Differential effect of equal copper, cadmium and nickel concentration on biochemical reactions in wheat seedlings

ARTICLE IN PRESS Ecotoxicology and Environmental Safety 73 (2010) 996–1003

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Differential effect of equal copper, cadmium and nickel concentration on biochemical reactions in wheat seedlings Ewa Gajewska n, Maria Sk"odowska ´dz´, Banacha 12/16, 90-237 Ło ´dz´, Poland Department of Plant Physiology and Biochemistry, University of Ło

a r t i c l e in f o

a b s t r a c t

Article history: Received 8 December 2009 Received in revised form 5 February 2010 Accepted 8 February 2010 Available online 4 March 2010

Influence of 75 mM copper (Cu), cadmium (Cd) and nickel (Ni) on growth, tissue metal accumulation, non-protein thiols (NPT) and glutathione (GSH) contents, membrane damage, lipid peroxidation and protein oxidation as well as protease, glutathione S-transferase (GST) and peroxidase (POD) activities were studied in the shoots and roots of wheat seedlings after 7 days of metal exposure. The greatest growth reduction was found in response to Cu treatment; however accumulation of this metal in the wheat tissues was the lowest compared to the other metals used. All metals caused enhancement of electrolyte leakage from cells as well as increased lipid peroxidation and protein carbonylation. Proteolytic activity was enhanced only in Cu-exposed seedlings and in the roots it coincided with elevated protein carbonylation. The most pronounced increase in POD activity in the shoots was found after Ni treatment while in the roots in response to Cu. In contrast to Cu, application of Cd and Ni resulted in accumulation of NPT and induction of GST activity, which suggested involvement of these mechanisms in metal tolerance in wheat. & 2010 Elsevier Inc. All rights reserved.

Keywords: Cadmium Copper Glutathione S-transferase Non-protein thiols Nickel Oxidative stress Peroxidase Wheat

1. Introduction Excess concentrations of heavy metals, both being micronutrients, such as Cu or Ni, and non-essential elements, such as Cd, are toxic for plants. Although influence of metals on plants has been extensively studied the mechanisms of their phytotoxicity are not fully understood. A growing body of evidence indicates that heavy metal toxicity may be related, at least partly, to oxidative stress (Dietz et al., 1999). In a cell oxidative stress manifests itself in the elevated concentrations of oxidatively injured macromolecules such as lipids and proteins. Lipid peroxidation considerably increases permeability of cell membranes leading to the alterations in their physiological functions and consequently to their damage (Stark, 2005). Protein carbonylation involves the modification of the side chains of certain amino acids resulting in the production of ketone or aldehyde derivatives. Formation of carbonyl groups alters the biological role of proteins and in the case of enzymatic ones it often leads to the inhibition of their activity. Protein carbonylation, due to its irreversibility, is considered to be one of the most serious modifications of the cell macromolecules (Møller et al., 2007). Plant cells may protect themselves against the excessive accumulation of abnormal or damaged oxidatively modified proteins by activation of their proteolytic system. It is considered that

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intracellular level of oxidized proteins reflects the balance between the rate of protein oxidation and degradation of oxidized proteins by proteases (Pena et al., 2008). Accumulation of oxidatively modified compounds including lipid peroxides and protein carbonyls has been reported for plants exposed to various heavy metals, both redox-active and nonredox-active ones (Kova´cˇik et al., 2008; Pena et al., 2008). Depending on their chemical nature heavy metals can induce oxidative stress in different ways. Copper, being able to catalyze the Fenton/Haber-Weiss reaction, is directly involved in the generation of hydroxyl radicals (OH.), which easily react with a variety of macromolecules causing their oxidation. Nickel, despite being a transition metal, does not seem to be an effective catalyst of the mentioned reaction due to its relatively high oxidation/ reduction potential (Leonard et al., 2004). Cadmium, a nontransition metal, is considered non-redox-active. Literature data indicate that all heavy metals, irrespective of their redox properties, may indirectly enhance reactive oxygen species level in plant cells (Dietz et al., 1999). Some metals, e.g. Cu and Cd, have been reported to stimulate oxidative stress by induction of lipoxygenase activity (Sko´rzyn´ska-Polit et al., 2006). To counteract detrimental effects of heavy metals, plants possess a complex defense system comprising a variety of metalbinding compounds, low molecular antioxidants, antioxidative enzymes, and those participating in detoxification processes. One of the most important mechanisms of plant protection against heavy metals is binding of metal ions to non-protein thiols (NPT). This group of compounds includes sulphur-rich oligopeptides

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called phytochelatins as well as glutathione (GSH), homoglutathione or free cysteine (Gupta et al., 2004). Apart from being a substrate for the synthesis of phytochelatins GSH itself has been reported to act as a heavy metal chelator (Burford et al., 2005). It is also a very important low-molecular antioxidant participating in the glutathione–ascorbate cycle, which plays a key role in the regulation of H2O2 content in plant cells. Glutathione S-transferase (EC. 2.5.1.18) catalyzes the conjugation of different electrophilic substrates to GSH. The resulting conjugates, being better water soluble and less toxic than the original compounds, are transported to a vacuole for further processing or degradation (Marrs, 1996). GSTs are considered to play an important role in detoxification of xenobiotics as well as endogenous compounds, including toxic products derived from oxidatively modified lipids and proteins (Edwards and Dixon, 2004). Marrs and Walbot (1997) proposed that in plants subjected to heavy metal stress GTS-like protein may participate in the transport of a phytochelatin–metal complex to a vacuole. Induction of GST activity has been found in response to a variety of environmental stress factors, including heavy metals (Dixit et al., 2001; Edwards and Dixon, 2004; Gajewska and Sk"odowska, 2008). Peroxidase (POD, EC 1.11.1.7.) reduces H2O2 using various reductants, e.g. phenolic compounds. This enzyme is involved in several processes of cell wall strengthening such as lignification, cross linking of hydroxyproline rich proteins and feruloylated polysaccharides (Gaspar et al., 1991). The purpose of our work was to compare the influence of equal concentration (75 mM) of metals differing in their redox potentials: Cu, Cd and Ni on wheat shoots and roots. To get better insight into the mechanisms of Cu, Cd and Ni toxicity in wheat, apart from their effect on growth and accumulation in the wheat tissues we determined the ability of these metals to cause oxidative destruction of lipids and proteins as well as to activate defense reactions, including NPT production and induction of protease, GST and POD activities.

997

temperature and electrical conductance (EC1) of liquid was determined. Then, the samples, still in the same solution, were incubated in a boiling water bath for 10 min to kill the tissue completely and conductance (EC2) was measured again. The electrolyte leakage (EL) was calculated according to the following formula: EL= (EC1/EC2)  100%. 2.4. Lipid peroxidation The quantity of lipid peroxidation products was measured in terms of thiobarbituric acid reacting substances (TBARS) content according to the modified method of Yagi (1976). The samples were homogenized (1:10 w/v) in a mortar with 50 mM sodium phosphate buffer pH 7.0. The obtained homogenate (1 cm3) was mixed with 1 cm3 of TBA solution (29 mM TBA in 8.75 M acetic acid) and heated at 95 1C for 1 h. After cooling, 3.5 cm3 of n-butanol was added and the tubes were vigorously shaken. After centrifugation (10,000  g, 10 min) the fluorescence of the resulting organic layer was measured at 531 nm (excitation) and 553 nm (emission) using Hitachi fluorescence F-2500 spectrometer (Hitachi Ltd., Tokyo, Japan). The concentration of TBARS was estimated by referring to a standard 1,1,3,3-tetraetoxypropane. The level of lipid peroxides was expressed in nmoles of TBARS per g FW. 2.5. Protein oxidation Protein oxidation, given as protein carbonyl groups (CO) content, was measured by reaction with 2,4-dinitrophenylhydrazine (DNPH) according to Levine et al. (1990). The samples were homogenized (1:5 w/v) in a mortar with 50 mM potassium phosphate buffer pH 7.5 containing 1 mM EDTA, 2 mM dithiothreitol, 0.2% (v/v) Triton X-100 and 1 mM phenylmethane sulphonyl fluoride (PMSF). After centrifugation (20,000  g, 20 min) the supernatant was incubated with 1% (w/v) streptomycin sulphate for 15 min to remove nucleic acids. After centrifugation (6000  g, 10 min) the obtained supernatant (0.4 cm3) was mixed with 0.6 cm3 10 mM DNPH in 2 M HCl. The blank was incubated with 2 M HCl. After 1 h of incubation at room temperature proteins were precipitated with 10% (w/v) trichloroacetic acid (TCA). The pellets were washed 3 times with ethanol:ethyl acetate (1:1) mixture to remove free DNPH. The final protein pellets were dissolved in 6 M guanidine hydrochloride in 20 mM potassium phosphate pH 2.3 for 30 min at 37 1C. After centrifugation (6000  g, 10 min) absorbance was measured at 370 nm using UV–vis Helios Gamma spectrophotometer (Thermo Electron Corp., Cambridge, UK); the same equipment was used in all other spectrophotometric assays. Protein recovery was estimated for each sample by measuring the absorption at 280 nm. Carbonyl groups content was calculated using a molar absorption coefficient for aliphatic hydrazones (22 mM  1 cm  1) and expressed in nmoles of CO per mg protein.

2. Material and methods

2.6. Non-protein thiols content

2.1. Plant material and growth conditions

NPT content was assayed using 5,50 -dithiobis-(2-nitrobenzoic acid) (DTNB) according to Israr et al. (2006). Fresh tissue was homogenized (1:10 w/v) in a mortar with 5% sulphosalicylic acid and the homogenate was centrifuged at 20,000  g for 20 min. The assay mixture (1.65 cm3) consisted of the obtained supernatant, 0.1 M sodium phosphate buffer pH 7.0, 0.5 mM EDTA and 0.25 mM DTNB. After 10 min of incubation at room temperature the absorbance was measured at 412 nm. The concentration of NPT was estimated by referring to a standard curve prepared using GSH and expressed in mmoles per g FW. Total glutathione content was determined according to the method of Brehe and Burch (1976). Fresh tissue was homogenized (1:10 w/v) in an ice-cold mortar with 50 mM sodium phosphate buffer pH 7.0 containing 1 mM EDTA and the homogenate was centrifuged at 20,000  g for 20 min. The reaction mixture (1.5 cm3) consisted of 100 mM sodium phosphate buffer pH 7.2, the obtained supernatant, 4.86 mM EDTA, 0.02% (w/v) bovine serum albumin, 0.227 mM DTNB, 0.2 mM NADPH and 0.05 U glutathione reductase. The reaction was run for 15 min and the absorbance was measured at 412 nm. GSH concentration was expressed in mmoles per g FW.

Seeds of wheat (Triticum aestivum L., cv. Zyta) provided by Hodowla Ros´lin Strzelce Sp. z o.o., Poland, were germinated in Petri dishes for 2 days. Then 30 seedlings were transferred into 330 cm3 of the diluted (1:4) Hoagland nutrient solution (Hoagland and Arnon, 1950) containing 75 mM Cu, Cd or Ni, supplied as sulphates. The seedlings grown in the nutrient solution not supplemented with the metals served as control. The seedlings were grown in a controlled climate room at 24 1C with 16-h photoperiod (175 mmol m  2 s  1 PPDF). After 7 days the shoots and roots were harvested and their length, Cu, Cd, Ni, NPT and GSH contents, electrolyte leakage, lipid peroxidation and protein oxidation as well as proteolytic activity and the activities of GST and POD were estimated. 2.2. Estimation of Cu, Cd and Ni concentrations Metal contents in the wheat shoots and roots were determined by atomic absorption spectrometry using Varian SpectrAA 300 spectrometer (Varian Australia Pty. Ltd., Mulgrave, Australia) equipped with a deuterium lamp for background correction and an air/acetylene flame. Concentrations of Cu, Cd and Ni were estimated at 324.8, 228.8 and 232 nm, respectively. Samples were prepared by wet digestion of oven-dried tissue in HNO3:HClO4 (4:1, v/v) solution at 140 1C. Metal concentrations in wheat tissues were expressed in mg per g DW. 2.3. Estimation of cell electrolyte leakage Extent of membrane damage was evaluated indirectly by conductometric measurement of solute leakage from cells according to the method of Masood et al. (2006) with some modifications. Samples of shoot and root tissue (0.2 g) were washed in deionised water and then placed in flasks containing 20 cm3 of deionised water. The samples were shaken gently (100 rpm) for 2 h at room

2.7. Proteolytic activity Proteolytic activity was assayed with azocasein as a substrate as described by Wang et al. (2004) with some modifications. Plant tissue was homogenized (1:5 w/v) in a mortar using 50 mM Tris–HCl buffer pH 7.5 containing 1 mM dithiotreitol and 5 mM b-mercaptoethanol. After centrifugation (20,000  g, 20 min) the obtained supernatant was used for the determination of proteolytic activity. The reaction mixture (1.2 cm3) consisted of 50 mM Tris–HCl buffer pH 7.5, 0.075% (w/v) azocasein and the supernatant. After 4 h of incubation at 37 1C the reaction was stopped by the addition of 0.6 cm3 10% TCA. Then the samples were centrifuged (4000  g, 10 min) and absorbance of the supernatant was measured at 366 nm. Reference samples in which TCA was added before the addition of the supernatant were run simultaneously. The proteolytic activity was expressed in

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units, each representing the amount of enzyme causing an increase in absorbance by 0.01 per 1 h.

Table 2 Concentrations of Cu, Cd and Ni in wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. Data are means7 SD, n =5.

2.8. POD and GST extraction and assays Treatment Fresh tissue was homogenized (1:10 w/v) in an ice-cold mortar using 50 mM sodium phosphate buffer pH 7.0 containing 1 mM EDTA and 1% polyvinylpyrrolidone. After centrifugation (20,000  g, 20 min) the supernatant was used for the determination of POD and GST activities. POD activity was measured by the method of Maehly and Chance (1954). The assay mixture contained 50 mM sodium acetate buffer pH 5.6, 5.4 mM guaiacol, 15 mM H2O2 and the enzyme extract. The increase in absorbance due to the oxidation of guaiacol to tetraguaiacol (e = 26.6 mM  1 cm  1) was monitored for 4 min at 470 nm. Reference samples lacking H2O2 were run simultaneously. POD activity was expressed in units, each representing the amount of enzyme catalyzing the formation of 1 mmole of tetraguaiacol per minute. GST activity was measured by the method of Habig et al. (1974) using CDNB (1-chloro-2,4-dinitrobenzene) as a substrate. The reaction mixture contained 100 mM potassium phosphate buffer pH 6.25, 0.75 mM CDNB, 30 mM GSH and the enzyme extract. The increase in absorbance due to the formation of the conjugate (e = 9.6 mM  1 cm  1) between GSH and CDNB was monitored at 340 nm. The reaction mixture without the enzyme extract was used as blank. GST activity was expressed in units, each representing the amount of enzyme catalyzing the formation of 1 nmole of S-conjugates per minute. Protein content in the samples was determined by the method of Bradford (1976), with a standard curve prepared using bovine serum albumin.

3. Results 3.1. Growth and metal accumulation Treatment with the heavy metals resulted in a significant decrease in wheat growth. After application of Cu, Cd and Ni shoot length was reduced by 51%, 48% and 33%, respectively (Table 1). Roots showed much higher sensitivity to the metals used exhibiting decrease in length by 91%, 63% and 72%, respectively. Additionally, browning of the roots was observed in the case of Cu-treated seedlings (data not shown). Root length to shoot length ratio was reduced by 81%, 29% and 59% after Cu, Cd and Ni treatments, respectively (Table 1). Growth inhibition was accompanied by a considerable accumulation of the metals in the tissues of wheat seedlings (Table 2). The lowest tissue metal accumulation was found in the case of seedlings treated with Cu. Concentration of this metal in the wheat shoots and roots was about 3 and 42 times higher than that in the control, respectively. The most marked increase in the tissue metal content was observed after treatment with Cd, whose concentration in the shoots and roots over 1240 and 1900 times exceeded that of the control, respectively. In the seedlings exposed to Ni, concentration of this metal in the shoots and roots was 47 and 309 times higher Table 1 Growth of wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. Data are means 7 SD, n= 20. Treatment

Shoot length (mm)

Root length (mm)

Root/shoot ratio

Control Cu Cd Ni

150.07 17.4 73.57 7.3nnn 78.37 6.1nnn 101.27 9.2nnn

420 7 45.2 40 7 3.8nnn 156 7 16.1nnn 116 7 12.4nnn

2.80 0.54 1.99 1.15

nnn

indicate values that differ significantly from the control at P o 0.001.

Root metal content (mg g  1 DW)

Cu Control Cu-treated

7.48 70.65 21.49 71.04nnn

14.60 7 1.08 618.597 59.12nnn

Cd Control Cd-treated

0.234 70.035 290.84 725.73nnn

0.889 7 0.099 1717.597 110.51nnn

Ni Control Ni-treated

2.46 70.15 115.35 715.88nnn

2.95 7 0.13 910.63 7 88.02nnn

nnn

indicate values that differ significantly from the control at P o0.001.

Table 3 Accumulation of Cu, Cd and Ni in wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. n= 5. Treatment

Metal accumulation in shoot (% of total metal content in a seedling)

Metal accumulation in root (% of total metal content in a seedling)

Root/shoot ratio of metal content in a seedling

Cu Cd Ni

3.4 14.5 11.2

96.6 85.5 88.8

28.78 5.91 7.89

2.9. Statistical analysis The results presented are the means of 5 independent experiments. For length estimation 4 plants were measured per experiment (n= 20), for other parameters a single sample was analyzed per experiment (n= 5). Sample variability was estimated by standard deviation of the mean. The statistical differences between control and treatment mean values were determined by Student’s paired t-test. Differences at P o0.05 were considered significant.

Shoot metal content (mg g  1 DW)

than that in the control, respectively. Regardless of the metal used, most of the given metal taken up by the plant was accumulated in roots; however differences in the distribution of the absorbed metal between shoots and roots could be observed (Table 3). While Cd and Ni concentrations in the roots were approximately 6–8 higher than those in the shoots, in the case of plants treated with Cu concentration of this metal in the roots almost 29 times exceeded that in the above-ground parts, constituting 96.6% of the total amount of Cu taken up by the seedlings.

3.2. Electrolyte leakage, oxidative stress and proteolytic activity Metal treatment considerably enhanced electrolyte leakage from the wheat cells, indicating increased permeability of cellular membranes (Fig. 1). In the shoots the most pronounced augmentation of electrolyte leakage, by 127%, was found in response to Cu application. Cd and Ni induced similar enhancement of electrolyte leakage from the wheat shoots, by 27% and 36%, respectively. In the roots all metals used caused approximately 30% increase in electrolyte leakage. Exposure of the wheat seedlings to the metals induced oxidative stress, which was evidenced by increased oxidation of lipids and proteins. In the shoots the most pronounced lipid peroxidation was found after treatment with Cu and it was higher than in the control by 111% (Fig. 2). Application of Cd and Ni resulted in the enhancement of TBARS content by 44% and 61%, respectively. In the roots all metals similarly influenced lipid peroxidation causing its increase by 25–30% in comparison to the control. In the shoots, regardless of the metal used, 30–40% enhancement of protein carbonylation was observed (Fig. 3). However, in the roots statistically significant increase in protein carbonyl groups content was found only after application of Cu and it exceeded the control value by 34%.

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12

180 Shoots

Shoots

10

150

8

6 **

**

4

2

TBARS content (nmol g-1 FW)

*** Electrolyte leakage (%)

999

*** 120 ***

* 90

60

30 0 Control

Cu

Cd

Ni

0

18

Control Roots

15

Cd

Ni

Roots **

**

**

12

40 TBARS content (nmol g-1 FW)

Electrolyte leakage (%)

Cu

50

9

6

3

*

* *

30

20

10 0 Control

Cu

Cd

Ni

Fig. 1. Electrolyte leakage from wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. Bars represent SD of means. n =5. nn, nnn indicate values that differ significantly from the control at Po 0.01 and P o0.001, respectively.

0 Control

Cu

Cd

Ni

Fig. 2. Lipid peroxidation in wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. Bars represent SD of means. n= 5. n, nnn indicate values that differ significantly from the control at P o0.05 and P o 0.001, respectively.

Proteolytic activity increased by 81% and 126% in the Cuexposed wheat shoot and roots, respectively, while it remained unaltered in the seedlings subjected to Cd and Ni stress (Fig. 4).

3.3. Non-protein thiols content Treatment with Cd resulted in over 2.5-fold and 10.5-fold increase in total NPT content in the wheat shoots and roots, respectively (Fig. 5). Application of Cu did not significantly influence total NPT concentration in either of the studied organs, while Ni stress led to over 2-fold enhancement of these compounds level in the roots. Apart from NPT concentration we also assayed the total content of GSH, which in the control shoots and roots constituted almost 60% of the total NPT. Treatment with Cd led to significant decrease in GSH concentration in the shoots, by 56% compared to the control (Fig. 5). On the contrary, exposure to Cu and Ni did not alter GSH content in these organs. In the roots application of Cd and Ni resulted in almost 5-fold and 2.4-fold increase in GSH content, while Cu did not influence its level.

3.4. GST and POD activities In both organs GST activity was not influenced by Cu treatment (Fig. 6). On the contrary, Cd and Ni induced GST activity in the shoots by 139% and 163% and in the roots by 207% and 94%, respectively. In the shoots POD activity exhibited over 5.5-fold and 11-fold enhancement after exposure to Cd and Ni, respectively (Fig. 7). In the roots statistically significant induction of POD activity, by 74% and 33%, was observed after treatment with Cu and Cd, respectively.

4. Discussion The most common response of plants to stress conditions, including excess concentrations of heavy metals, is restriction of growth. Plant tolerance to heavy metals is usually estimated on the basis of the degree of their root or shoot growth inhibition by the metal present in a nutrient solution (Michalak and Wierzbicka,

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20

8 Shoots

CO content (nmol mg-1 protein)

**

**

**

12

8

4

Proteolytic activity (U mg-1 protein)

Shoots

16

6 **

4

2

0

0 Control

Cu

Cd

Control

Ni

18

Cd

Ni

Cd

Ni

Roots

Roots Proteolytic activity (U mg-1 protein)

15 CO content (nmol mg-1 protein)

Cu

80

* 12

9

6

***

60

40

20

3 0 Control

0 Control

Cu

Cd

Ni

Fig. 3. Protein oxidation in wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. Bars represent SD of means. n= 5. n, nn indicate values that differ significantly from the control at P o 0.05 and Po 0.01, respectively.

1998; Wang and Zhou, 2005). Treatment of the wheat seedlings with equal concentration of Cu, Cd and Ni revealed differential sensitivity of the cultivar used, Zyta, to these three metals. Judging from the extent of length reduction after 7 days of metal exposure Cu appeared to be the most toxic, causing the greatest decrease in shoot and root lengths. Copper-induced inhibition of shoot growth was only slightly more pronounced than that caused by Cd. However, in the roots Cu led to over 90% reduction in length. Our observations are consistent with those of Wang and Zhou (2005), who also reported higher sensitivity of wheat to Cu compared to Cd. Literature data indicate that tolerance to heavy metals depends on plant species studied and may be even organ-dependent. Treatment of spinach with equal Cd and Ni doses had similar inhibitory effect on its leaves growth (Mishra and Agrawal, 2006), while in our work the above-ground parts of the wheat seedlings showed lower sensitivity to Ni compared to Cd. Shoots of cabbage exposed to equal Cd and Ni concentrations responded similarly

Cu

Fig. 4. Proteolytic activity in wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. Bars represent SD of means. n= 5. nn, nnn indicate values that differ significantly from the control at P o 0.01 and Po 0.001, respectively.

to wheat, while cabbage roots showed higher tolerance to Ni, which was in contrast to our observations for wheat (Pandey and Sharma, 2002). Results of our study showed that there was no relation between the amount of the uptaken metals and their inhibitory effect on wheat growth. The greatest decrease in length found in the Cu-treated seedlings was accompanied by the lowest accumulation of this metal in the wheat tissues, compared to Ni and Cd. All the metals exhibited low translocation to aboveground parts of the wheat seedlings being mostly retained by the roots. This difference in distribution of the uptaken metals between root and shoot was probably responsible for differential growth inhibition of these organs. Regardless of the metal used, growth reduction was more pronounced in roots than in shoots, which was reflected by considerably decreased values of the root/ shoot length ratio. Moreover, it could be noticed that decline in the root/shoot length ratio was related to increase in the root/ shoot ratio of the given metal concentration in the seedling.

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1001

250 Total NPT

Shoots

Shoots

*

200 GST activity (U mg-1 protein)

NPT content (µmol g -1 FW)

3

Total GSH

*** 2

1

***

150

100

50 *** 0 Control

Cu

Ni

0 Control

8

Roots

Cu

Cd

Ni

600 Roots

*** 500

6

**

4

2

***

*** ***

GST activity (U mg-1 protein)

NPT content (µmol g -1 FW)

Cd

400

**

300

200

100

0 Control

Cu

Cd

Ni

Fig. 5. Total non-protein thiols and total glutathione contents in wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. Bars represent SD of means. n= 5. nnn indicate values that differ significantly from the control at P o0.001.

The lowest root/shoot length ratio was found in Cu-treated seedlings, which exhibited the highest root/shoot metal content ratio. To estimate the extent of oxidative stress in the metal-treated wheat seedlings we determined the concentrations of TBARS and protein carbonyls indicating the degree of lipid and protein oxidation, respectively. The results revealed that all metals used in our experiment caused oxidative stress; however its intensity was only partly associated with the chemical nature of the metals. Copper, exhibiting the highest redox activity, was the only metal inducing protein carbonylation in the wheat roots. Similarly, in the shoots, the most pronounced lipid peroxidation was detected in response to Cu application. However, no such relation was found considering the level of protein carbonylation in the shoots and lipid peroxidation in the roots, where all three metals showed a similar effect. Contrary to the wheat shoots, in the case of sunflower leaves much higher increase in protein carbonyls level was found after treatment with Cu than Ni used at the same dose of 100 mM (Pena et al., 2008). In contrast to wheat roots, those of chamomile plants treated with equal concentration of Cu, Cd and Ni (120 mM) showed increased lipid peroxidation only after Cu exposure (Kova´cˇik et al., 2006, 2008, 2009). Apart from the

0 Control

Cu

Cd

Ni

Fig. 6. Glutathione S-transferase activity in wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. Bars represent SD of means. n= 5. n, nn, nnn indicate values that differ significantly from the control at Po 0.05, P o 0.01 and Po 0.001, respectively.

unclear relation between the intensity of oxidative damage found in the wheat seedlings and redox properties of the metals used, our experiment also showed that the intensity of oxidative stress was independent of concentration of the uptaken metals and was not closely associated with the degree of growth inhibition. Heavy metals have been reported to disturb structure and functioning of cell membrane, which was ascribed to the oxidative modification of macromolecules, mainly lipids, as well as the metalmediated alterations in membrane lipid composition (Quartacci et al., 2001; Liu et al., 2004). In our experiment the degree of cell membrane damage estimated on the basis of electrolyte leakage from the cells was similar to the changes in TBARS content, which suggested that increased membrane permeability resulted mostly from lipid peroxidation. In the roots of Cu-exposed wheat enhanced protein carbonylation coincided with induction of protease activity, while in the case of the other metals used neither accumulation of carbonylated proteins nor activation of proteolysis was observed in these

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12 Shoots

POD activity (U mg-1 protein)

10

**

8

6 ** 4

2

0 Control

Cu

Cd

Ni

24 Roots

POD activity (U mg-1 protein)

20 *

16

12 ** 8

4

0 Control

Cu

Cd

Ni

Fig. 7. Peroxidase activity in wheat shoots and roots after 7 days of treatment with 75 mM Cu, Cd and Ni. Bars represent SD of means. n= 5. n, nn indicate values that differ significantly from the control at P o 0.05 and Po 0.01, respectively.

organs. This could suggest involvement of proteases (slightly basic, pH 7.5) in the protection of wheat roots against Cu-generated oxidative stress. However, in the shoots where all the metals used led to comparable increase in protein carbonyl groups content proteolysis was induced exclusively after treatment with Cu. Similarly, no tight correlation between the degree of protein oxidation and protease activity was found in sunflower cotyledons exposed to heavy metals, including Cu, Cd and Ni (Pena et al., 2006). Pena et al. (2008) demonstrated that various fractions of proteases may differently respond to heavy metal stress. In the Cu-treated sunflower leaves exhibiting elevated level of carbonylated proteins these authors found increased basic (pH 8) protease activity, while acid (pH 5.5) and neutral (pH 7) protease activities were significantly lower compared to the control. Increased synthesis of non-protein thiol compounds is a common defense response of plants to heavy metal stress. Cadmium is considered to be the most effective inductor of the

accumulation of NPT, mostly phytochelatins (Gupta et al., 2004). In the wheat shoots only this metal caused increment in the total NPT concentration. It was accompanied by a significant decline in total GSH content, which may suggest that other thiols, possibly phytochelatins, were responsible for the observed accumulation of total NPT. An increase in NPT concentration together with depletion of glutathione pool under Cd stress has been previously reported (Romero-Puertas et al., 2007). Lowered GSH content might have been related to its utilization in phytochelatin synthesis as well as in the defense reactions mediated by GSHdependent enzymes, e.g. GST, which showed enhanced activity in Cd-treated wheat shoots. Compared to the shoots, wheat roots responded to Cd stress with much higher accumulation of NPT, which seemed to be partly due to increased synthesis of GSH, whose concentration increased after Cd application. However, since enhancement of total NPT largely exceeded that of GSH, content of other thiol compounds must have also increased in Cd-treated wheat roots. Increment in NPT content in the wheat roots was also found in response to Ni application. Contrary to the Cd-treated wheat seedlings in the case of the Ni-exposed ones GSH appeared to be the main thiol accumulated in the roots. It has been previously shown that Ni was a relatively weak activator of phytochelatin synthesis and phytochelatin-based sequestration is rather not essential for the detoxification of this metal (Schat et al., 2002; Gupta et al., 2004). In contrast to Cd and Ni, Cu application did not induce accumulation of NPT in the wheat seedlings. It might have been related to the relatively high concentration of this metal used in our experiment. Literature data indicate that Cu is able to increase NPT content in plants only when used in low doses (Sgherri et al., 2003). Exposure of plants to higher Cu doses may even lead to decrease in the concentrations of these compounds (Rama Devi and Prasad, 1998). Application of Cd and Ni led to a marked increase in GST activity, both in shoots and in roots of wheat seedlings. Since the observed enhancement of GST activity coincided with increased lipid peroxidation, induction of this enzyme activity in the wheat seedlings could have been associated with its role in the removal of highly reactive breakdown products of lipid peroxides. As mentioned earlier, significantly elevated lipid peroxidation was also found in response to Cu exposure; however, in contrast to Cd- and Ni-treated seedlings, it was not accompanied by increment in GST activity. Therefore, it is possible that there is a relation between high sensitivity of wheat seedlings to Cu stress and lack of induction of GST activity after this metal application. The activity of non-specific peroxidase has been reported to increase in response to various stress factors, including excess concentrations of heavy metals (Pandolfini et al., 1992; Dı´az et al., 2001). The metals used in our experiment had differential effects on POD activity in wheat shoot and root. Application of Cd led to increment in POD activity in both organs; however it was much more pronounced in the shoot. Contrary to Cu, which induced POD activity only in the root, Ni caused enhancement of this enzyme activity only in the shoot. In contrast, Pandolfini et al. (1992) reported increase in POD activity in both shoot and root of Ni-treated wheat; however the former organ showed much greater induction of this enzyme activity. Peroxidase-catalyzed reactions, including lignification and cross-links formation, result in decreased cell wall plasticity and consequently in its restricted elongation. Therefore, growth inhibition observed in plants subjected to heavy metal stress has been suggested to be connected with induction of POD activity (Chen et al., 2000; Dı´az et al., 2001). In Cu-treated chamomile, reduction in root growth was accompanied by brown colouration of this organ, increased POD activity and lignin accumulation (Kova´cˇik and Bacˇkor, 2008; Kova´cˇik and Klejdus, 2008). In agreement with these findings, in our work the highest induction of POD activity in the roots found

ARTICLE IN PRESS E. Gajewska, M. Sk!odowska / Ecotoxicology and Environmental Safety 73 (2010) 996–1003

after Cu application coincided with the greatest decrease in root growth as well as browning of the root system. This would imply association between the intensity of POD-mediated processes and growth inhibition. However, no such relation was observed in the wheat shoots. The most enhanced POD activity was detected in response to the application of Ni, which caused the lowest reduction in shoot length, compared to the other metals used.

5. Conclusion In conclusion, application of equal concentration of Cu, Cd and Ni differently influenced the growth of wheat seedlings with Cu, the redox-active metal, exhibiting the greatest toxicity. There was no relation between tissue accumulation of the metals and their inhibitory effect on wheat growth; however greater reduction in root length was clearly due to the retention of the uptaken metals mostly in this organ. All the metals used induced oxidative modifications of lipids and proteins; however intensity of these processes was not tightly associated either with the degree of growth reduction or with the metal absorption by the wheat tissues. Results of our study indicate that induction of GST activity and enhanced production of NPT can play an important role in tolerance of wheat to heavy metals.

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