Bot. Bull. Acad. Sin. (2004) 45: 291-299

Kuo and Kao — Antioxidant enzymes and Cd toxicity in rice seedling

Antioxidant enzyme activities are upregulated in response to cadmium in sensitive, but not in tolerant, rice (Oryza sativa L.) seedlings

Mei Chun KUO and Ching Huei KAO*

Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China

(Received January 14, 2004; Accepted May 3, 2004)

Abstract. Changes in H₂O₂ and malondialdehyde (MDA) contents and antioxidant enzyme activities in Cd-treated rice (Oryza sativa L.) seedlings of two cultivars were investigated. On treatment with CdCl₂, increases in H₂O₂ and MDA contents and antioxidant enzyme activities [speroxide dismutase (SOD), ascorbate peroxidase, glutathione reductase, catalase, and peroxidase (POX)] were observed in the leaves of Cd-sensitive cultivar (cv. Taichung Native 1, TN1) but not in Cd-tolerant cultivar (cv. Tainung 67, TNG67). The increased content of MDA and activities of SOD and POX preceded the occurrence of toxicity in CdCl₂-treated TN1 leaves. Pretreatment with abscisic acid (ABA) enhanced Cd tolerance and reduced Cd-induced increase in the content of MDA and increase in the activities of SOD and POX in TN1 leaves. Exogenous application of ABA biosynthesis inhibitor, fluridone, decreased Cd tolerance, increased the content of MDA, and increased the activities of SOD and POX in Cd-treated TNG67 leaves. Furthermore, fluridone's effects on toxicity, the content of MDA, and the activities of SOD and POX in Cd-treated TNG67 leaves were reversed by the application of ABA. In conclusion, the oxidative stress is differently expressed in TN1 and TNG67 rice seedlings in response to CdCl₂. Results also suggest that CdCl₂ causes an oxidative stress and CdCl₂-induced toxicity is mediated through oxidative stress in TN1 leaves.

Keywords: Abscisic acid; Cadmium; Oryza sativa L.; Oxidative stress.

Abbreviations: ABA, abscisic acid; APX, ascorbate peroxidase; CAT, catalase; Flu, fluridone; FW, fresh weight; GR, glutathione reductase; MDA, malondialdehyde; POX, peroxidase; SOD, superoxide dismutase; TN1, Taichung Native 1; TNG67, Tainung 67.

Introduction

Oxygen is essential for the existence of aerobic life, but toxic active oxygen species (AOS), which include the superoxide anion (O₂^-), hydroxyl radical (OH^.) and hydrogen peroxide (H₂O₂), are generated in all aerobic cells during metabolic processes (Foyer et al., 1994; Asada, 1999). Injury caused by these AOS, known as oxidative stress, is one of the major damaging factors in plants exposed to environmental stress. Plants cope with oxidative stress by using antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), peroxidase (POX), catalase (CAT), and the low molecular weight antioxidants, ascorbic acid and glutathione (Noctor and Foyer, 1998; Asada, 1999).

Cadmium (Cd), a heavy metal toxic to humans, animals, and plants is a widespread pollutant with a long biological half-life (Wagner, 1993). Three lines of evidence indicate that one mechanism of Cd toxicity is related to oxidative

stress in plant cells. First, Cd can promote the generation of AOS (Piqueras et al., 1999; Romero-Puertas et al., 1999; Chen et al., 2000; Shah et al., 2001; Sandalio et al., 2001; Schützendübel et al., 2001; Olmos et al., 2003). Second, Cd can inhibit or stimulate the activities of antioxidant enzymes (Shaw, 1995; Gallego et al., 1996; Chaoui et al., 1997; Dixit et al., 2001; Shah et al., 2001; Iannelli et al., 2002; León et al., 2002). Third, treatment with Cd results in cellular oxidative damage or lipid peroxidation (Shaw, 1995; Gallego et al., 1996; Chaoui et al., 1997; Lozano-Rodríguez et al., 1997; Dixit et al., 2001; Shah et al., 2001; Chien et al., 2002).

This study examines H₂O₂ content, lipid peroxidation, and antioxidant enzyme activities in two cultivars of rice (Oryza sativa L.) seedlings in response to Cd. One cultivar (cv. Taichung Native 1, TN1) is known to be sensitive to Cd, and the other (cv. Tainung 67, TNG67) is known to show significant tolerance to Cd (Hsu and Kao, 2003a). If a mechanism related to oxidative stress is involved in Cd toxicity, this mechanism should then be differently expressed in plants tolerant and sensitive to Cd. Therefore, the aim of this study was to investigate whether the oxidative stress mechanism is differently expressed in TN1 and TNG67 rice seedlings in response to Cd.

*Corresponding author. Tel: 886-2-23698159; Fax: 886-2-23620879; E-mail: kaoch@ntu.edu.tw

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

Materials and Methods

Plant Cultivation and Treatment

Two rice cultivars, an Indica type cultivar, TN1 and a Japonica cultivar, TNG67, obtained from Taiwan Agricultural Research Institute, Taichung, Taiwan, were used in this study. Seeds were sterilized with 2.5% (v/v) sodium hypochlorite for 15 min and washed extensively with distilled water. These seeds were then germinated in Petri dishes with wetted filter papers at 37°C in the dark. After 48 h incubation, uniformly germinated seeds were selected and cultivated in a 250 ml beaker containing half-strength Kimura B solution without aeration as described previously (Chu and Lee, 1989). The concentrations of N, P, K, S, Ca and Mg in half strength Kimura B solution were 11.5, 2.9, 7.2, 15.0, 7.4, and 8.7 p.p.m., respectively. The hydroponically cultivated seedlings were grown in a Phytotron (Agricultural Experimental Station, National Taiwan university, Taipei, Taiwan) with natural light at 30°C day (12 h)/25°C night (12 h) and 90% relative humidity. Twelve-day-old seedlings with three leaves were used in all experiments.

For Cd, abscisic acid (ABA), and fluridone (Flu) treatment, CdCl₂ (0.5 or 1.5 mM) , ABA (5 µM), and Flu (0.2 mM) were added directly to the culture solution during experiment.

Cd Determination

For determination of Cd, leaves or roots were dried at 65°C for 48 h. Dried material was ashed at 550°C for 20 h. The ash residue was incubated with 31% HNO₃ and 17.5 % (v/v) H₂O₂ at 72°C for 2 h, and dissolved in 0.1 N HCl. Cd was then quantified using an atomic absorption spectrophotometer (Model AA-6800; Shimadzu, Kyoto, Japan). The amount of Cd was expressed on the basis of dry weight (DW).

Determination of Chlorophyll, Protein, H₂O₂, and Lipid Peroxidation

Chlorophyll content was determined according to Wintermans and De Mots (1965) after extraction in 96% (v/v) ethanol. For protein determination, leaves were homogenized in a 50 mM sodium phosphate buffer (pH 6.8). The extracts were centrifuged at 17,600 g for 20 min, and the supernatants were used for determination of protein by the method of Bradford (1976) and antioxidant enzyme activities. The H₂O₂ content was measured colorometrically as described by Jana and Choudhuri (1981). H₂O₂ was extracted by homogenizing leaf tissue with phosphate buffer (50 mM, pH 6.5) containing 1 mM hydroxylamine. The homogenate was centrifuged at 6,000 g for 25 min. To determine H₂O₂ content, extracted solution was mixed with 0.1% titanium chloride in 20% (v/v) H₂SO₄. The mixture was then centrifuged at 6,000 g for 25 min. The absorbance was measured at 410 nm. The H₂O₂ content was calculated using the extinction coefficient 0.28 µmol^-1 cm^-1. Malondialdehyde (MDA), routinely used as an indicator of lipid peroxidation, was extracted with 5% trichloroace

tic acid and determined according Heath and Packer (1968). Chlorophyll, protein, H₂O₂, and MDA contents were expressed on the basis of initial fresh weight (FW).

Enzyme Assays

CAT activity was assayed by measuring the initial rate of disappearance of H₂O₂ (Kato and Shimizu, 1987). The decrease in H₂O₂ was followed as the decline in absorbance at 240 nm, and activity was calculated using the extinction coefficient (40 mM^-1 cm^-1 for at 240 nm) for H₂O₂ (Kato and Shimizu, 1987). POX activity was measured using a modification of the procedure of MacAdam et al. (1992). Activity was calculated using the extinction coefficient (26.2 mM^-1 cm^-1 at 470 nm) for tetraguaiacol. SOD was determined according to Paoletti et al. (1986). APX was determined according to Nakano and Asada (1981). The decrease in ascorbate concentration was followed as the decline in absorbance at 290 nm and activity was calculated using the extinction coefficient (2.8 mM^-1 cm^-1 at 290 nm) for ascorbate. GR was determined by the method Foster and Hess (1980). One unit of activity for CAT, POX, SOD, APX, and GR was defined as the amount of enzyme which degraded 1 µmol tetraguaiacol per min, inhibited by 50% the rate of NADH oxidation observed in control, degraded 1 µmol of ascorbate per min, and decreased 1 A₃₄₀ per min, respectively. Enzyme activities are expressed on the basis of mg protein.

Statistical Analysis

Statistical differences between measurements (n = 4) on different treatments or on different times were analyzed following Duncan's multiple range test or Student's t-test.

Results

TNG67 Is More Tolerant to Cd Than TN1

In plants, the most apparent symptom of Cd toxicity is chlorosis of the leaves (Das et al., 1997). When rice seedlings of TN1 and TNG67 were treated with 0.5 mM CdCl₂ for 7 d, leaf chlorosis was observed in TN1 seedlings, but not in TNG67 seedlings (Figure 1). In short term (3 d) experiments, chlorosis was first observed in the second leaf of TN1 seedlings. In the present investigation, unless otherwise indicated, Cd toxicity in the second leaf caused by 0.5 mM CdCl₂ was assessed by a decrease in chlorophyll and protein contents. Figure 2 shows the time courses of chlorophyll and protein contents in the second leaf of TN1 and TNG67 seedlings treated with or without 0.5 mM CdCl₂. It is clear that the decrease in chlorophyll and protein contents in TN1 leaves is more pronounced than in TNG 67 leaves, indicating that TNG67 seedlings are a Cd-tolerant cultivar while TN1 seedlings are Cd-sensitive. Figure 3 shows the changes in Cd content in rice seedling treated with 0.5 mM CdCl₂. Cd content in the second leaf and roots of TNG67 seedlings remained unchanged and slightly increased, respectively, after Cd treatment. In contrast, a marked increase in Cd content in Cd-treated TN1 leaves and roots was observed.

Kuo and Kao — Antioxidant enzymes and Cd toxicity in rice seedling

It seems that avoiding the build-up of Cd in the second leaf of TNG 67 prevents the toxic effect of Cd.

Oxidative Stress is Induced by Cd in TN1 but Not in TNG67 Leaves

MDA content in the second leaf remained unchanged in TN1 seedlings treated without CdCl₂ (Figure 4). It is clear that increase in MDA content in TN1 leaves induced by CdCl₂ was evident at 1 d after CdCl₂ treatment (Figure 4).

Figure 3. Changes in Cd content in the second leaf and roots of rice seedlings treated with or without CdCl₂ (0.5 mM). Bars represent standard errors (n = 4). Asterisks represent values significantly different at P < 0.05.

Figure 1. Effect of CdCl₂ (0.5 mM) on leaf chlorosis of Taichung Native 1 (TN1) and Tainung 67 (TNG67) rice seedlings. Picture was taken after 7 d of treatment.

Figure 2. Changes in chlorophyll and protein contents in the second leaf of rice seedlings treated with or without CdCl₂ (0.5 mM). Bars represent standard errors (n = 4). Asterisks represent values significantly different at P < 0.05.

Figure 4. Changes in H₂O₂ and MDA contents in the second leaf of rice seedlings treated with or without CdCl₂ (0.5 mM). Bars represent standard errors (n = 4). Asterisks represent values significantly different at P < 0.05.

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

However, no increase in MDA content in TNG 67 leaves was observed throughout the CdCl₂ treatment (Figure 4). This showed that the Cd toxicity in TN1 leaves was linked to lipid peroxidation. Lipid peroxidation is caused by AOS (Thompson et al., 1987). CdCl₂ treatment also caused an increase in H₂O₂ content in TN1 but not in TNG67 leaves (Figure 4). These results all support the involvement of AOS as the chemical species inducing Cd toxicity in TN1 leaves.

The striking increase in lipid peroxidation seen in TN1 leaves treated with CdCl₂ may be a reflection of the changes in antioxidant enzyme activities. As shown in Figures 5 and 6, CdCl₂-treated TN1 leaves had higher activities of SOD and POX than the controls at 1 d after treatment. Higher activities of GR, APX, and CAT activities of TN1 leaves was observed at 2 and 3 d, respectively, after treatment. However, no or a slight increase in antioxidant enzyme activities in Cd-treated TNG67 leaves was observed (Figures 5 and 6).

Oxidative Stress in TN1 Leaves Induced by Cd Precedes the Occurrence of Cd Toxicity

In order to know if increase in MDA content and antioxidant enzyme activities caused by CdCl₂ in TN1 leaves is a cause or a consequence of Cd toxicity, a short-term experiment (24 h) was conducted. As shown in Figure 7, increase in the content of MDA and the activities of SOD

Figure 6. Changes in the activities of CAT and POX in the second leaf of rice seedlings treated with or without CdCl₂ (0.5 mM). Bars represent standard errors (n = 4). Asterisks represent values significantly different at P < 0.05.

Figure 7. Changes in the contents of protein and MDA and the activities of SOD and POX in the second leaf of TN1 rice seedlings treated with or without CdCl₂ (0.5 mM) for 24 h. Bars represent standard errors (n = 4). Asterisks represent values significantly different at P < 0.05.

Figure 5. Changes in the activities of SOD, APX, and GR in the second leaf of rice seedlings treated with or without CdCl₂ (0.5 mM). Bars represent standard errors (n = 4). Asterisks represent values significantly different at P < 0.05.

Kuo and Kao — Antioxidant enzymes and Cd toxicity in rice seedling

and POX in TN1 seedlings treated with CdCl₂ was observed prior to the occurrence of Cd toxicity (decrease in protein content), indicating that the induction of oxidative stress in Cd-treated TN1 leaves is a cause of Cd toxicity.

Pretreatment with Abscisic Acid (ABA) Reduces Cd Toxicity and Cd-induced Oxidative Stress in TN1 Leaves

In a more recent work, we reported that ABA plays an important role in Cd tolerance and pretreatment of TN1 seedlings with ABA counteracting Cd-induced toxicity (Hsu and Kao, 2003a; b). If oxidative stress is an important mechanism in regulating Cd toxicity in TN1 leaves, then pretreatment with ABA is expected to reduce Cd toxicity and Cd-increased content of MDA and activities of SOD and POX in TN1 leaves. Since some chlorosis was observed in the second leaf of TN1 seedlings treated with ABA for 2 d, Cd toxicity was evaluated by using the third leaf and 1.5 mM CdCl₂. As expected, ABA pretreatment reduced Cd toxicity and reduced Cd-induced increase in the con

tent of MDA and increase in the activities of SOD and POX in the third leaf of TN1 seedlings (Figure 8 and 9).

Fluridone (Flu) Treatment Enhances Toxicity and Oxidative Stress in Cd-treated TNG67 Leaves

Previously, we have shown that treatment with Flu, an inhibitor of ABA biosynthesis, inhibited the increase in ABA content and enhanced toxicity in the second leaf of TNG67 seedlings treated with CdCl₂ (Hsu and Kao, 2003a; b). Here, we also observed that Flu enhanced toxicity, increased the content of MDA, and increased the activities of SOD and POX in Cd-treated TNG 67 leaves (Figures 10 and 11). It is interesting to note that the effect of Flu on Cd toxicity and oxidative stress in TNG67 can be reversed by the application of ABA (Figures 10 and 11).

Figure 8. Effect of ABA-pretreatment on chlorophyll and protein contents in the third leaf of TN1 rice seedlings treated with or without CdCl₂ (1.5 mM). Taichung Native 1 (TN1) rice seedlings were pretreated with ABA (5 µM) for 2 d and then treated with or without CdCl₂ (1.5 mM) for 2 d. Bars represent standard errors (n = 4). Values with the same letter are not significantly different at P < 0.05.

Figure 9. Effect of ABA-pretreatment on the content of MDA and the activities of SOD and POX in the third leaf of Taichung Native 1 (TN1) rice seedlings treated with or without CdCl₂ (1.5 mM). TN1 rice seedlings were pretreated with ABA (5 µM) for 2 d and then treated with or without CdCl₂ (1.5 mM) for 2 d. Bars represent standard errors (n = 4). Values with the same letter are not significantly different at P < 0.05.

Botanical Bulletin of Academia Sinica, Vol. 45, 2004

Discussion

Plants have a range of potential mechanisms at the cellular level that might be involved in detoxification and thus tolerance to heavy metals. These all appear to be involved primarily in avoiding the build-up of toxic concentrations at sensitive sites within the cell and thus preventing damage (Hall, 2002). Obviously Cd-tolerant plant must be able to prevent the absorption of excess Cd. Of the two rice cultivars used in the present study, more absorption of Cd was detected in Cd-sensitive TN1 seedlings than in Cd-tolerant TNG67 (Figure 3). Jarvis et al. (1976) examined the Cd distribution in 23 species and found that the higher up in the plant one checked, the lower the Cd concentration, which decreased in the following order; fibrous roots > storage roots > stems > leaves. We also observed that Cd content was higher in roots than leaves in both TN1 and TNG67 seedlings (Figure 3).

It has been documented that Cd can cause an increased production of AOS, including H₂O₂ (Piqueras et al., 1999; Romero-Puertas et al., 1999; Shah et al., 2001; Sandalio et al., 2001; Schützendübel et al., 2001; Olmos et al., 2003) and induce lipid peroxidation (Shaw, 1995; Gallego et al., 1996; Benavides and Tomaro, 1996; Chaoui et al., 1997;

Figure 11. Effect of fluridone (Flu, 0.2 mM) on the content of MDA and the activities of SOD and POX in the second leaf of Tainung 67 (TNG67) rice seedlings treated with CdCl₂ (0.5 mM) and ABA (5 µM). Chlorophyll and protein contents were measured after 2 d of treatments. Bars represent standard errors (n = 4). Values with the same letter are not significantly different at P < 0.05.

Lozano-Rodríquez et al., 1997; Dixit et al., 2001; Shah et al., 2001; Chien et al., 2002). This suggests that Cd treatment causes oxidative stress in plants. In the present study, five lines of evidence indicated that oxidative stress seems to be involved in toxicity of rice seedlings caused by CdCl₂. First, on treatment with CdCl₂, lipid peroxidation, H₂O₂ content, and antioxidant enzyme activities increased in the leaves of the Cd-sensitive TN1 but not in the Cd-tolerant TNG67 (Figures 4, 5, and 6). Second, Cd-increased lipid peroxidation, and the activities of SOD and POX preceded the appearance of Cd toxicity in TN1 leaves (Figure 7). Third, pretreatment with ABA enhanced Cd tolerance and reduced Cd-increased lipid peroxidation and the activities of SOD and POX in TN1 leaves (Figures 8 and 9). Fourth, exogenous application of ABA biosynthesis

Figure 10. Effect of fluridone (Flu, 0.2 mM) on the contents of chlorophyll and protein in the second leaf of Tainung 67 (TNG67) rice seedlings treated with CdCl₂ (0.5 mM) and ABA (5 µM). Chlorophyll and protein contents were measured after 2 d of treatments. Bars represent standard errors (n = 4). Values with the same letter are not significantly different at P<0.05.

Kuo and Kao — Antioxidant enzymes and Cd toxicity in rice seedling

H₂O₂ production in Cd-treated TN1 leaves was augmented by the stimulation of NADPH oxidase-like enzyme. However, this is likely to occur because Cd treatment resulted in an increase in H₂O₂ content and in SOD activity in TN1 leaves (Figures 4 and 5).

The most novel aspects of this report are the findings that ABA reduces oxidative stress indicators in Cd sensitive cultivar (TN1) and Flu increases indicators in Cd tolerant cultivar (TNG67) (Figures 9 and 11). The simplest explanation of this finding is that ABA closed stomata and reduced Cd uptake while Flu opened stomata and increased Cd uptake. Indeed, in our previous work, we have shown that exogenous ABA application could result in the decrease in transpiration rate and Cd content of TN1 seedlings and Flu treatment caused not only the block of ABA biosynthesis but also the increase in transpiration rate and Cd content in TNG67 seedlings (Hsu and Kao, 2003a).

In conclusion, we provide evidence that oxidative stress is differently expressed in TN1 and TNG67 rice seedlings in response to CdCl₂. Results also suggest that CdCl₂ causes oxidative stress and CdCl₂-induced toxicity is mediated through oxidative stress in TN1 leaves.

Acknowledgements. This work was financially supported by the National Science Council of the Republic of China.

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Kuo and Kao — Antioxidant enzymes and Cd toxicity in rice seedling