the importance of glutathione in defence against cadmium...

Glutathione and Cd Toxicity of Rice Seedlings 217

The Importance of Glutathione in Defence against Cadmium-induced Toxicity of

Rice Seedlings

Yun-Yang Chao1, Chwan-Yang Hong2, Chao-Yeh Chen1, and Ching Huei Kao1*

1 Department of Agronomy, National Taiwan University, Taipei 10617, Taiwan ROC

2 Department of Agricultural Chemistry and Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan ROC

ABSTRACT Cadmium (Cd)-sensitive and -tolerant rice

cultivars were used to study the role of reduced glutathione (GSH) in Cd-induced toxicity. Cd toxicity of rice seedlings was evaluated by the decrease in chlorophyll content and the increase in malondialehyde content in the second leaves. On treatment with 5 μM CdCl2 for 6 days, the content of GSH decreased in the second leaves of Cd-sensitive cultivar (cv. Taichung Native 1; TN1) but not in the Cd-tolerant cultivar (cv. Tainung 67; TNG67). Moreover, Cd-reduced GSH content was prior to Cd-decreased chlorophyll content in the second leaves of TN1 seedlings. Pretreatment of TN1 seedlings with 1 mM GSH for 6 h resulted in an increase in the contents of GSH and ascorbate (AsA) and the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in the second leaves of TN1 seedlings. Rice seedlings of TN1 pretreated with GSH were observed to reduce the subsequent Cd-induced toxicity. Exogenous application of the buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis, reduced the contents of GSH and AsA, the activities of GR and APX, and decreased Cd tolerance of TNG67 seedlings.

BSO effects on the contents of GSH and AsA, the activities of GR and APX, and Cd toxicity were reversed by the application of GSH. Based on the data obtained in this study, it could be concluded that GSH content plays a role in regulating Cd toxicity of rice seedlings. Key words: Cadmium, Glutathione, Oryza sativa

L., Oxidative stress.

穀胱甘肽可提高水稻鎘逆境之耐受性趙雲洋 1、洪傳揚 2、陳炤曄 1、高景輝 1*

1國立臺灣大學農藝學系 2國立臺灣大學農業化學系

摘要

本研究係以葉綠素含量降低及丙二醛含

量增加為鎘毒害之指標，來探討還原態穀胱

甘肽(GSH)在水稻幼苗鎘毒害上所扮演之角色。使用品種為對鎘耐受性高之臺農 67 號(TNG67)與對鎘敏感之臺中在來 1號(TN1)。TN1幼苗處理 5 μM氯化鎘 6 d後，GSH含量下降，而 TNG67 則不受影響。氯化鎘處理 TN1 幼苗過程中，GSH 含量先降低然後再降低葉綠素含量。TN1 幼苗前處理 GSH 6小時後，不僅增加 GSH及 ascorbate (AsA)含量，亦提高 ascorbate peroxidase (APX)及 glutathione reductase (GR)活性。 TN1 幼苗前處理 GSH 後可增加後續處理鎘的耐性。另一方面，TNG67 若先處理 GSH 合成抑制劑 buthionine sulfoximine (BSO)不僅

Research Article

* 通信作者, [email protected] 投稿日期：2011 年 10 月 14 日接受日期：2011 年 12 月 28 日作物、環境與生物資訊 8:217-228 (2011) Crop, Environment & Bioinformatics 8: 217-228 (2011) 189 Chung-Cheng Rd., Wufeng, Taichung 41362, TaiwanROC

Crop, Environment & Bioinformatics, Vol. 8, December 2011 218

降低 GSH 及 AsA 含量與 APX 及 GR 活性外，亦降低後續處理鎘的耐受性。外加 GSH 時可恢復 TNG67 因 BSO 降低的 GSH 及AsA 含量與 GR及 APX 活性。綜合上述結果，GSH 在水稻鎘逆境中扮演重要的調控角色。關鍵詞︰鎘、穀胱甘肽、水稻、氧化逆境。

INTRODUCTION Cadmium (Cd) is a trace element in all soils.

This metal enters the soils and water mainly from some anthropogenic activities, such as zinc smelting, nickel-Cd battery manufacture, fossil fuel combustion or application of phosphate fertilizers. It is taken up by plant roots and translocated to the aerial parts. Cd directly or indirectly inhibits physiological processes, like photosynthesis, respiration, cell elongation, plant-water relationship, nitrogen metabolism and mineral nutrition, resulting in poor growth and low biomass (Sanitá di Toppi and Gabrielli 1999).

Cd is a non-redox metal unable to produce reactive oxygen species (ROS) via Fenton and/or Haber-Weiss reactions. However, several lines of evidence have demonstrated that oxidative stress is a major component of Cd stress (Sandalio et al. 2001, Cho and Seo 2005, Hsu and Kao 2007). Plants detoxify ROS by the combination of antioxidants, such as ascorbate (AsA) and reduced glutathione (GSH), and antioxidative enzymes, such as superoxide dismutase, ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (Gratăo et al. 2005). AsA, GSH, APX, and GR constitute an AsA-GSH cycle to detoxify H2O2 (Gratăo et al. 2005).

GSH, the tripeptide γ-glutamylcysteinyl- glycine, is synthesized from two consecutive ATP-dependent reactions. In the first step γ-glutamylcysteine (γ-EC) is formed from L-glutamate and L-cysteine by γ-EC synthetase (γ-ECS). The second step is catalyzed by glutathione synthetase which adds glycine to the C-terminal of γ-EC forming GSH. When GSH is oxidized as part of its antioxidant activity, it forms glutathione disulfide (GSSG), the oxidized form of GSH. The GR reduces GSSG back to GSH at the expense of NADPH.

GSH is widely used as a marker of oxidative stress to plants. It has been shown that exposure to Cd resulted in a depletion of GSH in plant (Rauser 1987, Nocito et al. 2006, Metwally et al. 2005). It appears that the depletion of GSH is a critical step in Cd sensitivity. Cd-inhibited root growth of rice seedlings was observed to be counteracted by the addition of GSH (Chen and Kao 1995). On the other hand, buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, rendered rice roots susceptible to growth inhibition by Cd (Chen and Kao 1995). Rüegsegger et al. (1990) also demonstrated that inhibition of GSH biosynthesis reduces tolerance to Cd. It has been shown that Cd-tolerant tomato cells have elevated activity of γ-ECS, the first enzyme of GSH biosynthesis (Chen and Goldsbrough 1994). Metwally et al. (2005) were able to show that in response to Cd the more sensitive pea genotype had decreased level of GSH in their roots, whereas the more tolerant genotypes had increased root GSH level. Apparently the ability to synthesize GSH is crucial for protection against Cd. Freeman et al. (2004) also demonstrated that Ni tolerance and hyperaccumulation in Thlaspi species is linked to the constitutively elevated GSH biosynthesis. In a recent review, Sharma and Dietz (2009) stressed the importance of GSH in defence against metal-induced oxidative damage. It is known that reduction in GSH level in mutants or transgenic plants reduces Cd tolerance (Howden et al. 1995, Xiang et al. 2001). Transgenic plants over- accumulating GSH (Zhu et al. 1999) or with increased GSH recycling capacity via overexpression of GR (Pilon-Smits el al. 2000) exhibited an enhanced tolerance to Cd. However, there are other reports indicating that transgenic plants with elevated GSH conferred no or marginal additional tolerance (Arisi et al. 2000). Other functions for GSH include the formation of phytochelatins (PCs), which have affinity for heavy metals including Cd and are transported as complexes into the vacuole, thus allowing the plants to have some level of resistance to heavy metals (Sharma and Dietz 2006).

In terms of Cd toxicity (chlorophyll loss), it has been shown that rice seedlings of cultivar Tainung 67 (TNG67) are Cd-tolerant and those of cultivar Taichung Native 1 (TN1) are Cd-sensitive


(Hsu and Kao 2003). It appears that seedlings of these two rice cultivars with different tolerance to Cd provide a good system to study the role of GSH in regulating Cd-induced toxicity of rice plants. There is limited data on the effects of Cd on GSH levels in rice seedling. In the present study, cultivars of TN1 and TNG67 were used to examine the role of GSH in Cd toxicity of rice seedlings.

MATERIALS AND METHODS Plant materials and Cd treatment

Two rice (Oryza sativa L.) cultivars, an Indica type cultivar TN1 and a Japonica type cultivar TNG67, were adopted for this study. Seeds were sterilized with 3% sodium hypochlorite for 15 min, washed extensively with distilled water, and then germinated in Petri dishes with wetted filter papers at 37℃ in the dark. After 48 h incubation, uniformly germinated seeds were selected and cultivated in a beaker containing half-strength Kimura B solution, which contained the following macro- and micro-elements: 182.3 µM (NH4)2SO4, 91.6 µM KNO3, 273.9 µM MgSO4·7H2O, 91.1 µM KH2PO4, 182.5 µM Ca(NO3)2, 30.6 µM Fe-citrate, 0.25 µM H3BO3, 0.2 µM MnSO4·H2O, 0.2 µM ZnSO4·7H2O, 0.05 µM CuSO4·5H2O, and 0.07 µM H2MoO4 (Kimura et al. 1931). Since young rice seedlings were used for the present study, nutrient solution contains no silicon, although silicon is essential for growth of sturdy rice plants in the field. The nutrient solutions of pH 4.7 were replaced every 3 days. The hydroponically cultivated seedlings were grown in a Phytotron (Agricultural Experimental Station, National Taiwan University, Taipei, Taiwan) with natural sunlight at 30/25℃ day/night and 90% relative humidity. Four beakers were used for each treatment, with 20 seedlings in each beaker. Twelve-day-old seedlings with three leaves were then grown in the Kimura B solution with or without CdCl2 (5 μM) for 6 d. Cd toxicity (chlorosis, chlorophyll loss and lipid peroxidation) was first shown in the second leaves of rice seedlings (Hsu and Kao 2005). For this reason, unless otherwise indicated the second leaves of rice seedlings were used to perform all the chemical measurements and enzyme assays.

Growth response and Cd concentration At the end of treatment, the seedlings were

divided into three parts, i.e., shoot, adventitious roots, and primary roots. The lengths of shoots and primary roots and the FW and the DW of the shoots and roots (adventitious roots plus primary roots) were measured.

For determination of Cd, shoots and roots were dried at 65 ℃ for 48 h. Dried materials were ashed at 550℃ for 20 h. The ash residue was incubated with 31% HNO3 and 17.5% H2O2 at 72 ℃ for 2 h, and dissolved in distilled water. Cd concentration was then quantified using an atomic absorption spectrophotometer (Model AA-6800, Shimadzu, Kyoto, Japan). Amount of Cd was expressed on the basis of DW.

Measurements of chlorophyll and malondialdehyde (MDA)

The chlorophyll content was determined according to Wintermans and De Mots (1965) after extraction in 95% (v/v) ethanol. MDA was extracted with 5% (w/v) trichloroacetic acid and determined by thiobarbituric acid reaction as described by Heath and Packer (1968). Chlorophyll and MDA contents were expressed on the baisis of FW of the second leaves prior to Cd treatment.

Determinations of AsA, dehydroascorbate (DHA), GSH, GSSG, and PCs

Ascorbate and DHA contents in 5% trichloroacetic acid were determined as described by Law et al. (1983). The assay is based on the reduction of Fe3+ to Fe2+ by AsA. The Fe2+ then forms complexes with bipyridyl, giving a pink color that absorbs at 525 nm. GSH and GSSG contents in 3% sulfosalicylic acid extract were determined by the method of Smith (1985). The contents of GSH and GSSG were spectro- photometrically determined with an enzyme- recycling assay at 412 nm. The assay is based on sequential oxidation of GSH by 5, 5’-dithiobis- (2-nitrobenzoic acid) and reduction by NADPH in the presence of known amounts of GR. To quantify GSSG content, 2-vinylpridine was added to the extracts. Standard curves were generated with GSH and GSSG. AsA, DHA, GSH, and GSSG contents were expressed on the basis of FW of the second leaves prior to Cd treatment.


The difference between non-protein thiols and GSH was considered to represent PCs. The content of non-protein thiols was estimated according to Del Longo et al. (1993) with some modifications. Samples of shoots and roots (0.1 g) were homogenized in 1 mL of 5% sulfosalicylic acid and incubated in ice for 30 min. The extract was then centrifuged at 8000 x g for 15 min and the content of non-protein thiols was measured in the supernatant. The reaction mixture contained 100 μL of the supernatant, 1 mL of Tris-HCl (0.2 M, pH 8.2) and 75 μL 5, 5’-dithio-bis-(2- nitrobenzoic acid) (DTNB, 10 mM) and incubated for 20 min. After incubation, the absorbance was measured at 412 nm. An aliquot without DTNB was used to adjust the spectrophotometer to zero absorbance and GSH was used as standard. Values for the content of PCs are shown relative to the DW of roots and shoots.

Extraction and assays of APX and GR To measure APX and GR activities, leaf

tissues were homogenized with 0.1 M sodium phosphate buffer (pH 6.8) in a chilled pestle and mortar. For analysis of APX activity, 2 mM AsA was added to the extraction buffer. The homogenate was centrifuged at 12 000 x g for 20 min and the resulting supernatant was used for the determination of enzyme activity. The whole extraction procedure was carried out at 4℃ . APX activity was determined according to Nakano and Asada (1981). The decrease in AsA concentration was followed as a decline in the absorbance at 290 nm and activity was calculated using the extinction coefficient (2.8 mM-1cm-1 at 290 nm) for AsA. One unit of APX was defined as the amount of enzyme that breaks down 1 μmol of AsA per min. GR activity was determined by the method of Foster and Hess (1980). One unit of GR was defined as the amount of enzyme that decreases 1 absorbance min-1 at 340 nm. The activities of APX and GR were expressed on the basis of mg protein. The enzyme extracts were used for determination of protein by the method of Bradford (1976).

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. A P < 0.05 was considered statistically significant.

RESULTS Growth response and Cd concentration

To examine the effect of Cd on growth response, rice seedlings were grown in nutrient solution with or without 5 μM CdCl2 for 6 d. The length, fresh weight (FW) and dry weight (DW) of both shoots and roots of TN1 and TNG67 seedlings were reduced by CdCl2 (Table 1). Basically, the effects of Cd on growth response of TN1 were more pronounced than those of TNG67 (Table 1). The Cd concentration in shoots and roots of TNG67 was slightly increased after CdCl2 treatment (Table 1). In contrast, a marked increase in Cd concentration in Cd-treated shoots and roots of TN1 was observed (Table 1). Moreover, Cd concentration was significantly lower in shoots than in roots of TN1 seedlings (Table 1).

PC concentration To determine whether Cd induced the

accumulation of PCs, extracts obtained from shoots and roots of TN1 and TNG67 seedlings treated with or without 5 μM CdCl2 for 6 d were assayed for PC content. Exposure of TN1 seedling to CdCl2 resulted in a significant accumulation of PCs in shoots and roots, while Cd had slight effect on the content of PCs in both shoots and roots of TNG67 (Table 1).

Evaluation of Cd toxicity To evaluate the Cd toxicity of TNG67 and

TN1, seedlings were treated with 5 μM CdCl2 for 6 d. It was observed that Cd treatment resulted in a decrease in chlorophyll and an increase in MDA content in the second leaves of TN1 seedlings (Figs. 1A and B). However, Cd had no effect on chlorophyll and MDA contents in TNG67 leaves (Figs. 1C and D).

GSH content in TNG67 and TN1 leaves When seedlings were treated with 5 μM

CdCl2 for 6 d, it was observed that CdCl2 treatment resulted in a reduction in the content of GSH in the second leaves of TN1 seedlings (Fig. 2A). In contrast, no decrease in GSH content was observed in the second leaves of TNG 67


Table 1. Effect of CdCl2 on the length, fresh weight, and dry weight and the concentrations of Cd and phytochelatins (PCs) in roots and shoots of rice seedlings. Rice seedlings were treated with or without 5 μM CdCl2 for 6 days (n = 4). Asterisks represent values that are significantly different between –CdCl2 and + CdCl2 treatments at P ＜ 0.05.

Trait TN1

TNG67 -CdCl2 +CdCl2 -CdCl2 +CdCl2 Shoot length (cm) 14.9±0.2 13.2±0.2* 11.9±0.2 10.8±0.2* Root length (cm) 6.76±0.5 5.29±0.3* 10.2±0.5 8.87±0.3* Shoot FWx (mg seedling-1) 71.5±0.3 58.7±0.2* 56.4±0.2 50.8±0.1* Root FW (mg seedling-1) 50.5±0.3 42.8±0.2* 52.0±0.3 45.2±0.5* Shoot DW (mg seedling-1) 16.2±0.9 13.3±0.3* 13.1±0.6 11.9±0.9* Root DW (mg seedling-1) 7.57±0.3 6.57±0.1* 9.58±0.3 8.69±0.5* Shoot Cd concentration ( μg g-1 DW ) 0.64±0.24 5.89±0.7* 0.79±0.12 1.32±0.6* Root Cd concentration ( μg g-1 DW ) 0.78±0.09 11.8±1.7* 0.82±0.39 1.80±0.8* Shoot PCs content (μmol GSH eq.g-1 DW ) 16.5±1.9 22.9±2.3* 11.1±0.7 12.8±0.9 Root PCs content (μmol GSH eq.g-1 DW ) 2.52±0.7 5.72±0.9* 2.49±0.1 3.11±0.3* x FW: fresh weight; DW: dry weight.

Fig. 1. Effect of CdCl2 on the contents of chlorophyll (A and C) and MDA (B and D) in the second

leaves of TN1 and TNG67 seedlings, respectively. Rice seedlings were treated with 5 μM CdCl2 for 6 days. Bars indicate standard error (n = 4). Asterisks represent values that are significantly different between – CdCl2 and + CdCl2 at P < 0.05.


Fig. 2. Effects of CdCl2 on GSH content (A and C) and the GSH/GSSG ratio (B and D) in the second

leaves of TN1 and TNG 67 seedlings, respectively. Rice seedlings were treated with or without 5 μM CdCl2 for 6 days. Bars indicate standard error (n = 4). Asterisk represents values that are significantly different between – CdCl2 and + CdCl2 at P < 0.05.

seedlings (Fig. 2C). However, CdCl2 had no effect on the GSH/GSSG ratio in the second leaves of both TN1 and TNG67 seedlings (Figs. 2B and D).

To investigate the time courses of the contents of chlorophyll and GSH in the second leaves, TN1 seedlings were treated with or without 5 μM CdCl2. During the first 2 d, no significant difference could be found in chlorophyll content in TN1 leaves between +CdCl2 and -CdCl2 treatments, but subsequently chlorophyll content in Cd-treated leaves was lower (Fig. 3A). GSH content in Cd-treated leaves of TN1 seedlings was lower than their respective leaves at 2 d after treatment (Fig. 3B), indicating that Cd-reduced GSH content is prior to Cd-decreased chlorophyll content.

Pretreatment of TN1 seedlings with GSH GSH content (Fig. 4A) and the GSH/GSSG

ratio (Fig. 4B) were increased in the second leaves of TN1 seedling pretreated with 1 mM GSH for 6 h. It is also shown that the content of AsA (Fig.

4C), the AsA/DHA ratio (Fig. 4D), and the activities of APX (Fig. 4E) and GR (Fig. 4F) were increased in the second leaves of TN1 seedlings pretreated with GSH.

To test if GSH plays an important role in Cd-induced toxicity of TN1 seedlings, GSH- pretreated TN1 seedlings were then transferred to nutrient solution with or without CdCl2. It was observed that pretreatment of TN 1 seedlings with GSH exhibited a reduction of Cd-decreased chlorophyll content (Fig. 5A) and Cd-increased MDA content (Fig. 5B) in the second leaves of TN1 seedlings.

Pretreatment of TNG67 seedlings with BSO

GSH content (Fig. 6A) and the GSH/GSSG ratio (Fig. 6B) were decreased in the second leaves of Cd-tolerant TNG67 seedlings pretreated with 0.5 mM BSO, a specific inhibitor of γ-ECS, the first enzyme of GSH biosynthesis for 6 h. Results also


Fig. 3. Changes in the contents of chlorophyll (A) and GSH (B) in the second leaves of TN1 seedlings.

Rice seedlings were treated with or without 5 μM CdCl2. Bars indicate standard error (n = 4). Asterisks represent values that are significantly different between – CdCl2 and + CdCl2 at P < 0.05.

Fig. 4. Effect of GSH pretreatment on the contents of GSH (A) and AsA(C), the ratios of GSH/GSSG (B) and

AsA/DHA (D), and the activities of APX (E) and GR (F) in the second leaves of TN1 seedlings. TN1 rice seedlings were pretreated with 1 mM GSH for 6 h. Bars indicate standard errors (n = 4). Asterisks represent values that are significantly different between – GSH and + GSH at P < 0.05.


Fig. 5. Effect of CdCl2 on the contents of chlorophyll (A) and MDA (B) in the second leaves of TN1

seedlings pretreated with or without GSH. TN1 seedlings were pretreated with GSH for 6 h and then transferred to nutrient solution with or without 5 μM CdCl2 for 6 days. Bars indicate standard error (n = 4). Values with the same letter are not significantly different at P ＜ 0.05.

Fig. 6. Effects of BSO and BSO + GSH pretreatments on the contents of GSH (A) and AsA (C), the ratios

of GSH/GSSG (B) and AsA/DHA (D) and the activities of APX (E) and GR (F) in the second leaves of TNG67 seedlings. The concentrations of GSH and BSO are 1 mM and 0.5 mM, respectively. All measurements were made 6 h after pretreatment. Bars indicate standard errors (n = 4). Values with the same letter are not significantly different at P ＜ 0.05.


show that the content of AsA (Fig. 6C), the ratio of AsA/DHA (Fig. 6D), and the activities of APX (Fig. 6E) and GR (Fig. 6F) were decreased in leaves of TNG67 seedlings pretreated with BSO. The effect of BSO on the contents of GSH and AsA, the ratios of GSH/GSSG, and AsA/DHA, and the activities of APX and GR in the second leaves of TNG67 seedlings can be reversed by the application of 1 mM GSH (Fig. 6).

To confirm the involvement of GSH in Cd tolerance of TNG67 seedlings, BSO-pretreated TNG67 seedlings were then transferred to nutrient solution with or without CdCl2. It was observed that Cd treatment resulted in a decrease in chlorophyll content (Fig. 7A) and an increase in MDA content in the second leaves of TNG67 seedlings pretreated with BSO. Furthermore, the effects of BSO pretreatment on Cd-decreased chlorophyll content (Fig. 7A) and Cd-increased MDA content (Fig. 7B) in the second leaves of TNG67 seedlings can be rescued by the application of GSH.

DISCUSSION Cadmium treatment resulted in a significant

increase in Cd concentration in shoots and roots of TN1 seedlings (Table 1). The increase of Cd concentration in shoots and roots of TN1 seedlings is more pronounced than that of TNG67 (Table 1). In TN1 seedlings, the Cd concentration was lower in shoots than in roots, indicating that a higher proportion of the Cd taken up by TN1 remained in the roots. This is consistent with a number of previous reports (e.g. Jalil et al. 1994). Growth reduction caused by Cd has been reported previously (Chen and Kao 1995, Jalil et al. 1994). In the present study, we observed that growth reduction caused by Cd in TN1 seedlings is greater than that in TNG67 (Table 1). In plants, the most general symptom of Cd toxicity is chlorosis or chlorophyll loss (Das et al., 1997). In previous work, it has been demonstrated that Cd can induce oxidative stress in rice leaves, characterized by an increase in the content of MDA, an indicator of lipid peroxidation.

Fig. 7. Effect of CdCl2 on the contents of chlorophyll (A) and MDA (B) in the second leaves of

TNG67 seedlings pretreated with BSO and GSH + BSO for 6 h. TNG67 seedlings were pretreated with BSO and GSH + BSO for 6 h and then transferred to nutrient solution with or without 5 μM CdCl2 for 6 days. Bars indicate standard errors (n = 4). Values with the same letter are not significantly different at P ＜ 0.05.


Thus, we evaluated Cd toxicity by the decrease in chlorophyll content and the increase in MDA content. In this study, we observed that Cd decreased chlorophyll content and increased MDA content in the second leaf of leaves of TN1 seedlings, but not TNG67 (Fig. 1). Based on the data of growth response and toxicity caused by Cd obtained in the present study, we concluded that seedlings of TNG67 are more tolerant to 5 μM CdCl2 than those of TN1. Similar results have previously been reported using high CdCl2 concentrations (50 and 500 μM) (Hsu and Kao 2003, 2007). Moreover, the observed Cd tolerance of TNG67 is most likely due to a reduction of Cd uptake (Table 1).

GSH is synthesized in plants in an ATP-dependent two-step reaction catalyzed by the enzymes γ-ECS and glutathione synthetase. BSO is a specific and potent inhibitor of γ-ECS. BSO has been applied to plants to decrease GSH content. Here, we also observed that the addition of BSO to TNG67 seedlings decreased GSH content and the GSH/GSSG ratio in the second leaves (Figs 6A and B)

The present study indicated that GSH plays a role in the defence against Cd-induced toxicity of rice seedlings. This conclusion was based on the observations that (a) Cd treatment significantly decreased GSH content in leaves of the sensitive TN1 (Fig. 2A), but not in the tolerant TNG67 (Fig. 2C); (b) Cd-reduced GSH content in TN1 seedlings was prior to that Cd-decreased chlorophyll content (Figs. 3A and B); (c) GSH pretreatment increased endogenous GSH content (Fig. 4A) and caused a reduction in Cd toxicity of TN1 (Fig. 5); (d) BSO pretreatment reduced GSH content (Fig. 6), as well as Cd tolerance of TNG 67 seedlings (Fig. 7), and (e) the effect of BSO on GSH content and Cd toxicity of TNG67 seedlings was rescued by the application of GSH (Figs. 6A and 7). Our results indicate that the decline in GSH content is associated with Cd toxicity in TN1 rice seedlings and the maintenance of endogenous GSH biosynthesis under Cd stress is related to the tolerance of TNG67 rice seedlings. The protective effect of GSH against Cd toxicity has also been described previously (Metwally et al. 2005, Chen and Kao 1995, Chen and Goldsbrough 1994, Xiang et al. 2001, Zhu et al. 1999, Pilon-Smits et al. 2000). In a recent work, we also demonstrated that heat

shock-induced GSH accumulation in leaves increases Cd tolerance of TN1 rice seedlings (Chao et al. 2009).

The lower content of GSH in CdCl2-treated leaves of TN1 seedlings is possibly due to the reduction of the rate of GSH biosynthesis and production of PCs. However, the possibility that utilization and transport of GSH are altered by CdCl2 in TN1 leaves cannot be excluded.

In previous work, we demonstrated that, on treatment with CdCl2, the increase in the contents of H2O2 and MDA in the second leaves of TN1 seedlings (Kuo and Kao 2004, Hsu and Kao 2007). Thus, TNG67 seedlings experienced lower oxidative stress from Cd exposure than TN1. APX and GR, which are, respectively, the first and last enzymes in AsA-GSH cycle, are responsible for H2O2 detoxification. The involvement of GSH in inducing GR activity during chilling of maize has been described previously (Kocsy et al. 2001). From the present study, it is becoming evident that GSH is involved in regulating the content of AsA and the activities of APX and GR in rice leaves. This suggestion was based on the observations that (a) pretreatment with GSH resulted in an increase in the contents of GSH and AsA and the activities of APX and GR in TN1 leaves (Figs. 4A, C, E and F), (b) BSO pretreatment, which reduced the content of GSH (Fig. 6A), caused the decrease in the content of AsA and the activities of APX and GR in TNG67 leaves (Fig. 6C, E and F), and (c) the inhibitory effect of BSO in TNG67 leaves can be compensated by exogenous GSH (Figs. 6A, C, E and F). Thus, GSH in rice leaves can function to keep AsA-GSH cycle operating, thus contributing to the protection of subsequent Cd-induced oxidative injury.

The significance of PCs in Cd tolerance has been suggested (Sharma and Dietz 2006). However, the role of PCs in Cd tolerance is controversial. Several studies have shown that tolerant plants have higher content of PCs than sensitive ones (Rauser 1984). On the contrary, other experiments have shown that PCs are not responsible for the development of Cd tolerant phenotypes (Schat and Kalff 1992, Gupta et al. 2002, Hernández-Allica et al. 2006, Sun et al. 2007). Here, we show that Cd treatment markedly increased PC content in roots and shoots of TN1, a Cd sensitive cultivar, but not in TNG67, a Cd


tolerant cultivar (Table 1). It appears that Cd tolerance of TNG67 seedlings does not rely on the production of PCs.

ACKNOWLEDGMENTS This work was supported by a research grant

from the National Science Council of the Republic of China (NSC 96-2628-B-002-001).

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－編輯：楊純明

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