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The involvement of hydrogen peroxide in abscisic acid-induced activities
of ascorbate peroxidase and glutathione reductase in rice roots
Yu-Chang Tsai and Ching Huei Kao*
Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China; *Author for correspondence (e-mail: kaoch@ntu.edu.tw; phone: +886-2-23698159; fax: +886-23620879)
Received 18 May 2004; accepted 9 July 2004
Key words: Abscisic acid, Ascorbate peroxidase, Glutathione reductase, Hydrogen peroxide, Oryza sativa L.
Abstract
We have monitored the changes in antioxidant enzyme activities and H2O2concentrations in roots of rice
(Oryza sativa L., cv. Taichung Native 1) seedlings treated with exogenous abscisic acid(ABA). Decrease in superoxide dismutase (SOD) and catalase (CAT) activities was observed in rice roots in the presence of ABA. However, ascorbate peroxide (APX) and glutathione reductase (GR) activities were increased after the ABA treatment. ABA treatment resulted in an increase in H2O2 concentrations in rice roots.
Pre-treatment with dimethylthiourea, a chemical trap for H2O2, and diphenyleneiodonium chloride (DPI), a
well known inhibitor of NADPH oxidase, inhibited ABA-induced accumulation of H2O2 and
ABA-in-duced activities of APX and GR. ABA-inABA-in-duced accumulation of H2O2was found to be prior to
ABA-induced activities of APX and GR. Our results suggest that H2O2is involved in ABA-induced APX and
GR activities in rice roots.
Abbreviations: ABA – abscisic acid; APX – ascorbate peroxidase; CAT – catalase; DMTU – dim-ethylthiourea; DPI – diphenyleneiodonium chloride; DW – dry weight; GR – glutathione reductase; SOD – superoxide dismutase
Introduction
The plant hormone abscisic acid (ABA) is a ses-quiterpenoid synthesized from xanthophylls (Cre-elman 1989; Seo and Koshiba 2002) and appears to influence several physiological and develop-mental events (Creelman 1989; Kende and Ze-evaart 1997). It has been shown that ABA can increase the generation of active oxygen species (AOS) such as O2 and H2O2(Guan et al. 2000;
Pei et al. 2000; Jiang and Zhang 2002, 2003; Hung and Kao 2003) and enhance the activities of anti-oxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase,
ascorbate peroxidase (APX), and glutathione reductase (GR) in plant tissues (Anderson et al.1994; Bueno et al. 1998; Gong et al. 1998; Jiang and Zhang 2001, 2002; Hung and Kao 2003). Recently, many researchers have focused on functional aspects of H2O2 generation. H2O2is a
constituent of oxidative metabolism and is itself an AOS. Because H2O2 is a relatively stable and
diffusible through membrane, H2O2is thought to
constitute a general signal indicating cellular stress (Foyer et al. 1997; Neill et al. 2002). Earlier work demonstrated that ABA-induced expression of CAT gene in maize was mediated by increased H2O2 (Guan et al. 2000). H2O2 was
recently identified as a component of ABA sig-naling in guard cells (Zhang et al. 2001). It has also been shown that the ABA-induced antioxi-dant enzyme activities in maize leaves require the participation of H2O2 (Jiang and Zhang 2002,
2003).
We have previously shown that the reduction of root growth of rice seedlings by ABA is correlated with an increase in H2O2 concentration (Lin and
Kao 2001). In this study, an effort was made to discover a possible link between H2O2 and
anti-oxidant enzyme activities in roots of rice seedlings exposed to ABA. First of all, the change in the concentration of H2O2 and the activities of
anti-oxidant enzymes such as SOD, CAT, APX and GR in roots of rice seedlings were monitored after the treatment of ABA. H2O2-manipulators, such
as diphenyleneiodonium chloride (DPI), a well known inhibitor of NADPH oxidase (O2
syn-thase) (Levine et al. 1994; Bolwell et al. 1998; Papadakis and Roubelakis-Angelakis 1999; Pei et al. 2000; Orozco-Cardenas et al. 2001; Jiang and Zhang 2003), and dimethylthiourea (DMTU), a trap of H2O2(Levine et al. 1994; Rao et al. 1997;
Casano et al. 2001), were then used. The manip-ulation of H2O2 concentrations in the
ABA-trea-ted roots may help to assess the possible link between H2O2 and ABA-enhanced antioxidant
enzyme activities.
Materials and methods Plant material
Rice (Oryza sativa L., cv. Taichung Native 1) seeds were sterilized with 2.5% sodium hypochlorite for 15 min and washed extensively with distilled wa-ter. In order to get more uniformly germinated seeds, rice seeds in Petri dish (20 cm) containing distilled water were pre-treated at 37C for 1-day in darkness. Uniformly germinated seeds were se-lected and transferred to a Petri dish (9.0 cm) containing two sheets of Whatman No.1 filter paper moistened with 10 ml of distilled water for 2 days. Roots of 2-day-old seedlings were then treated with distilled water, ABA (9 lM) or H2O2
(10 mM). 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. For the
experiments examining the role of H2O2 in
regu-lating ABA-induced APX and GR activities in roots, DPI (0.1 lM) and DMTU (5 mM) were used. Each Petri dish contained 20 germinated seeds. Each treatment was replicated four times. The germinated seeds were allowed to grow at 27C in darkness.
H2O2determination
The H2O2 concentration was colorimetrically
measured as described by Jana and Choudhuri (1981). H2O2was extracted by homogenising roots
with 3 ml of phosphate buffer (50 mM, pH 6.8) including 1 mM hydroxylamine. The homogenate was centrifuged at 6000· g for 25 min. To deter-mine H2O2 concentrations, 3 ml of extracted
solution was mixed with 1 ml of 0.1% titanium chloride (Aldrich) in 20% (v/v) H2SO4 and the
mixture was then centrifuged at 6000· g for 15 min. The intensity of yellow colour of super-natant was measured at 410 nm. H2O2
concen-tration was calculated using the extinction coefficient 0.25 lmol 1cm 1. H2O2concentration
was expressed on the basis of tissue dry weight (DW).
Enzyme extraction and assays
For extraction of enzymes, root tissues were homogenized with 0.1 M sodium phosphate buffer (pH 6.8) in a chilled pestle and mortar. For anal-ysis of APX activity, 2 mM AsA was added to the extraction buffer. The homogenate was centri-fuged at 12,000· g for 20 min and the resulting supernatant was used for determination of enzyme activity. The whole extraction procedure was car-ried out at 4C. CAT activity was assayed by measuring the initial rate of disappearance of H2O2(Kato and Shimizu 1987). The decrease in
H2O2was followed as the decline in optical density
at 240 nm, and activity was calculated using the extinction coefficient (40 mM 1cm 1 at 240 nm) for H2O2. One unit of CAT was defined as the
amount of enzyme, which degrades 1 nmol H2O2
per min. SOD was determined according to Pao-letti et al. (1986). One unit of SOD was defined as the amount of enzyme that inhibits by 50% the rate of NADH oxidation observed in blank. APX
was determined according to Nakano and Asada (1981). The decrease in ascorbate concentration was followed as a decline in optical density at 290 nm and activity was calculated using the extinction coefficient (2.8 mM 1cm 1at 290 nm) for ascorbate. One unit of APX was defined as the amount of enzyme that degrades 1 lmol of ascorbate per min. GR was determined by the method of Foster and Hess (1980). One unit of GR was defined as the amount of enzyme that de-creases 1 A340 per min. Activities of all enzymes
were expressed on the basis of DW.
Statistical analysis
The results presented were the mean of four rep-licates. Means were compared by either Student’s t-test or Duncan’s multiple range test.
Results
The changes in antioxidant enzyme activities (SOD, CAT, APX and GR) in roots of rice
seedlings after treatment of 9 lM ABA are pre-sented in Figure 1. The ABA-treated roots had higher activities of APX and GR than the control (Figure 1b, c). On the contrary, the ABA-treated roots had lower activities of SOD and CAT than the control (Figure 1a, d).
ABA treatment caused an increase in H2O2
concentration in rice roots (Figure 2). The in-crease in H2O2was evident 8 h after treatment of
ABA. These results suggest that H2O2 may play
an important role in regulating the increase of APX and GR activities in rice roots treated with ABA.
To test whether H2O2 is involved in
ABA-in-duced APX and GR activities in roots of rice seedlings, DMTU, a chemical trap for H2O2
(Levine et al. 1994; Rao et al. 1997; Casano et al. 2001), was used. Roots of rice seedlings were pre-treated with or without DMTU for 12 h and then treated with 9 lM ABA for 24 h. As indicated in Figure 3a, when rice roots were pre-treated with DMTU, ABA-induced accumulation of H2O2 in
rice roots was significantly reduced. DMTU pre-treatment was also observed to be effective in inhibiting the increase in the activities of
Time (h) 0 4 8 12 16 20 24 APX (unit g -1DW) 0 100 200 300 400 Time (h) 0 4 8 12 16 20 24 CAT (unit g -1DW) 0 5 10 15 20 GR (unit g -1DW) 0 10 20 30 40 50 60 SOD (unit g -1DW) 0 20 40 60 80 100 ABA H2O A C D B * * * * * * * * *
Figure 1. Changes in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (CAT) in roots of rice seedlings in the presence or absence of ABA. Rice seeds were germinated in distilled water for 2 days and then were transferred to distilled water and 9 lM ABA, respectively. Asterisks indicate values that are significant at p < 0.05 by Student’s t-test.
APX and GR in rice roots caused by ABA (Figure 3b, c).
AOS originating from the plasma-membrane NADPH oxidase, which transfers electrons from cytoplasmic NADPH to O2to form O2 , followed
by dismutation of O2 to H2O2, has been a recent
focus in AOS signaling. In several model systems investigated in plants, the oxidative burst and the accumulation of H2O2appear to be mediated by
the activation of plasma-membrane NADPH oxi-dase complex (Pei et al. 2000; Orozco-Cardenas et al. 2001; Jiang and Zhang 2002, 2003). DPI, a chemical inhibitor of the NADPH oxidase com-plex found in mammalian neutrophils, inhibits the pathogen-, elicitor-, wound-, and ABA-induced accumulation of H2O2 in plants (Levine et al.
1994; Bolwell et al. 1998; Papadakis and Roube-lakis-Angelakis 1999; Pei et al. 2000; Orozco-Cardenas et al. 2001; Jiang and Zhang 2003). As shown in Figure 3a, when rice roots were pre-treated with 0.1 lM DPI, ABA-induced accumu-lation of H2O2 in rice roots was completely
inhibited. DPI also inhibited ABA-enhanced APX and GR activities in rice roots (Figure 3b, c).
To study the effect of exogenous H2O2, 10 mM
H2O2 was added to the root medium. The effect
of exogenous H2O2 on the changes in the
con-centrations of endogenous H2O2 and the
activi-ties of APX and GR in rice roots is shown in Figure 4. The results indicated that exogenous H2O2increased the concentrations of endogenous
H2O2 and the activities of APX and GR in rice
roots.
Discussion
The enzyme SOD brings about the dismutation of the radical O2 to H2O2, and plays an important
role in protecting cells against the toxic effect of O2 . It has been shown that ABA treatment results
in a significant increase in SOD activity in tobacco BY-2 cells (Bueno et al. 1998), maize seedlings (Jiang and Zhang 2001, 2002, 2003), and rice leaves Time (h) 0 4 8 12 16 20 24 H2 O2 ( mo l g -1 DW ) 0 10 20 30 40 50 ABA H2O * * * µ
Figure 2. Changes in H2O2 concentrations in roots of rice
seedlings in the presence or absence of ABA. Rice seeds were germinated in distilled water for 2 days and then were trans-ferred to distilled water and 9 lM ABA, respectively. Asterisks indicate values that are significant at p < 0.05 by Student’s t-test. H2O H2O DP I H2O DMTU H2O H2O AB A DPI AB A DM TU ABA GR (unit g -1DW) 0 10 20 30 40 50 H2 O2 ( µ mol g -1DW) 0 5 10 15 20 25 30 APX (unit g -1DW) 0 100 200 300 400 500 a a a a a a a a a a a a b b c c b b A B C
Figure 3. Effect of pre-treatments with dimethylthiourea (DMTU) and diphenyleneiodonium chloride (DPI) on the concentrations of H2O2and the activities of ascorbate
peroxi-dase (APX) and glutathione reductase (GR) in rice roots ex-posed to ABA. Two-day-old seedlings were pre-treated with 0.1 lM DPI and 5 mM DMTU, respectively, for 12 h and then treated with distilled water or 9 lM ABA for 24 h. Values with the same letter are not significantly different at p < 0.05, according to Duncan’s multiple range test.
(Hung and Kao 2003). However, we observed that treatment with 9 lM ABA caused a decrease in SOD activity in rice roots (Figure 1a). CAT is known to dismutate H2O2 into H2O and O2 in
plants. CAT activity appears to be increased by ABA in maize seedlings (Jiang and Zhang 2001, 2002, 2003). However, Bueno et al. (1998) dem-onstrated that CAT activity in tobacco BY-2 cells remained constant after the ABA treatment. In this study we observed that CAT was reduced in rice roots after 24 h of ABA treatment (Figure 1d).
The role of APX and GR in the H2O2
scav-enging in plant cells has been well established in
ascorbate–glutathione cycle (Foyer et al. 1997). APX activity in rice roots was increased by 9 lM ABA (Figure 1b). Similar results have been re-ported in maize seedlings (Jiang and Zhang 2001, 2002, 2003) and tobacco BY-2 cells (Bueno et al. 1998) subjected to ABA treatment. GR activity was increased in maize seedlings (Jiang and Zhang 2001, 2002, 2003) but was reduced in tobacco BY-2 cells after the ABA treatment (Bueno et al. 1998). In this study, we show that GR activity is enhanced by ABA in rice roots (Figure 1c).
ABA-induced H2O2 generation was first
ob-served in guard cells (Pei et al. 2000; Zhang et al. 2001). In subsequent work, ABA-induced in-creases in H2O2 have been reported for maize
seedlings (Jiang and Zhang 2002, 2003), rice leaves (Hung and Kao 2003) and rice roots (Lin and Kao 2001) (Figure 2). On the other hand, ABA de-creased the release of H2O2 from germinating
radish seeds (Schopfer et al. 2001). It appears that the increase in H2O2generation is not a common
response to ABA and this response is not confined to guard cells.
It has been shown that high concentration of DPI can affect other enzymes potentially involved in the generation of AOS, including extracellular peroxidase and nitric oxide synthase (Bolwell et al. 1998; Orozco-Cardenas et al. 2001; Schopfer et al. 2001). The fact that ABA-induced H2O2
accumu-lation can be inhibited by low concentration (0.1 lM) of DPI (Figure 3a) strongly suggests that ABA-dependent H2O2 generation originated, at
least in part, from plasma membrane NADPH oxidase. Since inhibition of CAT was inhibited 24 h after the ABA treatment (Figure 1d), the possibility that CAT may contribute to H2O2
generation at a later stage of the ABA treatment cannot be excluded, but seems unlikely.
The present study indicated that H2O2
partici-pated in the regulation of ABA-induced APX and GR activities in rice roots. This conclusion was based on observations that (a) ABA treatment induced generation of H2O2in rice roots, (b)
pre-treatment with DMTU inhibited ABA-induced generation of H2O2 and ABA-increased activities
of APX and GR in rice roots, (c) pre-treatment with DPI reduced ABA-induced generation of H2O2 and ABA-increased activities of APX and
GR in rice roots, and (d) exogenous application of H2O2increased the concentrations of endogenous
H2O2 and the activities in APX and GR in rice X Data H2 O2 ( µ mol g -1DW) 0 10 20 30 40 H2O2 H2O APX (unit g -1DW) 100 150 200 250 300 350 400 Time (h) 0 4 8 12 16 20 24 GR (unit g -1DW) 0 10 20 30 40 * * * * * * * *
Figure 4. Change in the concentrations of H2O2 and the
activities of ascorate peroxidase (APX) and glutathione reduc-tase (GR) in roots of rice seedlings in the presence or absence of external H2O2. Rice seeds were germinated in distilled water for
2 days and then were transferred to distilled water and 10 mM H2O2, respectively. Asterisks indicate values that are significant
roots. Since pre-treatment with DMTU and DPI completely reduced ABA-induced H2O2
genera-tion but only partially inhibited ABA-increased APX and/or GR in rice roots, suggesting that other signal molecules may also participate in regulating ABA-increased APX and GR activities in rice roots.
Acknowledgment
This work was supported by the National Science Council of the Republic of China (NSC 92-2313-B-002-001).
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