Bull Environ Contam Toxicol (2014) 93:618–624 DOI 10.1007/s00128-014-1361-z
Involvement of an Antioxidant Defense System in the Adaptive Response to Cadmium in Maize Seedlings (Zea mays L.) Xianghua Xu • Cuiying Liu • Xiaoyan Zhao Renying Li • Wenjing Deng
•
Received: 5 May 2014 / Accepted: 13 August 2014 / Published online: 26 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Chemical and biological analyses were used to investigate the growth response and antioxidant defense mechanism of maize seedlings (Zea mays L.) grown in soils with 0–100 mg kg-1 Cd. Results showed that maize seedlings have strong abilities to accumulate and tolerate high concentrations of Cd. For soil with 50 mg kg-1 Cd, the Cd contents in roots and shoots of maize seedlings are as large as 295.6 and 153.0 mg kg-1 DW, respectively, without visible symptoms of toxicity. Lower soil Cd concentrations lead to a decrease in reduced glutathione (GSH) content in leaves of maize seedlings, whereas higher soil Cd concentrations resulted in an increase in the activities of superoxide dismutase, guaiacol peroxidase, catalase, and ascorbate peroxidase. Maize seedlings have strong capacities to adapt to low concentrations of Cd by consuming GSH and to develop an antioxidative enzyme system to defend against high-Cd stress. Keywords Cadmium Oxidative stress Antioxidant enzymes Glutathione Maize seedlings Cadmium (Cd) is a metallic pollutant that enters the soil environment through various industrial and agricultural activities such as combustion of fossil fuels and use of agro-chemicals. It has been reported that *9.9–45.0 tons of Cd is discharged into the soil every year globally X. Xu (&) C. Liu X. Zhao R. Li Jiangsu Key Laboratory of Agricultural Meteorology, Nanjing University of Information Science and Technology, Nanjing, China e-mail: [email protected] W. Deng Department of Science and Environmental Studies, The Hong Kong Institute of Education, Tai Po, N.T., Hong Kong
123
(Kamnev and van der Lelie 2000). Cd pollution has been considered one of the most serious environmental problems worldwide. Consequently, cleanup of Cd-polluted soils becomes emergent and imperative. Phytoremediation has several benefits for managing heavy metal soil pollution at impacted sites as it is completed in situ, requires relatively few resources, and is environmentally sustainable. At present, there are basically two types of phytoextraction strategies available: one is using hyperaccumulators to extract metals from polluted soils because of very high metal concentration in shoots (Reeves and Baker 2000); another is the use of high biomass plants that are usually not metal-specific plants, and the low metal concentration in shoots can be compensated by the high biomass (Keller et al. 2003). Maize (Zea mays L.) is an important agricultural crop worldwide and it has been used in many studies for phytoremediation of heavy metal pollution due to the rapid growth and high biomass (Wang et al. 2007). However, information about the mechanisms of Cd tolerance in maize seedlings is still limited. Cd is not essential for plant growth, but it is easily taken up by roots and translocated into shoots by many plants species, thereby cause various symptoms of phytotoxicity such as chlorosis, photosynthesis inhibition, biomass reduction and ultimately plant death (Skro´zyn´ska-Polit et al. 2010; Zhang et al. 2013). At molecular level, Cd injury has been attributed to the accumulation of reactive oxygen species (ROS) (Li et al. 2012). Cd stress may disrupt the redox homeostasis of cells and cause the rapid formation of ROS, including superoxide radicals (O2 ), hydrogen peroxide (H2O2), and hydroxyl radicals (OH), which can affect lipids, proteins, carbohydrates, and nucleic acids and cause lipid peroxidation, membrane damage and enzyme inactivation, ultimately affecting the cell viability (Gill and Tuteja 2010). To quench ROS and
Bull Environ Contam Toxicol (2014) 93:618–624
overcome oxidative stresses, plants have developed a diverse array of defense strategies to protect themselves from ROS. These defense strategies mainly consist of antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and non-enzymatic antioxidants such as ascorbic acid (AsA), glutathione (GSH), tocopherol, and carotenoids (Gill and Tuteja 2010; Zhang et al. 2013). SOD is a key enzyme that catalyze the dismutation of O2 into H2O2 and O2. H2O2 is toxic to cells and can be further eliminated by CAT and POD through directly dissimulating H2O2 into H2O and O2. APX, GR and GSH are potential components of the ascorbate–glutathione (AsA–GSH) cycle and play a major role in the H2O2 scavenging pathways in chloroplasts as well as in cytosol (Gill and Tuteja 2010). The specific objectives of this study are: (1) to examine the uptake and accumulation of Cd by maize seedlings; (2) to investigate the effects of Cd toxicity on growth, chloroplast structure and lipid peroxidation; and (3) to evaluate the impacts of Cd on the activities of enzymatic antioxidants (SOD, POD, CAT and APX) and non-enzymatic antioxidants (GSH) in leaves of maize seedlings. Results from this study may provide insight into the tolerance mechanisms of maize seedlings in response to Cd stress.
Methods and Materials Soils used in this study were collected from the suburbs of Nanjing in Jiangsu Province of China; the soils were airdried, ground, and sieved with 3-mm meshes for the experiment. Basic physical and chemical properties of the soil were: pH 5.4, organic matter 1.66 %, and total Cd content 0.048 mg kg-1. 0.8 kg soil was mixed with CdCl2 solutions in ratios of 0, 0.3, 1.0, 3.0, 10, 20, 50, and 100 mg Cd kg-1 dry soil, respectively, packed into plastic pots (14 cm in diameter, 12 cm in height), and then incubated for 2 weeks. Each treatment was replicated three times. The real soil Cd concentrations were measured before sowing, and the deviations of measured Cd concentrations from the nominal concentrations were less all than 10 % (Table 1). For simplicity, the treatments are referred to according to their nominal concentrations in this study. Maize seeds (Zea mays L., cultivar Yedan 13) were obtained from Laizhou Academy of Agricultural Sciences, Shandong Province of China. Seeds were surface-sterilized with 1 % NaClO for 10 min, soaked in distilled water for 24 h, and then sown in soil at a depth of 0.5 cm. After emergence, the seedlings were thinned to 15 plants per pot and grown in a culture chamber with 25/20°C day/night temperature and 400 lmol m-2 s-1 photon flux intensity under 13/11 h day/night regime. The soil water holding
619 Table 1 Nominal and measured soil Cd concentrations (mean ± SD, n = 3) in each treatment (mg kg-1)
Nominal
Measured
0
0.048 ± 0.005
0.3
0.31 ± 0.04
1
0.92 ± 0.09
3
3.09 ± 0.27
10
9.77 ± 0.55
20
19.77 ± 1.12
50
49.30 ± 0.92
100
98.93 ± 5.12
capacity was maintained at 60 % throughout the growth period. Two weeks later, the shoots and roots were harvested individually. The root and shoot samples were washed with tap water and then with deionized water, and finally dried in oven at 80°C for 48 h. The dried samples were digested with concentrated HNO3/HClO4 (10:1, v/v). Cd concentration was determined using atomic absorption spectrophotometer (Thermo Sollar M6, USA). The standard sample of tomato leaves (ESP-1) purchased from the Institute for Environmental Reference Materials, Ministry of Environmental Protection of China was used as a reference material for quality control in this study. A blank was also prepared during the preparation of the samples, and duplicate analysis (three times) of all samples was also employed to minimize the analysis errors. Present measurement and reference value of the standard sample (ESP-1) were 0.75 ± 0.07 and 0.82 ± 0.09 lg g-1 for Cd, respectively. Detection limit of this method was 0.1 lg L-1. For transmission electron microscopy (TEM) analysis, pieces of leaf tissue were fixed in 2.5 % glutaraldehyde (v/v) in a 0.2 M sodium phosphate buffer (pH 7.2) at 4°C for 6–8 h, then buffer-washed and post-fixed in a 1 % osmium tetroxide buffer for 1–2 h. After buffer washing and dehydration in a gradient series of ethanol, the samples were infiltrated and embedded in Epson 812 resin. Ultrathin Sects. (70 nm) were prepared and mounted on copper grids, double stained with aqueous uranyl acetate and lead citrate, and viewed with a transmission electron microscope (Hitachi H-7650, Japan) at an accelerating voltage of 80 kV. Approximately 0.4 g of fresh leaves was homogenized with 2.0 mL of ice-cooled 100 mM sodium-phosphate buffer (pH 7.8) containing 0.1 mM EDTA and 1.0 % polyvinylpyrrolidone (PVPP). The homogenate was centrifuged at 12,000g for 20 min at 4°C. The supernatant was used for antioxidant enzyme determination. The SOD activity was measured spectrophotometrically at 560 nm based on the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Dhindsa et al. 1981). CAT activity was assayed by monitoring the disappearance of
123
620
Results and Discussion Cd concentrations in maize seedlings increased with increasing Cd concentration in the soil (Fig. 1). For soils containing 50 mg kg-1 Cd, the root and shoot Cd contents are as large as 295.6 and 153.0 mg kg-1 DW, respectively, but the biomass is still not affected by the elevated Cd (Table 2). This implied that the maize seedling had strong Cd accumulation and tolerance capacities, although Cd would be toxic to most plants when leaf Cd concentration is greater than 5–10 mg Cd kg-1 dry weight (White and Brown 2010). Considering the rapid growth and high biomass of maize, it would be a potential material for phytoremediation of soil Cd pollution (Wang et al. 2007). To better understand the tolerance and accumulation mechanisms of maize seedlings, the response of biomass, chloroplast ultrastructure, and biochemical parameters were further investigated in this study. Growth inhibition and biomass reduction are general responses of higher plants to heavy metal toxicity. The
123
600
-1
Cd content (mg kg DW)
H2O2 at 240 nm (Cakmak et al. 1993). POD activity was assayed by monitoring the change of absorbance at 470 nm caused by guaiacol oxidation (Cakmak et al. 1993). The activity of APX was measured by monitoring the decrease in the absorbance at 290 nm due to enzymatic oxidation of ascorbic acid by H2O2 (Asada 1984). Lipid peroxidation was determined by the estimation of malondialdehyde (MDA) content (Dhindsa et al. 1981). Approximately 0.25 g frozen leaves were homogenized in 5 mL 0.1 % trichloroacetic acid (TCA) in ice bath. The homogenate was centrifuged at 10,000g for 5 min. Equal volumes of supernatant and 0.5 % 2-thiobarbituric acid (TBA) in 20 % TCA were mixed, incubated at 95°C for 30 min and then quickly cooled. After centrifuging at 10,000g for 10 min, the absorbance of the supernatant was recorded at 532 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm. MDA content was determined using the extinction coefficient of 155 mM-1 cm-1. 0.1 g fresh leaves were homogenized with 2 mL of pre-cooled 100 mM phosphate–EDTA buffer (pH 8.0) containing 25 % metaphosphoric acid. The homogenate was centrifuged at 12,000g for 15 min, and the reduced glutathione (GSH) and oxidized glutathione (GSSG) contents were determined according to Hissin and Hilf (1976). The fluorescence intensity at 420 nm was recorded with the excitation at 350 nm by using fluorescence spectrophotometer (Hitachi 850, Japan). The results are presented as the mean ± standard deviation (SD). The differences between the control and each treatment were analyzed by one-way ANOVA followed by an LSD test using SPSS 11.5 statistical software.
Bull Environ Contam Toxicol (2014) 93:618–624
Shoot
450
Root
300 150 00
20
40
60
80
100
Cd treatment (mg kg -1) Fig. 1 The Cd content in the tissues of maize seedlings grown in Cdtreated soil for 14 days. The values are presented as the mean ± SD (n = 3)
Table 2 The effect of Cd on the biomass (fresh weight) of the shoots and the roots of maize seedlings Cd treatment (mg kg-1)
Shoot FW (g)
Root FW (g) 10.67 ± 0.23
0
10.77 ± 0.97
0.3
10.87 ± 1.41
12.30 ± 1.34
1
10.36 ± 0.73
10.24 ± 1.31
3
10.24 ± 1.27
11.07 ± 0.23
10
10.60 ± 0.65
10.25 ± 0.77
20 50
9.22 ± 1.99 9.30 ± 0.68
9.65 ± 1.53 10.20 ± 0.36
100
7.78 ± 0.67**
8.67 ± 0.82**
Data are presented as the mean ± SD (n = 3). Significant differences from the control are indicated as ** p \ 0.01
visual Cd toxicity symptoms like leaf spots were also observed for maize seedlings grown in soils with 50 and 100 mg kg-1 Cd contents (Fig. 2). Compared with the control, the shoot and root biomass (fresh weight, FW) decreased by 27.8 % and 18.7 %, respectively, as the Cd supply increased to 100 mg kg-1 soil (Table 2). The significant leaf spots and biomass reduction indicated that high Cd concentration produced toxic effects on the growth of maize seedlings. Anjum et al. (2011) found that the dry weight of mung bean (Vigna radiate) seedlings was reduced by 59.8 % at 100 mg Cd kg-1 soil; existing studies also showed that excessive application of Cd may lead to leaf chlorosis, senescence and growth inhibition (Skro´zyn´ska-Polit et al. 2010; Zhang et al. 2013). A possible explanation for the biomass reduction of maize seedlings may be related to its high Cd accumulation (Fig. 1). Nevertheless, no toxicity symptoms were observed for the maize seedlings when the soil Cd concentrations are less than 20 mg kg-1, implying that the maize seedlings tolerated Cd atis 20 mg kg-1 in this soil.
Bull Environ Contam Toxicol (2014) 93:618–624
The recently released results from the 1st Survey on Soil Pollution of China have shown that 82.8 % of the polluted samples are the result of inorganic pollutant, with the top three identified as Cd, Ni, and As (MEP and MLR 2014). Sewage irrigation is the major source of soil Cd pollution in China (Chen et al. 1999) and the Cd concentrations in Cd-polluted soils are generally less than 20 mg kg-1. For example, in the most typical Cd-polluted area of China, Zhangshi Sewage Irrigation Area in Shenyang, the soil Cd concentrations in the most severely polluted areas varies between 5 and 7 mg kg-1 (Wu et al. 1989); and the soil Cd concentrations in a field in Hunan Province of China that irrigated with waste water from ore dressing plant may reach up to 18.2 mg kg-1 (Wang and Zhang 2007). In the present study, the maize can tolerate 20 mg kg-1 Cd in soil and accumulate as large as 92.9 mg kg-1 Cd in shoots, without growth inhibition, toxicity symptoms, and biomass reduction. Therefore, the use of maize as a potential candidate for the remediation of Cd-polluted soils in China would be extremely promising. The ultrastructure of chloroplasts in maize leaves is presented in Fig. 3. The chloroplasts in maize leaves under the control and 20 mg kg-1 Cd treatment reveal a typical chloroplast structure where ellipsoidal shape with wellarranged thylakoid membranes of distinct grana and stroma regions can be intuitively observed (Fig. 3a, b). For maize seedlings grown in soil containing 50 mg kg-1 Cd, however, the chloroplasts had a lower development of grana and diminished stacking of thylakoids (Fig. 3c). When the
Fig. 2 The leaf symptoms of maize seedlings grown in soil with 0 (control a), 20 (b), 50 (c) and 100 (d) mg kg-1 Cd for 14 days
621
soil Cd concentration increased to 100 mg kg-1, the thylakoids were substantially swollen and damaged, thereby a significant reduction in chloroplast size (Fig. 3d). This indicated that Cd stress caused the imbalance synthesis of chloroplast lamellae leading to leaf chlorosis and senescence (Fig. 2). The MDA level is routinely used as an index of lipid peroxidation under stress conditions. The MDA content in leaves of maize seedlings increased significantly as the soil Cd concentration is greater or equal to 50 mg kg-1 (Fig. 4), suggesting that higher Cd levels stimulated the lipid peroxidation and resulted in growth inhibition (Table 2) and irreversible damage of leaf tissues (Figs. 2, 3). The significant growth inhibition, visible injuries, chloroplast damage, and changes in lipid peroxidation of maize seedlings under higher soil Cd treatments (50–100 mg kg-1) implied that the high Cd stress disrupted redox homeostasis of plant cells and caused ROS accumulation (Gill and Tuteja 2010; Anjum et al. 2011). Under the conditions of soil Cd concentration being less than 20 mg kg-1, however, the growth of maize seedlings was still not affected and the Cd content in the roots and shoots may reach to 193.5 and 92.9 mg kg-1 DW (Fig. 1), respectively, indicating that maize seedlings have developed a complex defense system inside the cell to fight against ROS and protect them from oxidative stress. Antioxidant enzymes, such as SOD, CAT, POD and APX, are involved in the processes of scavenging ROS (Gill and Tuteja 2010; Li et al. 2012). SOD act as the first line of defense against ROS by dismutating superoxide O2 to H2O2 and O2. The SOD activity under Cd concentrations ranging from 3 to 10 mg kg-1 increases with the elevated soil Cd concentration (Fig. 5), suggesting that the defense functions and physiological activities of the maize seedling cells were stimulated and the SOD activity was increased for reducing the rate of O2 generation and protecting the cell from the damage of increasing ROS levels. When the soil Cd was increased to 20–50 mg kg-1, the SOD activity turned to be normal and adaptation equilibrium of SOD is reached. However, the SOD activity began to decrease when the soil Cd concentration increase to 100 mg kg-1
Fig. 3 The chloroplast ultrastructure of leaves of maize seedlings exposed to 0 (control a), 20 (b), 50 (c) and 100 (d) mg Cd kg-1 soil for 14 days
123
622
Bull Environ Contam Toxicol (2014) 93:618–624
(Fig. 5). The decrease of SOD activity may be attributed to inhibition caused by the increasing H2O2, because Cu-SOD and Zn-SOD were inactive due to loss of Cu and Zn with increasing H2O2 (Aravind and Prasad 2003).This further verified that the SOD activity has a threshold and the protective function of SOD for membrane system is limited (Luo et al. 2011). The antioxidative network in plant cells is complex and highly interconnected. The balance between SOD and POD, APX, CAT activities may be crucial for determining the steady-state level of O2 and H2O2, and whether the accumulation of H2O2 affects the enzyme activity as well. The enhancement of CAT, POD and APX activities in maize seedling leaves (Fig. 5) showed that the three enzymes were functioning concurrently for removing H2O2 and preventing the formation of highly toxic OH. Meanwhile, the elevated CAT, POD and APX activities demonstrated that the accumulated H2O2 was not enough to cause toxicity. The elevated activities of SOD, POD, APX, and CAT in leaves of maize seedlings exposed to Cd illustrated a typical antioxidative defense mechanism, which may explain why the maize seedlings
-1
MDA content (nmol g FW)
60
∗ ∗∗
50 40 30 20 10 0
0
0.3
1
3
10
20
50
100
-1
Cd treatment (mg kg ) Fig. 4 MDA content in leaves of maize seedlings grown in Cdtreated soil for 14 days. The values are presented as the mean ± SD (n = 3). Significant differences from the control are indicated as *p \ 0.05, **p \ 0.01. 200
-1
U mg FW
SOD 160
1200
∗∗ ∗∗
POD 900
120
∗∗
80 40 0
0 0.3 1 3 10 20 50 100 -1
Cd treatment (mg kg )
∗∗ ∗
240
∗∗
C AT ∗∗
∗∗
4000
∗∗ ∗∗
180
AP X
∗
3000
600
120
2000
300
60
1000
0
0 0.3 1 3 10 20 50 100 -1
Cd treatment (mg kg )
Fig. 5 The response of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) in leaves of maize seedlings grown in Cd-treated soil for 14 days. The
123
can tolerant and accumulate considerably high concentrations of Cd. Depending on Cd concentration and plant species, Cd may inhibit or stimulate the activity of several antioxidative enzymes before any visible symptoms of toxicity appear (Anjum et al. 2011; Gill et al. 2012). Since the increase and decrease of antioxidant activities in response to Cd stress were also observed, it is conceivable that the antioxidant system, in addition to its function in detoxification, may also be a sensitive target of Cd toxicity in plants. This study showed that low Cd levels (1 mg kg-1) led to increased APX activity in maize leaves, this suggested that APX made an early adaptive response to Cd stress and may be a prospective biomarker of maize seedlings to soil Cd. The idea that APX is a potential biomarker of pollutant stress was also reported in several studies (Bonet et al. 2012; Song et al. 2012). GSH as a non-enzymatic antioxidant is often the first line of defense against oxidative stress. The present study showed that GSH of maize seedling leaves decreased significantly when the soil Cd concentration is less than 20 mg kg-1 (Fig. 6a). GSH is consumed by the AsA–GSH pathway as a substrate for glutathione peroxidase (GPX) and glutathione-S-transferases (GST), thereby is involved in the removal of ROS (Noctor et al. 2002). In addition, GSH is depleted during PCs synthesis because GSH is a precursor for the synthesis of phytochelatins (PCs), which can complex with Cd and reduce the toxicity of Cd2? to the cell. In this study, the decrease in GSH concentrations may be associated with the production of PCs. This finding is consistent with Cobbett (2000), who reported that a decrease in GSH content in Silene cucubalus under Cd stress was associated with the PCs synthesis. Hence, it could be inferred that the consumption of GSH to reduce the toxicity of Cd2? may be a primary mechanism for plants to adapt to low-concentration Cd stress. GSH is also acting as an important antioxidant to scavenge ROS through the oxidation of GSH to GSSG. When the soil Cd concentrations varied within 3–20 mg kg-1, the slightly increased levels of GSSG and the reduced GSH levels demonstrated a transformation from GSH to GSSG
0
0 0.3 1
3 10 20 50 100 -1
Cd treatment (mg kg )
0
∗ ∗
∗ ∗ ∗∗
0 0.3 1 3 10 20 50 100
Cd treatment (mg kg-1)
values are presented as the mean ± SD (n = 3). Significant differences from the control are indicated as *p \ 0.05, **p \ 0.01
A
∗∗
∗
-1
40 30
∗
20
∗
∗∗
10 0
∗
0
0.3
1
3
10
20
50 100 -1
Cd treatment (mg kg )
623 70 60
B
∗∗ ∗∗
50 40
∗
1.2
∗∗
∗∗
30 20
C
0.8
∗∗
0.6
∗∗
∗
0.4
∗
∗
50
100
∗∗ ∗∗
0.2
10 0
1.0
GSH/GSSG
50
GSSG content (µg g FW)
-1
GSH content (µg g FW)
Bull Environ Contam Toxicol (2014) 93:618–624
0
0.3
1
3
10
20
50 100 -1
Cd treatment (mg kg )
0.0 0
0.3
1
3
10
20 -1
Cd treatment (mg kg )
Fig. 6 The glutathione content in the leaves of maize seedlings grown in Cd-treated soil for 14 days. The values are presented as the mean ± SD (n = 3). Significant differences from the control are indicated as *p \ 0.05, **p \ 0.01
(Fig. 6b). For higher-concentration Cd treatment (50–100 mg kg-1), GSH levels in maize leaves increased sharply instead of continuing decline, suggests the presence of a new GSH supplement. Gill et al. (2012) also found that higher concentration of Cd would lead to increase in glutathione pool. The GSH/GSSG ratios decreased significantly under the low-Cd (0.3–20 mg kg-1) treatments (Fig. 6c), this may be partly caused by the low GSH levels, while the subsequent increase in GSH/GSSG ratio may be attributed to the establishment of GSH adaptive equilibrium. The normal GSH/GSSG ratio plays a key role in normal physiological conditions, and it is a potential indicator of oxidative stress (Nel et al. 2006). However, the mechanisms of disturbance in glutathione pool remain unclear, and the rationalities of this ratio as an index of oxidative stress still need to be confirmed. Conclusion Maize seedlings have a strong ability to accumulate and tolerate Cd. The plant has developed several strategies to adapt and defend against Cd toxicity. GSH provides a first defense line against Cd toxicity, and the maize seedlings can reduce the Cd toxicity through the GSH consumption at low Cd levels (0.3–10 mg Cd kg-1 soil). For high Cd stress (10–50 mg Cd kg-1 soil), maize seedlings developed an antioxidative enzyme response system through increasing the activities of SOD, POD, CAT and APX to scavenge ROS and reduce Cd toxicity. At very high Cd level (100 mg Cd kg-1 soil), however, the Cd toxicity may cause growth inhibition of maize seedlings and damages of chloroplast tissues, but the morphologic parameters such as biomass, leaf symptom and chloroplast ultrastructure appear to be insensitive to the Cd toxicity. Acknowledgments This project is supported by National Natural Science Foundation of China (No. 41101294) and Jiangsu Provincial Natural Science Foundation (No. BK2010572).
References Anjum NA, Umar S, Iqbal M, Khan NA (2011) Cadmium causes oxidative stress in mung bean by affecting the antioxidant enzyme system and ascorbate-glutathione cycle metabolism. Russ J Plant Physiol 58:92–99 Aravind P, Prasad MNV (2003) Zinc alleviates cadmium-induced oxidative stress in Ceratophyllum demersum L.: a free floating freshwater macrophyte. Plant Physiol Biochem 41:391–397 Asada K (1984) Chloroplasts: formation of active oxygen and its scavenging. Methods Enzymol 105:422–429 Bonet B, Corcoll N, Guasch H (2012) Antioxidant enzyme activities as biomarkers of Zn pollution in fluvial biofilms. Ecotox Environ Saf 80:172–178 Cakmak I, Strbac D, Marschner H (1993) Activities of hydrogen peroxide-scavenging enzymes in germinating wheat seeds. J Exp Bot 44:127–132 Chen H, Zheng C, Tu C, Zhu Y (1999) Heavy metal pollution in soils in China: status and countermeasures. Ambio 28(2):130–134 Cobbett CS (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physio 123:825–832 Dhindsa RS, Plumb-Dhindsa P, Thorpe TA (1981) Leaf senescence correlated: with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J Exp Bot 32:93–101 Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930 Gill SS, Khan NA, Tuteja N (2012) Cadmium at high dose perturbs growth, photosynthesis and nitrogen metabolism while at low dose it up regulates sulfur assimilation and antioxidant machinery in garden cress (Lepidium sativum L.). Plant Sci 182:112–120 Hissin PJ, Hilf R (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74:214–226 Kamnev AA, van der Lelie D (2000) Chemical and biological parameters as tools to evaluate and improve heavy metal phytoremediation. Biosci Rep 20:239–258 Keller C, Hammer D, Kayser A, Richner W, Brodbeck M, Sennhauser M (2003) Root development and heavy metal phytoextraction efficiency: comparison of different plant species in the field. Plant Soil 249:67–81 Li X, Zhang L, Li Y, Ma L, Bu N, Ma C (2012) Changes in photosynthesis, antioxidant enzymes and lipid peroxidation in soybean seedlings exposed to UV-B radiation and/or Cd. Plant Soil 352:377–387
123
624 Luo H, Li H, Zhang X, Fu J (2011) Antioxidant responses and gene expression in perennial ryegrass (Lolium perenne L.) under cadmium stress. Ecotoxicology 20:770–778 MEP (Ministry of Environmental Protection, PR China), MLR (Ministry of Land and Resources, PR China) (2014) Report of the national general survey on soil contamination (Jointly released by MEP and MLR on Apr. 17, 2014) Nel A, Xia T, Ma¨dler L, Li N (2006) Toxic potential of materials at the nanolevel. Science 311:622–627 Noctor G, Gomea L, Vanacker H, Foyer CH (2002) Interaction between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signaling. J Exp Bot 53:1283–1304 Reeves RD, Baker AJM (2000) Metal-accumulating plants. In: Raskin I, Ensley BD (eds) Phytoremediation of toxic metals using plants to clean up the environment. John Wily and Sons Inc., NY, pp 193–230 Skro´zyn´ska-Polit E, Dra˛z_ kiewicz M, Krupa Z (2010) Lipid peroxidation and antioxidative response in Arabidopsis thaliana exposed to cadmium and copper. Acta Physiol Plant 32:169–175
123
Bull Environ Contam Toxicol (2014) 93:618–624 Song H, Wang Y, Sun C, Wang Y, Peng Y, Cheng H (2012) Effects of pyrene on antioxidant systems and lipid peroxidation level in mangrove plants, Bruguiera gymnorrhiza. Ecotoxicology 21:1625–1632 Wang K, Zhang Y (2007) Investigation and evaluation on Cd pollution of the acidic farmland soils irrigated with Cd-polluted wastewater for 25 years. J Agro-Environ Sci 26(2):658–661 (in Chinese) Wang M, Zou J, Duan X, Jiang W, Liu D (2007) Cadmium accumulation and its effects on metal uptake in maize (Zea mays L.). Bioresour Technol 98:82–88 White PJ, Brown PH (2010) Plant nutrition for sustainable development and global health. Ann Bot 105:1073–1080 Wu Y, Chen T, Zhang X (1989) Ecological study on the Cd pollution in Zhangshi irrigation at Shenyang. Acta Ecologica Sinica 9(1):21–26 (in Chinese) Zhang B, Shang S, Zhang H, Jabeen Z, Zhang G (2013) Sodium chloride enhances cadmium tolerance through reducing cadmium accumulation and increasing anti-oxidative enzyme activity in tobacco. Environ Toxicol Chem 32:1420–1425
Involvement of an Antioxidant Defense System in the Adaptive Response to Cadmium in Maize Seedlings (Zea mays L.) Xianghua Xu • Cuiying Liu • Xiaoyan Zhao Renying Li • Wenjing Deng
•
Received: 5 May 2014 / Accepted: 13 August 2014 / Published online: 26 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Chemical and biological analyses were used to investigate the growth response and antioxidant defense mechanism of maize seedlings (Zea mays L.) grown in soils with 0–100 mg kg-1 Cd. Results showed that maize seedlings have strong abilities to accumulate and tolerate high concentrations of Cd. For soil with 50 mg kg-1 Cd, the Cd contents in roots and shoots of maize seedlings are as large as 295.6 and 153.0 mg kg-1 DW, respectively, without visible symptoms of toxicity. Lower soil Cd concentrations lead to a decrease in reduced glutathione (GSH) content in leaves of maize seedlings, whereas higher soil Cd concentrations resulted in an increase in the activities of superoxide dismutase, guaiacol peroxidase, catalase, and ascorbate peroxidase. Maize seedlings have strong capacities to adapt to low concentrations of Cd by consuming GSH and to develop an antioxidative enzyme system to defend against high-Cd stress. Keywords Cadmium Oxidative stress Antioxidant enzymes Glutathione Maize seedlings Cadmium (Cd) is a metallic pollutant that enters the soil environment through various industrial and agricultural activities such as combustion of fossil fuels and use of agro-chemicals. It has been reported that *9.9–45.0 tons of Cd is discharged into the soil every year globally X. Xu (&) C. Liu X. Zhao R. Li Jiangsu Key Laboratory of Agricultural Meteorology, Nanjing University of Information Science and Technology, Nanjing, China e-mail: [email protected] W. Deng Department of Science and Environmental Studies, The Hong Kong Institute of Education, Tai Po, N.T., Hong Kong
123
(Kamnev and van der Lelie 2000). Cd pollution has been considered one of the most serious environmental problems worldwide. Consequently, cleanup of Cd-polluted soils becomes emergent and imperative. Phytoremediation has several benefits for managing heavy metal soil pollution at impacted sites as it is completed in situ, requires relatively few resources, and is environmentally sustainable. At present, there are basically two types of phytoextraction strategies available: one is using hyperaccumulators to extract metals from polluted soils because of very high metal concentration in shoots (Reeves and Baker 2000); another is the use of high biomass plants that are usually not metal-specific plants, and the low metal concentration in shoots can be compensated by the high biomass (Keller et al. 2003). Maize (Zea mays L.) is an important agricultural crop worldwide and it has been used in many studies for phytoremediation of heavy metal pollution due to the rapid growth and high biomass (Wang et al. 2007). However, information about the mechanisms of Cd tolerance in maize seedlings is still limited. Cd is not essential for plant growth, but it is easily taken up by roots and translocated into shoots by many plants species, thereby cause various symptoms of phytotoxicity such as chlorosis, photosynthesis inhibition, biomass reduction and ultimately plant death (Skro´zyn´ska-Polit et al. 2010; Zhang et al. 2013). At molecular level, Cd injury has been attributed to the accumulation of reactive oxygen species (ROS) (Li et al. 2012). Cd stress may disrupt the redox homeostasis of cells and cause the rapid formation of ROS, including superoxide radicals (O2 ), hydrogen peroxide (H2O2), and hydroxyl radicals (OH), which can affect lipids, proteins, carbohydrates, and nucleic acids and cause lipid peroxidation, membrane damage and enzyme inactivation, ultimately affecting the cell viability (Gill and Tuteja 2010). To quench ROS and
Bull Environ Contam Toxicol (2014) 93:618–624
overcome oxidative stresses, plants have developed a diverse array of defense strategies to protect themselves from ROS. These defense strategies mainly consist of antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and non-enzymatic antioxidants such as ascorbic acid (AsA), glutathione (GSH), tocopherol, and carotenoids (Gill and Tuteja 2010; Zhang et al. 2013). SOD is a key enzyme that catalyze the dismutation of O2 into H2O2 and O2. H2O2 is toxic to cells and can be further eliminated by CAT and POD through directly dissimulating H2O2 into H2O and O2. APX, GR and GSH are potential components of the ascorbate–glutathione (AsA–GSH) cycle and play a major role in the H2O2 scavenging pathways in chloroplasts as well as in cytosol (Gill and Tuteja 2010). The specific objectives of this study are: (1) to examine the uptake and accumulation of Cd by maize seedlings; (2) to investigate the effects of Cd toxicity on growth, chloroplast structure and lipid peroxidation; and (3) to evaluate the impacts of Cd on the activities of enzymatic antioxidants (SOD, POD, CAT and APX) and non-enzymatic antioxidants (GSH) in leaves of maize seedlings. Results from this study may provide insight into the tolerance mechanisms of maize seedlings in response to Cd stress.
Methods and Materials Soils used in this study were collected from the suburbs of Nanjing in Jiangsu Province of China; the soils were airdried, ground, and sieved with 3-mm meshes for the experiment. Basic physical and chemical properties of the soil were: pH 5.4, organic matter 1.66 %, and total Cd content 0.048 mg kg-1. 0.8 kg soil was mixed with CdCl2 solutions in ratios of 0, 0.3, 1.0, 3.0, 10, 20, 50, and 100 mg Cd kg-1 dry soil, respectively, packed into plastic pots (14 cm in diameter, 12 cm in height), and then incubated for 2 weeks. Each treatment was replicated three times. The real soil Cd concentrations were measured before sowing, and the deviations of measured Cd concentrations from the nominal concentrations were less all than 10 % (Table 1). For simplicity, the treatments are referred to according to their nominal concentrations in this study. Maize seeds (Zea mays L., cultivar Yedan 13) were obtained from Laizhou Academy of Agricultural Sciences, Shandong Province of China. Seeds were surface-sterilized with 1 % NaClO for 10 min, soaked in distilled water for 24 h, and then sown in soil at a depth of 0.5 cm. After emergence, the seedlings were thinned to 15 plants per pot and grown in a culture chamber with 25/20°C day/night temperature and 400 lmol m-2 s-1 photon flux intensity under 13/11 h day/night regime. The soil water holding
619 Table 1 Nominal and measured soil Cd concentrations (mean ± SD, n = 3) in each treatment (mg kg-1)
Nominal
Measured
0
0.048 ± 0.005
0.3
0.31 ± 0.04
1
0.92 ± 0.09
3
3.09 ± 0.27
10
9.77 ± 0.55
20
19.77 ± 1.12
50
49.30 ± 0.92
100
98.93 ± 5.12
capacity was maintained at 60 % throughout the growth period. Two weeks later, the shoots and roots were harvested individually. The root and shoot samples were washed with tap water and then with deionized water, and finally dried in oven at 80°C for 48 h. The dried samples were digested with concentrated HNO3/HClO4 (10:1, v/v). Cd concentration was determined using atomic absorption spectrophotometer (Thermo Sollar M6, USA). The standard sample of tomato leaves (ESP-1) purchased from the Institute for Environmental Reference Materials, Ministry of Environmental Protection of China was used as a reference material for quality control in this study. A blank was also prepared during the preparation of the samples, and duplicate analysis (three times) of all samples was also employed to minimize the analysis errors. Present measurement and reference value of the standard sample (ESP-1) were 0.75 ± 0.07 and 0.82 ± 0.09 lg g-1 for Cd, respectively. Detection limit of this method was 0.1 lg L-1. For transmission electron microscopy (TEM) analysis, pieces of leaf tissue were fixed in 2.5 % glutaraldehyde (v/v) in a 0.2 M sodium phosphate buffer (pH 7.2) at 4°C for 6–8 h, then buffer-washed and post-fixed in a 1 % osmium tetroxide buffer for 1–2 h. After buffer washing and dehydration in a gradient series of ethanol, the samples were infiltrated and embedded in Epson 812 resin. Ultrathin Sects. (70 nm) were prepared and mounted on copper grids, double stained with aqueous uranyl acetate and lead citrate, and viewed with a transmission electron microscope (Hitachi H-7650, Japan) at an accelerating voltage of 80 kV. Approximately 0.4 g of fresh leaves was homogenized with 2.0 mL of ice-cooled 100 mM sodium-phosphate buffer (pH 7.8) containing 0.1 mM EDTA and 1.0 % polyvinylpyrrolidone (PVPP). The homogenate was centrifuged at 12,000g for 20 min at 4°C. The supernatant was used for antioxidant enzyme determination. The SOD activity was measured spectrophotometrically at 560 nm based on the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Dhindsa et al. 1981). CAT activity was assayed by monitoring the disappearance of
123
620
Results and Discussion Cd concentrations in maize seedlings increased with increasing Cd concentration in the soil (Fig. 1). For soils containing 50 mg kg-1 Cd, the root and shoot Cd contents are as large as 295.6 and 153.0 mg kg-1 DW, respectively, but the biomass is still not affected by the elevated Cd (Table 2). This implied that the maize seedling had strong Cd accumulation and tolerance capacities, although Cd would be toxic to most plants when leaf Cd concentration is greater than 5–10 mg Cd kg-1 dry weight (White and Brown 2010). Considering the rapid growth and high biomass of maize, it would be a potential material for phytoremediation of soil Cd pollution (Wang et al. 2007). To better understand the tolerance and accumulation mechanisms of maize seedlings, the response of biomass, chloroplast ultrastructure, and biochemical parameters were further investigated in this study. Growth inhibition and biomass reduction are general responses of higher plants to heavy metal toxicity. The
123
600
-1
Cd content (mg kg DW)
H2O2 at 240 nm (Cakmak et al. 1993). POD activity was assayed by monitoring the change of absorbance at 470 nm caused by guaiacol oxidation (Cakmak et al. 1993). The activity of APX was measured by monitoring the decrease in the absorbance at 290 nm due to enzymatic oxidation of ascorbic acid by H2O2 (Asada 1984). Lipid peroxidation was determined by the estimation of malondialdehyde (MDA) content (Dhindsa et al. 1981). Approximately 0.25 g frozen leaves were homogenized in 5 mL 0.1 % trichloroacetic acid (TCA) in ice bath. The homogenate was centrifuged at 10,000g for 5 min. Equal volumes of supernatant and 0.5 % 2-thiobarbituric acid (TBA) in 20 % TCA were mixed, incubated at 95°C for 30 min and then quickly cooled. After centrifuging at 10,000g for 10 min, the absorbance of the supernatant was recorded at 532 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm. MDA content was determined using the extinction coefficient of 155 mM-1 cm-1. 0.1 g fresh leaves were homogenized with 2 mL of pre-cooled 100 mM phosphate–EDTA buffer (pH 8.0) containing 25 % metaphosphoric acid. The homogenate was centrifuged at 12,000g for 15 min, and the reduced glutathione (GSH) and oxidized glutathione (GSSG) contents were determined according to Hissin and Hilf (1976). The fluorescence intensity at 420 nm was recorded with the excitation at 350 nm by using fluorescence spectrophotometer (Hitachi 850, Japan). The results are presented as the mean ± standard deviation (SD). The differences between the control and each treatment were analyzed by one-way ANOVA followed by an LSD test using SPSS 11.5 statistical software.
Bull Environ Contam Toxicol (2014) 93:618–624
Shoot
450
Root
300 150 00
20
40
60
80
100
Cd treatment (mg kg -1) Fig. 1 The Cd content in the tissues of maize seedlings grown in Cdtreated soil for 14 days. The values are presented as the mean ± SD (n = 3)
Table 2 The effect of Cd on the biomass (fresh weight) of the shoots and the roots of maize seedlings Cd treatment (mg kg-1)
Shoot FW (g)
Root FW (g) 10.67 ± 0.23
0
10.77 ± 0.97
0.3
10.87 ± 1.41
12.30 ± 1.34
1
10.36 ± 0.73
10.24 ± 1.31
3
10.24 ± 1.27
11.07 ± 0.23
10
10.60 ± 0.65
10.25 ± 0.77
20 50
9.22 ± 1.99 9.30 ± 0.68
9.65 ± 1.53 10.20 ± 0.36
100
7.78 ± 0.67**
8.67 ± 0.82**
Data are presented as the mean ± SD (n = 3). Significant differences from the control are indicated as ** p \ 0.01
visual Cd toxicity symptoms like leaf spots were also observed for maize seedlings grown in soils with 50 and 100 mg kg-1 Cd contents (Fig. 2). Compared with the control, the shoot and root biomass (fresh weight, FW) decreased by 27.8 % and 18.7 %, respectively, as the Cd supply increased to 100 mg kg-1 soil (Table 2). The significant leaf spots and biomass reduction indicated that high Cd concentration produced toxic effects on the growth of maize seedlings. Anjum et al. (2011) found that the dry weight of mung bean (Vigna radiate) seedlings was reduced by 59.8 % at 100 mg Cd kg-1 soil; existing studies also showed that excessive application of Cd may lead to leaf chlorosis, senescence and growth inhibition (Skro´zyn´ska-Polit et al. 2010; Zhang et al. 2013). A possible explanation for the biomass reduction of maize seedlings may be related to its high Cd accumulation (Fig. 1). Nevertheless, no toxicity symptoms were observed for the maize seedlings when the soil Cd concentrations are less than 20 mg kg-1, implying that the maize seedlings tolerated Cd atis 20 mg kg-1 in this soil.
Bull Environ Contam Toxicol (2014) 93:618–624
The recently released results from the 1st Survey on Soil Pollution of China have shown that 82.8 % of the polluted samples are the result of inorganic pollutant, with the top three identified as Cd, Ni, and As (MEP and MLR 2014). Sewage irrigation is the major source of soil Cd pollution in China (Chen et al. 1999) and the Cd concentrations in Cd-polluted soils are generally less than 20 mg kg-1. For example, in the most typical Cd-polluted area of China, Zhangshi Sewage Irrigation Area in Shenyang, the soil Cd concentrations in the most severely polluted areas varies between 5 and 7 mg kg-1 (Wu et al. 1989); and the soil Cd concentrations in a field in Hunan Province of China that irrigated with waste water from ore dressing plant may reach up to 18.2 mg kg-1 (Wang and Zhang 2007). In the present study, the maize can tolerate 20 mg kg-1 Cd in soil and accumulate as large as 92.9 mg kg-1 Cd in shoots, without growth inhibition, toxicity symptoms, and biomass reduction. Therefore, the use of maize as a potential candidate for the remediation of Cd-polluted soils in China would be extremely promising. The ultrastructure of chloroplasts in maize leaves is presented in Fig. 3. The chloroplasts in maize leaves under the control and 20 mg kg-1 Cd treatment reveal a typical chloroplast structure where ellipsoidal shape with wellarranged thylakoid membranes of distinct grana and stroma regions can be intuitively observed (Fig. 3a, b). For maize seedlings grown in soil containing 50 mg kg-1 Cd, however, the chloroplasts had a lower development of grana and diminished stacking of thylakoids (Fig. 3c). When the
Fig. 2 The leaf symptoms of maize seedlings grown in soil with 0 (control a), 20 (b), 50 (c) and 100 (d) mg kg-1 Cd for 14 days
621
soil Cd concentration increased to 100 mg kg-1, the thylakoids were substantially swollen and damaged, thereby a significant reduction in chloroplast size (Fig. 3d). This indicated that Cd stress caused the imbalance synthesis of chloroplast lamellae leading to leaf chlorosis and senescence (Fig. 2). The MDA level is routinely used as an index of lipid peroxidation under stress conditions. The MDA content in leaves of maize seedlings increased significantly as the soil Cd concentration is greater or equal to 50 mg kg-1 (Fig. 4), suggesting that higher Cd levels stimulated the lipid peroxidation and resulted in growth inhibition (Table 2) and irreversible damage of leaf tissues (Figs. 2, 3). The significant growth inhibition, visible injuries, chloroplast damage, and changes in lipid peroxidation of maize seedlings under higher soil Cd treatments (50–100 mg kg-1) implied that the high Cd stress disrupted redox homeostasis of plant cells and caused ROS accumulation (Gill and Tuteja 2010; Anjum et al. 2011). Under the conditions of soil Cd concentration being less than 20 mg kg-1, however, the growth of maize seedlings was still not affected and the Cd content in the roots and shoots may reach to 193.5 and 92.9 mg kg-1 DW (Fig. 1), respectively, indicating that maize seedlings have developed a complex defense system inside the cell to fight against ROS and protect them from oxidative stress. Antioxidant enzymes, such as SOD, CAT, POD and APX, are involved in the processes of scavenging ROS (Gill and Tuteja 2010; Li et al. 2012). SOD act as the first line of defense against ROS by dismutating superoxide O2 to H2O2 and O2. The SOD activity under Cd concentrations ranging from 3 to 10 mg kg-1 increases with the elevated soil Cd concentration (Fig. 5), suggesting that the defense functions and physiological activities of the maize seedling cells were stimulated and the SOD activity was increased for reducing the rate of O2 generation and protecting the cell from the damage of increasing ROS levels. When the soil Cd was increased to 20–50 mg kg-1, the SOD activity turned to be normal and adaptation equilibrium of SOD is reached. However, the SOD activity began to decrease when the soil Cd concentration increase to 100 mg kg-1
Fig. 3 The chloroplast ultrastructure of leaves of maize seedlings exposed to 0 (control a), 20 (b), 50 (c) and 100 (d) mg Cd kg-1 soil for 14 days
123
622
Bull Environ Contam Toxicol (2014) 93:618–624
(Fig. 5). The decrease of SOD activity may be attributed to inhibition caused by the increasing H2O2, because Cu-SOD and Zn-SOD were inactive due to loss of Cu and Zn with increasing H2O2 (Aravind and Prasad 2003).This further verified that the SOD activity has a threshold and the protective function of SOD for membrane system is limited (Luo et al. 2011). The antioxidative network in plant cells is complex and highly interconnected. The balance between SOD and POD, APX, CAT activities may be crucial for determining the steady-state level of O2 and H2O2, and whether the accumulation of H2O2 affects the enzyme activity as well. The enhancement of CAT, POD and APX activities in maize seedling leaves (Fig. 5) showed that the three enzymes were functioning concurrently for removing H2O2 and preventing the formation of highly toxic OH. Meanwhile, the elevated CAT, POD and APX activities demonstrated that the accumulated H2O2 was not enough to cause toxicity. The elevated activities of SOD, POD, APX, and CAT in leaves of maize seedlings exposed to Cd illustrated a typical antioxidative defense mechanism, which may explain why the maize seedlings
-1
MDA content (nmol g FW)
60
∗ ∗∗
50 40 30 20 10 0
0
0.3
1
3
10
20
50
100
-1
Cd treatment (mg kg ) Fig. 4 MDA content in leaves of maize seedlings grown in Cdtreated soil for 14 days. The values are presented as the mean ± SD (n = 3). Significant differences from the control are indicated as *p \ 0.05, **p \ 0.01. 200
-1
U mg FW
SOD 160
1200
∗∗ ∗∗
POD 900
120
∗∗
80 40 0
0 0.3 1 3 10 20 50 100 -1
Cd treatment (mg kg )
∗∗ ∗
240
∗∗
C AT ∗∗
∗∗
4000
∗∗ ∗∗
180
AP X
∗
3000
600
120
2000
300
60
1000
0
0 0.3 1 3 10 20 50 100 -1
Cd treatment (mg kg )
Fig. 5 The response of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) in leaves of maize seedlings grown in Cd-treated soil for 14 days. The
123
can tolerant and accumulate considerably high concentrations of Cd. Depending on Cd concentration and plant species, Cd may inhibit or stimulate the activity of several antioxidative enzymes before any visible symptoms of toxicity appear (Anjum et al. 2011; Gill et al. 2012). Since the increase and decrease of antioxidant activities in response to Cd stress were also observed, it is conceivable that the antioxidant system, in addition to its function in detoxification, may also be a sensitive target of Cd toxicity in plants. This study showed that low Cd levels (1 mg kg-1) led to increased APX activity in maize leaves, this suggested that APX made an early adaptive response to Cd stress and may be a prospective biomarker of maize seedlings to soil Cd. The idea that APX is a potential biomarker of pollutant stress was also reported in several studies (Bonet et al. 2012; Song et al. 2012). GSH as a non-enzymatic antioxidant is often the first line of defense against oxidative stress. The present study showed that GSH of maize seedling leaves decreased significantly when the soil Cd concentration is less than 20 mg kg-1 (Fig. 6a). GSH is consumed by the AsA–GSH pathway as a substrate for glutathione peroxidase (GPX) and glutathione-S-transferases (GST), thereby is involved in the removal of ROS (Noctor et al. 2002). In addition, GSH is depleted during PCs synthesis because GSH is a precursor for the synthesis of phytochelatins (PCs), which can complex with Cd and reduce the toxicity of Cd2? to the cell. In this study, the decrease in GSH concentrations may be associated with the production of PCs. This finding is consistent with Cobbett (2000), who reported that a decrease in GSH content in Silene cucubalus under Cd stress was associated with the PCs synthesis. Hence, it could be inferred that the consumption of GSH to reduce the toxicity of Cd2? may be a primary mechanism for plants to adapt to low-concentration Cd stress. GSH is also acting as an important antioxidant to scavenge ROS through the oxidation of GSH to GSSG. When the soil Cd concentrations varied within 3–20 mg kg-1, the slightly increased levels of GSSG and the reduced GSH levels demonstrated a transformation from GSH to GSSG
0
0 0.3 1
3 10 20 50 100 -1
Cd treatment (mg kg )
0
∗ ∗
∗ ∗ ∗∗
0 0.3 1 3 10 20 50 100
Cd treatment (mg kg-1)
values are presented as the mean ± SD (n = 3). Significant differences from the control are indicated as *p \ 0.05, **p \ 0.01
A
∗∗
∗
-1
40 30
∗
20
∗
∗∗
10 0
∗
0
0.3
1
3
10
20
50 100 -1
Cd treatment (mg kg )
623 70 60
B
∗∗ ∗∗
50 40
∗
1.2
∗∗
∗∗
30 20
C
0.8
∗∗
0.6
∗∗
∗
0.4
∗
∗
50
100
∗∗ ∗∗
0.2
10 0
1.0
GSH/GSSG
50
GSSG content (µg g FW)
-1
GSH content (µg g FW)
Bull Environ Contam Toxicol (2014) 93:618–624
0
0.3
1
3
10
20
50 100 -1
Cd treatment (mg kg )
0.0 0
0.3
1
3
10
20 -1
Cd treatment (mg kg )
Fig. 6 The glutathione content in the leaves of maize seedlings grown in Cd-treated soil for 14 days. The values are presented as the mean ± SD (n = 3). Significant differences from the control are indicated as *p \ 0.05, **p \ 0.01
(Fig. 6b). For higher-concentration Cd treatment (50–100 mg kg-1), GSH levels in maize leaves increased sharply instead of continuing decline, suggests the presence of a new GSH supplement. Gill et al. (2012) also found that higher concentration of Cd would lead to increase in glutathione pool. The GSH/GSSG ratios decreased significantly under the low-Cd (0.3–20 mg kg-1) treatments (Fig. 6c), this may be partly caused by the low GSH levels, while the subsequent increase in GSH/GSSG ratio may be attributed to the establishment of GSH adaptive equilibrium. The normal GSH/GSSG ratio plays a key role in normal physiological conditions, and it is a potential indicator of oxidative stress (Nel et al. 2006). However, the mechanisms of disturbance in glutathione pool remain unclear, and the rationalities of this ratio as an index of oxidative stress still need to be confirmed. Conclusion Maize seedlings have a strong ability to accumulate and tolerate Cd. The plant has developed several strategies to adapt and defend against Cd toxicity. GSH provides a first defense line against Cd toxicity, and the maize seedlings can reduce the Cd toxicity through the GSH consumption at low Cd levels (0.3–10 mg Cd kg-1 soil). For high Cd stress (10–50 mg Cd kg-1 soil), maize seedlings developed an antioxidative enzyme response system through increasing the activities of SOD, POD, CAT and APX to scavenge ROS and reduce Cd toxicity. At very high Cd level (100 mg Cd kg-1 soil), however, the Cd toxicity may cause growth inhibition of maize seedlings and damages of chloroplast tissues, but the morphologic parameters such as biomass, leaf symptom and chloroplast ultrastructure appear to be insensitive to the Cd toxicity. Acknowledgments This project is supported by National Natural Science Foundation of China (No. 41101294) and Jiangsu Provincial Natural Science Foundation (No. BK2010572).
References Anjum NA, Umar S, Iqbal M, Khan NA (2011) Cadmium causes oxidative stress in mung bean by affecting the antioxidant enzyme system and ascorbate-glutathione cycle metabolism. Russ J Plant Physiol 58:92–99 Aravind P, Prasad MNV (2003) Zinc alleviates cadmium-induced oxidative stress in Ceratophyllum demersum L.: a free floating freshwater macrophyte. Plant Physiol Biochem 41:391–397 Asada K (1984) Chloroplasts: formation of active oxygen and its scavenging. Methods Enzymol 105:422–429 Bonet B, Corcoll N, Guasch H (2012) Antioxidant enzyme activities as biomarkers of Zn pollution in fluvial biofilms. Ecotox Environ Saf 80:172–178 Cakmak I, Strbac D, Marschner H (1993) Activities of hydrogen peroxide-scavenging enzymes in germinating wheat seeds. J Exp Bot 44:127–132 Chen H, Zheng C, Tu C, Zhu Y (1999) Heavy metal pollution in soils in China: status and countermeasures. Ambio 28(2):130–134 Cobbett CS (2000) Phytochelatins and their roles in heavy metal detoxification. Plant Physio 123:825–832 Dhindsa RS, Plumb-Dhindsa P, Thorpe TA (1981) Leaf senescence correlated: with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J Exp Bot 32:93–101 Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930 Gill SS, Khan NA, Tuteja N (2012) Cadmium at high dose perturbs growth, photosynthesis and nitrogen metabolism while at low dose it up regulates sulfur assimilation and antioxidant machinery in garden cress (Lepidium sativum L.). Plant Sci 182:112–120 Hissin PJ, Hilf R (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74:214–226 Kamnev AA, van der Lelie D (2000) Chemical and biological parameters as tools to evaluate and improve heavy metal phytoremediation. Biosci Rep 20:239–258 Keller C, Hammer D, Kayser A, Richner W, Brodbeck M, Sennhauser M (2003) Root development and heavy metal phytoextraction efficiency: comparison of different plant species in the field. Plant Soil 249:67–81 Li X, Zhang L, Li Y, Ma L, Bu N, Ma C (2012) Changes in photosynthesis, antioxidant enzymes and lipid peroxidation in soybean seedlings exposed to UV-B radiation and/or Cd. Plant Soil 352:377–387
123
624 Luo H, Li H, Zhang X, Fu J (2011) Antioxidant responses and gene expression in perennial ryegrass (Lolium perenne L.) under cadmium stress. Ecotoxicology 20:770–778 MEP (Ministry of Environmental Protection, PR China), MLR (Ministry of Land and Resources, PR China) (2014) Report of the national general survey on soil contamination (Jointly released by MEP and MLR on Apr. 17, 2014) Nel A, Xia T, Ma¨dler L, Li N (2006) Toxic potential of materials at the nanolevel. Science 311:622–627 Noctor G, Gomea L, Vanacker H, Foyer CH (2002) Interaction between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signaling. J Exp Bot 53:1283–1304 Reeves RD, Baker AJM (2000) Metal-accumulating plants. In: Raskin I, Ensley BD (eds) Phytoremediation of toxic metals using plants to clean up the environment. John Wily and Sons Inc., NY, pp 193–230 Skro´zyn´ska-Polit E, Dra˛z_ kiewicz M, Krupa Z (2010) Lipid peroxidation and antioxidative response in Arabidopsis thaliana exposed to cadmium and copper. Acta Physiol Plant 32:169–175
123
Bull Environ Contam Toxicol (2014) 93:618–624 Song H, Wang Y, Sun C, Wang Y, Peng Y, Cheng H (2012) Effects of pyrene on antioxidant systems and lipid peroxidation level in mangrove plants, Bruguiera gymnorrhiza. Ecotoxicology 21:1625–1632 Wang K, Zhang Y (2007) Investigation and evaluation on Cd pollution of the acidic farmland soils irrigated with Cd-polluted wastewater for 25 years. J Agro-Environ Sci 26(2):658–661 (in Chinese) Wang M, Zou J, Duan X, Jiang W, Liu D (2007) Cadmium accumulation and its effects on metal uptake in maize (Zea mays L.). Bioresour Technol 98:82–88 White PJ, Brown PH (2010) Plant nutrition for sustainable development and global health. Ann Bot 105:1073–1080 Wu Y, Chen T, Zhang X (1989) Ecological study on the Cd pollution in Zhangshi irrigation at Shenyang. Acta Ecologica Sinica 9(1):21–26 (in Chinese) Zhang B, Shang S, Zhang H, Jabeen Z, Zhang G (2013) Sodium chloride enhances cadmium tolerance through reducing cadmium accumulation and increasing anti-oxidative enzyme activity in tobacco. Environ Toxicol Chem 32:1420–1425
