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Responses of seedling growth and antioxidant activity to excess iron and copper in Triticum aestivum L.
Ecotoxicology and Environmental Safety 86 (2012) 47–53
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Frontier Article
Responses of seedling growth and antioxidant activity to excess iron and copper in Triticum aestivum L. Xiaoning Li a, Haizhen Ma a, Pengxiang Jia a, Juan Wang a, Lingyun Jia a, Tengguo Zhang a, Yingli Yang a,n, Haijian Chen b, Xia Wei b a b
Life Science of College, Northwest Normal University, Lanzhou 730070, Gansu, China The Center for Spreading Agricultural Technology, Xigu District, Lanzhou 730060, China
a r t i c l e i n f o
abstract
Article history: Received 19 July 2012 Received in revised form 7 September 2012 Accepted 8 September 2012 Available online 29 September 2012
The purpose of this study was to analyze phytotoxicity mechanism involved in root growth and to compare physiological changes in the leaves of wheat seedlings exposed to short term iron (Fe) and copper (Cu) stresses (0, 100, 300 and 500 mM). All applied Fe or Cu concentrations reduced root and shoot lengths, but seed germination was inhibited by Cu only at 500 mM. Analyses using fluorescent dye 20 ,70 -dichlorodihydrofluorescein diacetate indicated enhanced H2O2 levels in seedling roots under Fe and Cu treatments. Cu stress at the same concentration induced a great reduction in cell viability and a strong damage on membrane lipid in the roots with respect to Fe treatment. Significant increases in the total chlorophyll (chl) content including chl a and chl b were observed in response to higher Fe concentrations, whereas the highest Cu concentration (500 mM) led to significant decreases in the total chl content including chl a. Additionally, leaf peroxidase (POD) and ascorbate peroxidase (APX) were stimulated by Fe stress, but the highest Fe concentration exhibited inhibitory effect on leaf APX activity. In contrast, copper treatment resulted in an elevation in leaf catalase and POD activities. Therefore, H2O2 content in the leaves associated with copper was significantly lower than that with iron at the same concentration. & 2012 Elsevier Inc. All rights reserved.
Keywords: Wheat Seed germination Seedling growth Antioxidant enzymes Lipid peroxidation
1. Introduction Enhanced industrial and mining activities have contributed to the increasing occurrence of heavy metals including iron (Fe) and copper (Cu) in ecosystems. Heavy metals are also added to soils from different human activities. Fe and Cu are essential mineral nutrients for plant growth at low concentrations, which plays key roles in photosynthetic and respiratory electron transport chains, even in ethylene sensing, cell wall metabolism and oxidative stress protection (Connolly and Guerinot, 2002; Yruela, 2009). However, excessive quantities of Fe and Cu can lead to phytotoxicity such as leaf chlorosis and growth inhibition (Bouazizi et al., 2010). The study of Mehraban et al. (2008) showed the maximal plant growth at iron concentration of 10 and 50 mg L 1 but a growth inhibition at 250 and 500 mg L 1. In general, germinating seeds are more sensitive to metal elements than mature plants, and the inhibition of the germination has become an important biomarker as a plant response to heavy-metal stress. Even though the influences of excess iron and copper on plant are investigated in a number of plant species (Nenova,
n
Corresponding author. E-mail address: [email protected] (Y. Yang).
0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.09.010
2006; Street and Kulkarn, 2007; Posmyk and Kontek, 2009), there are fewer reports on the precise mechanisms of the negative effects due to heavy metal stress. Additionally, inhibited biosynthesis of chlorophyll (chl) is a frequent symptom of metal toxicity (Van Assche and Clijsters, 1990; Sandalio et al., 2001). Iron and copper are essential for photosynthesis. Especially in plant tissues, up to 80 percent of iron and more than half of copper are found in the chloroplasts ¨ (Hansch and Mendel, 2009). However, photosynthesis is sensitive to Fe and Cu excess. Several studies have proposed that the toxicity of Fe and Cu may be explained by the reduction of chl content in plants (Shakya et al., 2008; Xing and Huang, 2010; Brahim and Mohamed, 2011). Besides, Fe and Cu excess can act catalytically via Haber–Weiss and Fenton reactions to generate ROS including superoxide, hydroxyl ¨ et al., 2001; radicals and hydrogen peroxide (H2O2) (Hegedus Connolly and Guerinot, 2002). Increased ROS accumulation within plant cells has the potential to cause oxidative injury by provoking macromolecule damage and membrane lipid oxidation, thus distributing metabolic pathways and affecting plant growth and development (Connolly and Guerinot, 2002; Posmyk and Kontek, 2009). To control the level of ROS and to protect the cells against oxidative damage initiated by ROS, some plants have established protective mechanisms such as antioxidant enzyme system. The correlation
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between antioxidant capacity and copper tolerance has been demonstrated in a large number of plants (Boojar and Goodarzi, 2007; Posmyk and Kontek, 2009). Contrary to these findings, Cu-treated leaves exhibited decreased activities of POD and APX in Brassica campestris ssp. chinensis Makino (Li et al., 2009). Apparently, the mechanisms of antioxidantive responses due to excess Fe or Cu are not yet fully understood. The study of physiological and ecological processes in response to heavy metal stresses has great significance for revealing the mechanism of plant anti-heavy metal and/or the mechanisms of the negative effects of metals. Compared to the studies of Cu phytotoxicity, to date scarce information has been reported on Fe toxicity in plants. This study aimed to analyze the mechanisms of Fe or Cu toxicity involved in the growth of wheat roots, and to evaluate the levels of chl and lipid peroxidation as well as the activities of antioxidative enzymes in the leaves of wheat seedlings exposed to short term Fe and Cu stresses.
2.5. Cell viability Wheat roots treated with different Fe or Cu concentrations were used to determine the loss of cell viability by Evans blue staining (Zanardo et al., 2009). Fresh roots were incubated for 15 min with 30 mL of 0.25 percent Evans blue solution, washed for 30 min to remove excess and unbound dye. After excised root tips (3 cm) were soaked in 3 mL of N,N-dimethylformamide for 50 min at room temperature, the absorbance of released Evans blue was measured at 600 nm, using deionized water as a blank. The loss of cell viability was expressed as the absorbance at 600 nm of treated roots in relation to untreated roots.
2.6. Chlorophyll content measurement Chl was extracted from plant leaves with 80 percent acetone, and chl content was determined spectrophotometrically at 663 and 646 nm according to Lichtenthaler (1987).
2.7. Antioxidant enzyme activities measurement
The seeds were surface-sterilized with 0.1 percent HgCl2 for 10 min, then soaked in water for 24 h. The plump and excellent seeds were plated in Petri dishes with two filter-paper discs containing 1/4 Hoagland medium supplemented with 100, 300 and 500 mM Fe or Cu from FeCl3 and CuCl2 respectively and germinated for four days at 257 1.5 1C in the darkness in an incubator (LRH-250A, made in Guangdong Medical Instruments Factory, China). Seed germination was defined as a root length of 1.0 mm or more. For root and shoot growth experiments, forty seedlings were cultivated in Petri dishes containing 6 mL of 1/4 Hoagland soluble containing 100, 300 and 500 mM FeCl3 or CuCl2 for six days at 25 72.5 1C under a light irradiance of 300 mmol m 2 s 1 (12 h light:12 h dark cycles). All assays were replicated at least three times to minimize experimental errors; each replicate was carried out on fifty seeds for germination and forty seedlings for growth measurement.
Fresh leaves were ground with chilled NaH2PO4/Na2HPO4 buffer (PBS, 50 mM, pH 7.8) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA) and one percent polyvinylpyrrolidone (PVP), centrifuged at 12,000 rpm min 1 for 30 min, and the supernatant was collected for the protein assay and the determination of CAT and POD activities. The amount of proteins was estimated according to the method proposed by Bradford (1976) using bovine serum albumin as a standard. CAT activity was analyzed according to Aebi (1984). The enzyme extraction was added to 3 mL 50 mM PBS buffer (pH 7.0). After 5 min incubation at 25 1C, the reaction was started by the addition of 6 mM H2O2 and the absorbance changes were recorded at 240 nm for 2 min. An absorbance change of 0.1 unit min 1 was defined as 1 unit (U) of CAT activity. POD activity was measured following a modification of the method of Rao and Paliyath (1996). The enzyme extraction was mixed with the reaction mixture containing 50 mM PBS (pH 7.0) and 20 mM guaiacol. After pre-incubation at 25 1C for 5 min, 6 mM H2O2 was added to initiate the reaction. Changes of the absorbance at 470 nm within 2 min were recorded for calculating POD activity. One unit of POD activity was defined as an absorbance change of 0.01 unit min 1. Plant material was ground in chilled 50 mM PBS buffer (pH 7.0) containing 1 mM EDTA-Na2 and 1 mM ascorbate (ASA). After centrifugation for 20 min at 10,000 rpm, the supernatant (enzyme extraction) was collected for the measurement of APX activity performed as described by Nakano and Asada (1981) with some modifications. A reaction mixture consist of 50 mM PBS (pH 7.0), 0.5 mM ASA, 3 mM H2O2 and the enzyme extraction, and the changes in the absorbance at 290 nm were recorded at 25 1C for 1 min after the addition of H2O2. One unit of APX activity was defined as an absorbance change of 0.1 unit min 1. The specific enzyme activity for all enzymes was expressed as U mg 1 protein.
2.3. Iron and copper content determination
2.8. Statistical analysis
1/4 Hoagland mediums supplemented with 0, 100, 300 and 500 mM FeCl3 or CuCl2 were acidified by HNO3 (one percent, v/v) and the actual concentrations of Fe or Cu were determined by flame Atomic Absorption Spectrophotometry (WFX210, China). The roots and leaves of wheat seedlings were prepared for Fe and Cu measurements according to Achary et al. (2012) with some modifications. Plant material was thoroughly washed with deionized water and dried to constant weight at 80 1C. The dry sample was dissolved in a solution containing 12 mL of concentrated nitric acid (HNO3), 4 mL hydrofluoric acid and 4 mL of H2O2 (30 percent, v/v), and was digested in a closed microwave digestion system (Multiwave 3000, Anton paar, Austria ) for 2.5 h. The chilled sample was transferred to polytetrafluoroethylene beaker, and was then evaporated to dryness. The resulting ash residue was dissolved in HNO3 (one percent, v/v), and total Fe and Cu contents were measured with atom absorption spectrophotometer.
All values were represented by an average of at least three replicate measurements7 standard error (SE). Statistical comparisons were carried out using SPSS 13.0 software, and significant differences were indicated by different letters (p o 0.05).
2. Materials and methods 2.1. Plant material Wheat (Tritium aestivum) cv. Ningchun four seeds were purchased from Gansu Agricultural University, in Lanzhou, China.
2.2. Seed germination and seedling growth experiment
2.4. Analysis of H2O2 content and lipid peroxidation The fluorescence intensity of H2O2 level in roots was visualized using a fluorescent dye 20 ,70 -Dichlorodihydrofluorescein diacetate (H2DCFDA) as described by Pei and Murata (2000). Roots were immersed in 50 mM H2DCFDA in 10 mM Tris– HCl buffer (pH 7.2) containing 50 mM KCl for 20 min, and were washed three times in Tris–HCl buffer to remove excess dye. Then, examinations of H2DCFDA fluorescence were observed using a Leica DMIRB inverted fluorescent microscope. H2O2 content in leaves was quantified by the method of Sergiev and Alexieva (1997) in homogenates prepared with 0.1 percent trichloroacetic acid as described in detail previously (Yang et al., 2011). Lipid peroxidation in roots or leaves was estimated from the amount of MDA determined by the thiobarbituric acid reaction as described in detail previously (Yang et al., 2011).
3. Results 3.1. Iron and copper contents The actual concentrations of Fe in the experimental solutions of FeCl3 prepared at 0, 100, 300 and 500 mM were 5.6, 11.2, 22.3 and 33.5 mg L 1, and the actual Cu concentrations in the experimental solutions of CuCl2 prepared at 0, 100, 300 and 500 mM were 0.006, 6.3, 19.1 and 31.7 mg L 1, respectively. In addition, iron and copper levels in the roots and leaves of wheat seedlings are shown in Table 1. Fe levels in the roots significantly elevated with increasing treatment concentrations, reaching almost 9.23-, 14.55- and 93.7-fold higher content at 100, 300 and 500 mM Fe dose, respectively. In comparison with the control, leaf Fe content exhibited an insignificant enhancement at 100 mM Fe, but Fe at 300 and 500 mM resulted in notably enhanced Fe content in the leaves. Similarly, Cu treatment led to increased Cu levels in the roots in a concentration-dependent manner, whereas compared
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Table 1 Changes of iron (mg g 1 DW) and copper (mg g 1 DW) levels in wheat seedling after treatment with different Fe and Cu concentrations. FeCl3 (iron content)
Treatment concentration (mM)
0 100 300 500
CuCl2 (copper content)
Root
Shoot
Root
Shoot
0.147 0.014a 1.33 7 0.044b 2.087 0.043c 13.40 7 0.12d
0.0647 0.008a 0.0867 0.011a 0.187 0.016b 1.187 0.029c
0.049 70.004a 0.17 70.009b 2.11 70.034c 5.29 70.076d
0.0767 0.002a 0.0837 0.003a 0.117 0.004b 0.137 0.004c
Value within each column marked with different letters means significant difference at po 0.05.
Table 2 Effects of iron and copper on seed germination and seedling growth in wheat. Treatment FeCl3 concentration (mM) Germination rate (percent) 0 100 300 500
96.67 7 0.02a 95.33 7 0.007a 96.00 7 0.01a 92.00 7 0.02a
CuCl2 Root length (cm)
Shoot length (cm)
Germination Root length rate (percent) (cm)
Shoot length (cm)
8.07 70.04a 11.15 7 0.05a 96.67 70.02a 8.07 70.04a 11.15 7 0.05a 6.84 70.03b 10.25 7 0.08a 94.67 70.01a 1.45 70.02b 8.78 7 0.14b 6.59 70.11c 9.44 7 0.08b 95.33 70.02a 0.84 70.01c 7.25 7 0.09c 3.50 70.05d 7.307 0.06c 86.00 70.01b 0.56 70.01d 6.68 7 0.05d
Value within each column marked with different letters means significant difference at po 0.05.
with the roots, the increased rate of copper content was lower in the leaves (Table 1). 3.2. Germination and growth parameters Table 2 shows the effects of FeCl3 and CuCl2 on seed germination and seedling growth. The seeds exposed to different Fe concentration as well as 100 and 300 mM copper for four days exhibited good germination, whereas a significant reduction in germination rate was observed when the concentration of copper was increased to 500 mM (Table 2). Both Fe and Cu inhibited significantly the growth of roots and shoots, and the reduction in the root length was more prominent in comparison with the shoot length. Additionally, the growth of seedling was more sensitive to copper toxicity than to Fe stress. For example, the decreased rates of roots and shoots were about 18 and 15 percent in wheat seedlings treated by 300 mM Fe, respectively; in contrast, the length of roots and shoots reduced about 90 and 35 percent as compared with control value under 300 mM Cu stress. 3.3. H2O2 generation and MDA level in roots As illustrated in Fig. 1A, a faint fluorescent signal was detected in untreated root. When the seedlings were treated with different iron concentrations for six days, H2O2 fluorescent signal in the root tissues significantly increased as compared with the control, and the maximal strong fluorescence was observed in wheat roots treated with 500 mM Fe (Fig. 1D). Similarly, the treatment of wheat seedlings with different Cu concentrations led to strong fluorescent signal in wheat roots in a concentration-dependent manner (Fig. 1E, F, and G), and the level of fluorescent signal was higher in Cu-treated roots than that in Fe-treated roots. As shown in Fig. 2, the constitutive MDA content was 8.23 mM g 1 FW in seedling roots. An insignificant increase was observed in the amount of MDA after treatment with 100 mM Fe for six days, but 300 and 500 mM Fe resulted in a significant elevation in this index content, as compared with the control. By contrast, increasing CuCl2 concentrations effectively induced MDA accumulation in wheat roots and the maximal MDA content
reached approximately 230 percent of the control after treatment with 500 mM Cu for six days. In addition, the amount of MDA in copper-treated roots was always higher than that in irontreated ones. 3.4. Cell viability in roots The loss of cell viability was evaluated by the Evans blue straining in wheat roots with increasing Fe or Cu concentrations (Fig. 3). A slight but not significant decrease in cell viability (permeable for the dye Evans Blue) was observed in the roots treated with 100 mM Fe, but 300 and 500 mM Fe stress induced a significant reduction of cell viability in the roots. Similarly, exposure to different Cu concentrations led to the reduction of cell viability, and the most remarkable reduction was found at 500 mM Cu treatment (Fig. 3). 3.5. Chlorophyll content in leaves After exposure of wheat seedlings to 100 mM Fe for six days, the concentrations of chl a and the total chl were not significantly changed, while an observable elevation in chl b content was found; 300 and 500 mM Fe resulted in significant increases in the levels of these indexes (Tables 2 and 3). By contrast, 100 mM Cu treatment induced significant increases in chl b and the total chl contents, while the reduction in chl a and the total chl contents was significant in response to 500 mM Cu treatment. 3.6. CAT, POD and APX activities in leaves Fe and Cu effects on leaf CAT activity in wheat seedlings are shown in Table 4. After treatment with different Fe concentrations for six days, leaf CAT activity remained unchanged in comparison with the control. However, leaf CAT activity increased by 124, 134 and 120 percent of the control values in the seedling exposed to 100, 300 and 500 mM Cu treatment for six days, respectively. The treatment of wheat seedlings with Fe led to a remarkable elevation of leaf POD activity (Table 4). In untreated seedlings,
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Fig. 1. H2O2 fluorescent signal in wheat roots treated with different Fe and Cu. (A) Control; (B) 100 mM Fe; (C) 300 mM Fe; (D) 500 mM Fe; (E) 100 mM Cu; (F) 300 mM Cu; and (G) 500 mM Cu.
Fig. 2. MDA level in wheat roots treated with different Fe and Cu concentrations. Values represent mean (7 SE). Different letters on bars indicate significant difference (Po 0.05) as compared with the control.
leaf POD activity was 9.04 U mg 1 protein. Compared to the control, wheat seedlings exhibited about 3.25-, 4.37- and 4.74fold enhancement in leaf POD activity after treatment with 100, 300 and 500 mM Fe for six days, respectively. Similarly, significantly increased POD activity was observed with increasing Cu concentration, but the increased rates of enzyme activity was lower than those in response to Fe (Table 4). In comparison with the control, a significant elevation in leaf APX activity was found when the seedlings were treated with 100 and 300 mM Fe, while 500 mM Fe treatment resulted in about 28 percent decrease in this enzyme activity. By contrast, no changes of APX activity were found in copper-treated leaves (Table 4). 3.7. H2O2 and MDA levels in leaves H2O2 generation in the leaves of wheat seedlings after treatment with FeCl3 and CuCl2 is shown in Table 5. Compared to the
Fig. 3. Loss of cell viability of root tips in wheat seedlings treated with different Fe and Cu concentrations. Values represent mean ( 7 SE). Different letters on bars indicate significant difference (Po 0.05) as compared with the control.
control, the amount of H2O2 increased about 68 and 79 percent in response to 100 and 300 mM Fe stresses, while the increased rate decreased under 500 mM Fe stress (about 125 percent of the control value). In contrast, H2O2 content significantly increased in response to 100 mM Cu treatment, while decreased with Cu at 500 mM (Po0.05), and also an insignificant change was observed in H2O2 level under 300 mM Cu stress. Table 5 shows that different FeCl3 and CuCl2 concentrations induced the changes of MDA level in the leaves of wheat seedlings. After the seedlings were exposed to 100, 300 and 500 mM Fe for six days, a significant increase in MDA content was observed, and the increased rates remained constant (about 34 percent enhancement) as compared to the control. When the seedlings were treated with 100, 300 and 500 mM Cu, there was about 26, 29 and 11 percent increase in leaf MDA content in comparison with control seedlings, respectively.
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Table 3 Effects of iron and copper on chlorophyll (chl) content (mg g 1 FW) in wheat seedlings. Treatment concentration (mM)
0 100 300 500
CuCl2
FeCl3 Chl a
Chl b
Total chl
Chl a
Chl b
Total chl
8.097 0.06a 8.127 0.13a 10.047 0.004c 9.267 0.004b
1.51 70.002a 1.67 70.003b 2.04 70.02d 1.84 70.01c
9.60 70.06a 9.79 70.13a 12.08 70.01c 11.10 70.01b
8.09 70.06b 8.48 70.01b 7.79 70.01b 6.84 70.003a
1.517 0.002a 1.897 0.19b 1.557 0.02a 1.547 0.01a
9.60 7 0.06b 10.37 7 0.03c 9.34 7 0.02 b 8.38 7 0.01a
Value within each column marked with different letters means significant difference at p o 0.05. Table 4 Changes of catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) activities (U mg 1 protein) in the leaves of wheat seedlings under iron and copper treatments. Treatment concentration (mM)
0 100 300 500
CuCl2
FeCl3 CAT
POD
APX
CAT
POD
APX
0.21 70.04a 0.21 70.01a 0.22 70.01a 0.23 70.01a
9.05 7 0.22a 29.42 7 0.22b 39.50 7 1.10 c 42.84 7 0.74c
0.99 70.02a 1.19 70.03c 1.12 70.01c 0.70 70.01b
0.21 70.04a 0.26 70.01b 0.28 70.02b 0.25 70.01b
9.057 0.22a 12.70 7 0.64b 21.49 7 2.48c 13.32 7 1.96b
0.99 70.02a 0.95 70.01a 1.06 70.04a 0.96 70.01a
Different letters in each column indicate significant statistical differences (P o0.05).
Table 5 Changes of H2O2 (ng g 1 FW) and MDA (mmol g 1 FW) levels in the leaves of wheat seedling after treatment with different Fe and Cu concentrations. FeCl3
Treatment concentration (mM)
H2O2
MDA
H2O2
MDA
0 100 300 500
13.17 7 0.38a 21.29 7 0.60c 23.29 7 0.50c 16.83 7 0.26b
27.07 70.54a 35.94 72.12b 36.22 70.19b 35.89 71.84b
13.17 7 0.38a 15.44 7 0.16b 12.92 7 0.82a 12.08 7 0.21a
27.07 7 0.18a 34.04 7 0.14b 35.18 7 0.20b 30.38 7 0.03b
CuCl2
Value within each column marked with different letters means significant difference at p o0.05.
4. Discussion Previous studies showed that iron and copper excess induce the inhibitory effects on seed germination and early seedling growth of plants (Nenova, 2006; Posmyk and Kontek, 2009). Differentially, in B. volubilis increasing Cu concentrations did not affect seed germination, and even significantly stimulated the growth of seedlings (Street and Kulkarn, 2007). Jiang and Liu (2001) also found that Cu stimulated the root growth of Zea mays L.. The present data showed that increasing concentrations of Fe and lowering concentrations of Cu (100 and 300) had no significant effect on the germination of wheat seeds, only Cu at 500 mM inhibited this parameter. In contrast, all applied Fe or Cu concentrations reduced root and shoot lengths, and the toxicity of these two metals is more pronounced in roots than in shoots, which can be explained by the facts that plant roots are the first point of contact for heavy metal toxic factors (Kabir and Iqbal, 2008) and that iron and copper contents were higher in the roots than in the leaves in wheat seedlings (Table 1). In addition, seedling growth was more sensitive to copper toxicity than to Fe stress, which might be associated with the stresses of different strength applied in this study. The phytotoxicity of heavy metals arises partly from the excessive generation of ROS including H2O2. For instance, copper induces secondary oxidative stress by importing the formation of harmful ROS (Posmyk and Kontek, 2009). Analyses using fluorescent dye H2DCFDA showed elevated H2O2 levels in the root tissue with increasing Fe and Cu concentrations. Furthermore, we
found that the positive effect of copper stress on H2O2 generation was stronger than that of iron treatment and that the maximal strong fluorescence was caused in 500 mM Cu-treated roots (Fig. 1G). In agreement with the studies of Ruley et al. (2004), in this study increasing H2O2 levels were correlated with the reduction of root growth due to Fe and Cu exposure. Generally MDA, a biomarker of lipid peroxidation, is closely correlated with the level of oxidative stress in plants when exposed to different environmental stresses (Koca et al., 2007). Significant decrease in MDA content under Cu stress was observed by Xu et al. (2011). The present findings correspond with the results of other authors who have observed increased MDA content in various plants under iron and copper excess (Boojar and Goodarzi, 2007; Sinha and Basant, 2009; Zhao and Liu, 2010). Moreover, the elevation of MDA content in this study showed a negative correlation with root length with increasing Fe and Cu concentrations. This further supported the conclusion that the inhibition of root growth was possibly attributed to increased H2O2 generation in response to Fe and Cu exposure because excessive ROS accumulation within plant cells can lead to membrane lipid oxidation, thus affecting plant growth and development (Connolly and Guerinot, 2002; Posmyk and Kontek, 2009). On the contrary, the study of Gajewska et al. (2006) indicated that the inhibition of wheat root growth by 200 mM nickel was not related to lipid peroxidation, Viability staining of root tip cells was used as indicators for Fe and Cu toxicity. The present data showed that lower Fe concentration did not affect cell viability in the root tips but higher Fe dose reduced cell viability. Similarly, exposure to different Cu concentrations significantly increased the number of dead cells within roots (Fig. 3). Moreover, the cell viability in wheat roots was more sensitive to Cu treatment than to Fe stress. These results indicated that Fe and Cu increased the number of dead cells in root tips, which appeared to be associated with the reduction of root growth and development in wheat seedlings. According to Nenova (2006), Fe deficiency caused a decrease of photosynthetic pigments in Pea plants, but excess Fe resulted in risen pigment concentrations. Additionally, no significant difference of chl content and serious decrease of chl synthesis due to iron and copper excess were observed in other plants (Kampfenkel et al., 1995; Shakya et al., 2008; Xing and Huang, 2010; Brahim and Mohamed, 2011). In this study, significant
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increases in the total chl including chl a and chl b were observed in wheat seedlings exposed to increasing Fe concentrations; in contrast, 100 mM Cu led to significant increases in the amount of chl b and the total chl, whereas wheat seedlings exhibited notable decreases in chl a and the total chl contents in response to the highest Cu concentration. Consistent with our results, the total Chl and Chl a contents significantly decreased in Ceratophyllum demersum L. under Cu treatment, while the change in Chl b was not significant (Rama Devi and Prasad, 1998). These findings can be explained by the study of Van Assche and Clijsters (1990) indicating that copper may reduce chl content by inhibiting the pigment biosynthesis and decreasing the photosynthetic electron transport. Besides, chl loss can be related to membrane oxidative damage produced by oxidative stress (Aarti et al., 2006) because increased H2O2 and MDA levels in this investigation were observed in the leaves of Cu-treated seedlings. The photosynthesis efficiency is one of the most important determinants of plant growth, and the photosynthesis efficiency increased with an increase in the content of chl (An and Zhou, 2009). Therefore, another reason for growth reduction in wheat seedlings exposed to Cu may be due to the effect of Cu on the amount of chl involved in the changes of the photosynthesis rate, whereas iron may produce growth reduction by interfering with other cellular processes. In agreement with Cu-inhibitory mechanism, Nichols et al. (2000) demonstrated that the decrease of dry biomass in Salvinia minima seedlings can be attributed to a metal-induced reduction of photosynthetic pigments. According to Boojar and Goodarzi (2007), the elevation of antioxidative enzyme activities is one component of the tolerant adaptation of plants to heavy metal toxicity. POD, CAT and APX constitute main H2O2 scavenging system in cells. In wheat leaves the increased POD activity in response to Fe and Cu stress supported previous suggestion to POD activity as a biomarker for metal toxicity in plants (Metwally et al., 2005). The stimulation of POD has also been reported in the leaves or shoots of various plant species subjected to Cu and Fe treatments (Mehraban et al., 2008; Verma et al., 2011; Wang et al., 2011). These findings indicated that POD plays an important role not only in scavenging H2O2 but also in protecting plant seedlings from oxidative damage during the treatment of heavy metals. CAT is frequently used by cells to rapidly catalyze the decomposition of H2O2 into less reactive gaseous oxygen and water molecules (Tayefi-Nasrabadi et al., 2011). Previous studies showed that low Cu concentration increased CAT activity but high Cu level inhibited this enzyme in Medicago sativa (Wang et al., 2011). Differently, a decrease or no variation of CAT activity was observed in the leaves and shoots of some plants under Cu excess (Bouazizi et al., 2010; Thounaojam et al., 2012). Despite an iron-containing enzyme, the activity of CAT significantly decreased in rice plant under iron excess (Mehraban et al., 2008). On contrary to these studies, in wheat seedlings leaf CAT activity was stimulated under all applied Cu concentrations, whereas Fe treatment did not affect this enzyme, indicating that leaf CAT in wheat seedlings is sensitive to copper stress and appears to be an efficient scavenger of H2O2 under copper treatment. APX is one of the major components of ASH–GSH cycle where it functions to prevent the accumulation of toxic levels of H2O2 in photosynthetic organisms (Thounaojam et al., 2012). Wang et al. (2011) reported that APX activity increased at lower Cu concentrations and reached a maximum at 30 mM of Cu in M. sativa. And also, increased activity of APX was observed in sweet potato grown in the 9.0 mM iron solution (Amstad et al., 1994). Moreover, iron triggers a rapid induction of APX gene expression in Brassica napus (Vansuyt et al. (1997)). In this study, leaf APX activity was higher with 100 and 300 mM Fe than 500 mM Fe, but no significant changes in APX activity was observed in Cu-treated leaves. Because of the
unchanged APX but increased POD, and especially because of the increased CAT, H2O2 content in the leaves associated with copper was significantly lower than that with iron at the same concentration (Table 5). Moreover, significant increase in MDA content indicated increased ROS generation in the leaves of wheat seedlings under Fe and Cu treatments.
5. Conclusions The present results may suggest that the increased H2O2 generation, thus causing oxidative damage, and the reduction of cell viability in the root tips are responsible for inhibitory mechanism of root growth in response to Fe and Cu stresses in plants. Significant increases in the total chl content including chl a and chl b were observed in wheat seedlings exposed to increasing Fe concentrations, whereas the highest Cu content led to significant decreases in these parameters. With regard to seedling growth, cell viability in the root tips, chl content and oxidative stress, Cu was found to be more toxic to wheat than Fe in all applied concentration ranges. Among antioxidant enzymes, leaf CAT and POD appear to play key roles in antioxidative protective mechanisms in wheat seedlings exposed to copper toxicity, but leaf POD in response to all Fe concentrations and APX due to lower Fe dose show important function in scavenging H2O2, which can explain the fact that H2O2 content in the leaves of wheat seedlings associated with copper was lower than that with iron at the same concentration. In addition, the increased MDA levels in response to iron and copper treatments were also observed in the leaf tissue.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 31160088), the Program for New Century Excellent Talents in University of China and Science and Technology Development Plan Program of Lanzhou City (No. 2011-1-142). References Aarti, P.D., Tanaka, R., Tanaka, A., 2006. Effects of oxidative stress on chlorophyll biosynthesis in cucumber (Cucumis sativus) cotyledons. Physiol. Plant. 128, 186–197. Achary, V.M.M., Patnaik, A.R., Panda, B.B., 2012. Oxidative biomarkers in leaf tissue of barley seedlings in response to aluminum stress. Ecotoxicol. Environ. Saf. 75, 16–26. Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Amstad, P., Moret, R., Cerutti, P., 1994. Glutathione peroxidase compensates for the hypersensitivity of Cu, Zn-superoxide dismutase overproducers to oxidant stress. J. Biol. Chem. 269, 1606–1609. An, J., Zhou, Q., 2009. Ecotoxicological effects of typical personal care products on seed germination and seedling development of wheat (Triticum aestivum L.). Chemosphere 76, 1428–1434. Boojar, M.M.A., Goodarzi, F., 2007. The copper tolerance strategies and the role of antioxidative enzymes in three plant species grown on copper mine. Chemosphere 67, 2138–2147. Bouazizi, H., Jouili, H., Geitmann, A., El Ferjani, E., 2010. Copper toxicity in expanding leaves of Phaseolus vulgaris L.: antioxidant enzyme response and nutrient element uptake. Ecotoxicol. Environ. Saf. 73, 1304–1308. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brahim, L., Mohamed, M., 2011. Effects of copper stress on antioxidative enzymes, chlorophyll and protein content in Atriplex halimus. Afr. J. Biotechnol 10, 10143–10148. Connolly, E.L., Guerinot, M., 2002. Iron stress in plants, reviews. Genome Biol. 3, 1–4. Gajewska, E., Slaba, M., Andrzejewska, R., Sklodowska, M., 2006. Nickel-induced inhibition of wheat root growth is related to H2O2 production, but not to lipid peroxidation. Plant Growth Regul. 49, 95–103.
X. Li et al. / Ecotoxicology and Environmental Safety 86 (2012) 47–53
¨ Hansch, R., Mendel, R.R., 2009. Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl). Curr. Opin. Plant Biol. 12, 259–266. ¨ A., Erdei, S., Horva´th, G., 2001. Comparative studies of H2O2 detoxifying Hegedus, enzymes in green and greening barley seedlings under cadmium stress. Plant Sci. 160, 1085–1093. Jiang, W., Liu, D., 2001. Effects of copper on root growth, cell division, and nucleolus of zea mays. Biol. Plant. 44, 105–109. Kabir, M., Iqbal, M.Z., 2008. Reduction in germination and seedling growth of Thespesia populnea L., caused by lead and cadmium treatments. Pak. J. Bot. 40, 2419–2426. Kampfenkel, K., Van Montagu, M., Inze, D., 1995. Effects of iron excess on Nicotiana plumbaginifolia plants (implications to oxidative stress). Plant Physiol. 107, 725–735. Koca, H., Ozdemir, F., Turkan, I., 2007. Effect of salt stress on chlorophyll fluorescence, lipid peroxidation, superoxide dismutase and peroxidase activities of cultivated tomato (L. esculentum) and its wild relative (L. pennellii). Environ. Exp. Bot. 60, 344–351. Li, Y., Song, Y., Shi, G.J., Wang, J.J., Hou, X.L., 2009. Response of antioxidant activity to excess copper in two cultivars of Brassica campestris ssp. chinensis Makino. Acta Physiol. Plant. 31, 155–162. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382. Mehraban, P., Zadeh, A.A., Sadeghipour, H.R., 2008. Iron toxicity in rice (Oryza sativa L.), under different potassium nutrition. Asian J. Plant Sci. 7, 251–259. Metwally, A., Safronova, V.I., Belimov, A.A., Dietz, K.J., 2005. Genotypic variation of the response to cadmium toxicity in Pisum sativum L. J. Exp. Bot. 56, 167–174. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by asorabate specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Nenova, V., 2006. Effect of iron supply on growth and photosystem II efficiency of pea plants. Gen. Appl. Plant Physiol., 81–90. Nichols, P.B., Couch, J.D., Al-Hamdani, S.H., 2000. Selected physiological responses of Salvinia minima to different chromium concentrations. Aquat. Bot. 68, 313–319. Pei, Z.M., Murata, Y., 2000. Calcium channels activated by hydrogen peroxide mediate abscisic acid signaling in guard cells. Nature 406, 731–734. Posmyk, M.M., Kontek, R., 2009. Antioxidant enzymes activity and phenolic compounds content in red cabbage seedlings exposed to copper stress. Ecotoxicol. Environ. Saf. 72, 596–602. Rama Devi, S., Prasad, M.N.V., 1998. Copper toxicity in Ceratophyllum demersum L. (Coontail), a free floating macrophyte: response of antioxidant enzymes and antioxidants. Plant Sci. 138, 157–165. Rao, M.V., Paliyath, G., 1996. Ultraviolet B and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol. 110, 125–136. Ruley, A.T., Sharma, N.C., Sahi, S.V., 2004. Antioxidant defense in a lead accumulating plant, Sesbania drummondii. Plant Physiol. Biochem. 42, 899–906.
53
Sandalio, L.M., Dalurzo, H.C., Gomez, M., Romero-Puertas, M.C., Del Rı´o, L.A., 2001. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 52, 2115–2126. Sergiev, I., Alexieva, V., 1997. Effect of spermine, atrazine and combination between them on some endogenous protective systems and stress markers in plants. C. R. Acad. Bulg. Sci. 51, 121–124. Shakya, K., Chettri, M.K., Sawidis, T., 2008. Impact of heavy metals (copper, zinc, and lead) on the chlorophyll content of some mosses. Arch. Environ. Contam. Toxicol. 54, 412–421. Sinha, S., Basant, A., 2009. Iron-induced oxidative stress in a macrophyte: a chemometric approach. Ecotoxicol. Environ. Saf. 72, 585–595. Street, R.A., Kulkarn, M.G., 2007. Toxicity of metal elements on germination and seedling growth of widely used medicinal plants belonging to Hyacinthaceae. Bull. Environ. Contam. Toxicol. 79, 371–376. Tayefi-Nasrabadi, H., Dehghan, G., Daeihassani, B., Movafegi, A., Samadi, A., 2011. Some biochemical properties of catalase from safflower (Carthamus tinctorius L. cv. M-CC-190). Afr. J. Agric. Res. 6, 5221–5226. Thounaojam, T.C., Panda, P., Mazumdar, P., Kumar, D., Sharma, G.D., Sahoo, L., Panda, S.K., 2012. Excess copper induced oxidative stress and response of antioxidants in rice. Plant Physiol. Biochem. 53, 33–39. Van Assche, F., Clijsters, H., 1990. Effects of metals on enzyme activity in plants. Plant Cell Environ. 13, 195–206. Vansuyt, G., Lopez, F., Inze´, D., Briat, J.F., Fourcroy, P., 1997. Iron triggers a rapid induction of ascorbate peroxidase gene expression in Brassica napus. FEBS Lett. 410, 195–200. Verma, J.P., Singh, V., Yadav, J., 2011. Effect of copper sulphate on seed germination, plant growth and peroxidase activity of Mung bean (Vigna radiata). Int. J. Bot. 7, 200–220. Wang, S.H., Zhang, H., Zhang, Q., Jin, G.M., Jiang, S.J., Jiang, D., He, Q.Y., Li, Z.P., 2011. Copper-induced oxidative stress and responses of the antioxidant system in roots of Medicago sativa. J. Agron. Crop Sci. 197, 418–429. Xing, W., Huang, W., 2010. Effect of excess iron and copper on physiology of aquatic plant Spirodela polyrrhiza (L.) Schleid. Environ. Toxicol. 25, 103–112. Xu, X.Y., Shi, G.X., Wang, J., Zhang, L.L., Kang, Y.N., 2011. Copper-induced oxidative stress in Alternanthera philoxeroides callus. Plant Cell, Tissue Organ Cult. (PCTOC) 106, 243–251. Yang, Y.L., Zhang, Y.Y., Wei, X.L., You, J., Wang, W.R., Lu, J., Shi, R.X., 2011. Comparative antioxidative responses and proline metabolism in two wheat cultivars under short term lead stress. Ecotoxicol. Environ. Saf. 74, 733–740. Yruela, I., 2009. Copper in plants: acquisition, transport and interactions. Funct. Plant Biol. 36, 409–430. Zanardo, D.I.L., Lima, R.B., Ferrarese, M.L.L., Bubna, G.A., Ferrarese-Filho, O., 2009. Soybean root growth inhibition and lignification induced by p-coumaric acid. Environ. Exp. Bot. 66, 25–30. Zhao, S., Liu, Q., 2010. Responses of root growth and protective enzymes to copper stress in turfgrass. Acta Biol. Cracov. Ser. Bot. 52, 7–11.
Contents lists available at SciVerse ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Frontier Article
Responses of seedling growth and antioxidant activity to excess iron and copper in Triticum aestivum L. Xiaoning Li a, Haizhen Ma a, Pengxiang Jia a, Juan Wang a, Lingyun Jia a, Tengguo Zhang a, Yingli Yang a,n, Haijian Chen b, Xia Wei b a b
Life Science of College, Northwest Normal University, Lanzhou 730070, Gansu, China The Center for Spreading Agricultural Technology, Xigu District, Lanzhou 730060, China
a r t i c l e i n f o
abstract
Article history: Received 19 July 2012 Received in revised form 7 September 2012 Accepted 8 September 2012 Available online 29 September 2012
The purpose of this study was to analyze phytotoxicity mechanism involved in root growth and to compare physiological changes in the leaves of wheat seedlings exposed to short term iron (Fe) and copper (Cu) stresses (0, 100, 300 and 500 mM). All applied Fe or Cu concentrations reduced root and shoot lengths, but seed germination was inhibited by Cu only at 500 mM. Analyses using fluorescent dye 20 ,70 -dichlorodihydrofluorescein diacetate indicated enhanced H2O2 levels in seedling roots under Fe and Cu treatments. Cu stress at the same concentration induced a great reduction in cell viability and a strong damage on membrane lipid in the roots with respect to Fe treatment. Significant increases in the total chlorophyll (chl) content including chl a and chl b were observed in response to higher Fe concentrations, whereas the highest Cu concentration (500 mM) led to significant decreases in the total chl content including chl a. Additionally, leaf peroxidase (POD) and ascorbate peroxidase (APX) were stimulated by Fe stress, but the highest Fe concentration exhibited inhibitory effect on leaf APX activity. In contrast, copper treatment resulted in an elevation in leaf catalase and POD activities. Therefore, H2O2 content in the leaves associated with copper was significantly lower than that with iron at the same concentration. & 2012 Elsevier Inc. All rights reserved.
Keywords: Wheat Seed germination Seedling growth Antioxidant enzymes Lipid peroxidation
1. Introduction Enhanced industrial and mining activities have contributed to the increasing occurrence of heavy metals including iron (Fe) and copper (Cu) in ecosystems. Heavy metals are also added to soils from different human activities. Fe and Cu are essential mineral nutrients for plant growth at low concentrations, which plays key roles in photosynthetic and respiratory electron transport chains, even in ethylene sensing, cell wall metabolism and oxidative stress protection (Connolly and Guerinot, 2002; Yruela, 2009). However, excessive quantities of Fe and Cu can lead to phytotoxicity such as leaf chlorosis and growth inhibition (Bouazizi et al., 2010). The study of Mehraban et al. (2008) showed the maximal plant growth at iron concentration of 10 and 50 mg L 1 but a growth inhibition at 250 and 500 mg L 1. In general, germinating seeds are more sensitive to metal elements than mature plants, and the inhibition of the germination has become an important biomarker as a plant response to heavy-metal stress. Even though the influences of excess iron and copper on plant are investigated in a number of plant species (Nenova,
n
Corresponding author. E-mail address: [email protected] (Y. Yang).
0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.09.010
2006; Street and Kulkarn, 2007; Posmyk and Kontek, 2009), there are fewer reports on the precise mechanisms of the negative effects due to heavy metal stress. Additionally, inhibited biosynthesis of chlorophyll (chl) is a frequent symptom of metal toxicity (Van Assche and Clijsters, 1990; Sandalio et al., 2001). Iron and copper are essential for photosynthesis. Especially in plant tissues, up to 80 percent of iron and more than half of copper are found in the chloroplasts ¨ (Hansch and Mendel, 2009). However, photosynthesis is sensitive to Fe and Cu excess. Several studies have proposed that the toxicity of Fe and Cu may be explained by the reduction of chl content in plants (Shakya et al., 2008; Xing and Huang, 2010; Brahim and Mohamed, 2011). Besides, Fe and Cu excess can act catalytically via Haber–Weiss and Fenton reactions to generate ROS including superoxide, hydroxyl ¨ et al., 2001; radicals and hydrogen peroxide (H2O2) (Hegedus Connolly and Guerinot, 2002). Increased ROS accumulation within plant cells has the potential to cause oxidative injury by provoking macromolecule damage and membrane lipid oxidation, thus distributing metabolic pathways and affecting plant growth and development (Connolly and Guerinot, 2002; Posmyk and Kontek, 2009). To control the level of ROS and to protect the cells against oxidative damage initiated by ROS, some plants have established protective mechanisms such as antioxidant enzyme system. The correlation
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between antioxidant capacity and copper tolerance has been demonstrated in a large number of plants (Boojar and Goodarzi, 2007; Posmyk and Kontek, 2009). Contrary to these findings, Cu-treated leaves exhibited decreased activities of POD and APX in Brassica campestris ssp. chinensis Makino (Li et al., 2009). Apparently, the mechanisms of antioxidantive responses due to excess Fe or Cu are not yet fully understood. The study of physiological and ecological processes in response to heavy metal stresses has great significance for revealing the mechanism of plant anti-heavy metal and/or the mechanisms of the negative effects of metals. Compared to the studies of Cu phytotoxicity, to date scarce information has been reported on Fe toxicity in plants. This study aimed to analyze the mechanisms of Fe or Cu toxicity involved in the growth of wheat roots, and to evaluate the levels of chl and lipid peroxidation as well as the activities of antioxidative enzymes in the leaves of wheat seedlings exposed to short term Fe and Cu stresses.
2.5. Cell viability Wheat roots treated with different Fe or Cu concentrations were used to determine the loss of cell viability by Evans blue staining (Zanardo et al., 2009). Fresh roots were incubated for 15 min with 30 mL of 0.25 percent Evans blue solution, washed for 30 min to remove excess and unbound dye. After excised root tips (3 cm) were soaked in 3 mL of N,N-dimethylformamide for 50 min at room temperature, the absorbance of released Evans blue was measured at 600 nm, using deionized water as a blank. The loss of cell viability was expressed as the absorbance at 600 nm of treated roots in relation to untreated roots.
2.6. Chlorophyll content measurement Chl was extracted from plant leaves with 80 percent acetone, and chl content was determined spectrophotometrically at 663 and 646 nm according to Lichtenthaler (1987).
2.7. Antioxidant enzyme activities measurement
The seeds were surface-sterilized with 0.1 percent HgCl2 for 10 min, then soaked in water for 24 h. The plump and excellent seeds were plated in Petri dishes with two filter-paper discs containing 1/4 Hoagland medium supplemented with 100, 300 and 500 mM Fe or Cu from FeCl3 and CuCl2 respectively and germinated for four days at 257 1.5 1C in the darkness in an incubator (LRH-250A, made in Guangdong Medical Instruments Factory, China). Seed germination was defined as a root length of 1.0 mm or more. For root and shoot growth experiments, forty seedlings were cultivated in Petri dishes containing 6 mL of 1/4 Hoagland soluble containing 100, 300 and 500 mM FeCl3 or CuCl2 for six days at 25 72.5 1C under a light irradiance of 300 mmol m 2 s 1 (12 h light:12 h dark cycles). All assays were replicated at least three times to minimize experimental errors; each replicate was carried out on fifty seeds for germination and forty seedlings for growth measurement.
Fresh leaves were ground with chilled NaH2PO4/Na2HPO4 buffer (PBS, 50 mM, pH 7.8) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA) and one percent polyvinylpyrrolidone (PVP), centrifuged at 12,000 rpm min 1 for 30 min, and the supernatant was collected for the protein assay and the determination of CAT and POD activities. The amount of proteins was estimated according to the method proposed by Bradford (1976) using bovine serum albumin as a standard. CAT activity was analyzed according to Aebi (1984). The enzyme extraction was added to 3 mL 50 mM PBS buffer (pH 7.0). After 5 min incubation at 25 1C, the reaction was started by the addition of 6 mM H2O2 and the absorbance changes were recorded at 240 nm for 2 min. An absorbance change of 0.1 unit min 1 was defined as 1 unit (U) of CAT activity. POD activity was measured following a modification of the method of Rao and Paliyath (1996). The enzyme extraction was mixed with the reaction mixture containing 50 mM PBS (pH 7.0) and 20 mM guaiacol. After pre-incubation at 25 1C for 5 min, 6 mM H2O2 was added to initiate the reaction. Changes of the absorbance at 470 nm within 2 min were recorded for calculating POD activity. One unit of POD activity was defined as an absorbance change of 0.01 unit min 1. Plant material was ground in chilled 50 mM PBS buffer (pH 7.0) containing 1 mM EDTA-Na2 and 1 mM ascorbate (ASA). After centrifugation for 20 min at 10,000 rpm, the supernatant (enzyme extraction) was collected for the measurement of APX activity performed as described by Nakano and Asada (1981) with some modifications. A reaction mixture consist of 50 mM PBS (pH 7.0), 0.5 mM ASA, 3 mM H2O2 and the enzyme extraction, and the changes in the absorbance at 290 nm were recorded at 25 1C for 1 min after the addition of H2O2. One unit of APX activity was defined as an absorbance change of 0.1 unit min 1. The specific enzyme activity for all enzymes was expressed as U mg 1 protein.
2.3. Iron and copper content determination
2.8. Statistical analysis
1/4 Hoagland mediums supplemented with 0, 100, 300 and 500 mM FeCl3 or CuCl2 were acidified by HNO3 (one percent, v/v) and the actual concentrations of Fe or Cu were determined by flame Atomic Absorption Spectrophotometry (WFX210, China). The roots and leaves of wheat seedlings were prepared for Fe and Cu measurements according to Achary et al. (2012) with some modifications. Plant material was thoroughly washed with deionized water and dried to constant weight at 80 1C. The dry sample was dissolved in a solution containing 12 mL of concentrated nitric acid (HNO3), 4 mL hydrofluoric acid and 4 mL of H2O2 (30 percent, v/v), and was digested in a closed microwave digestion system (Multiwave 3000, Anton paar, Austria ) for 2.5 h. The chilled sample was transferred to polytetrafluoroethylene beaker, and was then evaporated to dryness. The resulting ash residue was dissolved in HNO3 (one percent, v/v), and total Fe and Cu contents were measured with atom absorption spectrophotometer.
All values were represented by an average of at least three replicate measurements7 standard error (SE). Statistical comparisons were carried out using SPSS 13.0 software, and significant differences were indicated by different letters (p o 0.05).
2. Materials and methods 2.1. Plant material Wheat (Tritium aestivum) cv. Ningchun four seeds were purchased from Gansu Agricultural University, in Lanzhou, China.
2.2. Seed germination and seedling growth experiment
2.4. Analysis of H2O2 content and lipid peroxidation The fluorescence intensity of H2O2 level in roots was visualized using a fluorescent dye 20 ,70 -Dichlorodihydrofluorescein diacetate (H2DCFDA) as described by Pei and Murata (2000). Roots were immersed in 50 mM H2DCFDA in 10 mM Tris– HCl buffer (pH 7.2) containing 50 mM KCl for 20 min, and were washed three times in Tris–HCl buffer to remove excess dye. Then, examinations of H2DCFDA fluorescence were observed using a Leica DMIRB inverted fluorescent microscope. H2O2 content in leaves was quantified by the method of Sergiev and Alexieva (1997) in homogenates prepared with 0.1 percent trichloroacetic acid as described in detail previously (Yang et al., 2011). Lipid peroxidation in roots or leaves was estimated from the amount of MDA determined by the thiobarbituric acid reaction as described in detail previously (Yang et al., 2011).
3. Results 3.1. Iron and copper contents The actual concentrations of Fe in the experimental solutions of FeCl3 prepared at 0, 100, 300 and 500 mM were 5.6, 11.2, 22.3 and 33.5 mg L 1, and the actual Cu concentrations in the experimental solutions of CuCl2 prepared at 0, 100, 300 and 500 mM were 0.006, 6.3, 19.1 and 31.7 mg L 1, respectively. In addition, iron and copper levels in the roots and leaves of wheat seedlings are shown in Table 1. Fe levels in the roots significantly elevated with increasing treatment concentrations, reaching almost 9.23-, 14.55- and 93.7-fold higher content at 100, 300 and 500 mM Fe dose, respectively. In comparison with the control, leaf Fe content exhibited an insignificant enhancement at 100 mM Fe, but Fe at 300 and 500 mM resulted in notably enhanced Fe content in the leaves. Similarly, Cu treatment led to increased Cu levels in the roots in a concentration-dependent manner, whereas compared
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Table 1 Changes of iron (mg g 1 DW) and copper (mg g 1 DW) levels in wheat seedling after treatment with different Fe and Cu concentrations. FeCl3 (iron content)
Treatment concentration (mM)
0 100 300 500
CuCl2 (copper content)
Root
Shoot
Root
Shoot
0.147 0.014a 1.33 7 0.044b 2.087 0.043c 13.40 7 0.12d
0.0647 0.008a 0.0867 0.011a 0.187 0.016b 1.187 0.029c
0.049 70.004a 0.17 70.009b 2.11 70.034c 5.29 70.076d
0.0767 0.002a 0.0837 0.003a 0.117 0.004b 0.137 0.004c
Value within each column marked with different letters means significant difference at po 0.05.
Table 2 Effects of iron and copper on seed germination and seedling growth in wheat. Treatment FeCl3 concentration (mM) Germination rate (percent) 0 100 300 500
96.67 7 0.02a 95.33 7 0.007a 96.00 7 0.01a 92.00 7 0.02a
CuCl2 Root length (cm)
Shoot length (cm)
Germination Root length rate (percent) (cm)
Shoot length (cm)
8.07 70.04a 11.15 7 0.05a 96.67 70.02a 8.07 70.04a 11.15 7 0.05a 6.84 70.03b 10.25 7 0.08a 94.67 70.01a 1.45 70.02b 8.78 7 0.14b 6.59 70.11c 9.44 7 0.08b 95.33 70.02a 0.84 70.01c 7.25 7 0.09c 3.50 70.05d 7.307 0.06c 86.00 70.01b 0.56 70.01d 6.68 7 0.05d
Value within each column marked with different letters means significant difference at po 0.05.
with the roots, the increased rate of copper content was lower in the leaves (Table 1). 3.2. Germination and growth parameters Table 2 shows the effects of FeCl3 and CuCl2 on seed germination and seedling growth. The seeds exposed to different Fe concentration as well as 100 and 300 mM copper for four days exhibited good germination, whereas a significant reduction in germination rate was observed when the concentration of copper was increased to 500 mM (Table 2). Both Fe and Cu inhibited significantly the growth of roots and shoots, and the reduction in the root length was more prominent in comparison with the shoot length. Additionally, the growth of seedling was more sensitive to copper toxicity than to Fe stress. For example, the decreased rates of roots and shoots were about 18 and 15 percent in wheat seedlings treated by 300 mM Fe, respectively; in contrast, the length of roots and shoots reduced about 90 and 35 percent as compared with control value under 300 mM Cu stress. 3.3. H2O2 generation and MDA level in roots As illustrated in Fig. 1A, a faint fluorescent signal was detected in untreated root. When the seedlings were treated with different iron concentrations for six days, H2O2 fluorescent signal in the root tissues significantly increased as compared with the control, and the maximal strong fluorescence was observed in wheat roots treated with 500 mM Fe (Fig. 1D). Similarly, the treatment of wheat seedlings with different Cu concentrations led to strong fluorescent signal in wheat roots in a concentration-dependent manner (Fig. 1E, F, and G), and the level of fluorescent signal was higher in Cu-treated roots than that in Fe-treated roots. As shown in Fig. 2, the constitutive MDA content was 8.23 mM g 1 FW in seedling roots. An insignificant increase was observed in the amount of MDA after treatment with 100 mM Fe for six days, but 300 and 500 mM Fe resulted in a significant elevation in this index content, as compared with the control. By contrast, increasing CuCl2 concentrations effectively induced MDA accumulation in wheat roots and the maximal MDA content
reached approximately 230 percent of the control after treatment with 500 mM Cu for six days. In addition, the amount of MDA in copper-treated roots was always higher than that in irontreated ones. 3.4. Cell viability in roots The loss of cell viability was evaluated by the Evans blue straining in wheat roots with increasing Fe or Cu concentrations (Fig. 3). A slight but not significant decrease in cell viability (permeable for the dye Evans Blue) was observed in the roots treated with 100 mM Fe, but 300 and 500 mM Fe stress induced a significant reduction of cell viability in the roots. Similarly, exposure to different Cu concentrations led to the reduction of cell viability, and the most remarkable reduction was found at 500 mM Cu treatment (Fig. 3). 3.5. Chlorophyll content in leaves After exposure of wheat seedlings to 100 mM Fe for six days, the concentrations of chl a and the total chl were not significantly changed, while an observable elevation in chl b content was found; 300 and 500 mM Fe resulted in significant increases in the levels of these indexes (Tables 2 and 3). By contrast, 100 mM Cu treatment induced significant increases in chl b and the total chl contents, while the reduction in chl a and the total chl contents was significant in response to 500 mM Cu treatment. 3.6. CAT, POD and APX activities in leaves Fe and Cu effects on leaf CAT activity in wheat seedlings are shown in Table 4. After treatment with different Fe concentrations for six days, leaf CAT activity remained unchanged in comparison with the control. However, leaf CAT activity increased by 124, 134 and 120 percent of the control values in the seedling exposed to 100, 300 and 500 mM Cu treatment for six days, respectively. The treatment of wheat seedlings with Fe led to a remarkable elevation of leaf POD activity (Table 4). In untreated seedlings,
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Fig. 1. H2O2 fluorescent signal in wheat roots treated with different Fe and Cu. (A) Control; (B) 100 mM Fe; (C) 300 mM Fe; (D) 500 mM Fe; (E) 100 mM Cu; (F) 300 mM Cu; and (G) 500 mM Cu.
Fig. 2. MDA level in wheat roots treated with different Fe and Cu concentrations. Values represent mean (7 SE). Different letters on bars indicate significant difference (Po 0.05) as compared with the control.
leaf POD activity was 9.04 U mg 1 protein. Compared to the control, wheat seedlings exhibited about 3.25-, 4.37- and 4.74fold enhancement in leaf POD activity after treatment with 100, 300 and 500 mM Fe for six days, respectively. Similarly, significantly increased POD activity was observed with increasing Cu concentration, but the increased rates of enzyme activity was lower than those in response to Fe (Table 4). In comparison with the control, a significant elevation in leaf APX activity was found when the seedlings were treated with 100 and 300 mM Fe, while 500 mM Fe treatment resulted in about 28 percent decrease in this enzyme activity. By contrast, no changes of APX activity were found in copper-treated leaves (Table 4). 3.7. H2O2 and MDA levels in leaves H2O2 generation in the leaves of wheat seedlings after treatment with FeCl3 and CuCl2 is shown in Table 5. Compared to the
Fig. 3. Loss of cell viability of root tips in wheat seedlings treated with different Fe and Cu concentrations. Values represent mean ( 7 SE). Different letters on bars indicate significant difference (Po 0.05) as compared with the control.
control, the amount of H2O2 increased about 68 and 79 percent in response to 100 and 300 mM Fe stresses, while the increased rate decreased under 500 mM Fe stress (about 125 percent of the control value). In contrast, H2O2 content significantly increased in response to 100 mM Cu treatment, while decreased with Cu at 500 mM (Po0.05), and also an insignificant change was observed in H2O2 level under 300 mM Cu stress. Table 5 shows that different FeCl3 and CuCl2 concentrations induced the changes of MDA level in the leaves of wheat seedlings. After the seedlings were exposed to 100, 300 and 500 mM Fe for six days, a significant increase in MDA content was observed, and the increased rates remained constant (about 34 percent enhancement) as compared to the control. When the seedlings were treated with 100, 300 and 500 mM Cu, there was about 26, 29 and 11 percent increase in leaf MDA content in comparison with control seedlings, respectively.
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Table 3 Effects of iron and copper on chlorophyll (chl) content (mg g 1 FW) in wheat seedlings. Treatment concentration (mM)
0 100 300 500
CuCl2
FeCl3 Chl a
Chl b
Total chl
Chl a
Chl b
Total chl
8.097 0.06a 8.127 0.13a 10.047 0.004c 9.267 0.004b
1.51 70.002a 1.67 70.003b 2.04 70.02d 1.84 70.01c
9.60 70.06a 9.79 70.13a 12.08 70.01c 11.10 70.01b
8.09 70.06b 8.48 70.01b 7.79 70.01b 6.84 70.003a
1.517 0.002a 1.897 0.19b 1.557 0.02a 1.547 0.01a
9.60 7 0.06b 10.37 7 0.03c 9.34 7 0.02 b 8.38 7 0.01a
Value within each column marked with different letters means significant difference at p o 0.05. Table 4 Changes of catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) activities (U mg 1 protein) in the leaves of wheat seedlings under iron and copper treatments. Treatment concentration (mM)
0 100 300 500
CuCl2
FeCl3 CAT
POD
APX
CAT
POD
APX
0.21 70.04a 0.21 70.01a 0.22 70.01a 0.23 70.01a
9.05 7 0.22a 29.42 7 0.22b 39.50 7 1.10 c 42.84 7 0.74c
0.99 70.02a 1.19 70.03c 1.12 70.01c 0.70 70.01b
0.21 70.04a 0.26 70.01b 0.28 70.02b 0.25 70.01b
9.057 0.22a 12.70 7 0.64b 21.49 7 2.48c 13.32 7 1.96b
0.99 70.02a 0.95 70.01a 1.06 70.04a 0.96 70.01a
Different letters in each column indicate significant statistical differences (P o0.05).
Table 5 Changes of H2O2 (ng g 1 FW) and MDA (mmol g 1 FW) levels in the leaves of wheat seedling after treatment with different Fe and Cu concentrations. FeCl3
Treatment concentration (mM)
H2O2
MDA
H2O2
MDA
0 100 300 500
13.17 7 0.38a 21.29 7 0.60c 23.29 7 0.50c 16.83 7 0.26b
27.07 70.54a 35.94 72.12b 36.22 70.19b 35.89 71.84b
13.17 7 0.38a 15.44 7 0.16b 12.92 7 0.82a 12.08 7 0.21a
27.07 7 0.18a 34.04 7 0.14b 35.18 7 0.20b 30.38 7 0.03b
CuCl2
Value within each column marked with different letters means significant difference at p o0.05.
4. Discussion Previous studies showed that iron and copper excess induce the inhibitory effects on seed germination and early seedling growth of plants (Nenova, 2006; Posmyk and Kontek, 2009). Differentially, in B. volubilis increasing Cu concentrations did not affect seed germination, and even significantly stimulated the growth of seedlings (Street and Kulkarn, 2007). Jiang and Liu (2001) also found that Cu stimulated the root growth of Zea mays L.. The present data showed that increasing concentrations of Fe and lowering concentrations of Cu (100 and 300) had no significant effect on the germination of wheat seeds, only Cu at 500 mM inhibited this parameter. In contrast, all applied Fe or Cu concentrations reduced root and shoot lengths, and the toxicity of these two metals is more pronounced in roots than in shoots, which can be explained by the facts that plant roots are the first point of contact for heavy metal toxic factors (Kabir and Iqbal, 2008) and that iron and copper contents were higher in the roots than in the leaves in wheat seedlings (Table 1). In addition, seedling growth was more sensitive to copper toxicity than to Fe stress, which might be associated with the stresses of different strength applied in this study. The phytotoxicity of heavy metals arises partly from the excessive generation of ROS including H2O2. For instance, copper induces secondary oxidative stress by importing the formation of harmful ROS (Posmyk and Kontek, 2009). Analyses using fluorescent dye H2DCFDA showed elevated H2O2 levels in the root tissue with increasing Fe and Cu concentrations. Furthermore, we
found that the positive effect of copper stress on H2O2 generation was stronger than that of iron treatment and that the maximal strong fluorescence was caused in 500 mM Cu-treated roots (Fig. 1G). In agreement with the studies of Ruley et al. (2004), in this study increasing H2O2 levels were correlated with the reduction of root growth due to Fe and Cu exposure. Generally MDA, a biomarker of lipid peroxidation, is closely correlated with the level of oxidative stress in plants when exposed to different environmental stresses (Koca et al., 2007). Significant decrease in MDA content under Cu stress was observed by Xu et al. (2011). The present findings correspond with the results of other authors who have observed increased MDA content in various plants under iron and copper excess (Boojar and Goodarzi, 2007; Sinha and Basant, 2009; Zhao and Liu, 2010). Moreover, the elevation of MDA content in this study showed a negative correlation with root length with increasing Fe and Cu concentrations. This further supported the conclusion that the inhibition of root growth was possibly attributed to increased H2O2 generation in response to Fe and Cu exposure because excessive ROS accumulation within plant cells can lead to membrane lipid oxidation, thus affecting plant growth and development (Connolly and Guerinot, 2002; Posmyk and Kontek, 2009). On the contrary, the study of Gajewska et al. (2006) indicated that the inhibition of wheat root growth by 200 mM nickel was not related to lipid peroxidation, Viability staining of root tip cells was used as indicators for Fe and Cu toxicity. The present data showed that lower Fe concentration did not affect cell viability in the root tips but higher Fe dose reduced cell viability. Similarly, exposure to different Cu concentrations significantly increased the number of dead cells within roots (Fig. 3). Moreover, the cell viability in wheat roots was more sensitive to Cu treatment than to Fe stress. These results indicated that Fe and Cu increased the number of dead cells in root tips, which appeared to be associated with the reduction of root growth and development in wheat seedlings. According to Nenova (2006), Fe deficiency caused a decrease of photosynthetic pigments in Pea plants, but excess Fe resulted in risen pigment concentrations. Additionally, no significant difference of chl content and serious decrease of chl synthesis due to iron and copper excess were observed in other plants (Kampfenkel et al., 1995; Shakya et al., 2008; Xing and Huang, 2010; Brahim and Mohamed, 2011). In this study, significant
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increases in the total chl including chl a and chl b were observed in wheat seedlings exposed to increasing Fe concentrations; in contrast, 100 mM Cu led to significant increases in the amount of chl b and the total chl, whereas wheat seedlings exhibited notable decreases in chl a and the total chl contents in response to the highest Cu concentration. Consistent with our results, the total Chl and Chl a contents significantly decreased in Ceratophyllum demersum L. under Cu treatment, while the change in Chl b was not significant (Rama Devi and Prasad, 1998). These findings can be explained by the study of Van Assche and Clijsters (1990) indicating that copper may reduce chl content by inhibiting the pigment biosynthesis and decreasing the photosynthetic electron transport. Besides, chl loss can be related to membrane oxidative damage produced by oxidative stress (Aarti et al., 2006) because increased H2O2 and MDA levels in this investigation were observed in the leaves of Cu-treated seedlings. The photosynthesis efficiency is one of the most important determinants of plant growth, and the photosynthesis efficiency increased with an increase in the content of chl (An and Zhou, 2009). Therefore, another reason for growth reduction in wheat seedlings exposed to Cu may be due to the effect of Cu on the amount of chl involved in the changes of the photosynthesis rate, whereas iron may produce growth reduction by interfering with other cellular processes. In agreement with Cu-inhibitory mechanism, Nichols et al. (2000) demonstrated that the decrease of dry biomass in Salvinia minima seedlings can be attributed to a metal-induced reduction of photosynthetic pigments. According to Boojar and Goodarzi (2007), the elevation of antioxidative enzyme activities is one component of the tolerant adaptation of plants to heavy metal toxicity. POD, CAT and APX constitute main H2O2 scavenging system in cells. In wheat leaves the increased POD activity in response to Fe and Cu stress supported previous suggestion to POD activity as a biomarker for metal toxicity in plants (Metwally et al., 2005). The stimulation of POD has also been reported in the leaves or shoots of various plant species subjected to Cu and Fe treatments (Mehraban et al., 2008; Verma et al., 2011; Wang et al., 2011). These findings indicated that POD plays an important role not only in scavenging H2O2 but also in protecting plant seedlings from oxidative damage during the treatment of heavy metals. CAT is frequently used by cells to rapidly catalyze the decomposition of H2O2 into less reactive gaseous oxygen and water molecules (Tayefi-Nasrabadi et al., 2011). Previous studies showed that low Cu concentration increased CAT activity but high Cu level inhibited this enzyme in Medicago sativa (Wang et al., 2011). Differently, a decrease or no variation of CAT activity was observed in the leaves and shoots of some plants under Cu excess (Bouazizi et al., 2010; Thounaojam et al., 2012). Despite an iron-containing enzyme, the activity of CAT significantly decreased in rice plant under iron excess (Mehraban et al., 2008). On contrary to these studies, in wheat seedlings leaf CAT activity was stimulated under all applied Cu concentrations, whereas Fe treatment did not affect this enzyme, indicating that leaf CAT in wheat seedlings is sensitive to copper stress and appears to be an efficient scavenger of H2O2 under copper treatment. APX is one of the major components of ASH–GSH cycle where it functions to prevent the accumulation of toxic levels of H2O2 in photosynthetic organisms (Thounaojam et al., 2012). Wang et al. (2011) reported that APX activity increased at lower Cu concentrations and reached a maximum at 30 mM of Cu in M. sativa. And also, increased activity of APX was observed in sweet potato grown in the 9.0 mM iron solution (Amstad et al., 1994). Moreover, iron triggers a rapid induction of APX gene expression in Brassica napus (Vansuyt et al. (1997)). In this study, leaf APX activity was higher with 100 and 300 mM Fe than 500 mM Fe, but no significant changes in APX activity was observed in Cu-treated leaves. Because of the
unchanged APX but increased POD, and especially because of the increased CAT, H2O2 content in the leaves associated with copper was significantly lower than that with iron at the same concentration (Table 5). Moreover, significant increase in MDA content indicated increased ROS generation in the leaves of wheat seedlings under Fe and Cu treatments.
5. Conclusions The present results may suggest that the increased H2O2 generation, thus causing oxidative damage, and the reduction of cell viability in the root tips are responsible for inhibitory mechanism of root growth in response to Fe and Cu stresses in plants. Significant increases in the total chl content including chl a and chl b were observed in wheat seedlings exposed to increasing Fe concentrations, whereas the highest Cu content led to significant decreases in these parameters. With regard to seedling growth, cell viability in the root tips, chl content and oxidative stress, Cu was found to be more toxic to wheat than Fe in all applied concentration ranges. Among antioxidant enzymes, leaf CAT and POD appear to play key roles in antioxidative protective mechanisms in wheat seedlings exposed to copper toxicity, but leaf POD in response to all Fe concentrations and APX due to lower Fe dose show important function in scavenging H2O2, which can explain the fact that H2O2 content in the leaves of wheat seedlings associated with copper was lower than that with iron at the same concentration. In addition, the increased MDA levels in response to iron and copper treatments were also observed in the leaf tissue.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 31160088), the Program for New Century Excellent Talents in University of China and Science and Technology Development Plan Program of Lanzhou City (No. 2011-1-142). References Aarti, P.D., Tanaka, R., Tanaka, A., 2006. Effects of oxidative stress on chlorophyll biosynthesis in cucumber (Cucumis sativus) cotyledons. Physiol. Plant. 128, 186–197. Achary, V.M.M., Patnaik, A.R., Panda, B.B., 2012. Oxidative biomarkers in leaf tissue of barley seedlings in response to aluminum stress. Ecotoxicol. Environ. Saf. 75, 16–26. Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Amstad, P., Moret, R., Cerutti, P., 1994. Glutathione peroxidase compensates for the hypersensitivity of Cu, Zn-superoxide dismutase overproducers to oxidant stress. J. Biol. Chem. 269, 1606–1609. An, J., Zhou, Q., 2009. Ecotoxicological effects of typical personal care products on seed germination and seedling development of wheat (Triticum aestivum L.). Chemosphere 76, 1428–1434. Boojar, M.M.A., Goodarzi, F., 2007. The copper tolerance strategies and the role of antioxidative enzymes in three plant species grown on copper mine. Chemosphere 67, 2138–2147. Bouazizi, H., Jouili, H., Geitmann, A., El Ferjani, E., 2010. Copper toxicity in expanding leaves of Phaseolus vulgaris L.: antioxidant enzyme response and nutrient element uptake. Ecotoxicol. Environ. Saf. 73, 1304–1308. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brahim, L., Mohamed, M., 2011. Effects of copper stress on antioxidative enzymes, chlorophyll and protein content in Atriplex halimus. Afr. J. Biotechnol 10, 10143–10148. Connolly, E.L., Guerinot, M., 2002. Iron stress in plants, reviews. Genome Biol. 3, 1–4. Gajewska, E., Slaba, M., Andrzejewska, R., Sklodowska, M., 2006. Nickel-induced inhibition of wheat root growth is related to H2O2 production, but not to lipid peroxidation. Plant Growth Regul. 49, 95–103.
X. Li et al. / Ecotoxicology and Environmental Safety 86 (2012) 47–53
¨ Hansch, R., Mendel, R.R., 2009. Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl). Curr. Opin. Plant Biol. 12, 259–266. ¨ A., Erdei, S., Horva´th, G., 2001. Comparative studies of H2O2 detoxifying Hegedus, enzymes in green and greening barley seedlings under cadmium stress. Plant Sci. 160, 1085–1093. Jiang, W., Liu, D., 2001. Effects of copper on root growth, cell division, and nucleolus of zea mays. Biol. Plant. 44, 105–109. Kabir, M., Iqbal, M.Z., 2008. Reduction in germination and seedling growth of Thespesia populnea L., caused by lead and cadmium treatments. Pak. J. Bot. 40, 2419–2426. Kampfenkel, K., Van Montagu, M., Inze, D., 1995. Effects of iron excess on Nicotiana plumbaginifolia plants (implications to oxidative stress). Plant Physiol. 107, 725–735. Koca, H., Ozdemir, F., Turkan, I., 2007. Effect of salt stress on chlorophyll fluorescence, lipid peroxidation, superoxide dismutase and peroxidase activities of cultivated tomato (L. esculentum) and its wild relative (L. pennellii). Environ. Exp. Bot. 60, 344–351. Li, Y., Song, Y., Shi, G.J., Wang, J.J., Hou, X.L., 2009. Response of antioxidant activity to excess copper in two cultivars of Brassica campestris ssp. chinensis Makino. Acta Physiol. Plant. 31, 155–162. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382. Mehraban, P., Zadeh, A.A., Sadeghipour, H.R., 2008. Iron toxicity in rice (Oryza sativa L.), under different potassium nutrition. Asian J. Plant Sci. 7, 251–259. Metwally, A., Safronova, V.I., Belimov, A.A., Dietz, K.J., 2005. Genotypic variation of the response to cadmium toxicity in Pisum sativum L. J. Exp. Bot. 56, 167–174. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by asorabate specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Nenova, V., 2006. Effect of iron supply on growth and photosystem II efficiency of pea plants. Gen. Appl. Plant Physiol., 81–90. Nichols, P.B., Couch, J.D., Al-Hamdani, S.H., 2000. Selected physiological responses of Salvinia minima to different chromium concentrations. Aquat. Bot. 68, 313–319. Pei, Z.M., Murata, Y., 2000. Calcium channels activated by hydrogen peroxide mediate abscisic acid signaling in guard cells. Nature 406, 731–734. Posmyk, M.M., Kontek, R., 2009. Antioxidant enzymes activity and phenolic compounds content in red cabbage seedlings exposed to copper stress. Ecotoxicol. Environ. Saf. 72, 596–602. Rama Devi, S., Prasad, M.N.V., 1998. Copper toxicity in Ceratophyllum demersum L. (Coontail), a free floating macrophyte: response of antioxidant enzymes and antioxidants. Plant Sci. 138, 157–165. Rao, M.V., Paliyath, G., 1996. Ultraviolet B and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol. 110, 125–136. Ruley, A.T., Sharma, N.C., Sahi, S.V., 2004. Antioxidant defense in a lead accumulating plant, Sesbania drummondii. Plant Physiol. Biochem. 42, 899–906.
53
Sandalio, L.M., Dalurzo, H.C., Gomez, M., Romero-Puertas, M.C., Del Rı´o, L.A., 2001. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 52, 2115–2126. Sergiev, I., Alexieva, V., 1997. Effect of spermine, atrazine and combination between them on some endogenous protective systems and stress markers in plants. C. R. Acad. Bulg. Sci. 51, 121–124. Shakya, K., Chettri, M.K., Sawidis, T., 2008. Impact of heavy metals (copper, zinc, and lead) on the chlorophyll content of some mosses. Arch. Environ. Contam. Toxicol. 54, 412–421. Sinha, S., Basant, A., 2009. Iron-induced oxidative stress in a macrophyte: a chemometric approach. Ecotoxicol. Environ. Saf. 72, 585–595. Street, R.A., Kulkarn, M.G., 2007. Toxicity of metal elements on germination and seedling growth of widely used medicinal plants belonging to Hyacinthaceae. Bull. Environ. Contam. Toxicol. 79, 371–376. Tayefi-Nasrabadi, H., Dehghan, G., Daeihassani, B., Movafegi, A., Samadi, A., 2011. Some biochemical properties of catalase from safflower (Carthamus tinctorius L. cv. M-CC-190). Afr. J. Agric. Res. 6, 5221–5226. Thounaojam, T.C., Panda, P., Mazumdar, P., Kumar, D., Sharma, G.D., Sahoo, L., Panda, S.K., 2012. Excess copper induced oxidative stress and response of antioxidants in rice. Plant Physiol. Biochem. 53, 33–39. Van Assche, F., Clijsters, H., 1990. Effects of metals on enzyme activity in plants. Plant Cell Environ. 13, 195–206. Vansuyt, G., Lopez, F., Inze´, D., Briat, J.F., Fourcroy, P., 1997. Iron triggers a rapid induction of ascorbate peroxidase gene expression in Brassica napus. FEBS Lett. 410, 195–200. Verma, J.P., Singh, V., Yadav, J., 2011. Effect of copper sulphate on seed germination, plant growth and peroxidase activity of Mung bean (Vigna radiata). Int. J. Bot. 7, 200–220. Wang, S.H., Zhang, H., Zhang, Q., Jin, G.M., Jiang, S.J., Jiang, D., He, Q.Y., Li, Z.P., 2011. Copper-induced oxidative stress and responses of the antioxidant system in roots of Medicago sativa. J. Agron. Crop Sci. 197, 418–429. Xing, W., Huang, W., 2010. Effect of excess iron and copper on physiology of aquatic plant Spirodela polyrrhiza (L.) Schleid. Environ. Toxicol. 25, 103–112. Xu, X.Y., Shi, G.X., Wang, J., Zhang, L.L., Kang, Y.N., 2011. Copper-induced oxidative stress in Alternanthera philoxeroides callus. Plant Cell, Tissue Organ Cult. (PCTOC) 106, 243–251. Yang, Y.L., Zhang, Y.Y., Wei, X.L., You, J., Wang, W.R., Lu, J., Shi, R.X., 2011. Comparative antioxidative responses and proline metabolism in two wheat cultivars under short term lead stress. Ecotoxicol. Environ. Saf. 74, 733–740. Yruela, I., 2009. Copper in plants: acquisition, transport and interactions. Funct. Plant Biol. 36, 409–430. Zanardo, D.I.L., Lima, R.B., Ferrarese, M.L.L., Bubna, G.A., Ferrarese-Filho, O., 2009. Soybean root growth inhibition and lignification induced by p-coumaric acid. Environ. Exp. Bot. 66, 25–30. Zhao, S., Liu, Q., 2010. Responses of root growth and protective enzymes to copper stress in turfgrass. Acta Biol. Cracov. Ser. Bot. 52, 7–11.