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. 2005 Jan;137(1):317–327. doi: 10.1104/pp.104.045658

A Novel cis-Element That Is Responsive to Oxidative Stress Regulates Three Antioxidant Defense Genes in Rice1

Shigefumi Tsukamoto 1,2, Shigeto Morita 1,*, Etsuko Hirano 1, Hideki Yokoi 1, Takehiro Masumura 1, Kunisuke Tanaka 1
PMCID: PMC548862  PMID: 15618434

Abstract

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; Gasch et al., 2000; Desikan et al., 2001). In Escherichia coli, two regulons that are key components for the adaptation to oxidative stress have been characterized; these are the oxyR regulon, which is responsive to hydrogen peroxide, and the soxRS regulon, which is responsive to superoxide and nitric oxide (Pomposiello and Demple, 2001). Both regulons include multiple genes associated with ROS scavenging (katG for hydroperoxidase, ahpC for alkyl hydroperoxide reductase, gorA for glutathione reductase, sodA for manganese superoxide dismutase [SOD], and fldA and fldB for flavodoxins), repair of oxidative damage (nfo for endonuclease IV), and supplying reducing power (zwf for Glc-6-P dehydrogenase). These genes are regulated coordinately through activation of the redox sensitive transcription factors, OxyR and SoxR, by oxidation. Yeast (Saccharomyces cerevisiae) also has a redox-sensitive mechanism of regulation of antioxidant genes. A transcription factor, Yap1, that is activated by hydrogen peroxide (Delaunay et al., 2000) regulates the genes gsh1 (γ-glutamylcystein synthetase), glr1 (glutathione reductase), trx2 (thioredoxin), and tsa1 (thioredoxin peroxidase; Kuge and Jones, 1994; Wu and Moye-Rowley, 1994; Grant et al., 1996; Lee et al., 1999), and functions for the defense against oxidative stress (Kuge and Jones, 1994). Mammalian cells have the redox-responsive transcription factors AP-1 and NF-κB, which are activated by hydrogen peroxide and regulate genes involved in antioxidant defense, such as genes for γ-glutamylcystein synthetase, manganese SOD, and heme oxygenase (Rahman and MacNee, 2000). Higher plants are also thought to adapt to oxidative stress through coordinate regulation of a battery of antioxidant genes. Although some cis-elements responsive to ROS (ocs element, Chen and Singh, 1999; as-1 element, Garreton et al., 2002) and mitogen-activated protein (MAP) kinase cascades that are activated by oxidative stress (Kovtun et al., 2000; Samuel et al., 2000) have been characterized in higher plants, the details of the regulatory mechanisms of the antioxidant defense system are still unclear. So far, no redox regulatory system of antioxidant genes in higher plants has been reported.

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). In addition, we found that a 28-bp region of the 77-bp sequence described above is conserved among promoters of the sodCc1, trxh, and grx (glutaredoxin) genes in rice (Sha et al., 1997b), as shown in Figure 1A. These genes code for the components associated with oxidative stress defense. SOD catalyzes the dismutation of superoxide radical, which is the initiation reaction of ROS detoxification, and is a key component of ROS scavenging system (Bowler et al., 1994). Thioredoxin and glutaredoxin both have thioltransferase activity that catalyzes the reduction of protein disulfide (Holmgren, 1989). They are thought to function in antioxidative defense based on the finding that knockout mutants of yeast lacking thioredoxin and glutaredoxin are sensitive to oxidative stress (Luikenhuis et al., 1998; Izawa et al., 1999). In addition, thioredoxine and glutaredoxin are electron donors for the lipid peroxide-scavenging peroxidase, peroxiredoxin (Rouhier et al., 2002). Glutaredoxin also exhibits dehydroascorbate reductase activity, which functions in the recycling of the oxidized form of ascorbate (Wells et al., 1990; Sha et al., 1997a). Thioredoxin and glutaredoxin control the expressions of antioxidant genes by regulating the DNA-binding activities of redox-sensitive transcription factors such as AP-1, NF-κB, and OxyR (Hirota et al., 1997; Zheng et al., 1998; Harper et al., 2001). In addition, the thioltransferase activities of thioredoxin and glutaredoxin are involved in the repair of oxidized proteins. It has been demonstrated that thioredoxin and glutaredoxin regenerate oxidatively damaged proteins in vitro (Yoshitake et al., 1994), which suggests that they play a role in the repair of inactivated proteins under oxidative stress.

Figure 1.

Figure 1.

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A cis-element-like motif conserved on the promoter regions of three antioxidant genes in rice. A, Homologous sequences conserved on the promoter regions of sodCc1, trxh, and grx. sodCc1, Gene for cytosolic CuZn-SOD1; trxh, cytosolic thioredoxin gene; grx, glutaredoxin gene. The bold line above the sequence indicates the 28-bp sequence of the CORE motif. Conserved nucleotides between sodCc1 and trxh are shaded in gray boxes. The letters with black boxes indicate conserved nucleotides among sodCc1, trxh, and grx in the CORE motif. The numbers on both ends indicate the position of the sequences with respect to the translation start sites as +1. The accession numbers of sodCc1, trxh, and grx are L19435, D26547, and D86744, respectively. B, Sequences of mutated CORE motifs (M1, M2, and M3). M1, M2, and M3 are mutated versions of the CORE motif. Mutated nucleotides are shown in lowercase. The black arrows indicate a palindrome structure. The dashed arrows indicate a palindrome structure that contains mutated nucleotides.

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.

RESULTS

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 β-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; Kaminaka et al., 1999). Therefore, we treated the transformed calli with these chemicals and examined whether the 77-bp sequence had any effects on LUC gene expression. As shown in Figure 2A, the fusion promoter of the 77-bp sequence and CaMV 35S promoter (77HS-35SINTLUC+, black bar) responded to methyl viologen. Treatment with either 1 μm or 10 μm methyl viologen resulted in a 2.6- to 2.7-fold increase in LUC activity compared with the untreated control. However, no induction of LUC activity was observed by hydrogen peroxide or DTT treatment. The CaMV 35S promoter alone (35SINTLUC+, white bar) did not show any significant response to either of the treatments. These results demonstrate that the 77-bp sequence induces LUC gene expression in response to methyl viologen treatment, which suggests that the 77-bp sequence contains a cis-element that is responsive to methyl viologen-induced oxidative stress.

Figure 2.

Figure 2.

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The transient expression assay of a novel cis-element under oxidative stress condition. A, The response of promoter activity of a 77-bp homologous sequence to oxidative stress. A LUC construct, 77HS-35SINTLUC+, that is driven by a fusion promoter of the 77-bp sequence and the CaMV 35S promoter (black bars) and a control LUC construct, 35SINTLUC+, that is driven by the CaMV 35S promoter (white bars) were introduced into rice calli by particle bombardment. A GUS reporter construct (35SINTGUS) was also cointroduced as an internal control. The samples were treated with the chemicals indicated for 24 h, then subjected to LUC and GUS assay. Data are presented as relative LUC activities normalized with GUS activities. “ctrl” indicates samples without chemical treatments. Error bars indicate sd (n = 8–11). PQ, Methyl viologen. B, Changes in the promoter activity of the CORE motif in response to methyl viologen treatment. The transient expression assay was performed in rice seedlings using the LUC construct that is driven by a fusion promoter of CORE and the CaMV 35S promoter (COREd35SINTLUC+, black bars) and a control LUC construct (35SINTLUC+, white bars) as described above. A Renilla LUC construct (35SINTRluc) was used as an internal control. The transformed samples were treated with methyl viologen (PQ) for 24 h. “ctrl” indicates samples without methyl viologen treatments. Data are relative LUC activities normalized with Renilla LUC activities. Error bars indicate se (n = 3–4).

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.

Figure 3.

Figure 3.

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The response of promoter activities of mutated CORE motifs to methyl viologen. The transient expression assay was performed in rice seedlings with LUC constructs with a CORE motif (COREd35SINTLUC+) and mutated versions of CORE motifs (M1, M2, and M3). Transformed samples were treated with (black bars) or without (white bars) 5 μm methyl viologen for 24 h. Error bars indicate sd (n = 5–11).

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; Kaminaka et al., 1999), the expression profiles of the trxh and grx genes in response to oxidative stress are unclear. To examine whether these three genes respond to oxidative stress in vivo, we performed northern-blot analysis in methyl viologen-treated seedlings. The mRNA levels of sodCc1, trxh, and grx were increased by methyl viologen treatment (Fig. 4). The induction was observed by 4-h treatment and continued for at least 12 h. Although the induction of grx mRNA was less prominent compared with that of sodCc1 and trxh, induction kinetics were similar among the three genes. These results suggest that the three antioxidant defense genes are regulated coordinately in response to methyl viologen.

Figure 4.

Figure 4.

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Coordinate induction of three antioxidant genes by oxidative stress. Rice seedlings were treated with 10 μm methyl viologen (paraquat), and northern-blot analysis was performed with 20 μg of total RNA using gene-specific probes. The mRNA levels were quantified with a phosphor imager. The data were normalized with the mRNA levels of actin as an internal control.

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.

Figure 5.

Figure 5.

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Detection of nuclear proteins specifically bound to the CORE motif. A, Gel mobility shift assay using the CORE probe. Fifteen micrograms of nucleoproteins (lanes 2–6) from rice germinating embryos were incubated with 3 ng of 32P-labeled CORE probe at room temperature and then separated by PAGE. A nonlabeled probe was added as a competitor in the binding reaction (6 ng in lane 3, 12 ng in lane 4, and 24 ng in lane 5, respectively). Poly(dI-dC)·poly(dI-dC) was added by 30 ng in lanes 1 to 5 and 60 ng in lane 6, respectively. An asterisk indicates that poly(dI-dC)·poly(dI-dC) was used as a nonspecific competitor. The signal bands of specific complexes are indicated by arrowheads. B, Gel mobility shift assay using the mutant probes. Binding reactions were carried out with 15 μg of nucleoproteins, 3 ng of 32P-labeled CORE probe or mutant probes (M1, M2, and M3), and 50 ng of poly(dI-dC)·poly(dI-dC). C, Southwestern blot using the CORE probe. Ten or 15 μg of nucleoproteins from rice germinating embryos was fractionated by SDS-PAGE and then blotted onto a polyvinylidene difluoride membrane. The blot was probed with 32P-labeled CORE fragment or mutated CORE fragments (M1, M2, and M3).

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; Samuel et al., 2000). Hydrogen peroxide, superoxide, and ozone treatments activate a MAP kinase cascade, which is thought to regulate downstream stress-responsive genes. We hypothesized that sodCc1, trxh, and grx are regulated by a protein kinase cascade and examined the effects of two protein kinase inhibitors, staurosporine and PD98059. Staurosporine inhibits protein kinase A, protein kinase C, and Tyr kinase by competition with ATP. PD98059 [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one] inhibits MAP kinase kinase selectively (Dudley et al., 1995). The mRNA levels of sodCc1 and trxh were elevated by staurosporine and PD98059 irrespective of methyl viologen treatment (Fig. 6). The mRNA levels were increased 2.2- to 3.2-fold by both inhibitors without methyl viologen treatment and were similar to or higher than the transcript levels of plants treated with methyl viologen alone (1.9- to 2.0-fold). Although the induction was less pronounced, staurosporine and PD98059 also induced grx expression (1.7- and 1.4-fold, respectively) without methyl viologen treatment. These results suggest that a MAP kinase cascade is associated with the regulation of these three antioxidant defense genes in rice. We then performed a transient expression assay using PD98059-treated seedlings to examine whether the MAP kinase cascade is involved in the gene regulation mediated by CORE. The 35SINTLUC+ (control) and COREd35SINTLUC+, which is driven by the fusion promoter of CORE and the CaMV 35S promoter, were introduced into rice seedlings, respectively. The LUC activity of 35SINTLUC+ exhibited no significant change by PD98059 treatment. In the case of COREd35SINTLUC+, the LUC activity was increased by PD98059 treatment irrespective of methyl viologen treatment (Fig. 7). The induction was 2.2-fold by treatment with PD98059 alone, and 2.6-fold by PD98059 plus methyl viologen. The extent of the induction by the inhibitor is similar to that by methyl viologen alone (3.1-fold) observed in Figure 2B. Thus, we conclude that a MAP kinase cascade is associated with the regulatory mechanism of antioxidant genes via CORE.

Figure 6.

Figure 6.

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The effects of translation inhibitor and protein kinase inhibitors on mRNA levels of antioxidant genes. Rice seedlings were treated with the indicated inhibitors together with (black bars) or without (white bars) 10 μm methyl viologen for 6 h. The inhibitors used were 1% (w/v) cycloheximide (CHX), 0.5 μm staurosporine (st), and 100 μm PD98059 (PD). “ctrl” indicates samples without inhibitor treatments. Northern-blot analysis was performed and the data were quantified and normalized as described in Figure 4.

Figure 7.

Figure 7.

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Transient expression assays using MAP kinase kinase inhibitor. The transient expression assay was performed in rice seedlings using LUC plasmid with the CORE motif (COREd35SINTLUC+, black bars) and control LUC plasmid (35SINTLUC+, white bars) as described in Figure 2. The transformed samples were treated with 100 μm PD98059 (+PD) alone or together with 10 μm methyl viologen (+PQ+PD) for 6 h. The data are presented as relative values compared with the samples receiving no chemical treatment (ctrl). Error bars indicate sd (n = 3–4).

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).

Table I.

Rice genes possessing the CORE motif on the putative promoter regions

BAC/PAC Clone Position of CORE Motif Gene Description Putative Function
Expressed Genes with Known cDNAs or Expressed Sequence Tags
    OSJNBa0046G16 47025–46998 Multiple stress-responsive zinc-finger protein Stress defense
    OSJNBa0082M15 110303–330 Putative protein kinase Signal transduction
    OSJNBb0048O22 40809–836 Putative glycosyltransferase Metabolism, biosynthesis
    OSJNOa273B05 23437–464 Putative DNA repair protein Stress defense
    OJ1739D07 52111–085 Similar to Arabidopsis SABRE Regulation of cell expansion
    P0678F08 28340–367 Vacuolar-type H+-translocating inorganic pyrophosphatase Ion transport
    OJ1345_D02 130934–961 Unknown protein with SWIB/MDM2 domain Gene regulation
    OSJNBa0019D02 13929–956 Unknown protein
    P0655A07 31325–298 Unknown protein
    P0669G09 60825–798 Unknown protein
    P0702F05 31891–918 Unknown protein
Predicted Genes
    OJ1365_D05 138887–914 Hypothetical protein with protein kinase C phosphorylation site Signal transduction
    P0519E12 59947–920 Hypothetical protein with zinc-finger motif Transcription factor
    P0001B06 6272–299 Hypothetical protein with Dof domain Transcription factor
        2 Unknown protein genes
        17 Hypothetical protein genes
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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.

DISCUSSION

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) and the as-1 element in the tobacco GST gene (GNT35) that is responsible for induction by ROS (Garreton et al., 2002). In addition, the heat shock element of the cytosolic ascorbate peroxidase gene is involved in the regulation by superoxide (Storozhenko et al., 1998). Our findings suggest that CORE is involved in the coordinate regulation of sodCc1, trxh, and grx, and that these three genes are regulated by a common regulatory mechanism in response to oxidative stress. The database search of the rice genome sequence revealed that at least 11 expressed genes in rice possess the CORE motif on their putative promoter regions (Table I) and suggests that they might be regulated by CORE. Some of the genes are implicated in gene regulation, signal transduction, and stress defense. Among them a gene encoding multiple-stress responsive zinc-finger protein (bacterial artificial chromosome [BAC] clone OSJNBa0046G16) is of particular importance. This gene is essentially identical to the OSISAP1 gene reported by Mukhopadhyay et al. (2004) that is induced by cold, drought, and salt stress, and overexpression of OSISAP1 confers tolerance against those stresses in transgenic tobacco plants. Thus, OSISAP1 is demonstrated to function in the defense against multiple stresses. It is also supposed that a gene encoding putative DNA repair protein (fosmid clone OSJNOa273B05) is implicated in the oxidative stress defense by repairing oxidatively damaged DNA. A protein with SWIB/MDM2 domain (BAC clone OJ1345_D02) and putative protein kinase (BAC clone OSJNBa0082M15) might function in the gene regulation and signal transduction for antioxidant defense. The SWIB/MDM2 domain is known as a p53-binding domain that down-regulates the transactivation activity of p53 tumor suppressor protein (Kussie et al., 1996). It is possible that SWIB/MDM2-related protein might interact with other transcription factors to regulate defense response. The existence of the CORE motifs in H+-translocating inorganic pyrophosphatase gene (PAC clone P0678F08), a gene similar to Arabidopsis SABRE gene (BAC clone OJ1739D07), which regulates cell expansion in Arabidopsis (Aeschbacher et al., 1995), and putative glycosyltransferase gene (BAC clone OSJNBb0048O22) were not expected. Although it is unknown whether these genes have roles in antioxidant defense, it is suggested that numbers of genes involved in various biological processes might be regulated in response to oxidative stress via CORE. The results of the search for the CORE motif in the Arabidopsis genome and the promoter sequence of cytosolic SOD, trxh, and grx genes from other plants suggest that the CORE motif is not conserved in other plant species. It is possible that other plants also possess similar regulatory machinery responsive to oxidative stress as well as rice. But cis-elements that function in other species might have diverse sequences from that of CORE.

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). Recent reports have described the involvement of the MAP kinase cascade in ROS signaling. Activation of Arabidopsis MAP kinase kinase kinase ANP1 and MAP kinase AtMPK3 and 6 by hydrogen peroxide (Kovtun et al., 2000; Yuasa et al., 2001) and tobacco SIPK (salicylic-acid-induced protein kinase) by hydrogen peroxide, superoxide, and ozone (Samuel et al., 2000) have been observed. Like other stress-responsive MAP kinases, those MAP kinases responsive to oxidative stress are activated under a stressed condition. And the activation of the MAP kinase cascade initiated from ANP1 induces GST6 gene in Arabidopsis (Kovtun et al., 2000). Our data showed that an inhibitor of MAP kinase kinase elevated the expression of the three antioxidant genes (Fig. 6) and the activity of CORE (Fig. 7). This result suggests that a MAP kinase cascade might act as a negative regulator of CORE-mediated gene regulation in rice. And it is suggested that there might be two possibilities concerning the role of the kinase cascade. One is that CORE might respond to some stimuli other than oxidative stress and the kinase cascade might mediate the signal of such stimuli. And the other is that the MAP kinase cascade involved in the CORE-mediated regulation might mediate the oxidative stress signaling. In this case, it can be assumed that there might be multiple MAP kinase cascades for oxidative stress signaling.

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.

MATERIALS AND METHODS

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).

Plasmid Construction

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) into the XbaI and BamHI sites between the CaMV 35S promoter and GUS coding sequence to enhance reporter gene expression. The firefly and Renilla LUC (LUC and RLUC) reporter plasmids, 35SINTLUC+ and 35SINTRluc (S. Morita, unpublished data), were constructed by replacing the GUS-coding sequence of 35SINTGUS with a LUC- or RLUC-coding sequence (originating from pSP-luc+NF and pRluc-null, respectively; Promega, Madison, WI). The 77-bp homologous sequence of the rice sodCc1 gene was amplified by PCR using TSODP-1F primer (5′-AAGAGAAACAACATATTTAC-3′) and TSODP-3R primer (5′-ATTTAGATCGCTAAAAACAC-3′), and the fragment was cloned into the EcoRV site of pBluescriptSK+ (Stratagene, La Jolla, CA) by TA cloning. The 77-bp homologous sequence was excised and then integrated into the 5′ upstream region of the CaMV 35S promoter of 35SINTLUC+ at the PstI and HindIII sites to yield 77HS-35SINTLUC+. To integrate the CORE sequence into the 35SINTLUC+, a tandem dimer of CORE (COREd) was constructed by annealing two synthesized oligonucleotides, which yielded EcoRI and HindIII overhangs. Three mutated versions of CORE dimers were also constructed (M1d, M2d, and M3d). The sequences of the oligonucleotides were as follows: COREd plus strand, 5′-AGCTTAATAATTTATAAATAAAACTTTTATATAGATATCAATAATTTATAAATAAAACTTTTATATAG-3′; COREd minus strand, 5′-AATTCTATATAAAAGTTTTATTTATAAATTATTGATATCTATATAAAAGTTTTATTTATAAATTATTA-3′; M1d plus strand, 5′-AGCTTCCTCCGTTATAAATAAAACTTTTATATAGATATCCCTCCGTTATAAATAAAACTTTTATATAG-3′; M1d minus strand, 5′-AATTCTATATAAAAGTTTTATTTATAACGGAGGGATATCTATATAAAAGTTTTATTTATAACGGAGGA-3′; M2d plus strand, 5′-AGCTTAATAATTTAGCACGCCCCCTTTTATATAGATATCAATAATTTAGCACGCCCCCTTTTATATAG-3′; M2d minus strand, 5′-AATTCTATATAAAAGGGGGCGTGCTAAATTATTGATATCTATATAAAAGGGGGCGTGCTAAATTATTA-3′; M3d plus strand, 5′-AGCTTAATAATTTATAAAGCCAACTTGGCTATAGATATCAATAATTTATAAAGCCAACTTGGCTATAG-3′; and M3d minus strand, 5′-AATTCTATAGCCAAGTTGGCTTTATAAATTATTGATATCTATAGCCAAGTTGGCTTTATAAATTATTA-3′. After phosphorylation with T4 polynucleotide kinase, the fragments were cloned into the HindIII-EcoRI sites of pBluescriptSK+ to yield COREd/pBSK+, M1d/pBSK+, M2d/pBSK+, and M3d/pBSK+, respectively. Then the tandem dimers were excised by PstI and HindIII digestion and integrated into the 5′ upstream region of the CaMV 35S promoter of 35SINTLUC+. The resulting plasmids were named COREd35SINTLUC+, M1d35SINTLUC+, M2d35SINTLUC+, and M3d35SINTLUC+, respectively. The construction of the reporter plasmids was verified by sequencing.

Transient Expression Assay

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).

Northern-Blot Analysis

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). The trxh probe was prepared by PCR using primers based on the sequence of the full length cDNA of rice thioredoxin (Ishiwatari et al., 1995), and the grx probe was prepared by restriction digestion of the cDNA clone (Minakuchi et al., 1994). The blots were stripped and subsequently hybridized with rice actin cDNA (accession no. D15628; obtained from the Rice Genome Research Program, National Institute of Agrobiological Sciences, Tsukuba, Japan) as an internal control. The probes were labeled with a Bcabest Random Primer Labeling Kit (Takara Bio, Otsu, Japan). Hybridization was carried out at 42°C and washing was performed twice at 42°C with 3× SSC, 0.1% (w/v) SDS for the sodCc1, trxh and grx probes, and with 1× SSC, 0.1% (w/v) SDS for the actin probe. Hybridization signals were quantified by measuring the radioactivities of the signal bands using a phosphor imager (Molecular Imager GS-525; Bio-Rad).

Gel Mobility Shift Assay

Nuclear extracts were prepared from 4-d-old germinating embryos that were suspension-cultured in N6 medium as described previously (Morita et al., 1999). The samples were homogenized in an extraction buffer (50 mm Tris-HCl, pH 7.9, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 5 mm DTT, 1.6 mm salicylhydroxamic acid, and 1 μg mL−1 t-butylated hydroxytoluene) with physcotron homogenizer on ice. Homogenate was filtered through two layers of Miracloth (Calbiochem, La Jolla, CA), then centrifuged at 3,300g for 10 min at 4°C. The pellet of crude nuclei was suspended in the extraction buffer, and purified through a step gradient containing 40%, 60%, 80% (v/v) Percoll and 80% (w/v) Suc in the extraction buffer by centrifugation at 4,000g for 30 min at 4°C. The nuclear fraction was recovered from the interface between 80% Percoll and 80% Suc layers, and centrifuged at 3,300g for 30 min at 4°C after adding two volumes of the extraction buffer. The pellet of nuclei was suspended in a buffer A (25 mm Tris-HCl, pH 7.9, 5 mm MgCl2, 0.1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 mm DTT, 25% [v/v] glycerol, and 500 mm NaCl) and incubated for 30 min at 4°C with gentle stirring. Extracts were cleared by centrifugation at 25,000g for 30 min at 4°C, and dialyzed in buffer B (20 mm HEPES-KOH, pH 7.9, 100 mm KCl, 12.5 mm MgCl2, 0.2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 mm DTT, and 20% [v/v] glycerol) overnight at 4°C. The extracts were centrifuged at 25,000g for 30 min at 4°C, and stored at −80°C. The XbaI-XhoI fragments containing the CORE dimer or mutated dimers were excised from COREd/pBSK+, M1d/pBSK+, M2d/pBSK+, and M3d/pBSK+, respectively. The fragments were labeled with [α-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).

Southwestern-Blot Analysis

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).

Database Search for the CORE Motif

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).

Acknowledgments

The authors are grateful to the Rice Genome Research Program for providing rice actin cDNA.

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.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.045658.

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