Toxicity in leaves of rice exposed to cadmium is due to hydrogen peroxide accumulation

REGULAR ARTICLE

Toxicity in leaves of rice exposed to cadmium is due

to hydrogen peroxide accumulation

Yi Ting Hsu&Ching Huei Kao

Received: 1 May 2007 / Accepted: 11 July 2007 / Published online: 7 August 2007 # Springer Science + Business Media B.V. 2007

Abstract The production of H2O2 in detached rice

leaves of Taichung Native 1 (TN1) caused by CdCl2

was investigated. CdCl2 treatment resulted in H2O2

production in detached rice leaves. Diphenyleneiodo-nium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase (NOX), prevented CdCl2-induced

H2O2 production, suggesting that NOX is a H2O2

-genearating enzyme in CdCl2-treated detached rice

leaves. Phosphatidylinositol 3-kinase inhibitors wort-manin (WM) or LY294002 (LY) inhibited CdCl2

-inducted H2O2 production in detached rice leaves.

Exogenous H2O2 reversed the inhibitory effect of

WM or LY, suggesting that phosphatidylinositol 3-phosphate is required for Cd-induced H2O2

produc-tion in detached rice leaves. Nitric oxide donor sodium nitroprusside (SNP) was also effective in reducing CdCl2-inducing accumulation of H2O2 in

detached rice leaves. Cd toxicity was judged by the decrease in chlorophyll content. The results indicated that DPI, IMD, WM, LY, and SNP were able to reduce Cd-induced toxicity of detached rice leaves. Twelve-day-old TN1 and Tainung 67 (TNG67) rice seedlings were treated with or without CdCl2. In

terms of Cd toxicity (leaf chlorosis), it was observed that rice seedlings of cultivar TN1 are Cd-sensitive and those of cultivar TNG67 are Cd-tolerant. On treatment with CdCl2, H2O2 accumulated in the

leaves of TN1 seedlings but not in the leaves of TNG67. Prior exposure of TN1 seedlings to 45oC for 3 h resulted in a reduction of H2O2accumulation, as

well as Cd tolerance of TN1 seedlings treated with CdCl2. The results strongly suggest that Cd toxicity of

detached leaves and leaves attached to rice seedlings are due to H2O2accumulation.

Keywords Cadmium . Hydrogen peroxide .

NADPH oxidase . Oryza sativa . Phosphatidylinositol 3-kinase . Phosphatiylinositol 3-phosphate

Abbreviations ASC Ascorbate

DAB 3,3-Diaminobenzidine DMSO Dimethyl sulfoxide

DPI Diphenyleneiodonium chloride HS Heat shock

IMD Imidazole

LY 294002

NO Nitric oxide

NOX NADPH oxidase (EC 1.6.99.6) PI3K Phosphatidylinositol 3-kinase

PI3P Phosphatidylinostolinositol 3-phosphate ROS Reactive oxygen species

SNP Sodium nitroprusside

DOI 10.1007/s11104-007-9357-7

Responsible Editor: Fangjie J. Zhao. Responsible Editor: Fangjie J. Zhao. Y. T. Hsu

:

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

TN1 Taichung Native 1 TNG67 Tainung 67 WM Wortmannin

Introduction

Reactive oxygen species (ROS) have been character-ized as the key factor on the responses of plants to both biotic and abiotic stress (Apel and Hirt2004). Initially, ROS were only regarded as damaging to cells. More recently, ROS emerged as ubiquitous signaling mol-ecules participating in the recognition of and the response to stress factors (Foyer and Noctor2005).

Inside plant cells, ROS can be produced in chloroplasts, mitochondria, peroxisome, glyoxy-somes, byproducts of metabolic processes such as photosynthesis and respiration (Apel and Hirt 2004). Several biochemical mechanisms have been proposed to explain ROS production. Apoplastic H2O2

produc-tion can be mediated by cell-wall peroxidase, germin-like oxalate oxidases or amine oxidases (Apel and Hirt2004; Mittler et al.2004). However, it has been suggested that a NADPH oxidase (NOX), analogous to that which generated superoxide during the respiratory burst in mammalian phygocytes, can serve the source of the ROS detected in plants upon successful pathogen recognition (Auh and Murphy

1995; Doke 1983; Low and Merida 1996; Murphy and Auh 1996), in abscisic acid-mediated stomatal closure (Pei et al. 2000), in auxin-regulated gravi-tropic response (Joo et al.2005; Neill et al.2002), and under abiotic stresses such as temperature, wounding and ozone (Dat et al.1998; López-Delgado et al.1998; Orozco-Cárdenas and Ryan1999; Sharma et al.1996). Cadmium (Cd), a heavy metal toxic to humans, animals, and plants, is a widespread pollutant with a long biological half-life (Wagner 1993). It has been demonstrated that Cd can promote the generation of H2O2(Hsu and Kao2004; Kuo and Kao2004; Olmos

et al. 2003; Piqueras et al. 1999; Romero-Puertas et al.2004; Sandalio et al.2001; Schützendübel et al.

2001; Shah et al.2001). The involvement of NOX in Cd-induced H2O2 production has been suggested in

tobacco cells (BY-2 line; Olmos et al. 2003), pea leaves (Romero-Puertas et al. 2004), and pea roots (Rodriguez-Serrano et al.2006).

It has been shown that NOX is activated by binding phosphatidylinositol 3-phosphate (PI3P), a

product of phosphatidylinositol 3-kinase (PI3K), to the PX domain of p40phox (Ellson et al. 2001). In plant cells, PI3P is also known to be required for abscisic acid (ABA)-induced H2O2 production in

guard cells (Jung et al. 2002; Park et al. 2003) and in leaves (Hung and Kao 2004), methyl jasmonate-induced H2O2 production in leaves (Hung et al. 2006), and auxin-induced ROS production in roots (Joo et al. 2005).

We have previously shown that CdCl2 increases

the content of H2O2in detached leaves of rice cultivar

Taichung Native 1 (TN1; Hsu and Kao2004). In this article, we investigate the possibilities that NOX and PI3P, as found in animals cells and guard cells, activate H2O2 generation in CdCl2-treated detached

leaves of TN1 by using NOX inhibitors such as diphenyleneiodonium chloride (DPI) and imidazole (IMD), and PI3K inhibitors wortmanin (WM) and LY294002 (LY). Previously, we demonstrated that rice seedlings of cultivar TNG67 (TNG67) are more tolerant to Cd than those of cultivar TN1 (Hsu and Kao2003). Evidence has been provided to show that abscisic acid is involved in Cd tolerance of TNG67 rice seedlings (Hsu and Kao 2003). It appears that these two cultivars of rice seedlings with different tolerance to Cd provide a good system to study mechanism of Cd toxicity of rice plants. Thus, using detached rice leaves of TN1 and intact leaves attached to rice seedlings of TN1 and TNG67, the possible link of H2O2 between Cd and subsequent toxicity

(chlo-rophyll loss or leaf chlorosis) was also investigated.

Materials and methods Plant material and treatments

Rice (Oryza sativa L., cv. TN1, or TNG67) seeds were sterilized with 2.5% sodium hypochlorite for 15 min and washed extensively with distilled water. These seeds were then germinated in Petri dishes with wetted filter paper at 37°C under dark conditions. After 48 h incubation, uniformly germinated seeds were selected and cultivated in a 500 ml beaker containing half-strength Kimura B solution as de-scribed previously (Hsu and Kao 2003). The hydro-ponically cultivated seedlings were grown for 12 days in a Phytotron (Agricultural Experimental station, National Taiwan University, Taipei, Taiwan) with

natural sunlight at 30°C (day)/25°C (night) and 90% relative humidity. In our laboratory, detached leaves excised from the third leaf of TN1 rice seedlings have long been used as a model system for the studies of stress physiology. Thus, in experiments using de-tached rice leaves, the apical 3 cm of the third leaves of 12-day-old TN1 rice seedlings was used. A group of 10 segments was floated in a Petri dish containing 10 ml of test solution. Incubation was carried out at 27°C in the light (40 μmol m−2 s−1). Test solutions included CdCl2 (5 mM), H2O2 (1 mM), IMD

(50μM), nitric oxide (NO) donor, sodium nitroprus-side (SNP, 100 μM), and PI3K inhibitors, LY (100 μM) and WM (1 μM). LY and WM stock solutions were prepared in 100% dimethyl sulfoxide (DMSO). For experiments of using leaves attached to TN1 and TNG67 seedlings, CdCl2was added to

half-strength Kimura B solution at the time when the third leaf was fully expanded. CdCl2 concentrations at

0.5 mM and 30μM were applied over a short (2 days) and a longer period (6 days), respectively. For some experiments, seedlings exposed to 30°C and 45°C, respectively, for 3 h in darkness, to serve as non-heat shock (control) and heat shock (HS) treatments before the addition of CdCl2(0.5 mM or 30μM).

Evaluation of Cd toxicity

For detached rice leaves, Cd toxicity was judged by the decease in chlorophyll content. Based on our experience from the experiments of Cd effect on rice seedlings, chlorosis is first observed in the second leaf of TN1 seedlings. Thus, Cd toxicity in the second leaves attached to rice seedlings caused by excess Cd was assessed by chlorosis.

Determination of chlorophyll and H2O2

Chlorophyll content was determined according to Wintermans and De Mots (1965) after extraction in 96% (v/v) ethanol. H2O2was visually detected in the

leaves by using 3, 3-diaminobenzidine (DAB) as substrate (Orozco-Cárdenas and Ryan1999). Cd- and inhibitor-treated detached rice leaves were first rinsed with distilled water and were then supplied through the cut ends with DAB (1 mg ml−1) solution for 24 h under light at 27°C. Leaves were decolorized in boiling ethanol (95%) for 0.5 h. This treatment decolorized the leaves except for the brown

polymer-ization spots produced by DAB with H2O2. After

cooling, the leaves were extracted at room tempera-ture with fresh ethanol to visualize the brown polymerization product produced by DAB with H2O2. To verify the specificity of brown spots, before

staining with DAB some leaves were incubated for 2 h in 1 mM ascorbate (ASC), a H2O2scavenger. The

H2O2 staining was repeated four times with similar

results. The H2O2 content was also measured

color-imetrically as described by Jana and Choudhuri (1982). 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 H2O2content, the 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. Using this method, we found that absorbance increased linearly with the amount of H2O2 and addition of H2O2 to

extracts resulted in the predicated increase of absor-bance, i.e., added H2O2was fully recovered (data not

shown). The H2O2 content in leaf extracts was

calculated using an extinction coefficient of 0.28μmol−1cm−1.

Statistical analysis

Statistical differences between measurements (n=4) on different treatments or on different times were analyzed following LSD test.

Results

CdCl2increases H2O2production and decreases

chlorophyll in detached rice leaves

In the present study, H2O2 production was first

visualized by a histochemical method with 3,3-diaminobenzidine (DAB) that is based on the forma-tion by H2O2of brown polymerization product. The

development of DAB-H2O2 reaction product in

detached rice leaves could be prevented by H2O2

scavengers such as ASC, indicating that the DAB staining method for H2O2is specific (Fig.1a). CdCl2

treatment led to an accumulation of DAB-H2O2

reaction product (Fig.1a). When H2O2was measured

content in detached rice leaves (Fig.1b). As expected, CdCl2treatments resulted in a decrease in chlorophyll

content (Fig.1c).

The effect of NOX inhibitors

The role of NOX in the Cd-stimulated H2O2

production was investigated using NOX inhibitors such as diphenyleneiodonium chloride (DPI) and imidazole (IMD). When 1 μM DPI or 100 μM IMD

was added to detached rice leaves simultaneously with CdCl2, a reduction of Cd-induced H2O2

accu-mulation was observed (Fig. 1a,b). The decrease in chlorophyll content in detached rice leaves by CdCl2

was reduced by DPI and IMD (Fig.1c).

The effect of phosphatidylinositol 3-kinase inhibitors It has been shown that PI3P is important in NOX-mediated H2O2 production during ABA-induced

a

b

c

H2O (2h) ASC (2h) H2O DPI IMD WM LY

CdCl2 CdCl2 + DPI CdCl2 + IMD CdCl2 + WM CdCl2 + LY CdCl2 + WM + H2O2 CdCl2 + LY + H2O2 CdCl2 + SNP a H2 O2 ( μ mo l g -1 FW) 0 20 40 50 60 70 80 90 a a _a b c d d d CdC l2 CdC l2 + DPI CdC l2 + IM D CdC l2 + WM CdC l2 + LY CdC l2 + WM + H2 O2 CdC l2 + LY + H2 O2 CdC l2 + SN P e H2O C h lo rop h y ll (mg g -1 FW ) 0 1 2 3 4 5 b b _b b c c b CdC l2 CdC l2 + DPI CdC l2 + IMD CdC l2 + WM CdC l2 + LY CdC l2 + WM + H 2O 2 CdC l2 + LY + H2 O2 CdC l2 + SNP a d H2O C h loro phy ll (m g g -1 FW) 0 1 2 3 4 5 a a a _a a H2O _DPI IMD WM LY a SNP H2 O2 ( μ mol g -1 FW) 0 20 40 60 H2O DPI IMD WM LY _SNP a a a a a _a

Fig. 1 Effect of DPI, IMD, WM, LY, or SNP on the development of DAB-H2O2

reaction product (a), and the contents of H2O2(b) and

chlorophyll (c) in CdCl2

-treated detached rice leaves (cv. TN1) in the presence or absence of H2O2. The

con-centrations of CdCl2, DPI,

IMD, WM, LY, SNP, and ascorbate (ASC) were 5 mM, 10μM, 50 μM, 1μM, 100 μM, 100 μM, and 1 mM, respectively. DAB–H2O2reaction

prod-uct, for all treatments except ASC was visualized 24 h after treatment in the light. DAB–H2O2reaction

prod-uct for ASC treatment was visualized 2 h after treat-ment in the light. H2O2and

chlorophyll contents were measured 24 h after treat-ment in the light

stomatal closure (Jung et al. 2002), during ABA-promoted leaf senescence (Hung and Kao 2005), during methyl jasmonate-induced leaf senescence (Hung et al. 2006) and during auxin-induced root gravitropic responses (Joo et al. 2005). Thus, it is of great interest to understand whether PI3P is also important in CdCl2-induced H2O2production in rice

leaves. Wortmannin (WM) and LY294002 (LY) are inhibitors of PI3K, a product of which is PI3P. When detached rice leaves were treated with a solution WM (1 μM) or LY (100 μM), CdCl2-induced

accumula-tion of H2O2 in detached rice leaves was reduced

(Fig. 1a,b). WM or LY also reduced Cd-induced decrease in chlorophyll content (Fig.1c). Exogenous H2O2(1 mM) was observed to be able to reverse the

inhibitory effect of WM or LY on H2O2 production

and chlorophyll content (Fig.1a-c).

The effect of nitric oxide donor sodium nitroprusside Nitric oxide (NO) is a bioactive free radical implicated in a number of physiological functions, including intra-cellular mediation of some animal responses (Anbar1995). In plants, NO is involved in many physiological responses, such as pathogen response, programmed cell death, growth, germina-tion, root organogenesis, phytoalexin producgermina-tion, internal iron availability, and ABA-dependent sto-matal closure (Lamattina et al. 2003; Neill et al.

2003). We have previously demonstrated that Cd toxicity in detached rice leaves is reduced by NO (Hsu and Kao 2004). If H2O2is responsible for Cd

toxicity in detached rice leaves, then H2O2

accumu-lation is expected to be reduced by NO donor sodium nitroprusside SNP. As indicated in Fig. 1a, b, it is indeed the case. In the present study, we also show that the decrease in chlorophyll content in detached rice leaves caused by Cd was reduced by SNP (Fig.1c).

CdCl2induces H2O2accumulation in the leaves

of cultivar TN1 seedlings but not in cultivar TNG67 Figure 2 shows the effect of 0.5 mM CdCl2 on the

chlorosis of the second leaves of rice seedlings. It is clear that CdCl2treatment resulted in Cd toxicity of

the second leaves of TN1, but not in TNG67 seedlings (Fig. 2) at 2 days after Cd treatment. If H2O2 is important in regulating Cd toxicity, then

H2O2–DAB reaction product and H2O2 content are

expected to be more and higher, respectively, in CdCl2-treated TN1 seedlings than in TNG67. As

indicated in Fig.3, it is indeed the case.

The concentration of CdCl2used in the

aforemen-tioned study was 0.5 mM. We also conducted experi-ments with lower CdCl2 concentration, 30 μM,

applied over a longer period (6 days). Cd toxicity and H2O2 generation was also observed to be more

pronounced in TN1 seedlings than TNG67 treated with lower concentration (30 μM) CdCl2for 6 days

(Figs. 4 and 5). Thus, the responses to lower CdCl2

concentration are basically in accordance with those to higher CdCl2concentration.

TN1 TNG67

↑↑

CdCl2 – + – +

(0.5 mM, 2 d)

Fig. 2 Effect of CdCl2(0.5 mM) on the toxicity (chlorosis) in

the second leaves of TN1 and TNG67 rice seedlings. Rice seedlings were cultivated in half-strength Kimura B solution in a Phytotron with natural sunlight at 30°C (day)/25°C (night) and 90% relative humidity. CdCl2(0.5 mM) was added to

half-strength Kimura B solution at the time when the third leaves of both TN1 and TNG67 seedlings were fully expanded. Pictures were taken 2 days after the addition of CdCl2(0.5 mM). Arrow

Prior high temperature exposure of TN1 seedlings reduces Cd-induced H2O2 accumulation

Exposure in the range of 2 min to a few hours at temperature 5°C to 15°C above the normal growing temperature is usually called HS treatment. Prior exposure to HS has been shown to increase the tolerance of plants to subsequent Cd stress (Chen and

Kao1995; Orzech and Burke 1988). To test if prior high temperature exposure of TN1 seedlings affects subsequent Cd stress, TN1 seedlings were pretreated at 45°C for 3 h. It was observed that HS pretreatment induces tolerance of Cd stress (0.5 mM or 30μM) in TN1 seedlings (Figs. 6 and 8). Figures 7 and 9 also show that HS pretreatment resulted in the production of less H2O2in the second leaves of TN1 seedlings treated

with CdCl2than that non-HS (30°C) pretreatment.

Discussion

In the present study, 0.5 mM or 30 μM CdCl2was

used to evaluate Cd toxicity of leaves attached to rice

TN1 TNG67 CdCl2 – + – – – + (0.5 mM, 2 d) H2 O2 ( μ mol g -1 FW) 0 20 40 60 80 a bc c b

TN1

TNG67

b

a

Fig. 3 Effect of CdCl2 (0.5 mM) on DAB–H2O2 reaction

product (a) and H2O2content (b) in the second leaves of TN1

and TNG67 rice seedlings. Rice seedlings were cultivated in half-strength Kimura B solution in a Phytron with natural sunlight at 30°C (day)/25°C (night) and 90% relative humidity. CdCl2(0.5 mM) was added to half-strength Kimura B solution

at the time when the third leaves of both TN1 and TNG67 seedlings were fully expanded. The second leaves were excised to visualize H2O2–DAB reaction product and measure H2O2

content 2 days after the addition of CdCl2(0.5 mM)

TN1 TNG67

CdCl2 – + – +

(30 μM, 6 d)

Fig. 4 Effect of CdCl2(30μM) on the toxicity (chlorosis) in

the second leaves of TN1 and TNG67 rice seedlings. Rice seedlings were cultivated in half-strength Kimura B solution in a Phytotron with natural sunlight at 30°C (day)/25°C (night) and 90% relative humidity. CdCl2(30μM) was added to

half-strength Kimura B solution at the time when the third leaves of both TN1 and TNG67 seedlings were fully expanded. Pictures were taken 2 days after the addition of CdCl2(30μM). Arrow

seedlings. In a recent study on 64 soils (urban, forest, and agricultural soils) containing various levels of Cd contamination, free dissolved Cd concentrations ranged from 0.1 to 2,000 nM (Sauvé et al. 2000). Thus. The 30 μM CdCl2 used in some of our

experiments can be considered to be very high. Basically, rice seedlings in our study were considered to be suffering from acute Cd toxicity.

It has been shown that Cd is able to produce H2O2

(Hsu and Kao2004; Kuo and Kao2004; Olmos et al.

2003; Piqueras et al. 1999; Romero-Puertas et al.

2004; Sandalio et al.2001; Schützendübel et al.2001; Shah et al. 2001). Here, we also show that CdCl2

induced H2O2 production in detached rice leaves by

histochemistry with DAB (Fig. 1a) and colorimetric methods (Fig. 1b). Wounding is known to induce H2O2production (Orozco-Cárdenas and Ryan 1999).

When detached rice leaves are used, wounding is always a problem. However in the present study, each long and narrow rice leaf was cut transversely; thus the area of wounding was very small. Therefore, H2O2 generation of detached rice leaves induced by

CdCl2 is unlikely to be complicated the wounding

effect.

A role for plasma membrane NOX in the produc-tion of the H2O2 has been a recent focus in ROS

signaling (Sagi and Flurhr2006). Here, we show that DPI and IMD, inhibitors of NOX, reduced CdCl2

-induced H2O2 accumulation (Fig. 1a,b). It has been

shown that a high concentration of DPI can affect ← ← Pretreatment HS – – + + Treatment CdCl2 (0.5 mM, 2 d) – + – +

Fig. 6 Effect of HS pretreatment on the toxicity (chlorosis) in the second leaves of rice seedlings (cv. TN1) in the presence or absence of CdCl2(0.5 mM). Rice seedlings at the time when

the third leaves fully expanded were transferred to 30°C (non-HS) or 45°C ((non-HS) in the dark for 3 h. Rice seedlings were then cultivated in a Phytotron with natural sunlight at 30°C (day)/ 25°C (night) and 80% relative humidity. Pictures were taken 2 days after the addition of CdCl2 (0.5 mM) to half-strength

Kimura B solution. Arrow indicated the second leaves

TN1 TNG67 CdCl2 – + – + (30 μμM, 6 d)

a

TN1

TNG67

b

a

Fig. 5 Effect of CdCl2 (30 μM) on DAB–H2O2 reaction

product (a) and H2O2content (b) in the second leaves of TN1

and TNG67 rice seedlings. Rice seedlings were cultivated in half-strength Kimura B solution in a Phytron with natural sunlight at 30°C (day)/25°C (night) and 90% relative humidity. CdCl2(30μM) was added to half-strength Kimura B solution

at the time when the third leaves of both TN1 and TNG67 seedlings were fully expanded. The second leaves were excised to visualize H2O2–DAB reaction product and measure H2O2

other enzymes potentially involved in the production of ROS, including cell wall peroxidase and NO synthase (Bolwell et al. 1998). The fact that CdCl2-induced

H2O2 accumulation in detached rice leaves can be

inhibited by low concentration of DPI (1μM) and can be inhibited by both DPI and IMD (Fig.1a,b) strongly suggest that CdCl2-dependent H2O2 production

orig-inated, at least in part, from plasma membrane NOX. The involvement of NOX in Cd-induced H2O2

production has also been suggested in tobacco cells (Olmos et al.2003), pea leaves (Romero-Puertas et al.

2004), and pea roots (Rodriguez-Serrano et al.2006). It has been shown that zinc deficiency enhanced NAD(P)H-dependent superoxide radical production in

plasma membrane vesicles isolated from roots of bean plants (Pinton et al. 1994). Since cadmium toxicity has been shown to be associated zinc deficiency (Sandalio et al. 2001), thus the possibility that a Cd-induced reduction in Zn availability could stimulate superoxide radical production in leaves of rice seed-lings cannot be excluded.

The mechanism of ROS production and the molecules involved have been well investigated in animal cells, particularly in neutrophils. The NOX, which consists of many components, is responsible for ROS production in neutrophil cells and is activated by the binding of PI3P to one of the components (Ellson et al. 2001). In plant cells, PI3P is also known to be required for ABA-induced H2O2

production in guard cells (Jung et al.2002; Park et al.

2003) and in detached rice leaves (Hung and Kao

2005), methyl jasmonate-induced H2O2production in

detached rice leaves (Hung et al. 2006), and auxin-induced ROS production in roots (Joo et al.2005). It Pretreatment HS Treatment CdCl2 (0.5 mM, 2 d) Pretreatment HS Treatment CdCl2 (0.5 mM, 2 d) – – + + – + – + – – + + – + – + a H2 O2 ( μ mol g -1 FW) 0 20 40 60 a c bc b

b

a

Fig. 7 Effect of HS pretreatment on the H2O2–DAB reaction

product (a) and H2O2content (b) in the second leaves of rice

seedlings (cv. TN1) in the presence or absence of CdCl2

(0.5 mM). Rice seedlings at the time when the third leaves fully expanded were transferred to 30°C (non-HS) or 45°C (HS) in the dark for 3 h. Rice seedlings were then cultivated in a Phytotron with natural sunlight at 30°C (day)/25°C (night) and 80% relative humidity. The second leaves were were excised to visualize H2O2–DAB reaction product and measure H2O2

content, 2 days after the addition of CdCl2(0.5 mM) to

half-strength Kimura B solution

Pretreatment HS Treatment CdCl2 (30 μM, 6 d) – – + + – + – +

Fig. 8 Effect of HS pretreatment on the toxicity (chlorosis) in the second leaves of rice seedlings (cv. TN1) in the presence or absence of CdCl2(30μM). Rice seedlings at the time when the

third leaves fully expanded were transferred to 30°C (non-HS) or 45°C (HS) in the dark for 3 h. Rice seedlings were then cultivated in a Phytotron with natural sunlight at 30°C (day)/ 25°C (night) and 90% relative humidity. Pictures were taken 6 days after the addition of CdCl2(30 μM) to half-strength

appears that PI3P is important in CdCl2-induced H2O2

production based on two lines of evidence. First, LY or WM, inhibitor of PI3K, was able to reduce CdCl2

-induced H2O2 production (Fig.1a,b). Second,

exog-enous H2O2reversed the inhibitory effect of the PI3K

inhibitors on CdCl2-indcued H2O2 accumulation

(Fig. 1a,b). These results supported further that NOX is involved in CdCl2-induced H2O2production.

In neutrophlis, PI3P regulated H2O2 production by

binding the non-catalytic component p40phox of the NOX (Ellson et al. 2001). However, a rice homolog

of p40phox has not been reported. Therefore, the detailed mechanism of the action of PI3P during H2O2 production in the rice leaves needs further

investigation.

The fact that NOX and PI3K inhibitors, which reduced the Cd-induced H2O2production (Fig.1a,b),

were able to prevent Cd-decreased chlorophyll con-tent in detached rice leaves (Fig.1c) suggests that Cd toxicity of detached rice leaves is due to H2O2

accumulation.

Several reports convincingly demonstrate that NO is a able to counteract the toxicity of paraquat and diquat, which are known to generate ROS in potato and rice leaves (Beligni and Lamattina 1999; Hung et al. 2002), and block H2O2 production induced by

jasmonic acid in tomato leaves (Orozco-Cárdenas and Ryan 2002). Here we also showed that SNP, a NO donor, blocks H2O2 production and reduces Cd

toxicity (Fig. 1a,b). It has been shown that poly-amines are able to protect against oxidative damage caused by paraquat (Chang and Kao1997), acid rain (Velikova et al. 2000) and heavy metals such as Cd and Cu (Groppa et al. 2001). Recently, we reported that polyamines (spermidine and spermine) are able to protect Cd-induced toxicity of detached rice leaves and this protection is most likely related to the avoidance of H2O2 generation and Cd uptake

(Hsu and Kao2007). All these results support further that Cd toxicity is due to H2O2production in detached

rice leaves.In plants, the most general symptom of Cd toxicity is chlorosis (Das et al. 1997). Based on chlorosis and chlorophyll loss of the second leaves of rice seedlings, it was demonstrated that rice seedlings of cultivar TNG67 are more tolerant to Cd than those of cultivar TN1 (Hsu and Kao 2003; Kuo and Kao

2004). Evidence has also been provided to show that Cd content in the second leaf and roots of TNG67 seedlings remained unchanged and slightly increased, respectively, after Cd treatment (Hsu and Kao 2003; Kuo and Kao2004). In contrast, a marked increase in Cd content in Cd-treated TN1 leaves and roots was observed (Hsu and Kao 2003; Kuo and Kao 2004). We also observed that higher content of Cd in TN1 than TNG67 leaves results in more production of H2O2 in TN1 than TNG67 leaves (Kuo and Kao 2004). The fact that on treatment with CdCl2(0.5 mM

for 2 days or 30 μM for 6 days), the H2O2 content

increased in Cd-sensitive TN1 seedlings but not in Cd-tolerant TNG67 (Figs. 3 and5) supports the idea

Pretreatment HS Treatment CdCl2 (30 μμM, 6 d) Pretreatment HS Treatment CdCl2 (30 μM, 6 d) – – + + – + – + – – + + – + – + H2 O2 ( μ mol g -1 FW) 0 20 40 60 80 a c b c

b

a

Fig. 9 Effect of HS pretreatment on the H2O2–DAB reaction

product (a) and H2O2content (b) in the second leaves of rice

seedlings (cv. TN1) in the presence or absence of CdCl2

(30μM). Rice seedlings at the time when the third leaves fully expanded were transferred to 30°C (non-HS) or 45°C (HS) in the dark for 3 h. Rice seedlings were then cultivated in a Phytotron with natural sunlight at 30°C (day)/25°C (night) and 90% relative humidity. The second leaves were were excised to visualize H2O2–DAB reaction product and measure H2O2

content 6 days after the addition of CdCl2(30 μM) to

that Cd toxicity is due to H2O2accumulation caused

by Cd. Prior exposure to HS has been shown to increase tolerance of plants to subsequent Cd stress (Chen and Kao1995; Orzech and Burke1988). Here we also demonstrated that HS pretreatment of TN1 seedlings resulted in the reduction of H2O2

produc-tion as well as Cd toxicity of TN1 seedlings treated with CdCl2(Figs.6,7,8, and9). H2O2production is

believed to be the consequence of lower capacity of antioxidant system to avoid H2O2 accumulation. In

this connection, HS pretreatment may increase the capacity of antioxidant system to reduce H2O2

production under the subsequent Cd treatment. Our unpublished data indeed show that HS pretreatment of rice seedlings resulted in higher contents of reduced glutathione and ascorbate and higher activities of glutathione reductase and ascorbate peroxidase than non-HS.

Based on the results obtained from detached rice leaves and intact leaves attached to rice seedlings, we conclude that Cd toxicity in leaves of rice seedlings is due to H2O2 accumulation. This conclusion is

basically consistent with the results of Cho and Seo (2005), who reported that a lower H2O2accumulation

confers Cd-tolerance in Arabidopsis seedlings.

Acknowledgements This work was supported by a research grant from the National Science Council of the Republic of China (NSC 94-2313-B-002-028).

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