of the cellular con-

tent. The resulting cell walls were dried and treated according to the same protocol

described previously for the determination of the copper content in soluble fraction.

Quantification of H2O2

Hydrogen peroxide levels were determined according to Sergiev et al. [30]. Root tis-

sues (500 mg) were homogenized in an ice bath with 5 ml 0.1% (w/v) TCA. The

homogenate was centrifuged at 12,000 × g for 15 min and 0.5 ml of the supernatant

was added to 0.5 ml 10 mM potassium phosphate buffer (pH 7.0) and 1 ml 1M KI.

The absorbance of the supernatant was read at 390 nm and the content of H2O2

was extrapolated from a standard curve.

Malondialdehyde determination

The level of lipid peroxidation was evaluated by the thiobarbituric acid (TBA) reac-

tion. Fresh tissue was homogenized in 0.5% (w/v) TBA prepared in 30% (w/v) TCA

as described by Baccouche et al. [2]. The homogenate was incubated at 95 °C in a

water bath for 30 min and then cooled in an ice bath. After centrifugation at 10,000×g

for 10 min, the supernatant was measured at 532 nm and corrected by substracting

the non-specific absorbance at 600 nm. MDA concentration was calculated using an

extinction coefficient of 155 mM–1 cm–1.

Peroxidase extraction

Plant material was extracted in 50 mM K-Phosphate buffer (pH 7.0) containing 5

mM sodium ascorbate and 0.2 mM EDTA. The homogenate was centrifuged at

13,000 × g for 15 min. The resulting supernatant was considered as soluble enzymat-

ic fraction.

The pellet was rinsed several times with extraction buffer, stirred for 1hour in 1 M

NaCl (1.5 ml g–1 FW) in 50 mM K-phosphate buffer and centrifuged at 13,000 × g

for 15min; the supernatant was then desalted and concentrated. The resultant prepa-

ration was considered as ionically bound enzymatic fraction. All operations were

performed at 4 °C.

Soluble and ionic protein concentrations were determined according to Bradford

[8] using bovine serum albumin as a standard.

Oxidative stress in bean roots 235

Acta Biologica Hungarica 59, 2008

236 H. BOUAZIZI et al.

Acta Biologica Hungarica 59, 2008

Enzyme assays

Guaiacol peroxidase activities in both soluble and cell wall fractions were deter-

mined following the increase in absorbance at 470 nm by adding the enzymatic

preparation to 2 ml of 9 mM guaiacol and 10 mM hydrogen peroxide in 50 mM K-

phosphate buffer (pH 7.0) [10].

Coniferyl alcohol peroxidase activities in both soluble and ionically cell wall were

measured by monitoring the decrease in absorbance at 260 nm [25]. NADH-oxidase

activity was measured by monitoring the decrease in absorbance at 340 nm accord-

ing to Ishida et al. [16].

Transmission electron microscopy (TEM)

In situ localization of H2O2

Hydrogen peroxide production was assessed cytochemically via determination of

cerium perhydroxide formation after the reaction of CeCl3 with endogenous H2O2

[26] Fresh roots were cut into slices (2 mm2) and were incubated for 1 h in 5 mM

CeCl3 in 50 mM 3-(N-morpholino) propanesulphonic acid (MOPS) pH 7.2. After

incubation, sections were fixed in 2 % formaldehyde, 2.5% glutaraldehyde in 0.05 M

phosphate buffer (pH 7.2) (freshly prepared). Samples were washed twice in the

same buffer, dehydrated in a graded aceton series (25, 50, 70, 90, 95 and 100%).

Specimens were gradually embedded in Spurr’s resin.

Thin sections of embedded tissues were obtained on a Leica Ultracut microtome,

contrasted by uranyl acetate in ethanol, mounted on uncoated copper grids and

observed using a transmission electron microscope (JEOL-JEM-100 S) operated at

80 kV.

Statistical analysis

The results presented are the values ± standard error obtained from at least six repli-

cates, except for MET (n = 3). Significant differences between treated and control

plants are determined using the ANOVA test (P < 0.05).

RESULTS

Effect of copper excess on seedling growth and protein content

In order to determine the effect of copper excess on the morphological aspect, we

tried to highlight the effect of Cu excess on the production of fresh and dry biomass

and on the longitudinal growth.

Results presented in Fig. 1 show that exposure to 50 µM and 75 µM of CuSO4

induced a significant decrease in fresh matter (35% and 50%, respectively, compared

with the control). Also, dry matter was decreased by 27% and 43%, in Cu-treated

roots, respectively, with 50 and 75 µM of CuSO4 (Fig. 1B). Excess of copper caused

inhibition in root elongation. This inhibition reaches 43% in roots treated by 75 µM

of CuSO4 (Fig. 1C).

Oxidative stress in bean roots 237

Acta Biologica Hungarica 59, 2008

Fig. 1. Fresh and dry weight production (A, B), elongation of bean roots (C) exposed to 50 and 75 µM

of CuSO4 for 3 days. Data are mean values of three independent experiments. SE are indicated by

vertical bars

238 H. BOUAZIZI et al.

Acta Biologica Hungarica 59, 2008

Soluble protein content was reduced by 70% and 73% in roots, treated with 50 µM

and 75 µM of CuSO4, respectively (Fig. 2A). The cell wall bound proteins were

increased (31% compared with the control) in roots treated with 50 µM CuSO4 and

were decreased (27% compared with the control) in roots treated with 75 µM CuSO4

(Fig. 2B).

Fig. 2. Protein content: Soluble and Ionic fraction (A and B), total copper content (black columns), cop-

per content in the cell wall fraction (white columns) (C), H2O2 level (D), MDA accumulation (E) in bean

roots exposed to 50 and 75 µM of CuSO4 for 3 days. Data are mean values of three independent exper-

iments. SE are indicated by vertical bars

Copper content

When hydroponically grown beans were exposed to 50 and 75 µM of CuSO4, cop-

per was highly accumulated in root tissues.

Figure 2 C shows that the quantity of accumulated copper in the roots treated by

50µM of CuSO4 is sixty times that of the control. In the presence of 75 µM of CuSO4,

the quantity of accumulated copper exceeds 100 times that of the control. The analy-

sis of the parietal fraction of copper in the treated seedlings shows that this fraction

presents 40% of the total copper accumulated on the level of the roots treated by 50

µM of CuSO4. This fraction reaches 50% of total copper accumulated in 75 µM Cu-

treated roots.

H2O2 determination

Hydrogen peroxide levels were measured in the roots of bean seedlings treated with

50 µM and 75 µM of CuSO4. Figure (2D) shows that the copper excess causes a sig-

nificant production of hydrogen peroxide in the treated roots. We also noted that the

level of accumulated H2O2 increases with the concentration of metal in the nutrient

medium. H2O2 production was not detectable in control roots.

MDA determination

The level of malondialdehyde (MDA) was increased significantly in treated roots

(Fig. 2E). This increase reaches 100% in 75 µM Cu-treated roots and 32% in roots

treated by 50 µM of CuSO4. Copper induced a lipoperoxidation process in roots.

Effect of copper excess on peroxidase activities

Copper-treatment significantly enhanced the peroxidase activities in soluble and ion-

ically cell wall-bound fractions (Fig. 3). The data show a notable stimulation of sol-

uble and ionic guaiacol peroxidase activities. This stimulation reached 236% and

797% in the soluble fraction after treatment with 50 and 75 µM of CuSO4, respec-

tively (Figs 3A, B). Similarly, we noted a significant increase in soluble and ionic

alcohol peroxidase activities (Figs 3C, D).

The soluble NADH-oxidase activity was modulated by copper excess, when a sig-

nificant increase was detected in roots treated with 50 and 75 µM of CuSO4, this

stimulation is estimated to be 57% and 54%, respectively, compared with the control.

Also, we noted a stimulation in ionic NADH-oxidase activity. This stimulation is

estimated at 46.5% and 50%, respectively, compared to the control (Figs 3E, F).

Oxidative stress in bean roots 239

Acta Biologica Hungarica 59, 2008

240 H. BOUAZIZI et al.

Acta Biologica Hungarica 59, 2008

Determination and localization of H2O2 in situ

The histochemical assay is based on the determination of cerium perhydroxide after

the reaction of CeCl3 with endogenous H2O2 which produced electron-dense insolu-

ble precipitates. In a section of control bean roots, electron-dense precipitates of ceri-

um perhydroxides were not detectable in the cell wall of the rhizoderm or in the inter-

cellular spaces of the parenchyma (Figs 4A, D).

However, at 50 µM of CuSO4, H2O2 accumulation was evident in the cell wall of

rhizoderm and in the intercellular spaces of parenchyma (Figs 4, E). At 75 µM of

Fig. 3. Activities of soluble and ionic guaiacol (A, B), coniferyl alcohol peroxidases (C, D) and NADH-

oxidase (E, F) in bean roots exposed to 50 and 75 µM of CuSO4 for 3 days. Data are mean values of three

independent experiments. SE are indicated by vertical bars

Oxidative stress in bean roots 241

Acta Biologica Hungarica 59, 2008

Fig. 4. Cytochemical localization of Cu-induced H2O2 accumulation in cell wall, rhizoderme (A, B, C)

(×15,000) and intercellular spaces of parenchyma (D, E, F) (×15,000) in bean roots , exposed to 50 and

75 µM of CuSO4 for up to 3 days

242 H. BOUAZIZI et al.

Acta Biologica Hungarica 59, 2008

CuSO4, the electron-dense precipates were clearly visible in the apoplastic spaces of

rhizoderm, in the cell wall and in the apoplastic spaces of parenchyma (Figs 4C, F).

The size of electron-dense precipitates increased with the increase in Cu levels.

DISCUSSION

We examined the physiological responses of bean roots to copper excess, in particu-

lar the effect on peroxidase activities in soluble and cell wall bound fractions. The

effect of Cu2+ ions on soluble and ionic protein contents and H2O2 levels was also

studied. We studied the modulation of peroxidase response under stress application

and we investigated the response at ultrastructural level by visualizing the accumu-

lation and intracellular distribution of H2O2 by transmission electron microscopy

(TEM).

Our results confirm that excess of copper is a potent growth inhibitor. The pres-

ence of 50 µM or 75 µM of CuSO4 in the nutrient medium inhibited the production

of fresh and dry matter and reduced root elongation. The inhibition of growth can be

explained by the activation of peroxidases. These enzymes are involved in the degra-

dation of the endogenous indole-3-acetic acid [12] and in the lignification process

and cell wall cross linking [9].

Accordingly, our results showed the inhibition of growth in bean roots accompa-

nied by an accumulation of copper in both soluble and cell wall fractions. The data

show that copper level increased when Cu amounts increased in nutrient solution.

Also, we noted that the cell wall was the location of the highest Cu accumulation.

Almost half of absorbed copper was fixed to the cell wall. O’Neill et al. [23] showed

that the insoluble copper in cell wall must be bound to a polymeric structural com-

ponent. They suggested that one possible class of binding sites in the primary cell

wall is that comprising acidic polysaccharides, particularly pectins. But pectins do

not bind all the Cu2+ ions [22, 28, 37]. Copper can also be fixed on neutral polysac-

charides, like xyloglucan [17, 27]. Some authors suggested that Cu2+ can bind to cell

wall glycoprotein rich in His and/or Cys residues [31] and affinity for extensins has

been proposed [3, 7].

Fry et al. [11] suggested that the fixation of Cu2+ to the cell wall is likely to act as

a site-specific pro-oxidant, leading to the cleavage of polysaccharide chains. The

accumulation of copper in the cell wall and in the soluble fraction of roots induced

oxidative stress as is evident from increased levels of malondialdehyde (MDA) and

hydrogen peroxide (H2O2). MDA, a cellular indicator of lipid peroxidation was used

to assess oxidative stress. MDA and H2O2 accumulation increased with higher cop-

per concentration in the nutrient medium. The overproduction of H2O2 and accumu-

lation of MDA constitutes two of the detectable metabolic responses to the abiotic

stress especially to heavy metals stress.

Consistent with our results, some authors have reported that copper induced per-

oxidation of polyunsaturated fatty acids in membrane lipids and the generation of

malondialdehyde [19]. Lipid peroxidation process in plants can be initiated by

enzymes such as an á-dioxygenase, peroxidases or lipoxygenases [21].

The release of hydrogen peroxide radicals during abiotic stress is a characteristic

of oxidative stress. The H2O2 produced can play the role of a local signal leading to

cellular death. It can, in the same way, be diffused through the cells causing a change

in the redox status of the cellular membranes and their polarity; this would stimulate

the antioxidant mechanisms of defense [34].

The cytochemical localization of cerium perhydroxide precipitates in Cu-treated

bean roots demonstrated that H2O2 accumulation only occurred in the apoplastic

spaces and it coincided with the increase of the activity of soluble and ionic NADH-

oxidase and coniferyl alcohol peroxidase. The increase of these enzyme activities can

be explained by activation of lignification process in response to cupric stress [9, 18,

19]. In fact, NADH-oxidase could be the primary site of H2O2 generation [4, 26].

The stimulation of soluble and ionic guaiacol and coniferyl alcohol peroxidase

activities can be explained by the role of these enzymes in the elimination of hydro-

gen peroxide. Since these enzymes were reported to reduce H2O2 using phenolic

compounds as electron donors [2, 18, 24, 29]. The hydrogen peroxide would consti-

tute a signal of transduction. It was suggested that the increase in the content of this

radical stimulates the oxidative polymerization of certain organic defense molecules

in the cell wall which form a mechanical barrier towards the stress inducing agent

[33].

In conclusion, the present work has shown that the presence of excess Cu causes

oxidative stress in bean roots and subsequent increase in antioxidant responses due

to increased production of H2O2 and MDA. Accordingly, it was observed that excess

Cu in roots led to lipoperoxidation and production of H2O2 inducing changes in the

activities of soluble and ionic GPX, ACPX and NADH oxidase.

REFERENCES

1. Apel, K., Hirt, H. (2004) Reactive oxygen species: metabolism, oxidative stress, and signal traduc-

tion. Plant Biol. 55, 373–399.

2. Baccouche, S., Chaoui, A., El Ferjani, E. (1998) Nickel induced oxidative damage and antioxidant

responses in Zea mays shoots. Plant Physiol. Biochem. 36, 689–694.

3. Biggs, K. J., Fry, S. C. (1990) Solubilization of covalently bound extensin from capsicum cell walls.

Plant Physiol. 92, 197–204.

4. Bolwell, G. P. (1999) Role of active oxygen species and nitric oxide in plant defence response. Curr.

Opinion Plant Biol. 2, 287–294.

5. Bolwell, G. P., Wojatzek, P. (1997) Mechanisms for the generation of reactive oxygen species in plant

defence – a abroad perspective. Physiological and Molecular Plant Pathology. 51, 347–366.

6. Bolwell, G. P., Bindschedler, L. V., Blee, K. A., Butt, V. S., Davies, D. R., Gardner, S. L., Gerrish,

C., Minibayeva, F. (2002) The apoplastic oxidative brust in response to biotic stress in plants: a three-

component system, Journal of Experimental Botany 53, 1367–1376.

7. Bown, D. P., Bolwell, G. P., Gatehouse, J. A. ( 1993) Characterization of potato (Solanum tuberosum

L.) extensine- a novel extensin –like cDNA from dormant tubers. Gene 134, 229–233.

8. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of

protein utilizing the principal of protein-dye binding. Anal. Biochem. 72, 248–258.

Oxidative stress in bean roots 243

Acta Biologica Hungarica 59, 2008

244 H. BOUAZIZI et al.

Acta Biologica Hungarica 59, 2008

9. Chen, E. L., Chen, Y. A., Chen, L. M., Liu, Z. H. (2002) Effect of copper on peroxidase activity and

lignin content in Raphanus sativus. Plant Physiol. Biochem. 40, 439– 444.

10. Fielding, J. L., Hall, J. L. (1978) A biochemical and cytochemical study of peroxidase activity in

roots of Pisum sativum. J. Exp. Bot. 29, 979–986.

11. Fry, S. C., Miller, J. G., Dumville, J. C. (2002) A proposed role for copper ions cell wall loosening.

Plant and Soil 247, 57–67.

12. Gazaryon, I. G., Chubar, T. A., Mareeva, E. A., Lagrimini, L. M., Van Huystee, R. B., Thorneleley,

R. N. F. (1999) Aerobic oxidation of indole-3-acetic acid oxidation by peanut peroxidase.

Phytochemistry 51, 175–186.

13. Gupta, M., Cuypers, A. J., Vanngrosveld, Clijsters, H. (1990) Copper affects the enzymes of the

ascorbate-glutathione cycle and its related metabolites in the roots of Phaseolus vulgaris. Plant phys-

iol. 64, 418–423.

14. Halliwell, B., Gutteridge, J. M. C. (1984) Oxygen toxicity, oxygen radicals, transition metals and dis-

ease. Biochem. J. 219, 1–14.

15. Halliwell, B., Gutteridge, J. M. C. (1990) Role of free radicals and catalytic metalions in human dis-

ease; an overview. Meth.Enzymol. 186, 1–85.

16. Ishida, A., Ookubo., Ono, K. (1987) Formation of hydrogen peroxide by NAD(P)H oxidation with

isolated cell wall-associated peroxidase from cultured liverwort cells, Marchantia polymorpha L.

Plant Cell Physiol. 28, 723–726.

17. Jones, J. K. N., Stoodley, R. J. (1965) Fractionation using copper complexes, in: Methods in

Carbohydrate Chemistry, Vol 5. (Eds). RL Whistler, Academic Press, New York, pp 36–38.

18. Jouili, H., El Ferjani, E. (2003) Changes in antioxidant and lignifying enzyme activities in sunflower

roots (Helianthus annuus L.) stressed with copper excess, C. R. Biologies. 326, 639–644.

19. Jouili, H., El Ferjani, E. (2004) Effect of copper excess on superoxide dismutase, catalase and per-

oxidase activities in sunflower seedlings (Helianthus annuus L.). Acta physiologiae Plantarum 26,

29–35.

20. Mazhoudi, S., Chaoui, A., Gorbal, M. H., El Ferjani, E. (1997) Response of antioxidant enzymes to

excess copper in tomato (Lycopersion esculentum, Mill.). Plant Sci. 127, 182–186.

21. Mithöfer, A., Schulze, B., Boland, W. (2004) Biotic and heavy metal stress response in plants: evi-

dence for common signals, FEBS Letters. 566, 1–5.

22. Nikdel, S., Mackellar, D. G., Rezaaiyan, R. (1991) Analysis of mineral-content and amount of chelat-

ed minerals in citrus juice by inductively coupled plasma emission-spectroscopy. J. Agric. Food

Chem. 39, 1775–1775.

23. O’Neill, M., Albersheim, P., Darvill, A. G. (1990) The pectic polysaccharides of primary cell walls.

In: Ed. P. M. Dey. Methods in Plant Biochemistry. 2. Academic Press London, pp. 415–441.

24. Pandolfini, T., Gabbrielli R., Comparini, C. (1992) Nickel toxicity and peroxidase activity in

seedlings of Triticum aestivum L. Plant, Cell Environm. 15, 719–725.

25. Pedereńo, M. A., Barcelo, A. R., Sabatter, F., Munoz, R. (1989) Control by pH of cell wall peroxi-

dase activity involved in lignification. Plant Cell Physiol. 30, 237–241.

26. Ranieri, A., Castagna, A., Pacini, J., Baldan, B., Mensuali, S. Soldatin, G. F. (2003) Early production

and scavenging of hydrogen peroxide in the apoplast of sunflower plants exposed to ozone. Journal

of Experimental Botany 54, 2529–2540.

27. Rao, P. S. (1959) Tamarind. In Industrial Gums. Ed. R. L. Whistler. Academic Press New York, pp.

461–504.

28. Schlemmer, U. (1989) Studies of the binding of copper, zinc and calcium to pectin, alginate, car-

rageenan and gum in HCO3– CO2 buffer. Food Chem. 32, 223–234.

29. Schützendübel, A., Nikolova, I. P., Rudolf, C., Polle, A. (2002) Cadmium and H2O2-induced oxida-

tive stress in Populus×canescens roots. Plant Physiol. Biochem. 40, 577–584.

30. Sergiev, I., Alexieva, V., Karanov, E. (1997) Effect of spermine, atrazine and combination between

them on some endogenous protective systems and stress markers in plants. Compt. Rend. Acad. Bulg.

Sci. 51, 121–124.

31. Sommer-Knudsen, J., Bacic, A. (1997) A micro-scale method for determining relative metal-binding

affinities of proteins. Mol. Biotechnol. 8, 215–218.

32. Van Assche, F., Clijsters, H. (1990) Effects of metals on enzyme activity in plants. Plant Cell

Environ. 13, 195–206.

33. Velikova, V., Tsonev, T., Yordanov, T. (1999) Light- and CO2-responses of photosynthesis and

chlorophyll fluorescence characteristics in bean plants after simulated acid rain. Physiol. Plant. 107,

77–83.

34. Velikova, V., Yordanov, I., Edreva, Q. (2000) Oxidative and some antioxidant systems in acid rain-

treated bean plants. Protection role of exogenous polyamines, Plant Science 151, 59–66.

35. Vranová, E., Inzé, D., Van Breusegem, F. (2002) Signal transduction during oxidative stress, J. Exp.

Botany 53, 1227–1236.

36. Wang, H., Shan, X. B. Wen., Zhang, S., Wang, Z. (2004) Responses of antioxidative enzymes to

accumulation of copper in a copper hyperaccumulator of Commoelina communis, Archive of

Environmental Contamination and Toxicology 47, 185–192.

37. Wucherpfennig, K. (1992) Removing iron and copper ions out of beverages complexing agents.

Deut. Lebensm.-Rundschau. 88, 313–317.

38. Yruela, I. (2005). Copper in plants. Braz. J. Plant Physiol. 17, 145–156.

Oxidative stress in bean roots 245

Acta Biologica Hungarica 59, 2008