AOBPreview originally published online on January 4, 2007
Annals of Botany 2007 99(2):239-244; doi:10.1093/aob/mcl265
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Mutual Regulation of Arabidopsis thaliana Ethylene-responsive Element Binding Protein and a Plant Floral Homeotic Gene, APETALA2
1 Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2 Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan
3 Japan Science and Technology Agency (JST), CREST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
* For correspondence. E-mail mkawai{at}iam.u-tokyo.ac.jp
Received: 29 August 2006 Returned for revision: 5 October 2006 Accepted: 31 October 2006 Published electronically: 4 January 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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BACKGROUND AND AIMS: It has previously been shown that Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) contributed to resistance to abiotic stresses. Interestingly, it has also been reported that expression of ethylene-responsive factor (ERF) genes including AtEBP were regulated by the activity of APETALA2 (AP2), a floral homeotic factor. AP2 is known to regulate expression of several floral-specific homeotic genes such as AGAMOUS. The aim of this study was to clarify the relationship between AP2 and AtEBP in gene expression.
METHODS: Northern blot analysis was performed on ap2 mutants, ethylene-related Arabidopsis mutants and transgenic Arabidopsis plants over-expressing AtEBP, and a T-DNA insertional mutant of AtEBP. Phenotypic analysis of these plants was performed.
KEY RESULTS: Expression levels of ERF genes such as AtEBP and AtERF1 were increased in ap2 mutants. Over-expression of AtEBP caused upregulation of AP2 expression in leaves. AP2 expression was suppressed by the null-function of ethylene-insensitive2 (EIN2), although AP2 expression was not affected by ethylene treatment. Loss of AtEBP function slightly reduced the average number of stamens.
CONCLUSIONS: AP2 and AtEBP are mutually regulated in terms of gene expression. AP2 expression was affected by EIN2 but was not regulated by ethylene treatment.
Key words: APETALA2, Arabidopsis thaliana, AtEBP, ERF, EIN2, EIN3
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Co-ordinated regulation of gene expression is an essential biological event, especially when each transcriptional factor acts as a key regulator. In Arabidopsis, the floral meristem produces four concentric whorls of floral organs (sepals, petals, stamens and carpels). According to the ‘ABC’ model for the determination of floral organ identity, A activity specifies sepals, A and B activities lead to petals, B and C activities lead to stamens, and C activity specifies carpels (Weigel and Meyerowitz, 1994).
The APETALA2 (AP2) gene, which belongs to the A class of genes, exhibits several characteristics distinct from other ABC genes. Although most ABC genes contain a MADS domain, AP2 contains two APETALA2/ethylene-responsive element binding protein (AP2/EREBP) domains (Jofuku et al., 1994). The AP2 transcript is not observed in a region-specific pattern in the four wholes of flower, and is detected in other vegetative tissues (Jofuku et al., 1994; Okamuro et al., 1997). Recent reports showed that AP2 controlled seed mass (Jofuku et al., 1994, 2005; Ohto et al., 2005) and that expression of the AP2 protein was translationally regulated by the microRNA mi172 (Aukerman and Sakai, 2003; Chen, 2004). Thus, AP2 may play an important role in both floral and whole-plant development.
AP2 belongs to the AP2/EREBP family, one of the largest groups of plant transcriptional factors (Riechmann et al., 2000). It is known that AP2 suppresses expression of AGAMOUS, the C gene of a floral homeotic gene (Drews et al., 1991; Bomblies et al., 1999). In addition, AP2 regulates the expression of ethylene-responsive factor (ERF) genes containing one AP2/EREBP domain (Okamuro et al., 1997). However, the relationship of the transcriptional regulation between AP2 and ERF genes is not fully understood.
Previously, we characterized Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), one of the ERF genes, as a transcriptional activator (Ogawa et al., 2005). AtEBP is regulated by an ethylene signal (Büttner and Singh, 1997; Ogawa et al., 2005). It was clarified that AtEBP is regulated by EIN2, but not EIN3, suggesting that AtEBP expression is independently regulated under EIN3 in ethylene signalling. Interestingly, it was reported that AtEBP expression was regulated by AP2 (Okamuro et al., 1997). Nevertheless, relationships between AtEBP and AP2 in ethylene signal transduction have not been investigated in detail.
Here, we show that AP2 regulates ERF genes such as AtEBP and AtERF1, and the over-expression of AtEBP causes the accumulation of AP2 transcripts. The regulation of AP2 in ethylene signalling and the functional role of AtEBP in floral development are also demonstrated.
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MATERIALS AND METHODS |
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Plant materials
The ‘Columbia’ ecotype of Arabidopsis thaliana was used. All plants were cultivated in growth chambers at 23 °C under continuous light. Ethylene-related mutants, etr1-1, ein2-1, ein3-1 and ctr1-1, and a floral homeotic mutant, ap2-5, were obtained from the Arabidopsis Biological Resource Center (Columbus, OH, USA). Transgenic Arabidopsis plants over-expressing AtEBP were obtained as described previously (Ogawa et al., 2005).
A knockout plant of AtEBP with a T-DNA insert was obtained from the Torrey Mesa Research Institute, USA. The knockout plants were selected on Murashige–Skoog medium containing 2·4 µg mL–1 glufosinate ammonium. The T3 generation of the homozygous plants confirmed that T-DNA was inserted in the ORF of AtEBP by use of genomic PCR amplification analysis.
Northern blot analysis
Plant tissues were homogenized with liquid nitrogen in the extraction buffer [200 mM Tris–HCl (pH 8·0), 10 mM ethylenediaminetetraacetic acid, 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). With regard to the 32P-labelled probes, the 3'-untranslated region was used for AtERF1 (Fujimoto et al., 2000) and the C-terminals of the coding region, except the AP2/EREBP domain which was used for AP2 and AtEBP (Jofuku et al., 1994; Büttner and Singh, 1997).
Hybridization was performed in 10 % dextran sulfate solution containing 1M NaCl, 1 % SDS and 10 µg mL–1 heat-denatured salmon sperm DNA at 65 °C for overnight. Washing was performed with 2x SSC for 10 min, with 1x SSC containing 0·1 % SDS at 65 °C for 30 min and 0·1x SSC containing 0·1 % SDS at 65 °C for 30 min. The membranes were analysed using a BAS1500 imaging plate scanner (Fuji Film, Tokyo, Japan).
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RESULTS |
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Effects of AP2 on AtEBP expression
It has been reported that expression of the ERF genes including AtEBP is regulated by AP2 activity (Okamuro et al., 1997). This observation lead to the investigation of the transcriptional regulation of AP2/EREBP domain-containing genes in the current study. To investigate the relationships in the transcriptional regulation of AP2 and AtEBP, an analysis was made of the expression patterns of AP2 and ERF genes such as AtEBP and AtERF1 in different tissues (flowers, stems and leaves). To avoid cross-hybridization among AP2, AtEBP and AtERF1, each specific probe was used for Northern blot hybridization (Fig. 1). In the wild type (WT), the AtEBP mRNA level was high in leaves and low in flowers, while AP2 mRNA levels were low in all the tissues analysed (flowers, stems and leaves). There is a point-mutation in the AP2/EREBP domain of AP2 (residue Gly-159 to Glu) in the ap2-5 mutant, which leads to reduced transcriptional activity of AP2 (Jofuku et al., 1994). As a result, the floral homeotic phenotype was observed. To test whether AP2 activity affects the AtEBP expression, we investigated mRNA accumulation of AtEBP in ap2-5. In ap2-5, the AtEBP mRNA level was increased in flowers, leaves and stems. The AP2 mRNA level of ap2-5 was also increased, especially in flowers and leaves, suggesting that AP2 activity also suppresses its own AP2 gene expression. The expression pattern of AtERF1 was similar to that of AtEBP: the AtERF1 mRNA level of ap2-5 increased in flowers, leaves and stems compared with the WT. mRNA accumulation of these genes was examined in ap2-7, and similar results were obtained (data not shown). These results may suggest that AP2 represses the expression of ERF genes such as AtEBP, AtERF1 and the AP2 gene itself.
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Over-expression of AtEBP and upregulation of AP2 expression
In our previous work, it was demonstrated that AtEBP acts as a transcriptional activator (Ogawa et al., 2005). In fact, over-expression of AtEBP in Arabidopsis resulted in the upregulation of plant defence genes such as PDF1·2 and GST6. To determine the effects of AtEBP on the regulation of AP2 expression, mRNA accumulation of AP2 and AtERF1 was investigated in leaves of Arabidopsis plants over-expressing AtEBP. As shown in Fig. 2, the AP2 mRNA level was increased in Arabidopsis over-expressing AtEBP. The AtERF1 mRNA level was also increased in these lines. These results suggest that AtEBP upregulates the expression of AP2 and AtERF1 genes directly or indirectly.
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Analysis of the mRNA level of AP2 and other genes in ethylene mutants
AtEBP expression is regulated in the ethylene signalling pathway (Büttner and Singh, 1997; Ogawa et al., 2005). Since over-expression of AtEBP caused upregulation of AP2, it was of interest to test whether AP2 expression was also controlled through the ethylene signalling pathway. mRNA accumulation of AP2 was investigated in ethylene-related Arabidopsis mutants: ethylene resistant 1–1 (etr1-1), ethylene insensitive 2-1 (ein2-1) and ethylene insensitive 3–1 (ein3-1) mutants, which were isolated as ethylene-insensitive, and constitutive triple response 1-1 (ctr1-1), which was isolated as a constitutive active mutant in the ethylene signalling pathway. The results showed that low-levels of AP2 and AtERF1 mRNAs were detected in ein2-1 (Fig. 3); however, mRNA accumulation of these genes was not changed in ctr1-1 compared with the WT.
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AP2 expression after ethephone treatment was also analysed (Fig. 4). AtEBP expression was increased in WT and the ein3-1 mutant after ethephone treatment, suggesting that AtEBP expression was independent of the transcriptional control of EIN3. This result is consistent with our previous work (Ogawa et al., 2005). In contrast, ERF1 expression was increased in WT after ethephone treatment but not in ein3-1. It is known that ERF1 expression is transcriptionally controlled by functional EIN3 (Solano et al., 1998). AP2 expression was not changed by ethephone treatment.
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Floral phenotype in an AtEBP knockout plant
In order to understand the effect of AtEBP on plant development, a mutant line with a T-DNA insert in the AtEBP gene was analysed (Fig. 5A), and the AtEBP transcript was found to be lower in these plants (Fig. 5B). As shown in Fig. 6, the number of stamens in the AtEBP knockout plant was reduced compared with the WT. Five or four stamens were frequently observed in AtEBP knockout plants (approx. 20 % in 150 flowers in three independent experiments; Fig. 6E, F), although six stamens were observed in the WT and the vector control line (Fig. 6A, B). Such a phenotype was not observed in the AtEBP over-expression lines (Fig. 6C, D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Recent studies have shown that AP2 plays a global role not only in floral development but also in the control of seed mass (Jofuku et al., 1994, 2005; Okamuro et al., 1997; Ohto et al., 2005). In addition, AP2 expression is controlled transcriptionally and translationally in a co-ordinated manner. In particular, micro RNAs are thought to target mRNAs of AP2 and its homologs, thereby inhibiting the translation process (Aukerman and Sakai, 2003; Chen, 2004). However, the transcriptional regulation of AP2 has not been well understood (Okamuro et al., 1997).
The current study showed that AP2 activity repressed AtEBP, AtERF1 and AP2 expression. This is consistent with previous results showing that AP2 regulates its own AP2 expression (Okamuro et al., 1997; Chen, 2004) as well as other genes, such as the ERF genes.
In addition, over-expression of AtEBP increased the expression level of AP2. AtEBP is a transcriptional activator interacting with GCC-box, an ethylene-responsive element (Büttner and Singh, 1997). Although the over-expression of AtEBP up-regulated AP2 and AtERF1 expression, these promoters (
2·0 kb upstream from ATG) did not contain the GCC-box. Interestingly, analysis of tomato ERF Pti4 interacting with GCC-box revealed that Pti4 bound to promoters in the absence of GCC-box (Chakravarthy et al., 2003). Like Pti4, transcriptional regulation of the target genes of AtEBP may be complex.
Down-regulation of AP2 was observed in ein2-1. The null mutation of EIN2 resulted in a complete loss of responsiveness to ethylene, suggesting that EIN2 is essential in the ethylene signal pathway. However, AP2 expression was not induced by ethylene treatment or in ctr1-1, indicating that EIN2 is a receiver for various signals. It is known that EIN2 receives not only ethylene but also other signals, such as paraquat and jasmonic acid (Alonso et al., 1999). The N-terminal of EIN2 is thought to be necessary for ethylene responsiveness. On the other hand, the C-terminal of EIN2 is required for transducing the signal to the downstream components (Wang et al., 2002). Our observations suggested that the AP2 expression was induced via EIN2 but not by the ethylene signal (Fig. 7).
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The AP2 mRNA level did not change in the ein3-1 mutant. The position of EIN3 is a branch of the ethylene signalling pathway under EIN2. It is known that the sensitivity of ein3 mutants to ethylene is weaker than ein2 mutants (Wang et al., 2002). Previous studies reported that both EIN3-dependent and independent pathways exist downstream of EIN2 (Binder et al., 2004; Seifert et al., 2004). Furthermore, AtEBP expression is independently regulated under EIN3 in ethylene signalling (Ogawa et al., 2005; this study). In this study, AP2 was not induced by ethylene despite increasing expression of AtEBP. We suggest that these signal transductions compete with one another.
AtEBP knockout plants exhibited a weak floral phenotype with a lower number of stamens. An evaluation was also made of AtERF1 and AP2 expression in AtEBP knockout plants having the same level of WT (data not shown). The ctr1 mutants showed an earlier-maturing phenotype in the gyneocium compared with the flower, and ein mutants affect the maturation of the gyneocium (Kieber et al., 1993). Interestingly, the ant mutants show a similar phenotype to the AtEBP knockout plants (Elliott et al., 1996; Klucher et al., 1996). ANT is a member of the AP2/ERF family containing the AP2/EREBP domains.
Over-expression of AtEBP caused up-regulation of AP2 in leaves. Despite the accumulation of AP2 mRNA in transgenic Arabidopsis plants over-expressing AtEBP, no abnormal flowers were observed. Chen (2004) reported that micro RNAs control transcriptional regulation of AP2 expression. That is, most transgenic Arabidopsis plants over-expressing AP2 had normal flowers and only a fraction exhibited the agamous-like phenotype. However, over-expression of AP2 mutated at the target site of micro RNAs demonstrated a more severe floral phenotype. Accumulation of AP2 protein was detected only in transgenic plants over-expressing mutated AP2, not in normal AP2. Thus, we consider that accumulation of AP2 mRNA in Arabidopsis over-expressing AtEBP is not sufficient to change flower development.
This study has shown the mutual relationships between AP2 and AtEBP. AtEBP and functional EIN2 affected the transcriptional regulation of AP2. AtEBP contributed slightly to flower development, especially stamen development. Future reports in this series will focus on the homeotic role of AtEBP.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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We thank Dr Minori Uchimiya for editing the manuscript. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan, and CREST, JST, Japan.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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