Journal of Biological Sciences

Year: 2009 | Volume: 9 | Issue: 1 | Page No.: 44-50
DOI: 10.3923/jbs.2009.44.50

Activity of Antioxidant Enzymes in Response to Cadmium in Arabidopsis thaliana

A. Saffar, M.B. Bagherieh Najjar and M. Mianabadi

Abstract: The effects of the heavy metal cadmium (Cd⁺²) on growth and activities of the antioxidant enzymes, catalase (CAT), peroxidase (POD) and polyphenol oxidase (PPO) have been investigated in Arabidopsis thaliana L. seedlings. The concentration of 50 and 100 μM CdCl₂ was shown to strongly inhibit the growth of roots and lipid peroxidation. Lipid peroxidation of seedlings shoots rose with increasing concentrations of Cd⁺² as indicated by malondialdehyde (MDA) concentration. As Cd⁺² concentration increased, catalase (CAT) activity declined progressively, while peroxidase and polyphenol oxidase activity increased when compared to the untreated plants. Close correlations between increased MDA formation and decreased root growth as well as CAT activity suggests that lipid peroxidation might caused cell damage and death proposing that applied concentrations of Cd⁺² could be toxic to cells. It was also noted that Cd⁺²-induced cell injury and lipid peroxidation correlated with increased peroxidase and polyphenol oxidase activities, two antioxidant enzymes involved in polyphenol peroxidation as lignification substrates. Together, the results suggest that in Arabidopsis thaliana reactive oxygen species (ROS) could be induced by phytotoxic concentrations of Cd⁺² leading to increased POD and PPO activities which play a crucial role in detoxification of elevated concentrations of Cd⁺² possibly via lignifications and physical barrier formation.

Keywords: Arabidopsis, cadmium, polyphenol oxidase and peroxidase

INTRODUCTION

Any metallic chemical element that has relatively high density and is toxic for living organisms at low concentrations such as mercury (Hg), cadmium (Cd⁺²), arsenic (As), chromium (Cr), thallium (Ti) and lead (Pb) are called heavy metals. Small traces of heavy metal contamination in the environment can lead to poisoning levels because they tend to accumulate in biological systems over time (Lee et al., 2005). Cadmium toxic properties come from its chemical similarity to zinc, an essential micronutrient for plants, animals and humans. In humans, long-term exposure to cadmium may cause renal dysfunction, lung disease and cancer, as well as bone defects. Because cadmium and zinc (or occasionally lead) occur naturally within the raw ore, it is produced as an inevitable by-product of their refining. The most usage of cadmium is in nickel/cadmium batteries, as pigments, stabilizers for PVC, in alloys and electronic compounds, as an impurity in several products, including phosphate fertilizers, detergents and refined petroleum products. A high concentration of cadmium, copper and silver in waters has been identified as a potential health and environmental hazard (Hanzlík et al., 2004).

Cadmium is not only a plant nutrition element (Yang et al., 1997; Thevenod and Friedmann, 1999) but also is a hazardous heavy metal that has high mobility in soil-plant systems and can enter the food chain very easily (Nriagu and Pacyna, 1988).

In aerobic conditions, heavy metals may induce reactive oxygen species (ROS) such as O₂^-, H₂O₂, ^.OH, ¹O₂. In plants, ROS may cause damage to cell membranes, proteins, DNA replication and repair, as well as reduction of chloroplast pigments (Somashekaraiah et al., 1992; Drazkiewicz et al., 2004). Therefore, as the first level in the food chain, deduction of photosynthetic activity, leading to decline in crop yield happens by excess of cadmium (Ouzounidou, 1996; Maksymiec, 1997; Siedlecka et al., 2001).

Treatment of plants with Cd⁺² causes leaf roll, chlorosis, reduced growth (Krupa, 1988); as well as inhibition of chlorophyll synthesis and various reactions in Calvin cycle (Padmaja et al., 1990). The experimental evidences suggest that exposure of plants to Cd⁺² inhibits activities of some enzymes including hexokinase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase (Chugh and Sawhney, 1999), nitrate and nitrite reductase (Boussama et al., 1999; Kumar and Dubey, 1999) and enzymes involved in sulfate assimilation (Lee and Leustek, 1999). Many of these inhibitory effects are thought to be caused by ROS created by Cd⁺² (Sandalio et al., 2001).

Plants possess two very efficient antioxidant defense systems: (i) the enzymatic pathway including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), polyphenol oxidase (PPO), glutathione oxidase (GSHOx) and (ii) non-enzymatic antioxidant defense systems such as ascorbate, glutathione,α-tocopherol and carotenoids. Both of these pathways allow for scavenging of ROS leading to protection of plant cells from oxidative damage (Gratao et al., 2005; Blokhina et al., 2003).

In these studies, the effect(s) of cadmium, as a destructive heavy metal in environment, on some aspects of enzymatic anti-oxidant systems of Arabidopsis studied and showed that Arabidopsis can be employed as powerful indicator of Cd⁺² contamination in the environment.

MATERIALS AND METHODS

Plant materials and growth conditions: Arabidopsis thaliana L. wild type seeds were surface-sterilized by washing with 70% (v/v) ethanol for 2 min and sodium hypochlorite solution (20%) for 8 min in Gorgan University of Agricultural Sciences and Natural Resources (Golestan University) in Iran (2006-2007).

Seeds were then washed 3 times in sterile distilled water and germinated on agar plates containing MS (Murashige and Skoog, 1962) salts, supplemented with or without cadmium chloride (50, 85 and 100 mM). Plates were kept at 4°C in the dark for 4 days to synchronize germination then, transferred to a climate room at 22°C and 65% relative humidity with 16 h light (60 μmol m^-2 sec^-1)/ 8 h dark cycles. Twenty one day old plants were used for biochemical analysis.

Lipid peroxidation: Lipid peroxidation was measured by the thio-barbituric assay for malondialdehyde (MDA) as lipid peroxidation index (Shalata and Neumann, 2001) with some modifications as follows. 0.05 g of plant material was ground in 2 mL of 0.1% (w/v) cool trichloro-acetic acid (TCA) on ice. The homogenate was centrifuged at 10,000 x g for 5 min at 4°C and then 250 μL of supernatant was mixed with 2 mL TCA/TBA reagent (0.25% TBA containing 10% TCA). The mixture was incubated at 95°C for 30 min and kept on ice for 15 min. Then, it was centrifuged at 10,000 x g for 10 min. Absorbance of sample was read at 532, 600 and 440 nm. Lipid perixidation were calculated (Du and Bramlage, 1992) as the malonyldialdehyde (MDA) equivalents according to this equation:

LP (nmol mL^-1) = [[(A₅₃₂ -A₆₀₀) -[(A₄₄₀-A₆₀₀) (MA of sucrose at 532 nm /MA of sucrose at 440 nm)]]/157000]10⁶

Trypan blue exclusion and cell viability: Cell viability was measured by trypan blue exclusion (Pasqualini et al., 2003). Leaves were plunged in lactophenol trypan blue (30 mL ethanol, 10 mL glycerol, 10 mL lactic acid, 10 mg trypan blue and 10 mL distilled water) and boiled at 95°C for 2-3 min and then at room temperature for 1 h. Samples were transferred into hydrate chloral solution (2.5 g mL^-1) and boiled for decolorized about 20 min. After several exchanges of hydrate chloral solution, samples were equilibrated in 50% (w/v) glycerol and visually observed under stereomicroscopy; non-viable cells were blue-stained.

Enzyme activities assay: For preparation of crude enzyme extracts, a 0.05 g sample of fresh leaves was ground in 2 mL of 0.1 M cool phosphate buffer (pH 6.8) on ice bath (Kar and Mishra, 1976). Crude extract was centrifuged at 15,000 x g for 15 min at 4°C. The supernatant was used for catalase, peroxidase and polyphenol oxidase activity assays. The protein concentration of the supernatant was measured according to Lowry et al. (1951). Catalase and peroxidase activities were measured according to Erdeil et al. (2002) as follows:

Catalase: The assay mixture (3 mL) contained 15 mM H₂O₂, 50 mM phosphate buffer (pH 6.8) and 100 μL enzymes extract. The decline at absorbance in 40 nm was scanned automatically with a spectrophotometer (Shimidzo UV-160) in kinetic mode. Molar extension coefficient of catalase is 40 L mM^-1cm^-1.

Peroxidase: The assay mixture (3 mL) contained 20 mM guaiacol, 25 mM phosphate buffer (pH 6.8), 40 mM H₂O₂, 10 μL enzyme extract. Guaiacol peroxidase activity was scanned in 470 nm; molar extension coefficient of peroxidase is 26.6 L mM^-1cm^-1.

Polyphenol oxidase: The assay mixture (3 mL) of contained 10 mM pirogalol, 25 mM phosphate buffer (pH 6.8) and 200 μL enzyme extract. Enzyme activity determined in 420 nm, its molar extension coefficient is 2.47 L mM^-1cm^-1.

Statistical analysis: Leaves of all developmental stages of control and treated plants were sampled separately for analyses and the experiments were repeated four times. The data presented in figures are the mean of four independent experiments with calculated standard deviations. A Duncan test was used to identify statistical differences between pairs of means at a confidence level of 95% for each set of data.

RESULTS AND DISCUSSION

The effect(s) of cadmium on Arabidopsis thaliana growth: Arabidopsis thaliana was cultured on MS medium with different concentration of cadmium. The result shown, increased concentration of cadmium affects on Arabidopsis thaliana growth parameters like as fresh weight, length of roots, chlorophyll content and etc. Figure 1 showed the samples with the best difference among them. The Fig. 2 (average of measurement) revealed cadmium inhibited the growth of Arabidopsis thaliana both fresh weight and length of roots (100 μM). Also, high concentration of cadmium decreased amount of lateral roots of plants (Fig. 2).

Lipid peroxidation and cell death: Malonyldialdehyde (MDA) content as lipid peroxidation index of twenty-one day-old Arabidopsis thaliana under the effect of cadmium was established. Present results that showed MDA content increased in treated plants. Its maximum was belonging to treated plant grown on media completed with 100 μM of cadmium compared to control (Fig. 3). The results of cell membranes damage is structure and function perturbation and finally cell death. Trypan blue staining showed the most coloring as cell death parameter was occurred under the effect of 100 μM of cadmium and shown significance increase compared with control (Fig. 4).

Antioxidant enzymes activity: The activity of catalase, guaiacol peroxidase and polyphenol oxidase assessed in Arabidopsis thaliana treated with cadmium and control. Cd⁺² treated plants showed statistically significant decrease in catalase activity compared with controls (Fig. 5). Whereas, increasing Cd⁺² concentration in medium was causing enhanced the activity of peroxidase and exposure to 100 μM Cd⁺² were caused a marked increased enzyme activity with statistically significant between treated plant and control. However, the plant exposure to Cd⁺² don`t show the alterations but only on 100 μM Cd⁺² supplement, PPO activity of Arabidopsis thaliana seedling statistically significant increased.

The results showed that oxidative stress was produced in Arabidopsis thaliana seedling under the effect of Cd⁺², hazardous heavy metal. ROS can react with amino acids, unsaturated fatty acids and other molecules to cause peroxidation of essential lipids in the membranes and proteins and etc. Lipid peroxidation damages membrane integrity (cell death) resulting in increases in the permeability of the plasma membrane, which leads to leakage of K⁺ ions and other solutes so plant growth was inhibited.

Figure 1 shown, Cd⁺² excess in medium were caused inhibition of roots growth, reduction of roots number and fresh weight as growth parameters. It was thinking reduction of surface area of leaves and photosynthesis affects on plant growth. It also has been indicated the high concentration of Cd⁺² probably could inhibits essential metal absorption like Fe⁺² and Zn⁺² and then plant growth (Rogers et al., 2000). Although, chlorophyll content wasn`t measured in treated plant but chlorophyll reduction become observable especially on 100 μM of Cd concentration. Therefore, reduction of chlorophyll content and photosynthesis could be other reason for inhibition of plant growth. In the other Cd⁺² treated plants, destruction of chlorophyll or disturbance of chlorophyll biosynthesis introduced as an important agent of reducing of photosynthesis and plant growth (Somashekaraiah et al., 1992; Barcelo et al., 1988). A significant reduction of total root length of Arabidopsis thaliana seedling under the effect of Cd⁺² was shown (Fig. 1), this is under the effect of inhibition of cell enlargement or cell division exposure to Cd⁺². Aidid and Okamoto (1993) reported that Cd⁺² is an irreversible inhibitor of proton pump responsible for elongation growth rate, turgor pressure and cell wall extensibility. So, reduction of cell enlargement caused the root growth of Arabidopsis thaliana to be inhibited.

According to these results, MDA content in Cd⁺² treated Arabidopsis thaliana seedling increased, this result is in agreement with data obtained on other Cd⁺² treated plant for example bean plants (Somashekaraiah et al., 1992), sunflower (Gallego et al., 1996) and maize and pea plants (Lozano-Rodrigues et al., 1997). MDA is one of several low molecular weight end products formed via decomposition of certain primary and secondary lipid peroxidation products and MDA enhancement is lipid peroxidation and oxidative stress indicator. So, phytotoxicity of Cd⁺² was stablished by enhancement of free radical and lipid peroxidation.

According to Mashoudi et al. (1997) responses of antioxidative enzymes to metal stress like excess Cd⁺² vary among plant species and even different plant tissues. Moreover, H₂O₂ can accumulate in plant tissues where the antioxidant activities are suppressed and detoxify due to catalase and peroxidase activities. Cd⁺² significantly reduced Catalase activity in treated plants compared to control (Fig. 5). Such a result is in agreement with data obtained by Sandalio et al. ( 2001) in pea leaves, Somashekaraiah et al. (1992) in bean plants and Gallego et al. (1996) in sunflower. CAT activity doesn`t change after incubation with 100 μM Cd<sup>+2</sup>, this evidence indicated, direct addition of Cd<sup>+2</sup> don`t affect on CAT activity. Immunoaffinity purification of pea plant catalase with the anti-catalase antibody revealed the decline of CAT activity is under the effect of reduction of protein (Sandalio et al., 2001).

Cd²⁺ induced significantly enhancement in guaiacol peroxidase activity compared to control. Activation of peroxidase-dependent reactions involved in oxidation of many organic compounds like polyphenols in suberine and lignin deposition (Lagrimini, 1991; Schopfer, 1996; Quiroga et al., 2000; Lin and Kao, 2001). Schutzendubel et al. (2001) reported enhancement of peroxidase activity, oxidation of phenols, and lignification in Scots Pine Roots exposure to 50 μM Cd⁺². Lignification process as a defense response induced in response to pathogen invasion (Cosgrove, 1997), salinity stress (Lin and Kao, 2001), and heavy metal toxicity (Schutzendubel et al., 2001). Therefore, toxicity of Cd⁺² was decreased by peroxidase consumption of H₂O₂ (Wang et al., 2004) and lignification process as a physical barrier against heavy metals (Hegedus, 2001). The capability of adaptation to stress can relate to the increase or sustained ability to form defense barriers for reinforcing the cell structure by lignification-related enzymes. However, increase in the activities of lignification-related enzymes, particularly PPO activity, might be the result from prolonged heavy metal stress.

REFERENCES