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First published online June 24, 2005; 10.1104/pp.105.063586

Plant Physiology 138:1436-1445 (2005)
© 2005 American Society of Plant Biologists

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ENVIRONMENTAL STRESS AND ADAPTATION

Functional Analysis of Arabidopsis Ethylene-Responsive Element Binding Protein Conferring Resistance to Bax and Abiotic Stress-Induced Plant Cell Death1

Taro Ogawa, Ling Pan, Maki Kawai-Yamada, Li-Hua Yu, Saburo Yamamura, Tomotsugu Koyama, Sakihito Kitajima2, Masaru Ohme-Takagi, Fumihiko Sato and Hirofumi Uchimiya*

Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113–0032, Japan (T.O., L.P., M.K.-Y., L.-H.Y., H.U.); Iwate Biotechnology Research Center, Kitakami, Iwate 024–0003, Japan (S.Y., H.U.); Gene Function Research Laboratory, National Institute of Advanced Industrial Science and Technology, Tsukuba 305–8562, Japan (T.K., M.O.-T.); and Department of Applied Life Sciences, Kyoto University, Kitashirakawa, Sakyo, Kyoto 606–8502, Japan (S.K., F.S.)


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 
Arabidopsis (Arabidopsis thaliana) ethylene-responsive element binding protein (AtEBP) gene was isolated as a suppressor of Bax-induced cell death by functional screening in yeast (Saccharomyces cerevisiae). To further examine the cell death suppressive action of AtEBP in plant cells, we established transgenic tobacco (Nicotiana tabacum) plants overexpressing AtEBP as well as transgenic tobacco plants ectopically expressing mouse Bax protein under a dexamethasone-inducible promoter. We prepared the crosses of the selective lines of each transgenic plant, which were evaluated in terms of cell death suppression activity. Results indicate that AtEBP suppressed Bax-induced cell death in tobacco plants, an action also associated with a lowered level of ion leakage. Furthermore, tobacco Bright Yellow-2 cells overexpressing AtEBP conferred resistance to hydrogen peroxide (H2O2) and heat treatments. AtEBP protein localized in the nucleus and functioned as an in vivo transcription activator as confirmed in transient assays and experiments using stable transgenic system. Up-regulation of defense genes was observed in transgenic Arabidopsis plants overexpressing AtEBP. Based on the analysis of mRNA accumulation in ethylene-related mutants, the position of AtEBP in signaling pathway is presented.

The proapoptotic mammalian protein Bax can induce cell death in the budding yeast, Saccharomyces cerevisiae (Sato et al., 1994Go; Greenhalf et al., 1996Go; Zha et al., 1996Go) as well as in mammalian cells (Oltvai et al., 1993Go). Bax forms channels in the outer membrane of the mitochondrion and triggers the release of cytochrome c, which activates a series of caspases, initiating a cascade of protease activation. Commonalties in the mechanism of Bax-induced cell death were found in yeast and mammalian cells (for review, see Reed, 1997Go). Furthermore, Bax-induced cell death in yeast can be suppressed by expression of mammalian (Xu and Reed, 1998Go), and plant (Kawai et al., 1999Go; Kampranis et al., 2000Go; Pan et al., 2001Go) genes, respectively. In our previous work, we attempted to screen genes from an Arabidopsis (Arabidopsis thaliana) cDNA library, which suppressed the lethal effects of mouse Bax in yeast (Pan et al., 2001Go). The most abundant gene (82% of isolated genes) screened was found to be AtEBP, known as Arabidopsis ethylene-responsive element binding protein gene. AtEBP has one APETALA2/ethylene responsive element binding protein (AP2/EREBP) domain, which is known to bind GCC-box (AGCCGCC), an ethylene responsive cis-element (Allen et al., 1998Go). Büttner and Singh (1997)Go also isolated AtEBP as an in vitro interacting partner of OBF4/TGA4, a basic Leu zipper transcription factor. However, the biological significance of AtEBP in planta is not fully understood.

The AP2/EREBP family is a unique group of plant transcriptional factors. The AP2/EREBP domain consists of about 60 conserved amino acids (Allen et al., 1998Go). The APETALA2 family, containing two AP2/EREBP domains, regulates plant development (Jofuku et al., 1994Go; Elliot et al., 1996Go; Klucher et al., 1996Go; Boutilier et al., 2002Go). On the other hand, the ethylene-responsive factor (ERF) family, containing one AP2/EREBP domain, is one of the largest groups of transcriptional factors (Riechmann et al., 2000Go). These genes can work in various conditions: downstream of ethylene, jasmonate, and abscisic acid signaling pathways; and environmental-stress responses such as cold, dry, and salt (Ohme-Takagi and Shinshi, 1995Go; Stockinger et al., 1997Go; Finkelstein et al., 1998Go; Liu et al., 1998Go; Solano et al., 1998Go; Menke et al., 1999Go; van der Fits and Memelink, 2000Go, 2001Go; Gu et al., 2002Go). It is known that two types of transcriptional factors, namely a transcriptional activator and a repressor, are included in ERF family proteins (Fujimoto et al., 2000Go; Ohta et al., 2000Go, 2001Go). Furthermore, several ERF proteins are reported to interact with other proteins such as transcriptional factor, nitrilase-like protein, and ubiquitin-conjugated enzyme (Büttner and Singh, 1997Go; Xu et al., 1998Go; Koyama et al., 2003Go). These facts suggest that the physiological role of each ERF may be divergent.

In this study, we demonstrate that plant cells overexpressing AtEBP are resistant to Bax-induced cell death and abiotic stresses such as hydrogen peroxide (H2O2) and heat. Furthermore, AtEBP functions as a transcriptional activator as demonstrated in transient assays and experiments using stable transgenic systems. Based on the analysis of gene expression levels in ethylene-related mutants, we also present the position of AtEBP in the ethylene signaling pathway.


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 LITERATURE CITED
 

AtEBP Suppresses Bax-Induced Cell Death in Tobacco Plants

AtEBP gene was previously isolated as a suppressor of Bax-induced cell death by functional screening in yeast (Pan et al., 2001Go). To confirm that AtEBP suppresses cell death in plant cells, we established transgenic tobacco (Nicotiana tabacum) plants expressing AtEBP and ectopically expressing Bax using a dexamethasone (DEX)-inducible system (Aoyama and Chua, 1997Go; Fig. 1A). We generated five independent transgenic tobacco plants expressing AtEBP under cauliflower mosaic virus (CaMV) 35S promoter, followed by northern-blot analysis using AtEBP cDNA as a probe (data not shown). We selected two lines (EBP8 and EBP20) for the following experiments. We also generated about 20 independent transgenic tobacco plants ectopically expressing Bax. When transgenic Bax plants (2-week-old) were transferred to a medium containing 10 µM DEX, extensive etiolation at the whole-plant level was observed at 4 d after DEX treatment (Fig. 1B). One of these lines, namely Bax21, showed Bax expression immunologically at 6 h after DEX treatment (Fig. 1C). We also selected this line for the following cross experiment.



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  		Figure 1. Suppression of Bax-induced cell death in tobacco plants overexpressing AtEBP. A, Schematic diagrams of the DEX-inducible vector construct carrying Bax (pTA-Bax) and the constitutive expression vector construct carrying AtEBP (pBIG-AtEBP). 6 x UAS, Glucocoriticoid-regulated transcription factor-regulated promoter; Bax, mouse Bax-coding sequence; 3A, pea rbc-3A poly A addition sequence; E9, pea rbc-E9 poly A addition sequence; GVG, chimeric glucocoriticoid-regulated transcriptional factor; 35S, CaMV 35S promoter; {Omega}, tobacco mosaic virus transcriptional enhancer sequence; AtEBP, AtEBP coding sequence; NOS polyA, nopaline synthetase polyadenylation sequence. B, Bax-induced chlorosis in tobacco plant. Two-week-old tobacco plant was treated in a medium containing 10 µM DEX for 4 d. Bar = 1 cm. C, Western-blot analysis of Bax protein in transgenic tobacco plants. Two-week-old tobacco plants (Bax21 line) were treated in a medium containing 10 µM DEX for 1 d. Total proteins (20 µg) obtained from shoots of the plants at each time period (0, 6, 12, and 24 h) were used. D, a, RT-PCR analysis of AtEBP and Bax in the hybrid line. Two-week-old plants (Bax21 and Bax21 x EBP20) were treated in a medium containing 1 µM DEX for 24 h. Total RNAs from shoots of plants and a pair of specific oligonucleotide primers of AtEBP and Bax were used in RT-PCR analysis. b, Western-blot analysis of Bax proteins in the hybrid line. Two-week-old tobacco plants (Bax21 and Bax21 x EBP20) were treated in a medium containing 10 µM DEX for 2 d. Total proteins (20 µg) obtained from shoots of the plants at each time period (0, 24, and 48 h) were used. E, Repressed Bax-induced chlorosis in the hybrid line expressing AtEBP. Two-week-old tobacco plants (Bax21 and Bax21 x EBP20) were treated in a medium containing 10 µM DEX for 4 d. Bar = 1 cm. F, Bax-induced ion leakage in the hybrid line. Leaf sections obtained from 2-week-old tobacco plants (Bax21 and Bax21 x EBP20) were incubated with 0, 0.1, and 1.0 µM DEX in distilled water at 27°C. The resulting values are indicated as relatives. Data represent mean ± SE obtained from four independent measurements. G, Cytological observation of O2– and H2O2 production in tobacco lines. Two-week-old tobacco plants (Bax21, Bax21 x EBP20, and nontransgenic plant, SR1) were treated with 1 µM DEX for 7 h to induce Bax, and the detached leaves were stained by NBT (top) and DAB (bottom). Bar = 50 µm. The experiments were repeated at least three times, and representative photographs are shown.

 
To evaluate whether AtEBP suppresses Bax-induced cell death in plants, we prepared a hybrid line by crossing these transgenic tobacco plants expressing AtEBP (EBP20) with plants ectopically expressing Bax (Bax21). Only F1 hybrid plants were used in the following experiments. Constitutive expression of AtEBP and DEX-induced expression of Bax in the hybrid line were confirmed by reverse transcription (RT)-PCR (Fig. 1D, a). Furthermore, the expression of Bax protein in the hybrid line was also detected immunologically (Fig. 1D, b). Although an apparent chlorosis of leaves in the parent line was seen at 4 d after DEX treatment, leaves of the hybrid line retained green color (Fig. 1E). To quantitatively evaluate cell death, ion leakage from leaf discs was measured. Ion leakage has been used as an indicator of plant cell death (Mitsuhara et al., 1999Go; Rizhsky et al., 2002Go; Kawai-Yamada et al., 2004Go). The level of electrolyte leakage of each transgenic line was increased after DEX treatment, but the hybrid line showed lower levels of ion leakage compared with the parent line (Fig. 1F).

We already reported that production of reactive oxygen species (ROS) production was involved in cell death triggered by overexpression of Bax in Arabidopsis plants (Kawai-Yamada et al., 2004Go). To test whether or not AtEBP expression affects ROS production by Bax, these transgenic tobacco lines (Bax21 and Bax21 x EBP20) and nontransgenic tobacco (SR1) were treated with 1 µM DEX for 7 h and the leaves were stained with nitroblue tetrazolium (NBT) and 3, 3'-diaminobenzidine (DAB) for the detection of superoxide (O2–) and H2O2, respectively. As shown in Figure 1G, subcellular localizations of O2– and H2O2 were observed in tobacco plant cells expressing Bax despite AtEBP expression. NBT and DAB failed to stain nontransgenic tobacco plant cells.


Tobacco Bright Yellow-2 Cells Overexpressing AtEBP Confer Resistance to H2O2 and Heat Stresses

We established tobacco Bright Yellow-2 (BY-2) cells overexpressing AtEBP by the Agrobacterium-mediated method (Shaul et al., 1996Go). AtEBP expression was detected only in the cell lines transformed with a vector possessing AtEBP (Fig. 2A).



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  		Figure 2. Resistance of tobacco BY-2 cells expressing AtEBP to H2O2 and heat. A, RNA gel-blot analysis of tobacco BY-2 cells expressing AtEBP. Total RNAs (10 µg) were obtained from 4-d-old transgenic tobacco BY-2 cells. The full-length coding region of AtEBP was used as a probe. C1 and C2, Vector control lines; T1 and T2, cell lines expressing AtEBP under CaMV 35S promoter. B and C, Quantification of dead cells after H2O2 (B) or heat (C) treatments. Ten-day-old transgenic BY-2 cells were treated with each concentration of H2O2 (0, 2, and 5 mM) for 4 h, followed by Evans blue staining. Dye bound to dead cells was quantified by A600. Ten-day-old transgenic BY-2 cells were also treated with 55°C for 2 h. These data present mean ± SD obtained from three independent measurements.

 
Using such cell lines, we evaluated H2O2-induced cell death. Ten-day-old cells were transferred to a fresh medium and then treated with H2O2 (0, 2, and 5 mM) for 4 h. The extent of cell death was determined by selective staining of dead cells with Evans blue by measurement of A600 (Fig. 2B). Compared with the control lines (C1 and C2), repression of cell death in the transgenic lines (T1 and T2) was apparent at 2 and 5 mM H2O2.

A brief heat treatment of suspension-cultured cells has also previously been demonstrated to induce cell death evident by apoptotic phenotype such as cell shrinkage and DNA laddering (McCabe et al., 1997Go; Swidzinski et al., 2002Go). We also investigated the resistance to heat stress using the same transgenic lines. Namely, 10-d-old cells were incubated at 55°C for 2 h and kept for 12 h at 27°C and shrunken cells were counted under a microscope (Fig. 2C). The control lines showed 50% to 80% of shrunken cells, whereas the transgenic lines showed only 30% to 40% of shrunken cells.


Localization of Green Fluorescence Protein-AtEBP Fusion Protein Localizes in the Nucleus, and AtEBP Acts as a Transcriptional Activator

A putative nuclear localization signal was found in front of the AP2/EREBP domain of AtEBP protein. We previously reported that the nuclear localization of AtEBP was essential for the cell death suppression activity in yeast cells (Pan et al., 2001Go). To clarify the localization of AtEBP in plant cells, a plasmid possessing green fluorescence protein (GFP) gene fused to AtEBP under CaMV 35S promoter was constructed, which was then bombarded to the epidermal cells of onion (Allium cepa). As shown in Figure 3A, the fluorescence of GFP-AtEBP was localized exclusively in the nucleus, whereas the fluorescence of GFP alone was observed in the whole cell.



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  		Figure 3. Cellular localization of GFP-AtEBP and transactivation of GAL4-responsive transcription by AtEBP fused with the DNA binding domain of GAL4. A, Nuclear localization of GFP-AtEBP in plant cells. The plasmids, 35S-GFP (left) and 35S-GFP-AtEBP (right), were bombarded to onion epidermal cells. At 24 h after incubation, GFP fluorescence was observed under a fluorescent microscope. B, Schematic diagram of the reporter and effecter constructs used in the transient expression assay. The reporter construct contains five copies of the GAL4-binding sites (5 x GAL4) upstream of a minimal TATA region (starting position –46) of CaMV 35S promoter (TATA), the firefly gene for LUC, and the terminator of nopaline synthase (T-NOS). The effecter plasmid contains the DNA-binding domain of yeast GAL4 protein (GAL4-DB) and AtEBP driven by CaMV 35S promoter. The translational enhancer sequence from tobacco mosaic virus ({Omega}) was located upstream of the site of translational initiation. C, Relative LUC activities in Arabidopsis leaves after cotransfection with the effecter and reporter plasmids. LUC activities are expressed relative to values obtained with the reporter plasmid alone (None, set arbitrarily at 1). Data represent mean ± SD of three independent measurements.

 
To characterize AtEBP in terms of transcriptional activity, we performed a transient expression assay using a GAL4-responsive reporter system. The reporter construct contained Luciferase (LUC) under five copies of the GAL4-binding sites and TATA region of CaMV 35S promoter. The effecter construct contained the DNA-binding domain of yeast GAL4 protein fused to full length of AtEBP driven by CaMV 35S promoter (Fig. 3B). Bombardment of plasmids into Arabidopsis leaves augmented LUC activity, and the activity was further augmented with increased addition of the effecter plasmids (Fig. 3C).


AtEBP Up-Regulates the Expression of Reporter Gene Possessing the GCC-Box in the Promoter

Next we established a genetic system in order to determine whether AtEBP controls transcription through GCC-box in the promoter of target genes. We previously obtained three transgenic tobacco lines (NsERF2 pro-GUS, NsERF3 pro-GUS, and NsERF4 pro-GUS) possessing {beta}-glucuronidase (GUS) gene fused with the promoters of NsERF2/3/4 from Nicotiana sylvestris, respectively (Kitajima et al., 2000Go). The promoter of NsERF3 contains two GCC-box sequences and that of NsERF2 contains a putative binding sequence of EIN3/TEIL (Kosugi and Ohashi, 2000Go), but that of NsERF4 does not contain any known binding site related to the ethylene signaling (Fig. 4A). We also generated transgenic tobacco plants expressing AtEBP under CaMV 35S promoter.



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  		Figure 4. Up-regulation of the reporter gene possessing GCC-box in transgenic tobacco plants expressing AtEBP. A, Schematic diagram of the structures of NsERF2/3/4 pro-GUS. The promoter regions of NsERF2 (NsERF2 pro), NsERF3 (NsERF3 pro), and NsERF4 (NsERF4 pro) were fused with GUS gene and the terminator of nopaline synthase (T-NOS). Translational initiation is indicated as +1. B, RT-PCR analysis of AtEBP in the hybrid lines. AtEBP expression in shoots of 2-week-old hybrid lines was confirmed by RT-PCR using a pair of specific oligonucleotide primers of AtEBP. SR1, Nontransgenic tobacco plants; EBP8, a transgenic tobacco line expressing AtEBP; NsERF2/4 pro-GUS, transgenic tobacco lines expressing GUS under NsERF2/4 promoter lacking GCC-box; NsERF3 pro-GUS, a transgenic tobacco line expressing GUS under NsERF3 promoter containing GCC-box (Kitajima et al., 2000Go). C, Analysis of GUS activity in each hybrid line. GUS activities were measured using shoots of 2-week-old tobacco plants. Data represent mean ± SD obtained from at least four independent measurements. Numerals correspond to Figure 2B. Plus sign (+) and minus sign (–), presence or absence of AtEBP expression or GCC-box, respectively.

 
To obtain hybrid plants, transgenic tobacco line expressing AtEBP (EBP8) and NsERF2/3/4 pro-GUS lines were crossed to each other. AtEBP expression in the hybrid plants (F1) was confirmed by RT-PCR (Fig. 4B). Accordingly, we measured GUS activities in these hybrid lines. The results indicated that the hybrid line (NsERF3 pro-GUS x EBP8) containing the GCC-box in the promoter and expressing AtEBP, showed enhanced GUS activity compared with the control line (NsERF3 pro-GUS x SR1; Fig. 4C). In contrast, despite AtEBP expression, the other hybrid lines (NsERF2/4 pro-GUS x EBP8) lacking GCC-box in the promoter did not show any change of GUS activity compared with the control lines (NsERF2/4 pro-GUS x SR1). These results suggest that AtEBP functions as a transcriptional activator through the GCC-box in plant cells.


AtEBP Overexpression Causes the Up-Regulation of Defense Genes

In order to identify the genes that are regulated by AtEBP in plant cells, several transgenic Arabidopsis lines overexpressing AtEBP under CaMV 35S promoter were established. Using such lines, we investigated the expression of defense genes including pathogenesis-related (PR) genes. As shown in Figure 5, expressions of PDF1.2, a plant defensin gene (Penninckx et al., 1996Go), and GST6 (Chen et al., 1996Go), which is related to ROS detoxification, were increased in Arabidopsis lines overexpressing AtEBP. On the other hand, the expressions of PR-1/2/3 in Arabidopsis lines overexpressing AtEBP were not different from the control line. The data were obtained as replicates with the same results.



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  		Figure 5. Up-regulation of defense genes in Arabidopsis lines overexpressing AtEBP. Total RNAs (10 µg) obtained from shoots of 35-d-old plants were loaded. The coding region of AtEBP excepting AP2/EREBP domain, and the 3'-UTR of PR-1, PR-2, PR-3, PDF1.2, and GST6 were used as probes. A gel stained with ethidium bromide (which indicates rRNA) is shown as a control.

 

The Position of AtEBP in the Ethylene Signaling Pathway

Many isolated ethylene-related mutants have been analyzed for their epistasis relationships, and the framework of the ethylene signaling pathway is almost established (for review, see Ecker, 1995Go; Wang et al., 2002Go). Nevertheless, the precise positions of over 100 ERF genes, including AtEBP in the ethylene signaling pathway, have not been identified, except for ERF1 (Solano et al., 1998Go). To clarify the position of AtEBP in the ethylene signaling pathway, we analyzed mRNA accumulation in ethylene-related mutants such as ethylene-insensitive mutants (ethylene resistant 1-1 [etr1-1], ethylene insensitive 2-1 [ein2-1], and ethylene insensitive 3-1 [ein3-1]), a constitutive active mutant in the ethylene signal transduction (constitutive triple response 1-1 [ctr1-1]), and an ethylene overproducing mutant (ethylene overproducer 3-1 [eto3-1]). As shown in Figure 6, the level of AtEBP mRNA was lower in ein2-1 but higher in ctr1-1 and equal in etr1-1, ein3-1, and eto3-1 than in wild type. ERF1 mRNA was detected only in ctr1-1. Same results of the data were obtained in the separate experiments. Thus, AtEBP may be located downstream of EIN2 and CTR1, but not under EIN3.



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  		Figure 6. Comparison of AtEBP mRNA accumulation in ethylene-related mutants. Total RNAs (10 µg) from shoots of 30-d-old plants were loaded. The coding region of AtEBP excepting AP2/EREBP domain and the 3'-UTR of ERF1 were used as probes. A gel stained with ethidium bromide (which indicates rRNA) is shown as a control.

 

   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 LITERATURE CITED
 

AtEBP Confers Resistance to Cell Death

We demonstrated previously that AtEBP suppressed Bax-induced cell death in yeast, where nuclear transport of AtEBP was essential (Pan et al., 2001Go). However, we were unable to identify the precise mechanism associated with this phenomenon. Thus, this study was designed to determine whether or not AtEBP confers the suppressive ability of Bax-induced plant cell death. The research showed that overexpression of AtEBP confers resistance to not only Bax-induced cell death at the whole-plant level but also H2O2- and heat-induced cell death in plant cells.

Both ROS generation and the release of cytochrome c from the mitochondria cause the activation of cell death in mammalian cells (Green and Reed, 1998Go). ROS production by ecotopic expression of Bax occurs in plant cells (Kawai-Yamada et al., 2004Go, and this study). Following Bax-induced ROS production, cell death at the whole-plant level with cell shrinkage, membrane destruction, and other apoptotic phenotype has been observed (Kawai-Yamada et al., 2001Go, 2004Go). Such phenotypes of plant cells were also observed by exogenous following treatments of H2O2 and heat (Levine et al., 1996Go; McCabe and Leaver, 2000Go; Houot et al., 2001Go; Swidzinski et al., 2002Go). Thus, both Bax expression and exogenous treatments (H2O2 and heat) induce a similar cell death phenomenon.

The scheme of Bax-induced cell death is consistent with our previous reports (Kawai-Yamada et al., 2001Go, 2004Go). In tobacco plants, Bax-induced ROS generation was observed at 7 h after DEX treatment, loss of membrane permeability shown as ion leakage was observed up to 24 h after DEX treatment, and an apparent chlorosis of leaves was observed at 4 d after DEX treatment. In Arabidopsis, ROS generation, ion leakage, and chlorosis by Bax were also observed in similar time scale (Kawai-Yamada et al., 2004Go). These data indicate that the lethal effect of Bax protein is common in different plant species.

AtEBP expression did not influence the process of O2– and H2O2 production caused by Bax, but the phenomena of ion leakage and apparent chlorosis of leaves were late or repressed compared to the control line. We assume that the suppressive activity of AtEBP on ROS-induced cell death is due to the regulation of ROS-related genes (Fig. 7). Consistent with this conclusion, overexpression of AtEBP resulted in up-regulation of GST6, which is related to ROS metabolism. It may be interesting to point out that other plant GST homologs were also isolated as functional suppressors of Bax-induced cell death in yeast (Kampranis et al., 2000Go; Pan et al., 2001Go).



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  		Figure 7. Proposed model for the position and role of AtEBP in the ethylene signaling pathway. This study suggests that AtEBP does not seem to interact with EIN3 and that AtEBP regulates the expression levels of several plant defense genes such as PDF1.2 and GST6 as indicated by white arrow.

 

AtEBP Is an in Vivo Transcriptional Activator

It was demonstrated that AtEBP can bind GCC-box in vitro (Büttner and Singh, 1997Go). To clear the function of AtEBP as a transcriptional factor, we performed series of the following experiments. Microscopic observation of GFP-AtEBP fusion protein confirmed that AtEBP localizes in the nucleus. The transient expression assay by bombardment also suggested that AtEBP is a transcriptional activator. Furthermore, genetic experiments using transgenic tobacco plants expressing AtEBP and constructs possessing respective reporter genes suggested that AtEBP up-regulates the reporter gene possessing the GCC-box in the promoter.

Overexpression of AtEBP coordinately accumulated PDF1.2 and GST6 mRNAs. PDF1.2 is a typical downstream gene of the ethylene/jasmonic acid signaling (Penninckx et al., 1996Go, 1998Go). The GCC-box in the promoter of PDF1.2 is known to be important for transcriptional regulation by methyl jasmonic acid (Brown et al., 2003Go). Indeed, overexpression of ERF genes such as Arabidopsis ERF1 and AtERF2, and tomato (Lycopersicon esculentum) Pti4 up-regulated PDF1.2 expression (Gu et al., 2002Go; Brown et al., 2003Go; Lorenzo et al., 2003Go). Thus, it is likely that AtEBP promotes transcription of PDF1.2 through in vivo binding to the GCC-box. However, PDF1.2 expression was not always increased according to a level of AtEBP mRNA. An appropriate dosage of AtEBP expression may stimulate PDF1.2 expression, whereas excess expression may inhibit this expression by unknown mechanism. On the other hand, GST6 expression level was dependent on the level of AtEBP expression. GST6 is one of H2O2- and salicylic acid-inducible genes (Chen et al., 1996Go; Chen and Singh, 1999Go). The GST6 promoter 2.0 kb upstream from ATG site did not possess GCC-box. However, the cis-element of TGA4, a basic Leu zipper transcriptional factor known to interact with AtEBP (Büttner and Singh, 1997Go), was found in the promoter. AtEBP and TGA4 may regulate the expression of several genes cooperatively. Some concerns regarding equivocal gene expression lead us to conclude that we cannot exclude the possibility of physiological and environmental status of plant materials, which may affect to RNA-blot analysis. Further exploitation of such problems not only by transcript analysis but also by protein analysis needs to be pursued.


Expression Pattern of AtEBP

Various expression patterns were reported in ERF genes, although these genes are known to be regulated by the same hormone, ethylene. For instance, Solano et al. (1998)Go found rapid induction of ERF1 in response to ethylene gas within 15 min. ERF1 was constitutively expressed in ctr1, and ethylene induction of ERF1 was totally dependent on a functional EIN3 protein (Fig. 7). In the case of AtERF genes, Fujimoto et al. (2000)Go also demonstrated late induction of AtERF1, AtERF2, and AtERF5 at 12 h after ethylene treatment.

Analysis of AtEBP expression in ethylene-related mutants indicated that AtEBP is located downstream of CTR1 and EIN2, but not ETR1 and EIN3 (Fig. 7). Since four ETR1 homologs and five EIN3 homologs are found in Arabidopsis genome (Riechmann et al., 2000Go; Wang et al., 2002Go), AtEBP expression may be directed by these genes. The accumulation of AtEBP mRNA in eto3-1 was almost equaled to that in wild type, because eto3-1 mutant was overproducing only at the seedling stage (Woeste et al., 1999Go). One-month-old mature plants were used in that experiment, and the amount of ethylene may not be overproduced in eto3-1.

ERF1, a key factor of the ethylene signaling pathway, is known to be located the downstream of EIN3 (Fig. 7). Solano et al. (1998)Go showed that transgenic Arabidopsis overexpressing EIN3 expressed ERF1 mRNA highly, supporting the position of ERF1 being a direct target of EIN3. Transgenic Arabidopsis overexpressing ERF1 showed constitutive ethylene responses such as inhibition of hypocotyl elongation, while transgenic plants overexpressing AtEBP did not show such phenotype (data not shown). Thus, AtEBP and ERF1 may be distinctly separate from each other.

In conclusion, we demonstrated in this study that AtEBP conferred the resistance to Bax-induced cell death in tobacco plants and the resistance to exogenous treatments such as H2O2 and heat in plant cells. Further studies are planned to dissect the molecular regulation controlled by AtEBP and to clear the link between the physiological and developmental phenotype.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
LITERATURE CITED
 

Plant Materials

The Columbia ecotype of Arabidopsis (Arabidopsis thaliana), and tobacco (Nicotiana tabacum) cv Petit Havana SR1 and cv Samsun NN were used. All plants were cultivated in growth chambers at 23°C (Arabidopsis) and 27°C (tobacco) under continuous light. The ethylene-related mutants, etr1-1, ein2-1, ein3-1, and eto3-1, were obtained from the Arabidopsis Biological Resource Center (Columbus, OH).

Suspension-cultured cells of tobacco BY-2 were cultured weekly in liquid Murashige and Skoog (1962)Go medium supplemented with Suc (30 g/L), KH2PO4 (200 mg/L), thiamine hydrochloride (1 mg/L), and 2,4-dichlorophenoxyacetic acid (0.2 mg/L) as described previously (Nagata et al., 1992Go). To induce cell death by H2O2, 10-d-old tobacco BY-2 cells were treated with H2O2 (0, 2, and 5 mM) and cultured under continuous shaking at 27°C for 4 h. The viability of BY-2 cells was measured by the addition of 0.05% Evans blue (Nakalai, Kyoto) for 15 min, followed by extensive wash with distilled water to remove unbound dye. Dye bound to dead cells was solubilized with 50% methanol and 1% SDS for 30 min at 50°C, and quantified by A600. To induce cell death by heat treatment, 10-d-old tobacco BY-2 cells were treated with 55°C for 2 h and returned to 27°C for 12 h. The percentage of shrunken cells was calculated by observations of the cells under a microscope with cytoplasmic shrinkage.


Transformation of Arabidopsis, Tobacco Plants, and Tobacco BY-2 Cells

The open reading frame of AtEBP was inserted into plasmids, pBIN (CaMV 35S) and pBIG (CaMV 35S-{Omega}), and the open reading frame of mouse Bax was inserted into the DEX-inducible vector pTA7002 as described previously (Kawai-Yamada et al., 2001Go). The resulting plasmid, pBIN-AtEBP, was transformed into Arabidopsis plants (Bechthold et al., 1993Go) and tobacco BY-2 cells (Shaul et al., 1996Go) by Agrobacterium-mediated transformation. The plasmids, pBIG-AtEBP and pTA-Bax, were transformed into tobacco cv Petite Havana SR1 as described by Gallois and Marinho (1995)Go. Transgenic tobacco (cv Samsun NN) plants, NsERF2 pro-GUS, NsERF3 pro-GUS, and NsERF4 pro-GUS (Kitajima et al., 2000Go) were also used.


Northern-Blot Analysis

Plant tissues were homogenized with liquid nitrogen in the extraction buffer (200 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl, 0.1% SDS, and 0.1% mercapthoethanol). Total RNAs (10 µg) were fractionated on 1.2% agarose gel containing 5% formaldehyde, and transferred to a nylon membrane (Biodyne B, Pall, Washington, NY). The 32P-labeled probes, the 3'-untranslated region (UTR) were used for PR-1 (Metzler et al., 1991Go), PR-2 (Uknes et al., 1992Go), PR-3 (Samac et al., 1990Go), PDF1.2 (Penninckx et al., 1996Go), and GST6 (Chen et al., 1996Go). The full-length coding region was used for AtEBP in the experiments using BY-2 cells, shown in Figure 2A, and tobacco plants, and the coding region of AtEBP (Büttner and Singh, 1997Go), except for AP2/EREBP domain, was used for the other experiments using Arabidopsis plants.

Hybridization was performed in 10% dextran sulfate solution containing 1 M NaCl, 1% SDS, and 10 µg/mL heat-denatured salmon sperm DNA at 65°C for overnight. Washing was performed with 2x SSC for 10 min, with 1x SSC with 0.1% SDS at 65°C for 30 min, and with 0.1x SSC with 0.1% SDS at 65°C for 30 min. The membranes were analyzed by a BAS1500 imaging plate scanner (Fuji Photo Film, Tokyo).


Western-Blot Analysis

Plant tissues were homogenized in the extraction buffer (50 mM HEPES, pH 8.0, 2 mM EDTA, 330 mM sorbitol, and 0.8% mercaptoethanol). Total proteins (20 µg per lane) separated by SDS-15% PAGE were transferred onto a polyvinylidene fluoride membrane (Milipore, Belford, MA). After blocking with phosphate-buffered saline buffer (137 mM NaCl, 8.10 mM Na2HPO4, 2.68 mM KCl, and 1.47 mM KH2PO4) containing 5% skim milk for overnight at 4°C. The membranes were treated with a polyclonal antibody for Bax (09–499, Upstate Biotechnology, Lake Placid, NY), followed by treatment with horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000 dilution; Amersham Pharmacia Biotech, Piscataway, NJ). Detection was accomplished by an enhanced chemiluminescence kit (Amersham Pharmacia) with exposure to x-ray film (Fuji Photo Film).


Analysis of Ion Leakage

Three leaf discs from 2-week-old tobacco plants were floated on 2 mL water with different concentrations (0, 0.1, and 1.0 µM) of DEX. Following incubation at 27°C, the conductivity of the bathing solution was measured with an electrical conductivity meter (B-173, Hitachi, Tokyo).


Histochemical Detection of O2– and H2O2

Histochemical detection of O2– and H2O2 was performed by treating leaves with NBT, as described by Rao and Davis (1999)Go, and with DAB, as described by Thordal-Christensen et al. (1997)Go. At 7 h after treatment with 1 µM DEX, leaves detached from seedlings were used. For NBT staining, samples were vacuum infiltrated with 10 mM NaN3 in 10 mM potassium phosphate buffer, pH 7.8, for 2 min, and incubated in 0.1% NBT in 10 mM potassium phosphate buffer, pH 7.8, for 30 min at room temperature. For DAB staining, samples were incubated with 0.1% DAB in 10 mM potassium phosphate buffer, pH 7.8 for 6 h. These stained samples were cleared by boiling in acetic acid:glycerol:ethanol (1:3:3, [v/v/v]) solution before photographs were taken.


Transient Expression Assay

To express GFP-AtEBP fusion protein in plant cells, AtEBP was inserted into the GFP cassette vector, pUC18-35S-GFP (Niwa et al., 1999Go) at the downstream of GFP in frame. The resulting plasmid (35S-GFP-AtEBP, 0.8 µg) was transfected into the onion epidermis with the bombardment method by described by Ohta et al. (2000)Go. After 24 h of incubation, GFP fluorescence was observed using a fluorescence microscopy with a filter set providing 455 to 490 nm excitation and emission above 515 nm.

In cotransfection assays, the reporter plasmid (1.6 µg) and the effecter plasmid (1.2 µg) were bombarded into Arabidopsis leaves in each experiment. The pUC18 was used to adjust the total amount of bombarded DNA. LUC assay was performed with the Dual-Luciferase Reporter Assay System and a luminescence reader (TD-20/20; Promega, Madison, WI). To normalize values after each transfection, 0.4 µg of the plasmid pPTRL, which included LUC from Renilla under the control of the CaMV 35S promoter, was used as an internal control (Fujimoto et al., 2000Go; Ohta et al., 2001Go). Normalized LUC activity recorded after transfection with the reporter plasmid alone was set arbitrarily at 1.


GUS Assay

GUS assay was performed by using the substrate, 4-methylumbelliferyl-{beta}-D-glucuronide (Jefferson et al., 1987Go). Briefly, leaves were homogenized in the extraction buffer (50 mM phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% sarcosyl, and 10 mM mercapthoethanol). After the crude solution was incubated with 4-methylumbelliferyl-{beta}-D-glucuronide at 37°C for 30 min, the reaction was stopped by the addition of Na2CO3 solution. The amount of 4-methylumbelliferone, the resulting product, was measured with a fluorescence spectrophotometer (F4500, Hitachi) with excitation at 365 nm and emission at 455 nm.


Distribution of Materials

Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. H. Shinshi for providing us with the plasmid containing the tobacco mosaic virus enhancer.

Received March 30, 2005; returned for revision March 30, 2005; accepted April 13, 2005.


   FOOTNOTES
 
1 This work was supported by Research for the Future from the Japan Society for the Promotion of Science. Back

2 Present address: Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki Sakyo-ku, Kyoto 606–8585, Japan. Back

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.063586.

* Corresponding author; e-mail uchimiya{at}iam.u-tokyo.ac.jp; fax 81–3–5841–8466.


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