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First published online December 23, 2004; 10.1104/pp.104.045658 Plant Physiology 137:317-327 (2005) © 2005 American Society of Plant Biologists
A Novel cis-Element That Is Responsive to Oxidative Stress Regulates Three Antioxidant Defense Genes in Rice1Laboratory of Genetic Engineering, Graduate School of Agriculture, Kyoto Prefectural University, Shimogamo, Sakyo, Kyoto, 606–8522, Japan (S.T., S.M., E.H., H.Y., T.M., K.T.); and Basic Research Division, Kyoto Prefectural Institute of Agricultural Biotechnology, Seika, Soraku, Kyoto, 619–0244, Japan (S.M., T.M., K.T.)
All organisms have defense systems against oxidative stress that include multiple genes of antioxidant defense. These genes are induced by reactive oxygen species under condition of oxidative stress. In this study, we found that a 28-bp motif is conserved on the promoter regions of three antioxidant defense genes in rice (Oryza sativa): cytosolic superoxide dismutase (sodCc1), cytosolic thioredoxin (trxh), and glutaredoxin (grx). We demonstrated that the 28-bp sequence acts as a cis-element responsive to oxidative stress by transient expression assay and designated it as CORE (coordinate regulatory element for antioxidant defense). The CORE was activated by methyl viologen treatment and induced a 3.1-fold increase in expression of the reporter gene, but it did not respond to hydrogen peroxide. The expressions of the sodCc1, trxh, and grx genes were coordinately induced by methyl viologen, suggesting that multiple genes involved in antioxidant defense are controlled by a common regulatory mechanism via CORE. Application of the mitogen-activated protein kinase kinase inhibitor caused the constitutive induction of the sodCc1, trxh, and grx genes and the activation of CORE without methyl viologen treatment. These results indicate that a mitogen-activated protein kinase cascade is involved in the gene regulation mediated by CORE.
In living cells, reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radical are produced as harmful by-products through aerobic metabolism. An excess of ROS causes cellular damage through oxidation of biomolecules including nucleic acids, proteins, and lipids. Especially in higher plants, the photosynthetic electron transport system produces ROS when CO2 fixation is limited by environmental stress conditions (Asada, 1999  ). In spite of their harmful effects, ROS are known as important signaling molecules for regulation of antioxidant defense (Karpinski et al., 1997; Morita et al., 1999), defense response to pathogens (Lamb and Dixon, 1997), and cell death (Jabs et al., 1996; Bethke and Jones, 2001). Therefore, the cellular ROS level is kept under tight control.
All organisms have defense systems against oxidative stress. These systems include multiple genes that are induced by ROS in response to oxidative stress (Demple and Amabile-Cuevas, 1991
We have been characterizing the genes involved in ROS scavenging systems and antioxidant defense systems in rice (Oryza sativa). We previously showed that the promoter regions of sodCc1 (the gene for cytosolic SOD) and trxh (the gene for cytosolic thioredoxin) have a 77-bp conserved sequence (Sakamoto et al., 1995
The 28-bp homologous sequence that is conserved on the promoter regions of sodCc1, trxh, and grx has a characteristic structure. There is a palindrome consisting of a 19-bp nucleotide on the 3' half of the 28-bp sequence (Fig. 1B). It is probable that the 28-bp homologous sequence functions as a cis-element and regulates three antioxidant genes in rice. Here, we examine the above hypothesis and reveal the existence of a novel cis-element that is responsive to methyl viologen-induced oxidative stress. We also demonstrate that a MAP kinase cascade is associated with the regulation of the antioxidant genes via this novel cis-element.
Characterization of a Novel cis-Element Responsive to Oxidative Stress
To examine whether the 77-bp homologous sequence conserved on the rice sodCc1 and trxh promoters acts as a cis-element, we constructed a luciferase (LUC) reporter plasmid harboring the 77-bp sequence on the promoter region and performed a transient expression assay with this plasmid. A LUC reporter plasmid, 35SINTLUC+ (S. Morita, unpublished data) that is driven by the cauliflower mosaic virus (CaMV) 35S promoter was used as a control plasmid, and the 77-bp sequence of the sodCc1 gene was fused to the 5' upstream region of the CaMV 35S promoter on the 35SINTLUC+ to yield 77HS-35SINTLUC+. Each LUC construct was introduced into rice calli by particle bombardment. A
In the 77-bp sequence described above, there is a 28-bp sequence conserved between the sodCc1 and trxh genes, which are 97% homologous (Fig. 1A). This sequence is also conserved in the grx promoter region (which is 75% homologous to that of sodCc1). We postulated that this sequence functions as a cis-element for the methyl viologen response and designated it as CORE (coordinate regulatory element for antioxidant defense). To test whether CORE is involved in the gene regulation, we constructed a LUC reporter plasmid that is driven by a fusion promoter of CORE and the CaMV 35S promoter (COREd35SINTLUC+) by integration of the CORE tandem dimer into the 5' upstream region of the CaMV 35S promoter of 35SINTLUC+. The COREd35SINTLUC+ was introduced into rice seedlings together with a Renilla LUC reporter (35SINTRLUC) as an internal control. The transformed samples were treated with methyl viologen for 24 h, then subjected to dual LUC assay. The results are shown in Figure 2B as relative LUC activities normalized with Renilla LUC activities. The induction of LUC gene expression by methyl viologen treatment was observed in the samples transformed with the reporter plasmid with the CORE dimer. There was no significant increase in LUC activity in the case of the control plasmid, indicating that the induction of LUC by methyl viologen was dependent on the CORE sequence. There was a 2.2- to 3.1-fold increase in LUC activity by 1 to 10 µM methyl viologen treatment in the presence of the CORE dimer. To determine which region within the 28-bp sequence is necessary for methyl viologen induction, three mutated CORE sequences (M1–M3; Fig. 1B) were used for transient expression assay. As described above, the 28-bp sequence of the CORE motif contains a characteristic palindrome. M1 has mutated nucleotides in the 5' end of the 28-bp sequence and has an intact palindrome structure. M2 and M3 have mutations in the palindrome structure. M2 has mutations that eliminate the palindrome completely. However, M3 has a palindrome structure whose sequence is different from that of CORE. Three mutants of the CORE tandem dimer were fused to the 5' upstream region of the CaMV 35S promoter of 35SINTLUC+, respectively. Although the LUC activity was induced by 5 µM methyl viologen treatment in the case of the reporter constructs with intact CORE sequence, no significant induction was observed in the transient expression assay using either of the mutant plasmids (Fig. 3). These results indicate that the 28-bp portion of the CORE sequence acts as a cis-element responsive to methyl viologen.
Induction of the Three Antioxidant Genes by Superoxide in Vivo
Our results suggest that CORE is involved in the regulation of the sodCc1, trxh, and grx genes. Although we previously demonstrated the induction of rice sodCc1 by methyl viologen (Sakamoto et al., 1995
Characterization of CORE-Binding Nucleoproteins To examine the existence of transcription factor(s) that binds to CORE and regulates the expression of antioxidant defense genes, a gel mobility shift assay was performed using the CORE motif as a probe. Crude nuclear extracts were prepared from rice germinating embryo treated with or without methyl viologen, and 32P-labeled CORE probe was added to form DNA-protein complexes. Poly(dI-dC)·poly-(dI-dC) was added to suppress nonspecific binding. To ensure the specificity of nucleoprotein binding, nonlabeled CORE fragment was added as a competitor. Two signal bands of specific bindings were observed (Fig. 5A, lane 2). However, there was no difference in band patterns between the extracts from the methyl viologen-treated and nontreated samples (data not shown). The signals of nucleoproteins that bind to CORE were attenuated by addition of an unlabeled competitor in a dose-dependent manner (lanes 3–5). A signal band with low mobility was detected in lanes 2 to 5. This band disappeared by addition of twice the amount of poly(dI-dC)·poly(dI-dC) (lane 6), indicating that it is a nonspecific signal. When the mutated CORE fragments (M1–M3) were used as probes, the intensities of the signal bands were lowered compared with the CORE probe (Fig. 5B). These results demonstrate the existence of nucleoproteins that recognize and bind to the CORE motif specifically. Southwestern-blot analysis was also performed to characterize the molecular masses of the CORE-binding proteins. One major signal band and three minor bands were detected (Fig. 5C), indicating that there are four nucleoproteins interacting with the CORE sequence directly. When the mutant probes were used, the signal bands disappeared completely (M1) or were attenuated (M2 and M3; Fig. 5C). Thus, the specificity of the detected bands was confirmed. The molecular masses of the proteins were 24.4, 21.8, 18.5, and 11.3 kD, respectively.
Association of MAP Kinase Cascade to CORE-Mediated Gene Regulation
To gain insight into the regulatory mechanisms of sodCc1, trxh, and grx, we examined the effects of cycloheximide and protein kinase inhibitors on the expression of these genes. Northern-blot analysis was performed using rice seedlings that were treated with cycloheximide or protein kinase inhibitors. Cycloheximide treatment inhibited the methyl viologen induction of the three antioxidant genes (Fig. 6). These results showed that de novo protein synthesis is necessary for the induction of these antioxidant genes in response to methyl viologen. Moreover, cycloheximide treatment repressed the basal mRNA level of grx but not those of sodCc1 and trxh, indicating that protein synthesis is required for maintaining the basal level of grx expression under an unstressed condition. It has been shown that a MAP kinase cascade is involved in the signaling of oxidative stress in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum; Kovtun et al., 2000
Existence of the CORE Motifs in the Rice Genome Sequence Our results suggest that CORE regulates three antioxidant defense genes in response to oxidative stress. To examine whether the CORE motif is conserved in other genes in rice, we searched for the motif in the rice genome sequence. A BLAST analysis and a database search of the RiceGAAS (Rice Genome Automated Annotation System) revealed that there are 33 CORE motifs existing on the putative promoter regions of the annotated genes in the rice genome (Table I). Among them, 11 genes were confirmed as expressed genes by known cDNAs or expressed sequence tags, suggesting that there are numbers of genes regulated by CORE. The predicted functions of these genes are gene regulation (unknown protein with SWIB/MDM2 domain), signal transduction (putative protein kinase), stress defense (multiple stress-responsive zinc-finger protein, DNA repair protein), metabolism (putative glycosyltransferase), regulation of cell expansion (a protein similar to Arabidopsis SABRE), and ion transport (vacuolar-type H+-translocating inorganic pyrophosphatase). Three predicted genes are also implicated in signal transduction (a protein with protein kinase C phosphorylation site) and transcriptional regulation (proteins with zinc-finger motif and Dof domain).
We also performed a search for the CORE motif in the Arabidopsis genome sequence, which revealed that there were no sequences homologous to the 28-bp CORE motif nor the 19-bp palindromic structure conserved in Arabidopsis. The promoter sequences of 11 defense genes that are up-regulated in the Arabidopsis mutant with reduced chloroplastic SOD activity (Rizhsky et al., 2003 ) and Arabidopsis genes that are induced by methyl viologen treatment (op den Camp et al., 2003) were analyzed, but no sequences homologous to the CORE motif were found. The promoters of cytosolic SOD, trxh, and grx genes from other plant species were also examined, and they didn't show significant homology to the CORE motif. Thus, it is probable that the CORE motif is not conserved in other plant species.
In this study, we identified a novel cis-element that is responsive to oxidative stress. Three antioxidant genes in rice were shown to contain a conserved CORE in their promoter regions (Fig. 1A) that induced a 3.1-fold increase in the reporter gene expression by methyl viologen treatment (Fig. 2B). The 28-bp portion of the CORE motif is required for methyl viologen induction, and mutation in any part of this motif caused loss of induction (Fig. 3). The CORE is a highly AT-rich motif and is a completely novel cis-element. It has no similarity to currently known cis-element motifs listed in the plant cis-acting elements databases PlantCARE (http://intra.psb.ugent.be:8080/PlantCARE/) and PLACE (http://www.dna.affrc.go.jp/htdocs/PLACE/). Because the 77-bp homologous sequence including CORE was not responsive to hydrogen peroxide (Fig. 2A), we suppose that the CORE does not respond to hydrogen peroxide. The role of hydrogen peroxide as a signal in abiotic and biotic stress defense such as acclimation to chilling and high light, pathogen defense response, wounding response, and cell death, has been well documented (Vranova et al., 2002 ). It seems that CORE does not function in such defense systems that hydrogen peroxide acts as a signal. Although the molecular species of ROS that invoke the activation of CORE is unclear, it is most likely that CORE responds to superoxide because methyl viologen treatment is supposed to enhance superoxide production within plants. Superoxide is also known to trigger cellular redox responses. In E. coli, the soxRS regulon is induced by superoxide through the activation of a redox-regulated transcription factor, SoxR (Pomposiello and Demple, 2001). The role of superoxide in plant cell death has been well characterized in the Arabidopsis mutant lsd1, in which superoxide is thought to trigger a runaway cell death (Jabs et al., 1996), and rcd1 mutant in which superoxide induces cell death but hydrogen peroxide has no effects (Overmyer et al., 2000). Thus, there are several signaling pathways responsive to superoxide specifically. The CORE might have a regulatory role in such pathways under condition of oxidative stress. It is also an intriguing question whether CORE responds to other oxidative stresses such as UV irradiation and ozone exposure. Various environmental stresses such as cold, drought, and high light are known to enhance ROS production within plants, and CORE might be involved in the defense against environmental stresses. Currently, we haven't tested those stresses for investigating the stress response of CORE, because the molecular species of ROS produced by those stresses are not identified and the direct effects of them are unclear. Therefore, we used chemical treatments with methyl viologen and hydrogen peroxide as model stress conditions for examining the function of CORE. Studying the effects of other environmental stresses is important for understanding the physiological role of the CORE and remains to be a subject for future analyses.
There are two cis-elements reported so far that are responsive to ROS. Those are the ocs-element that mediates the induction of the glutathione S-transferase GST6 gene in Arabidopsis by hydrogen peroxide (Chen and Singh, 1999 The induction of sodCc1, trxh, and grx genes by methyl viologen should be referred to as a late response, since all three inductions were observed after 4 h of methyl viologen treatment (Fig. 4). Also, the application of cycloheximide inhibited the induction of antioxidant genes (Fig. 6). This result suggests that the methyl viologen induction described in this study is not a direct and rapid response, and that multiple steps are involved in the signaling and regulatory pathway. Northern-blot analysis (Figs. 4 and 6) revealed that grx showed a slightly different expression pattern from that of sodCc1 and trxh. The extent of methyl viologen induction of grx was lower than that of sodCc1 and trxh (Fig. 4). And cycloheximide treatment suppressed the expression of grx gene, whereas it had no effects on the basal expression levels of other two genes (Fig. 6). These results suggest that there might be different regulatory mechanisms for those genes in addition to CORE-mediated regulation and that the 77-bp homologous sequence conserved between sodCc1 and trxh genes, which is not conserved in grx gene, might be associated with the different regulations. In our gel mobility shift assay, two specific complexes of CORE and nuclear proteins were observed (Fig. 5A). Southwestern blotting also revealed several nuclear proteins directly bound to the CORE motif (Fig. 5C). The difference of the number of detected bands in the gel mobility shift assay and the southwestern blotting suggests that the multiple nucleoproteins may form a complex that binds to CORE. Methyl viologen treatment did not show any effects on the profile of CORE-binding proteins (data not shown), suggesting that oxidative stress does not change the binding capacity of the protein factors but rather modifies the conformation of binding proteins or the structure of the binding complexes.
We also revealed that a MAP kinase cascade is associated with the regulation via CORE (Figs. 6 and 7). The MAP kinase cascade is a well-known signaling component conserved from yeast to higher organisms. Plant MAP kinases are activated by various extracellular stimuli, such as pathogen infection, wounding, osmotic stress, cold, drought, and UV irradiation, and activated kinases regulate downstream target genes (Zhang and Klessig, 2001 Currently, the molecular mechanism of signal transduction from the MAP kinase cascade to CORE is unknown. Further analysis of transcription factors that bind to the CORE motif will clarify the mechanism of defense response against oxidative stress in higher plants. It would also help us to improve stress tolerance in plants by up-regulating a set of antioxidant genes by single gene manipulation.
Plant Materials and Growth Conditions
Rice (Oryza sativa) L. cv Nipponbare seedlings were grown hydroponically under a 16-h-light/8-h-dark cycle at 150 µmol photons m–2 s–1 for 7 d at 28°C. Rice callus was induced from scutella by incubating sterilized mature seeds on N6 agar plates containing 2 mg L–1 2,4-dichlorophenoxyacetic acid for 3 weeks in the dark at 28°C. Then, the callus was suspension-cultured in G liquid medium containing 2 mg L–1 2,4-dichlorophenoxyacetic acid in the dark at 28°C. Rice germinating embryos were prepared in suspension cultures as described previously (Morita et al., 1999
A GUS reporter plasmid, 35SINTGUS (S. Morita, unpublished data), was constructed from pBI221 (CLONTECH, Palo Alto, CA) by integration of the first intron of the rice sodCc2 gene (accession no. L19434; Sakamoto et al., 1995
The LUC reporter plasmids described above were introduced into calli or seedlings by particle bombardment using a Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, Hercules, CA) following the manufacturer's instruction. The 35SINTGUS or 35SINTRluc, both of which are driven by the CaMV 35S promoter, was cointroduced as an internal control. Plant samples were bombarded with 1.0-µm gold particles from 6 cm distance with 1,100-psi rupture discs. After bombardment, the samples were treated with chemicals (methyl viologen, hydrogen peroxide, DTT, PD98059, or staurosporine) for 6 to 24 h, then washed twice with H2O and ground in liquid nitrogen. Proteins were extracted with Passive Lysis buffer (Promega). Dual LUC assays were performed using a Dual-Luciferase Reporter Assay system (Promega) following the manufacturer's instruction. LUC and Renilla LUC activities were measured for 10 s, respectively, with a luminometer (Lumat LB 9507; EG&G Berthold, Wildbad, Germany). The GUS assay was performed as described previously (Sakamoto et al., 1995
Rice seedlings were treated with chemicals by immersing roots in chemical. Total RNA was prepared by the guanidine method with CsCl ultracentrifugation. Twenty micrograms total RNA was denatured with formamide, then separated through 1.2% (w/v) agarose gel and transferred to a nitrocellulose membrane (Hybond-C extra; Amersham Biosciences, Piscataway, NJ). The blots were probed with a 32P-labeled rice sodCc1 3'-untranslated region fragment (accession no. L19435), full-length cDNA encoding rice thioredoxin (accession no. D21836), and rice glutaredoxin (accession no. X77150). The probe for sodCc1 was prepared as described previously (Kaminaka et al., 1999
Nuclear extracts were prepared from 4-d-old germinating embryos that were suspension-cultured in N6 medium as described previously (Morita et al., 1999
Nucleoprotein separated by SDS-PAGE thorough 12% (w/v) gel was transferred to a polyvinylidene difluoride (PVDF) membrane (Immun-Blot PVDF Membrane; Bio-Rad). The membrane was blocked in a buffer C (25 mM HEPES-KOH, pH 7.9, 100 mM KCl, and 0.5 mM DTT) containing 5% (w/v) BSA for overnight at 4°C. The membrane was washed with buffer C containing 0.25% (w/v) BSA. The binding reaction was carried out using 20 ng of 32P-labeled probe for 8 h at room temperature in 2.5 mL of buffer C containing 0.25% (w/v) BSA and 10 µg mL–1 poly(dI-dC)·poly(dI-dC). The membrane was washed twice with buffer C for 30 min at room temperature. Then, the signals were visualized using a Molecular Imager (Bio-Rad).
The CORE motifs existing in the rice (O. sativa subsp. japonica) genome sequence were searched by BLAST analysis using RiceBLAST (http://riceblast.dna.affrc.go.jp/). The motifs found by this analysis were then examined whether they are located on the putative promoter regions of annotated genes using the RiceGAAS database (http://ricegaas.dna.affrc.go.jp/rgadb/). We defined the region of 100 bp to 1.5 kb upstream of coding sequence as a putative promoter region in this analysis. The search for the CORE motif in the Arabidopsis genome sequence was performed by BLAST analysis (http://www.Arabidopsis.org/Blast/). The promoter sequences of cytosolic SOD genes from maize (accession nos. U34726 and U34727), Nicotiana plumbaginifolia (L08253), tomato (X87372), sweet potato (L36229), Populus tremuloides (AF016893), trxh gene from tobacco (Z11803), and grx gene from Deschampsia antarctica (Antarctic hairgrass; AY323230) were analyzed by GENETYX software (GENETYX Corporation, Tokyo).
The authors are grateful to the Rice Genome Research Program for providing rice actin cDNA. Received April 30, 2004; returned for revision August 17, 2004; accepted September 20, 2004.
1 This work was supported by Grants-in-Aid for Scientific Research (nos. 10460149 and 11740448) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Rice Genome Research Program (grant no. MP2121) from the Ministry of Agriculture, Forestry and Fisheries of Japan. 
2 Present address: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1–3–1 Kagamiyama, Higashi-Hiroshima, 739–8526, Japan. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.045658. * Corresponding author; e-mail shigeto{at}kab.seika.kyoto.jp; fax 81–75–703–5675.
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ABSTRACT
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-glutamylcystein synthetase), glr1 (glutathione reductase), trx2 (thioredoxin), and tsa1 (thioredoxin peroxidase; Kuge and Jones, 1994
B, which are activated by hydrogen peroxide and regulate genes involved in antioxidant defense, such as genes for 
-glucuronidase (GUS) reporter plasmid, 35SINTGUS, was cointroduced as an internal control, and the LUC activity of each sample was normalized by GUS activity. We previously revealed that the rice cytosolic sodCc1 gene is induced by treatment with methyl viologen, hydrogen peroxide, or dithiothreitol (DTT; Sakamoto et al., 1995
-32P] dCTP by the Klenow fragment of DNA polymerase I. A nuclear extract containing 15 µg protein and 3 ng of 32P-labeled probes was mixed into the buffer B, and the binding reaction was carried out for 20 min at room temperature. Poly(dI-dC)·poly(dI-dC) was added for preventing nonspecific binding as indicated in the figure legend. The samples were fractionated through 12% (w/v) polyacrylamide gel in a running buffer of 25 mM Tris, 190 mM Gly, and 1 mM EDTA at 4°C. The gels were dried using a gel drier (Bio-Rad), and the signals were visualized using a Molecular Imager (Bio-Rad). 
