Differential Expression of Two Cytosolic Ascorbate Peroxidases and Two Superoxide Dismutase Genes in Response to Abiotic Stress in Rice

Rice Science, 2011, 18(3): 157−166 Copyright © 2011, China National Rice Research Institute Published by Elsevier BV. All rights reserved

Differential Expression of Two Cytosolic Ascorbate Peroxidases and Two Superoxide Dismutase Genes in Response to Abiotic Stress in Rice Shigeto MORITA1, 2, Shinya NAKATANI1, Tomokazu KOSHIBA3, Takehiro MASUMURA1, 2, Yasunari OGIHARA1, 4, Kunisuke TANAKA1 (1Laboratory of Genetic Engineering, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8522, Japan; 2Basic Research Division, Kyoto Prefectural Institute of Agricultural Biotechnology, Seika, Soraku, Kyoto 619-0244, Japan; 3Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan; 4Kihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan)

Abstract: Superoxide dismutase (SOD) and ascorbate peroxidase (APX) play central roles in the pathway for scavenging reactive oxygen species in plants, thereby contributing to the tolerance against abiotic stress. Here we report the responses of cytosolic SOD (cSOD; sodCc1 and sodCc2) and cytosolic APX (cAPX; OsAPX1 and OsAPX2) genes to oxidative and abiotic stress in rice. RNA blot analyses revealed that methyl viologen treatment caused a more prominent induction of cAPXs compared with cSODs, and hydrogen peroxide treatment induced the expression of cAPXs whereas cSODs were not affected. These results suggest that cAPXs play more important roles in defense against oxidative stress compared with cSODs. It is noted that cSODs and cAPXs showed coordinate response to abscisic acid treatment which induced both sodCc1 and OsAPX2. However, cSODs and cAPXs responded differentially to drought, salt and chilling stress, which indicates that cSOD and cAPX genes are expressed differentially in response to oxidative and abiotic stress in rice. Key words: ascorbate peroxidase; superoxide dismutase; reactive oxygen species; abiotic stress; rice

Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radical are highly reactive, toxic intermediates of oxygen metabolism. They are inevitably produced within living cells and cause severe damages through oxidation of cellular components including nucleic acids, proteins and lipids. In higher plants, the photosynthetic electron transport system in chloroplasts produces ROS even under optimal conditions (Asada, 1999). The production of ROS in chloroplasts is enhanced when CO2 fixation is limited under abiotic stress such as high light, drought and chilling. Therefore, plants suffer from the toxicity of ROS under prolonged stress conditions, and protection against photooxidative damages is a key component of stress tolerance in plants. Plants have antioxidant defense systems that include multiple enzymes of ROS-scavenging pathways. Among those enzymes, superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC Received: 14 October 2010; Accepted: 4 May 2011 Corresponding author: Shigeto MORITA ([email protected])

1.11.1.11) play central roles in ROS scavenging (Noctor and Foyer, 1998; Asada, 1999). SOD catalyzes the decomposition of superoxide to hydrogen peroxide and molecular oxygen (Mittler et al, 2004). SOD isoforms are classified by their metal cofactor and subcellular localizations: cytosolic CuZn-SOD, chloroplastic CuZnSOD, peroxisomal CuZn-SOD, mitochondrial Mn-SOD, and chloroplastic Fe-SOD (Mittler et al, 2004). APX is a major enzyme functioning in the scavenging of hydrogen peroxide in plant cells (Noctor and Foyer, 1998; Asada, 1999). It uses ascorbate as a specific electron donor and catalyzes the reduction of hydrogen peroxide to water. APX is also divided into distinct isoforms according to their subcellular localization, namely, chloroplasts, microbodies, mitochondria and cytosol (Shigeoka et al, 2002; Mittler et al, 2004). SOD and APX isoforms are both encoded by small multigene families, and rice genome encodes seven SOD (Table 1) and eight APX genes (Teixeira et al, 2004). These isoforms are expressed differentially in response to various stress (Sakamoto et al, 1995;

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Kaminaka et al, 1999a; Teixeira et al, 2006), which suggests that each isoform has a divergent function in plant cells and that complex regulation of multiple isoforms is required for fine control of the cellular ROS levels. Among the multiple SOD and APX isoforms localized in different subcellular compartments, the transcript levels of cytosolic SODs (cSODs) and cytosolic APXs (cAPXs) are higher than any other SOD and APX isoforms in rice leaves, according to the RiceXPro expression profile database (http:// ricexpro.dna.affrc.go.jp/index.html) (Sato et al, 2011), which suggests that cSODs and cAPXs play major roles in defense against oxidative stress. Also, the importance of cAPX in photooxidative stress defense is confirmed by the analysis of an Arabidopsis knockout mutant of cytosolic APX1, revealing that cAPX is required in protecting chloroplasts from light stress despite its cytosolic localization (Davletova et al, 2005). Therefore, we focus on the expression of cSOD and cAPX genes in rice and investigate the stress responses of these genes. We previously found that sodCc1 and sodCc2 showed different responses to abscisic acid (ABA), drought and salt stress (Sakamoto et al, 1995; Kaminaka et al, 1999a). sodCc2 was induced by ABA, drought and NaCl treatment, whereas sodCc1 did not show significant induction by these treatments. Because SOD and APX catalyze the sequential reaction in the ROS-scavenging pathway, it is thought that coordinate expression of these two enzymes is important for efficient scavenging of ROS. This notion is supported by a number of reports describing the increase in SOD and APX activities under chilling (Schöner and Krause, 1990), high light (Cakmak and Marschner, 1992; Mishra et al, 1993), drought (Mittler and Zilinskas, 1994), and ozone fumigation (Conklin and Last, 1995; Rao et al, 1996).

In this study, we investigated the responses of two cSOD and two cAPX genes to ABA treatment, oxidative and abiotic stress, in order to examine whether the expression of these antioxidant enzymes is regulated coordinately.

MATERIALS AND METHODS Rice materials and growth conditions Rice (Oryza sativa L. cv Nipponbare) seeds were imbibed at 28 °C overnight, and then grown hydroponically with water under a 16 h light/8 h dark cycle at a light intensity of 150 μmol/(m2·s) for 8–14 d at 28 °C. Shoots were sampled and frozen in liquid nitrogen immediately, and stored at -80 °C until use. Germinating rice embryos were prepared in suspension cultures as described previously (Morita et al, 1999). Chemical and stress treatments Treatment of seedlings with methyl viologen, hydrogen peroxide, ABA, or NaCl was carried out hydroponically by immersing the roots in a solution of each chemical. Drought stress treatment was performed by withholding water at 28 °C for 24 h. For chilling stress treatment, plants were transferred to a growth chamber at 4 °C and incubated for 12 h. All the stress treatments described above were carried out under constant illumination [150 μmol/(m2·s)]. Treatment of germinating embryos with methyl viologen or hydrogen peroxide was performed as described previously (Morita et al, 1999). cDNA cloning and sequence analysis The full length cDNA of OsAPX2 was obtained

Table 1. SOD isoforms in rice. Gene

RAP-DB gene ID

Accession no. of cDNA

sodCc1 sodCc2 perSOD sodCp sodA1 sodB sodB2

Os03g0351500 Os07g0665200 Os03g0219200 Os08g0561700 Os05g0323900 Os06g0143000 Os06g0115400

D00999 (Sakamoto et al, 1992), AK061662 D01000 (Sakamoto et al, 1992), AK243377 AK073785 D85239 (Kaminaka et al, 1997), AK059841 L19436 (Sakamoto et al, 1993), AK070528 AB014056 (Kaminaka et al, 1999b), AK062073 AK111656

Putative subcellular localization cytosol cytosol peroxisome chloroplast mitochondria chloroplast chloroplast

Shigeto MORITA, et al. Differential Expression of cAPX and cSOD Genes under Abiotic Stress in Rice

by screening of a cDNA library prepared from 14 day-old green seedlings using a full length cDNA of OsAPX1 (previously called APXa; accession No. D45423) as a probe (Morita et al, 1997). The nucleotide sequence of the positive clone was confirmed with an ABI PRISM 310 genetic analyzer (Applied Biosystems, USA). RNA blot analysis Total RNA was isolated from seedlings by the guanidine thiocyanate/CsCl-ultracentrifugation method (Chirgwin et al, 1979) and from germinating embryos by the SDS-phenol method (Shirzadegan et al, 1991). The RNA (15–20 µg) was denatured with formamide, then fractionated through 1.2% agarose gels and transferred to nitrocellulose membranes (Hybond-C extra; GE Healthcare UK Ltd, UK), or directly blotted onto nitrocellulose membranes with a slot blotter (Bio-Dot microfiltration apparatus; Bio-Rad, USA). The blots were probed with 3′-UTR fragments of rice cAPX and cSOD cDNAs (accession Nos. D00999 and D01000, respectively) (Sakamoto et al, 1992). The 3′-UTR probes of rice cSOD were prepared by PCR as described previously (Kaminaka et al, 1999a). The cAPX probes were prepared by PCR using the following primer pairs: 5′-GAGGTTTCTAGTCTACTACTGC-3′ and 5′-GGATGCAGCATTGCAGTTGAGC-3′ for OsAPX1, 5′-GAAGCCTTTAGAGAGCGGGATA-3′ and 5′-AT CTTGACAGCAAATAGCTTGG-3′ for OsAPX2. The blots were stripped and subsequently hybridized with rice Actin cDNA (accession No. D15628) as an internal control. The insert DNA of the Actin cDNA clone was excised by restriction digestion and used for the probe. The probes were labeled with 32P using a BcaBEST Labeling Kit (Takara Bio, Japan). Hybridization was carried out at 42 °C and washing was performed twice at 42 °C for 20 min, with 2 × SSC, 0.1% SDS for the SOD and APX probes and 0.5 × SSC, 0.1% SDS at 55 °C for the Actin probe. Hybridization signals were visualized by autoradiography or a phosphorimager (Molecular Imager GS-525; Bio-Rad). The transcript level was quantified by measuring the radioactivity of the signal bands using the phosphorimager (Molecular Imager GS-525) or measuring the signal intensities using the KODAK 1D Image Analysis Software

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(Eastman Kodak, USA). Western blot analysis and APX gel assay In order to confirm the reactivity of the antibody used for detection of rice cAPX proteins, recombinant OsAPX1 protein was expressed in E. coli. The coding sequence of OsAPX1 was excised from the cDNA plasmid (Morita et al, 1997) by Sac I and Kpn I digestion, and cloned into an expression vector pQE32 (Qiagen, USA). The expression of the recombinant protein was induced in the E.coli strain JM109 by addition of 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) and subsequent incubation at 28 °C for 5 h. Total protein extract of the culture was fractionated by SDS-PAGE, and subjected to western blot analysis as described below. For analysis of rice cAPXs, protein extracts were prepared from rice seedlings by grinding on ice in an extraction buffer containing 50 mmol/L potassium phosphate (pH 7.6), 0.1 mmol/L EDTA, 0.1% Triton X-100, and 5 mmol/L ascorbic acid. The homogenate was centrifuged at 20 000×g for 15 min at 4 °C, and the supernatant was recovered. Protein content was determined by the Bradford method. Proteins were fractionated by SDS-PAGE, or native PAGE on a 10% gel at 4 °C. APX activity was detected by activity staining on native gels as described previously (Mittler and Zilinskas, 1993). Western blotting was performed using the rabbit antiserum against maize cAPX (Koshiba, 1993) as a primary antibody at a 1: 4 000 dilution. For detection of the signal, alkaline phosphatase-conjugated secondary antibody (Promega, USA), 5-bromo-4chloro-3-indolyl phosphate, and nitroblue tetrazolium were used (BCIP/NBT Color Development Substrate; Promega). The signal intensities of activity staining and western blotting were quantified using the KODAK 1D Image Analysis Software (Eastman Kodak, USA). Statistical analysis Data were statistically analyzed by t-test for pairwise comparison of treated samples and untreated controls or Dunnett’s test for comparison of treated samples against the initial untreated samples. The difference at P<0.05 was considered as significant.

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RESULTS Responses of rice cSODs and cAPXs to oxidative stress The transcript levels of rice cSOD (sodCc1 and sodCc2) and cAPX genes (OsAPX1 and OsAPX2) were examined by northern blot or RNA slot blot analyses using 3′-UTR fragments as gene-specific probes. First, we investigated the oxidative stress response by treating rice seedlings with methyl viologen (a compound that generates superoxide within chloroplasts) or hydrogen peroxide. As shown in Fig. 1, methyl viologen treatment caused a significant induction of cAPXs, and a slight induction

of cSODs as well. Meanwhile, hydrogen peroxide caused a slight induction of cAPXs but not of cSODs. We also examined the oxidative stress response of germinating embryos in suspension culture (Fig. 2). In this case, methyl viologen and hydrogen peroxide caused a significant increase in cAPX transcripts. However, methyl viologen treatment caused only a slight induction of cSODs, and hydrogen peroxide treatment failed to induce cSODs. Thus, these results revealed that oxidative stress induced cAPX genes more prominently than cSOD genes. Responses of rice cSODs and cAPXs to ABA, drought, salt and chilling stress We previously observed that sodCc1 and sodCc2 showed different responses to ABA (Sakamoto et al,

Fig. 1. Changes in transcript levels of the cSOD and cAPX genes in response to oxidative stress in rice seedlings. A, Responses of the cSOD genes; B, Responses of the cAPX genes. Fourteen-day-old green seedlings were treated with 10 μmol/L methyl viologen (MV) or 1 mmol/L hydrogen peroxide (H2O2). Northern blot analysis was performed with 15–20 μg of total RNA using gene-specific probes. The results were quantified, and the transcript levels of cSODs and cAPXs were normalized with that of Actin. Error bars indicate deviation (n=2). The asterisks indicate statistical significance (Dunnett’s test: P<0.05).

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Fig. 2. Changes in transcript levels of the cSOD and cAPX genes in response to oxidative stress in germinating rice embryos. A, Responses of the cSOD genes; B, Responses of the cAPX genes. Stress treatment and northern blot analysis were performed as described in Fig. 1. ‘Control’ indicates the samples without stress treatments. Error bars indicate the SD (n=3). The asterisks indicate statistical significance (t-test: P<0.05).

1995; Kaminaka et al, 1999a). In the present study, we examined whether OsAPX1 and OsAPX2 also responded to ABA in a similar manner. RNA slot blot analysis of ABA-treated seedlings revealed that OsAPX1 and sodCc2 were induced by ABA (Fig. 3). In contrast, the transcript levels of sodCc1 and OsAPX2 were not elevated significantly. ABA is a well-known signal transducer under drought, salt and chilling stress (Leung and Giraudat, 1998; Finkelstein et al, 2002). A number of genes involved in stress adaptation are regulated by ABA under these stress conditions (Leung and Giraudat, 1998). Therefore, we investigated the responses of cSOD and cAPX genes to drought, salt and chilling stress (Fig. 4). Drought treatment caused an induction of sodCc2 instead of sodCc1, as observed previously (Kaminaka et al, 1999a). In contrast, the expression of OsAPX1 and OsAPX2 was not up-regulated during the stress. Salt stress caused no significant increase in cSOD and cAPX transcripts. Chilling treatment caused a decrease in transcript levels of sodCc1, sodCc2 and OsAPX2, but did not affect the expression of OsAPX1. These results revealed that cSODs and cAPXs were expressed differentially in response to the abiotic stress, and also suggested that the responses of cAPX genes were different in ABA and abiotic stress

treatments. Expression of cAPX protein in response to ROS and ABA In this study, we found that the transcript level of cAPXs was elevated by methyl viologen, hydrogen

Fig. 3. Effects of ABA treatment on transcript levels of the cSOD and cAPX genes in rice. A, Transcript levels of the cSOD genes; B, Transcript levels of the cAPX genes. Ten-day-old rice seedlings were treated with 0.1 mmol/L ABA, and RNA slot blot analysis was performed with 20 μg of total RNA using gene-specific probes. The results were quantified, and the transcript levels of cSOD and cAPX were normalized with that of Actin. Error bars indicate the SE (n=3). The asterisks indicate statistical significance (Dunnett’s test: P<0.05).

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Fig. 4. Effects of drought, salt and chilling treatments on transcript levels of the cSOD and cAPX genes in rice. A, Transcript levels of the cSOD genes; B, Transcript levels of the cAPX genes. Ten-day-old rice seedlings were treated with drought, salt (100 mmol/L NaCl), or chilling (4 °C) stress for the indicated periods. In the case of chilling treatment, plants were subsequently recovered at 28 °C for 12 h. RNA slot blot analysis was performed as described in Fig. 3. ‘R’ indicates the recovered sample. Error bars indicate the SE (n=3−4). The asterisks indicate statistical significance (Dunnett's test: P<0.05).

peroxide and ABA. In order to examine whether the cAPX protein level and the activity were also elevated by these treatments, we carried out APX activity staining and western blot analyses. We used the antiserum against maize cAPX (Koshiba, 1993) for western analysis, and its reactivity with rice cAPX was confirmed since the antiserum cross-reacted with a recombinant OsAPX1 protein produced in E. coli (data not shown). Also, the anti-maize cAPX antiserum detected a single major band with an apparent molecular mass of 27 kDa in rice leaf extract (data not shown). Since the calculated molecular mass of OsAPX1 and OsAPX2 were quite similar (27.2 kDa and 27.1 kDa, respectively), it was likely that these two proteins were not separated by SDS-PAGE. In an attempt to separate OsAPX1 and OsAPX2, we fractionated protein extracts by native PAGE. As a result, a single band was detected by activity staining on a native gel, and a major band with similar mobility was observed by western blotting (Fig. 5). Since OsAPX1 and OsAPX2 were highly homologous to each other with 83.9% homology in amino acid sequence, we assumed that the two cAPXs were not separated in this experiment and that the major band represents both of the cAPX isozymes. The signal

intensities of the major band were quantified, and regarded as cAPX activity and protein level (Fig. 5). The results indicate that hydrogen peroxide caused elevations of both cAPX activity and protein level. ABA also caused slight increase in the activity and protein level. However, in the case of methyl viologen treatment, the cAPX activity and protein level did not increase significantly. Thus, the elevation of the transcript level of cAPXs did not lead to an increase in APX activity under methyl viologeninduced oxidative stress.

DISCUSSION In this study, we revealed that rice cSOD and cAPX genes were coordinately induced by ABA treatment (Fig. 3). However, cSOD and cAPX genes responded differentially to oxidative stress (methyl viologen and hydrogen peroxide) (Figs. 1 and 2) and abiotic stress (drought and chilling) (Fig. 4). cAPXs were induced more prominently than cSODs by oxidative stress (Figs. 1 and 2), which suggests that cAPX plays a more important role in defense against oxidative stress than cSOD.

Shigeto MORITA, et al. Differential Expression of cAPX and cSOD Genes under Abiotic Stress in Rice

Fig. 5. Changes in the activity and protein level of cAPX in response to oxidative stress and ABA treatments. Rice seedlings (8–14 days old) were treated with 10 μmol/L methyl viologen (MV) (A), 1 mmol/L hydrogen peroxide (H2O2) (B) or 0.1 mmol/L ABA (C) for the indicated periods. The soluble protein (10 μg per lane) was fractionated by native PAGE, and subsequently analyzed by APX activity staining and western blotting with anti-cAPX antiserum. Images are representative results of activity staining and western blot analysis (upper and lower panels, respectively). The activity and protein level of cAPX (filled and grey bars, respectively) are shown in graphs. Error bars indicate the SE (n=3). The asterisks indicate statistical significance (Dunnett's test: P<0.05).

We also revealed that ABA treatment caused the induction of sodCc2 and OsAPX1 (Fig. 3), indicating that these genes were regulated coordinately by ABA. Previous reports suggested the involvement of ROS in the ABA signaling pathway regulating the expression of antioxidant enzymes. Guan et al (2000) suggested that the induction of the maize catalase Cat1 gene by ABA was mediated by hydrogen peroxide. Jiang and Zhang (2002) suggested that water stress-induced accumulation of ABA in maize leaves triggered the increased generation of ROS, which led to the elevation of the activities of SOD, APX, catalase and glutathione reductase. We observed that ROS treatments induced sodCc1 and OsAPX2, which were not responsive to ABA, as well as ABA-responsive sodCc2 and OsAPX1 (Figs.1 and 2). If the ABA induction of sodCc2 and OsAPX1 is mediated by ROS as suggested in maize, oxidative stress responses of

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the ABA-responsive genes and non-responsive ones have to be regulated by different mechanisms. Since NAD(P)H oxidase was suggested to be involved in ABA-induced ROS production (Jiang and Zhang, 2002), sodCc2 and OsAPX1 might respond to ROS generated by NAD(P)H oxidase. In our experiment, sodCc1 and sodCc2 were both down-regulated by the chilling treatment (Fig. 4), whereas previous study reported that sodCc1 was up-regulated by low temperature (Lee et al, 2009). Also, salt stress-induced up-regulation of OsAPX2 reported by Teixeira et al (2006) was not observed in our results (Fig. 4). Although the possible reasons for these discrepancies are unclear, the intensities of stress treatments may be different between our study and the previous ones because of the differences in experimental conditions. It is also noted that OsAPX2 was down-regulated by the chilling treatment (Fig. 4). It is likely that decreased expression of cAPX leads to the accumulation of hydrogen peroxide, which acts as a signal of chilling stress and triggers the induction of defense genes (Prasad et al, 1994). This idea is consistent with a previous observation that cytosolic APX1-deficient soybean cultivars have higher cold tolerance than APX1-expressing ones (Funatsuki et al, 2003). These results suggest that down-regulation of cAPX may contribute to the adaptation to chilling stress. Previous studies and our current results revealed that OsAPX1 and OsAPX2 showed different expression patterns in response to environmental cues, which suggests that these genes have different physiological roles. Previous proteomics analysis of rice seedlings showed that the OsAPX1 protein was expressed in etiolated seedlings (Komatsu et al, 1999). OsAPX1 was also highly expressed in roots (Teixeira et al, 2006), suggesting its function in non-photosynthetic tissues. Interestingly, OsAPX1 was induced by infection with a blast pathogen, Magnaporthe grisea (Agrawal et al, 2003). Therefore, OsAPX1 might function in biotic stress defense. In contrast, OsAPX2 was up-regulated by salinity (Teixeira et al, 2006) and wounding (Agrawal et al, 2003). The enzymatic properties of recombinant cAPX proteins indicated that OsAPX2 had a higher specific activity [20 mmol/(L·min·mg)] and higher affinity for ascorbate

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(Km for ascorbate: 1 mmol/L) than OsAPX1 [specific activity: 15 mmol/(L·min·mg), Km for ascorbate: 4 mmol/L] (Lu et al, 2005). Therefore, OsAPX2 functions more effectively than OsAPX1 in the scavenging of hydrogen peroxide. In addition, the analysis of transgenic Arabidopsis plants overexpressing OsAPX1 or OsAPX2 revealed that OsAPX2-expressing lines showed higher tolerance to salt stress than OsAPX1expressing lines (Lu et al, 2007). Taken together, these observations suggest that OsAPX2 plays a major role in the stress defense compared with OsAPX1. In our experiment, hydrogen peroxide and ABA caused an increase in the cAPX protein level and cAPX activity in accordance with an elevation of the transcript level of cAPX genes (Fig. 5). However, the increase in the transcript level was not directly correlated with the protein level and cAPX activity under the methyl viologen treatment (Fig. 5), which suggests two possible models of regulation. One possibility is that cAPX is regulated posttranscriptionally at the level of protein synthesis, as in the case of the drought stress response of cAPX in pea (Mittler and Zilinskas, 1994). The other possibility is that inactivation and concomitant degradation of cAPX protein might occur during the methyl viologen treatment. Because APX is irreversibly inactivated in the absence of ascorbate (Asada, 1999), APX activity is lowered under severe stress conditions due to the oxidation of ascorbate (Shikanai et al, 1998). In our experiment, decreases in the cAPX protein level and activity were not observed by the methyl viologen treatment, suggesting that the elevation of cAPX transcript level contributed to maintaining the cAPX protein level and activity under the inhibitory condition of severe oxidative stress. We previously found a novel cis-element designated as CORE, which was responsive to methyl viologen on the promoters of sodCc1, cytosolic thioredoxin trxh, and glutaredoxin genes in rice (Tsukamoto et al, 2005). Although CORE is not conserved in rice cAPX genes, there is a possibility that it might be indirectly involved in the regulation of cAPX genes through an intermediate regulatory factor that is controlled by CORE. It is also likely that there are distinct cis-elements regulating the oxidative stress response of cAPX genes other than CORE. We previously

revealed that hydrogen peroxide is involved in the oxidative stress signaling that leads to the induction of cAPX in rice (Morita et al, 1999). Currently, two ciselements, ocs and as-1, are known to be responsive to hydrogen peroxide (Chen and Singh, 1999; Garreton et al, 2002), although these cis-element motifs are not found on the promoter regions of OsAPX1 or OsAPX2. Therefore, there must be unknown cis-elements regulating the hydrogen peroxide-mediated induction of cAPXs. The identification of such cis-elements and elucidation of the regulatory mechanisms of cAPX genes remains a subject for future analyses.

ACKNOWLEDGEMENTS The authors are grateful to the Rice Genome Research Program (the National Institute of Agrobiological Sciences, Tsukuba, Japan) for provision of the rice Actin cDNA. This work was supported by the Grants-in-Aid for Scientific Research (Grant No. 10460149 to K.T. and Grant No. 11740448 to S.M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant from the Rice Genome Research Program (Grant No. MP2106 to K.T.) from the Ministry of Agriculture, Forestry and Fisheries of Japan.

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