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Sep 16, 2011 - Shih-Chueh Cho • Yun-Yang Chao •. Chwan-Yang Hong • Ching Huei Kao. Received: 27 July 2011 / Accepted: 8 September 2011 / Published ...
Plant Growth Regul (2012) 66:27–35 DOI 10.1007/s10725-011-9625-7

ORIGINAL PAPER

The role of hydrogen peroxide in cadmium-inhibited root growth of rice seedlings Shih-Chueh Cho • Yun-Yang Chao • Chwan-Yang Hong • Ching Huei Kao

Received: 27 July 2011 / Accepted: 8 September 2011 / Published online: 16 September 2011  Springer Science+Business Media B.V. 2011

Abstract Cadmium (Cd) is readily taken up by the roots of rice seedlings, leading to growth reduction. H2O2 is a constituent of oxidative metabolism and is itself a reactive oxygen species. In this study, the participation of H2O2 in CdCl2-inhibited growth of rice roots was investigated. CdCl2 treatment increased H2O2 production in rice roots. CdCl2 treatment had no effect on the activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase, but inhibited the activity of catalase (CAT) in rice roots. CdCl2-inhibited root growth and -increased H2O2 content were lessened in the presence of diphenyleneiodonium chloride, an inhibitor of H2O2 generating NADPH oxidase. However, this stimulation of root growth in CdCl2-treated seedlings is small (about 5%). Calcium (Ca) is important in many physiological processes in plants. Attempts were also made to determine whether the action of Ca on CdCl2-inhibited growth of rice roots is associated with H2O2. CaCl2 application reduced the production of H2O2, the decrease in CAT activity, and the inhibition of root growth caused by CdCl2. The effects of CaCl2 application could be reversed by exogenous H2O2. Our results indicate that the Cd causes a decline in CAT and to a lower extent a stimulation of NADPH oxidase in rice roots, with the subsequent generation of H2O2, an agent responsible for growth inhibition.

S.-C. Cho  Y.-Y. Chao  C. H. Kao (&) Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China e-mail: [email protected] C.-Y. Hong Department of Agricultural Chemistry and Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, Republic of China

Keywords Cadmium  Calcium  Hydrogen peroxide  Rice  Root growth Abbreviations APX AsA CAT CM-H2DCFDA

DPI DW GR ROS SOD

Ascorbate peroxidase Ascorbic acid Catalase 5-(and-6)-Chloromethyl-20 ,70 dichlorodihydrofluorescin diacetate, acetyl ester Diphenyleneiodonium chloride Dry weight Glutathione reductase Reactive oxygen species Superoxide dismutase

Introduction During the past decades, many researches have focused on the functional aspects of H2O2. H2O2 is a constituent of oxidative metabolism and is itself a reactive oxygen species (ROS). The accumulation of H2O2 increases the probability of hydroxyl radical (OH•) formation via a Fenton-type reaction. This leads to the phenomenon known as oxidative stress (Foyer and Noctor 2000). Initially, H2O2 was only regarded as damaging to cells (Dat et al. 2000; Gechev et al. 2006). More recently, H2O2 emerged as ubiquitous signaling molecule participating in the recognition of and the response to stress factors (Foyer and Noctor 2005; Gechev et al. 2006). Several biochemical mechanisms have been proposed to explain H2O2 production. Antioxidant enzymes, including

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superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (CAT), and nonenzymatic antioxidants, such as ascorbate and glutathione, work in concert to detoxify O2-• and H2O2. The balance between SOD and the different H2O2-scavenging enzymes in cells is considered to be important in determining the steady state of H2O2 (Noctor and Foyer 1998). ROS originating from the plasma-membrane NADPH oxidase, which transfers electrons from cytoplasmic NADPH to O2 to form O2-•, followed by dismutation to H2O2, has been a focus in ROS signaling research (Doke 1983; Auh and Murphy 1995). NADPH oxidase, thus, represents another source for H2O2 production in plant cells. Cadmium (Cd) is a pollutant and its presence in the environment is essentially due to anthropogenic activities (Sanita´ di Toppi and Gabbrielli 1999). Major sources of Cd pollution are mainly from industrial processes and phosphate fertilizers. Because of its long biological half-life, Cd is highly toxic. Taken up in excess by plants, Cd directly or indirectly inhibits physiological processes, such as respiration, photosynthesis, cell elongation, plant-water relationships, nitrogen metabolism, and mineral nutrition, resulting in poor growth and low biomass (Sanita´ di Toppi and Gabbrielli 1999; Pa´l et al. 2006). It has been shown that Cd causes oxidative stress by generating ROS, including H2O2 (Sanita´ di Toppi and Gabbrielli 1999; Cho and Seo 2005; Hsu and Kao 2007). The involvement of NADPH oxidase in Cd-induced H2O2 production has been suggested in tobacco (BY-2 line; Olmos et al. 2003; Garnier et al. 2006), pea leaves (Romero-Puertas et al. 2004), and pea roots (Rodriguez-Serano et al. 2006). Ranieri et al. (2005) demonstrated that the high concentration of Cd causes a decline in the antioxidant protection of the roots of bread wheat, with the consequent generation of considerable amounts of H2O2. Cho and Seo (2005) reported that a lower H2O2 accumulation confers Cd-tolerance in Arabidopsis seedlings. However, this result is still debatable in that Garnier et al. (2006) have reported that, in tobacco (BY-2) cells, the production of H2O2 upon Cd treatment was not sufficient to trigger all the Cddependent deleterious effects. Calcium (Ca) is important in many physiological processes and is believed to act as a second messenger (Bush 1995). Our earlier work demonstrated that Ca can counteract the growth inhibition of rice roots induced by NaCl (Lin and Kao 1995). Several findings have shown that Ca can protect maize and Trifolium repens seedlings against Cd toxicity (El-Enany 1995; Wang and Song 2009). However, it is not known whether Ca protects rice roots from Cd toxicity. Nor do we know the possible mechanisms for the action of Ca in Cd-inhibited growth of rice roots. Rice is an important cereal crop in Asia. In Taiwan, inappropriate disposal of industrial waste has given rise to

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widespread Cd contamination of irrigated water (higher than 10 mg L-1) (Chen and Lee 1997). Thus, there is urgent need to study the mechanisms of Cd toxicity of rice plants. The present work was undertaken with the objective to examine the possible involvement of H2O2 in CdCl2inhibited growth of rice roots. Attempts were also made to determine whether the action of Ca on CdCl2-inhibited growth of rice roots is associated with H2O2.

Materials and methods Plant material and growth conditions Rice (Oryza sativa L., cv. Taichung Native 1, an Indica type) seeds were sterilized with 2.5% sodium hypochlorite for 15 min and washed extensively with distilled water. To obtain more uniformly germinated seeds, rice seeds in Petri dish (20 cm) containing distilled water were pretreated at 37C for 1 day under dark conditions. Uniformly germinated seeds were then selected and transferred to a Petri dish (9.0 cm) containing two sheets of Whatman No.1 filter paper (Whatman, UK) moistened with 10 mL of distilled water for 2 day. Two-day-old seedlings were then transferred to distilled water, CdCl2, CaCl2, H2O2 and diphenyleneiodonium chloride (DPI) at the desired concentration as specified in the individual experiments. Root growth of rice seedlings grown in distilled water is similar to that grown in medium containing inorganic salts; thus, seedlings grown in distilled water were used as the controls. Each Petri dish contained ten seedlings and each treatment was replicated four times. The seedlings were allowed to grow at 27C in darkness. The seminal roots of rice seedlings at the times specified in the individual experiments were used for analyses the growth of roots, the activities antioxidant enzymes (SOD, APX, GR and CAT), the concentrations of Cd and Ca, and the contents of H2O2. Root growth Root growth was judged by the length and DW of seminal roots. At the end of each treatment, the seminal roots were used to determine length and DW. For DW determination, the roots were dried at 65C for 48 h, by which time DW was constant. Determination of Cd and Ca For determination of Cd and Ca, root samples were dried at 65C for 2 days. Dried material was ashed at 550C for 4 days. The ash residue was incubated with 31% HNO3 and 17.5% H2O2 at 72C for 2 h, and dissolved in distilled water. Cd and Ca concentrations were then quantified using

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an atomic absorption spectrophotometer (Model AA-6800, Shimadzu, Kyoto, Japan) and expressed on the basis of DW. Detection of H2O2 The H2O2 was determined by fluorescence microscopy. H2O2 imaging was conducted according to Sandalio et al. (2008). Two-day-old rice seedlings were treated with 0.1 mM CdCl2 for various incubation times as specified in the individual experiments. For DPI experiments, roots of rice seedlings were treated with 10 lM DPI for 1 h followed by incubation with 0.1 mM CdCl2 for 6 h. After CdCl2 and DPI treatments, root segments (2 cm from the tip) were cut and placed on a Petri dish containing 1 mL Tris–HCl buffer (10 mM, pH 7.2) with 50 lM 5-(and-6)chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate (CM-H2DCFDA, dissolved in 0.0025% dimethyl sulfoxide) in the dark for 30 min. A Nikon SMZ 1500 stereoscopic fluorescent microscope was used for fluorescence images. Dye excitation was at 495 nm; emitted light was detected at 515 nm. Images were captured with use of an Evolution MP cooled color CCD Camera (Evolution MP 5.1, Media Cybernetics, MD, USA) and analyzed by use of the QCapture Pro 6.0 software (QImaging Corp., BC, Canada). For the in situ histochemical detection of H2O2, root segments stained with CM-H2DCFDA were cut into approximately 1-cm segments. The segments were embedded in 5% agar and then transversely cut into sections of 30–50 lm using a vibrating microslicer (DTK1000; Dosaka EM Co. Ltd, Kyoto, Japan). The transverse sections were observed with an Axio Scope A1 fluorescent microscope, following the manufacturer’s instructions (Carl Zeiss, Jena, Germany). Axio Scope A1 images were acquired with an AxioCam camera and processed with Axiovision software. In some experiments, the H2O2 content was measured spectrophotometrically after reaction with TiCl4 (Tsai et al. 2004). The reaction mixture consisted of 2 mL of 50 mM phosphate buffer (pH 6.8) root extract supernatant and 1 mL reagent [0.1% (v/v) TiCl4 in 20% (v/v) H2SO4]. The blank reaction consisted of 50 mM phosphate buffer in the absence of root extract. The absorbance was measured at 410 nm. The amount of H2O2 was calculated by use of a standard curve prepared with known concentrations of H2O2. The H2O2 content was expressed on the basis of DW. Enzyme extraction and assays Root samples were excised and immediately used for enzyme extraction. All operations were carried out at 4C.

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For extraction of enzymes, roots 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 ascorbic acide (AsA) was added to the extraction buffer. The homogenate was centrifuged at 12,000g. SOD activity was determined according to Paoletti et al. (1986). The reaction mixture (2.73 mL) contained 100 mM triethanolaminediethanolamine buffer (pH 7.4), 7.5 mM NADH, EDTA/ MnCl2 (100 mM/50 mM, pH 7.4), 10 mM 2-mercaptoethanol, and enzyme extract (0.2 mL). The reaction was started by the addition of NADH. The reaction was allowed to proceed for 10 min. The absorbance was measured at 340 nm. One unit of SOD was defined as the amount of enzyme that inhibited by 50% the rate of NADH oxidation observed in blank sample. CAT activity was assayed according to Kato and Shimizu (1987). The decrease in H2O2 was followed as the decline in the absorbance at 240 nm, and the activity was calculated using the extinction coefficient (40 mM-1 cm-1 at 240 nm) for H2O2. One unit of CAT was defined as the amount of enzyme which degraded 1 lmol H2O2 per min. 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-1 cm-1 at 290 nm) for AsA. One unit of activity for APX was defined as the amount of enzyme that degraded 1 lmol 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 decreased 1 absorbance min-1 at 340 nm. Protein content in roots of rice seedlings is very low. Thus, in the present study, enzyme activities were expressed on the basis of DW. Statistical analysis Statistical differences between measurements (n = 4) for different treatments or different times were analyzed following Duncan’s multiple range test or Student’s t-test. A P \ 0.05 was considered statistically significant.

Results CdCl2 inhibits the growth of rice roots Root growth was tracked by measuring length and DW of roots. Increasing concentration of CdCl2 from 0.05 to 0.2 mM progressively decreased root length (Fig. 1a) and DW (Fig. 1b). The decrease in length and DW of rice roots is closely associated with increase in Cd concentration in rice roots (Fig. 1c). Figure 2 shows the time courses of length and DW of rice roots treated with either distilled

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(Fig. 3a). Examination of transverse sections of rice roots taken at 1 cm from tip showed that Cd-induced H2O2 production was found mainly in the epidermis, exodermis, sclerenchyma, and the vascular cylinder (Fig. 3b). DPI effect The role of NADPH oxidase in the Cd-stimulated H2O2 was investigated using DPI, an inhibitor of NADPH oxidase. To test if DPI pretreatment affects the subsequent Cdinduced H2O2 production, rice roots were pretreated with 10 lM DPI for 1 h. It was observed that green fluorescence of H2O2 in rice roots treated with 0.1 mM CdCl2 for 6 h was significantly reduced by DPI pretreatment (Fig. 4). The H2O2 content shown in Fig. 5c was measured spectrophotometrically. Furthermore, H2O2 accumulation in rice roots treated with 0.1 mM CdCl2 for 12 h was reduced by a 12-h pretreatment of 1 lM DPI (Fig. 5c). The inhibition of length and DW of rice roots was lessened by DPI pretreatment (Fig. 5a, b). Calcium effect

Fig. 1 Effect of CdCl2 concentration on length (a), DW (b), and Cd concentration (c) of rice roots. Two-day-old rice seedlings were treated with different concentrations of CdCl2 (0–0.2 mM) for 2 days. Bars show means ± SE (n = 4). Values with the same letter are not significantly different at P \ 0.05

water or 0.1 mM CdCl2. The decrease in length and DW caused by CdCl2 was evident at 6 h after treatment (Fig. 2a, b). Changes in the activities of antioxidant enzymes To test the effect of CdCl2 on the activities of antioxidant enzymes in roots, 2-day-old rice seedlings were treated with 0.1 mM CdCl2 for 3, 6, 9, and 12 h. We observed that CdCl2 had no effect on the activities of SOD, APX and GR in rice roots (Fig. 2c–e). However, the activity of CAT was decreased by CdCl2 after 3 h of treatment in comparison with the control (Fig. 2f).

The influence of CaCl2 on Cd-induced H2O2 accumulation in rice roots is shown in Fig. 6. CaCl2 (5 mM) alone had no effect on CM-H2DCFDA fluorescence in rice roots. However, Cd-induced green fluorescence of H2O2 was reduced by 5 mM CaCl2 or by 2 mM AsA, a H2O2 scavenger (Fig. 6a). The H2O2 content shown in Fig. 6b was measured spectrophotometrically. Moreover, H2O2 accumulation in rice roots caused by CdCl2 was reduced by CaCl2 (Fig. 6b). Since the experiments were performed by adding CaCl2 and CdCl2 simultaneously, the influence of Ca on Cdincreased H2O2 content in rice roots was most likely due to competition between Ca and Cd leading to reduced Cd concentration in rice roots. Figure 7a shows that the increase in Cd concentration in rice roots caused by CdCl2 was reduced by CaCl2. Ca concentration was observed to be higher in rice roots treated with CaCl2 together with CdCl2 compared with rice roots treated with CdCl2 alone (Fig. 7b). Cd-inhibited growth of rice roots was significantly reduced by CaCl2 (Fig. 8a, b). Moreover, Cd-inhibited CAT activity in rice roots was greatly reduced by CaCl2 (Fig. 8c). The effect of CaCl2 on the reduction of Cdinhibited growth and CAT activity in rice roots was reversed by exogenously applied H2O2 (Fig. 8a–c).

H2O2 in rice roots in response to CdCl2 In vivo detection of H2O2 in rice roots was carried out using a fluorescent probe. CM-H2DCFDA was used to follow for H2O2 production. The H2O2 production is a fast process, occurring at the second hour after Cd treatment

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Discussion Cadmium is readily taken up by the roots of rice seedlings, leading to growth reduction (Chen and Kao 1995). It is

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Fig. 2 Changes in length (a), DW (b), and the activities of SOD (c), APX (d), GR (e), and CAT (f) of rice roots in the presence or absence of CdCl2. Two-day-old rice seedlings were treated with distilled water (-CdCl2) or 0.1 mM CdCl2 (?CdCl2). Bars show means ± SE (n = 4). Asterisks represent values that are significantly different between -CdCl2 and ?CdCl2 treatments at P \ 0.05

A

-CdCl2 Bright field

Fluorescence

Bright field

+CdCl 2 Bright field

H2O

H 2O

DPI

DPI

B

Bright field

Fluorescence

Fluorescence

Fluorescence

CdCl2

Fig. 3 Changes in H2O2 production in rice roots treated with or without CdCl2. a Two-day-old rice roots were treated with distilled water (-CdCl2) or 0.1 mM CdCl2 (?CdCl2) for 0–6 h. H2O2 was visualized at the approximately 5 mm from root tip. Experiments were repeated four times with similar results. Representative photographs of rice roots were shown. Bar 1 mm. b Fluorescence of CM-H2DCFDA (50 lM) in transverse section obtained from roots (1 cm from the tip) treated with 0.1 mM CdCl2 for 6 h. Experiments were repeated four times with similar results. Bar 100 lm

H 2O

CdCl 2

H 2O

CdCl 2

Fig. 4 Effect of DPI on the production of H2O2 in roots of rice seedlings in the presence or absence of CdCl2. Two-day-old rice seedlings were pretreated with distilled H2O or 10 lM DPI for 1 h and then transferred to H2O and 0.1 mM CdCl2 for 6 h, respectively. H2O2 was visualized at approximately 5 mm from root tip. Experiments were repeated four times with similar results. Representative photographs of rice root were shown. Bar 1 mm

evident that the pattern of root length of rice seedlings in response to CdCl2 is similar to that of DW of roots (Figs. 1, 2a, b). Thus, the length and DW of rice roots were used as an indicator of root growth in the present investigation. It has been shown that Cd is able to produce H2O2 (Cho and Seo 2005; Garnier et al. 2006). The results show that CdCl2 induced H2O2 production in rice roots by the fluorescent probe CM-H2DCFDA and spectrophotometric

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A

A

B

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H2O2 (µmol g DW)

B

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a b

20 15

c

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10 5 0

Fig. 5 Effect of DPI on length (a), DW (b), and H2O2 content (c) of rice roots in the presence or absence of CdCl2. Two-day-old rice seedlings were pretreated with distilled H2O or 1 lM DPI for 12 h and then transferred to distilled H2O and 0.1 mM CdCl2 for 12 h, respectively. The H2O2 content was determined spectrophotometrically. Bars show means ± SE (n = 4). Values with the same letter are not significantly different at P \ 0.05

method (Figs. 3, 4a, 6). The induction of H2O2 production by CdCl2 has also been observed in detached rice leaves and intact leaves attached to rice seedlings (Hsu and Kao 2007). To scavenge H2O2, plants posses a well organized antioxidant defense system. The steady-state level of H2O2 within cells is determined by an interplay between the activity of H2O2 generating mechanisms and the activity of H2O2 detoxifying enzymes. H2O2-detoxifying enzymes are composed of SOD, APX, GR, and CAT (Noctor and Foyer 1998; Mittler et al. 2004; Gill and Tuteja 2010). Catalase is a tetrameric iron porphyrin protein that catalyzes the dismutation of the H2O2 into water and oxygen. In the present study, It is shown that Cd inhibited the activity of CAT in rice roots (Fig. 2f). However, the activities of SOD, APX, and GR in rice were not affected

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CdCl2

_

_

+

CaCl2

_

+

_

+ +

Fig. 6 Effect of CaCl2 on the production of H2O2 in roots of rice seedlings in the presence or absence of CdCl2. a Two-day-old rice seedlings were treated distilled H2O, 5 mM CaCl2, 0.1 mM CdCl2, 5 mM CaCl2 ? 0.1 mM CdCl2, and 2 mM AsA ? 0.1 mM CdCl2 for 2 days. H2O2 was visualized at approximately 5 mm from root tip. Experiments were repeated four times with similar results. Representative photographs of rice roots were shown. Bar 1 mm. b Twoday-old rice seedlings were treated distilled H2O, 5 mM CaCl2, 0.1 mM CdCl2, and 5 mM CaCl2 ? 0.1 mM CdCl2 for 2 days. The H2O2 content was determined spectrophotometrically. Bars show means ± SE (n = 4). Values with the same letter are not significantly different at P \ 0.05

by CdCl2 (Fig. 2c–e). Thus, Cd-decreased CAT activity might be responsible for H2O2 production in rice roots. Possible underlying mechanisms for decreased CAT activity are postulated. Wronska-Nofer et al. (1999) hypothesized an interaction between Cd and the catalytic subunit of CAT as a possible underlying mechanism of the reduced CAT-activity. It was observed that Cd treatment resulted in Fe deficiency in rice roots (unpublished data), which might be the cause of a decreased CAT activity, since Fe is an essential element in its active center. Alternatively, the participation of oxidation of CAT protein and its degradation by proteolysis might also be the mechanism of Cd-decreased CAT activity (RomeroPuertas et al. 2002). Plasma-membrane NADPH oxidase is a protein that transfers electrons from cytoplasmic NADPH to an O2 to form O2-, followed by dismutation of O2- to H2O2 (Doke

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a 3.0

2.0

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c c 0.0

CdCl2 CaCl2

_

_

+

+

_

+

_

+

Fig. 7 Effect of CaCl2 on the concentrations of Cd (a) and Ca (b) in roots of rice seedlings in the presence or absence of CdCl2. Two-dayold rice seedlings were treated with distilled H2O, 5 mM CaCl2, 0.1 mM CdCl2, and 5 mM CaCl2 ? 0.1 mM CdCl2 for 2 days. Bars show means ± SE (n = 4). Values with the same letter are not significantly different at P \ 0.05

1983; Auh and Murphy 1995). The results of this study show that DPI, a NADPH oxidase inhibitor, at low concentration (1 lM) reduced CdCl2-induced H2O2 accumulation (Fig. 5c). This indicates that CdCl2-dependent H2O2 production in roots originated, at least in part, from plasmamembrane NADPH oxidase. The involvement of NADPH oxidase in Cd-induced H2O2 production has also been described previously (Olmos et al. 2003; Hsu and Kao 2007). However, Ranieri et al. (2005) demonstrated that H2O2 generation in bread wheat roots caused by the excess of Cd is due to NAD(P)H oxidation by peroxidases. Whether the production of H2O2 caused by Cd in rice roots is also mediated through NAD(P)H oxidation remains to be investigated. The data revealed that Cd inhibited root growth and increased H2O2 contents, and that these two Cd effets were lessened in the presence of DPI (Fig. 5a, b). However, this stimulation of root growth by DPI in Cd-treated seedlings is really small (about 5%). Therefore, the accumulation of the H2O2 in roots of Cd-treated rice seedlings is caused by both the inhibition of CAT and to a lower extent, by the stimulation of NADPH oxidase. Apoplastic H2O2 production can also be mediated by cell-wall peroxidase, germine-

Fig. 8 Effect of CaCl2 and H2O2 on length (a), DW (b), and CAT activity (c) in roots of rice seedlings in the presence or absence of CdCl2. Two-day-old rice seedlings were treated with distilled H2O, 5 mM CaCl2, 10 mM H2O2, 0.1 mM CdCl2, 0.1 mM CdCl2 ? 5 mM CaCl2, 0.1 mM CdCl2 ? 10 mM H2O2, and 0.1 mM CdCl2 ? 5 mM CaCl2 ? 10 mM H2O2 for 2 days. Bars show means ± SE (n = 4). Values with the same letter are not significantly different at P \ 0.05

like oxidases, or amine oxidases (Mittler et al. 2004). It is not known whether Cd will activate these enzymes in rice roots. Further work is necessary to clarify this possibility. It has been shown that application of Ca can protect against Cd toxicity (El-Enany 1995; Wang and Song 2009). In the present study, it was observed that addition of CaCl2 at a concentration of 5 mM significantly reduced Cdinduced H2O2 production in rice roots (Figs. 6a, b) and counteracted the Cd-inhibited growth of rice roots (Fig. 8a, b). We also observed that the Ca effect on Cd-inhibited growth of rice roots could be reversed by exogenous application of H2O2 (Fig. 8a, b). Moreover, the Cd concentration in rice roots was reduced by application of Ca

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(Fig. 7a). Ca channels have been characterized as being Cd permeant (White 2000; Perfus-Barbeoch et al. 2002). Thus, a direct Ca competition with Cd uptake may partly account for the effect of Ca on Cd-induced H2O2 production (Fig. 7). Ca application also diminished the Cd-decreased CAT activity in rice roots (Fig. 8c). This would explain why Ca is able to reduce Cd-induced H2O2 production. All these results support further that Cd-inhibited growth of rice roots is due to H2O2. Several studies have reported an association between increase in ionically bound peroxidase activity and the timing or location of cessation of cell growth (Gardiner and Cleland 1974; Goldberg et al. 1986; MacAdam et al. 1992). It appears that growth inhibition may result from cell wall stiffening process related to the formation of cross-linkages among cell wall polymers by peroxidase (Fry 1986). H2O2 is a necessary substrate for a cell wall stiffening process catalyzed by peroxidase (Schopfer 1996). A sufficient supply of H2O2 seems to be required for ensuring complete stiffening of the cell wall. Results observed in the present study suggest that NADPH oxidase, which shows sensitivity to DPI, is operating in rice roots treated with CdCl2. It appears that H2O2 in Cd-treated rice roots generated in the cell wall. Previous work has shown that ionically bound peroxidase activity in roots of rice seedlings is increased by CdCl2 (Chen and Kao 1995). Thus, the possibility that Cdinhibited growth of rice roots mediated through cell-wall stiffening cannot be excluded. Examination of the transverse section of rice roots, it was observed that H2O2 production was localized in the epidermis, exodermis, sclerenchyma, and vascular cylinder (Fig. 3b). It is not known whether these tissues are the target tissues for Cd-inhibited root growth. Future research on the importance of Cd-induced H2O2 production in the epidermis, exodermis, sclerenchyma, and/or vascular cylinder of rice roots is likely to be highly rewarding. Acknowledgments This work was supported by a research grant from the National Science Council of the Republic of China (NSC 100-2313-B-002-002).

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