We isolated genes encoding CE1 element binding factors (CEBFs) employing a yeast one-hybrid system. CEBFs belong to the AP2/ERF superfamily of transcription factors [18
]. The AP2/ERF proteins are classified into three families: AP2, ERF and RAV. Whereas AP2 and RAV family members possess an additional AP2 or B3 DNA-binding domain, ERF family members possess a single AP2/ERF domain. The ERF family is further divided into two subgroups, the DREB/CBF subfamily (group A) and the ERF subfamily (group B) [19
]. Among the 52 positive clones we analyzed, 39 encoded B group proteins (i.e., B-3 subfamily members), whereas 13 encoded A group proteins (i.e., A-6 subfamily members) (Table ).
The in vitro
binding sites of many AP2/ERF superfamily proteins have been studied in detail. The DRE core sequence, i.e., the binding site for DREB1A and DREB2A, which are representative members of the DREB/CBF subfamily, is A/GCCGAC [19
]. The GCC box core sequence, which is the consensus binding site for ERF family members, is AGCCGCC [24
]. Thus, the two sequences share the CCGNC consensus sequence, the central G being essential for high affinity binding. On the other hand, the core sequence of the CE1 element is CCACC, which differs from the DRE and the GCC box core sequences. The results of our one-hybrid screen indicate that a subset of AP2/ERF family members (i.e., at least ten B-3/B-2 subgroup members and three A-6 subgroup proteins) bind the CE1 element in yeast.
Several of the CEBFs have been reported as GCC box binding proteins. For example, the preferred in vitro
binding site of AtERF1, AtERF2 and AtERF5 is the wild type GCC box, AGCCGCC [25
]. Mutations of the Gs at the second and fifth positions reduced their binding activity to less than 10% of that obtained with the wild type sequence. Similarly, the mutation of the second G of the core sequence greatly reduced the in vitro
binding of RAP2.4 [26
]. However, in our one-hybrid screen, AtERF1, AtERF5 and RAP2.4 were isolated as multiple isolates (i.e., 4, 5 and 9 isolates, respectively). The result suggests that these proteins can interact with the non-GCC box sequence, CCACC, under physiological conditions (i.e. in yeast).
AP2/ERF proteins are involved in various cellular processes, including biotic and abiotic stress responses [18
]. Many DREB/CBF family proteins (e.g., DREB1A, DREB1B, DREB1C, DREB2A, RAP2.1 and RAP2.4) are involved in ABA-independent abiotic stress responses [19
], whereas ERF family members (e.g., ERF1, ORA59, AtERF2, AtERF4, AtERF14, and RAP2.3) are generally involved in ethylene and pathogen defense responses [18
]. In particular, several of the AP2/ERF proteins are involved in ABA response. ABI4, which belongs to the DREB/CBF subfamily, is a positive regulator of ABA and sugar responses [35
]. DREB2C and maize DBF1 are also positive regulators of ABA response [36
]. On the other hand, AtERF7 [38
], ABR1 [39
] and AtERF4 [34
] are ERF subfamily proteins that are negative regulators of ABA response.
To determine the in vivo
functions of CEBFs in ABA response, we generated their OX lines and acquired knockout lines for phenotype analysis when available. As mentioned above, several CEBFs (i.e., ERF1, AtERF2 and ORA59) are known to regulate defense responses. However, their involvement in ABA response and the functions of other CEBFs have not been reported yet. Here, we present our results obtained with CEBFs, AtERF13 and RAP2.4L. AtERF13 was found to possess very high transcriptional activity in yeast (Figure ) and localized in the nucleus. Its expression was limited to the shoot meristem region and young emerging leaves (Figure ), implying that it may play a role in shoot growth or development. Consistent with this notion, AtERF13 OX lines exhibited minor dwarfism (Figure ). The growth retardation observed in the OX lines may reflect the normal inhibitory role of AtERF13 or be the result of its ectopic overexpression. However, we think that AtERF13 probably play a role in growth regulation. Because we could not obtain its knockout lines, we prepared and analyzed its RNAi lines. Our results showed that the RNAi lines grew faster than wild type plants (Additional file 3
), suggesting that AtERF13 may inhibit seedling growth.
Overexpression of AtERF13 conferred ABA hypersensitivity during postgermination growth. As shown in Figure both shoot and root growth was severely inhibited by the low concentration of ABA, which had little effect on wild type seedling growth. Additionally, the AtERF13 OX lines were hypersensitive to glucose, whose effect is mediated by ABA. We did not carry out extensive expression analysis of ABA-responsive genes in AtERF13 OX lines. However, our limited target gene analysis showed that expression of several ABA-responsive genes was affected by AtERF13 (Figure ). Thus, our results strongly suggest that AtERF13 may be involved in ABA response. As mentioned in the Results, we did not observe distinct phenotypes with AtERF13 RNAi lines except faster seedling growth, presumably because of the functional redundancy among CEBFs.
In the case of RAP2.4L, we did not observe changes in ABA sensitivity in its OX lines, although we observed up-regulation of several ABA-responsive genes (Figure ). However, the transgenic lines were glucose-hypersensitive, suggesting that it may be involved in sugar response (Figure ). We also analyzed its knockout lines, but did not observe distinct phenotypes (not shown). RAP2.4 is the closest homologue of RAP2.4L; therefore, we also analyzed its OX and knockout phenotypes. We did not observe alterations in ABA response in either the OX or the knockout lines of RAP2.4 (not shown). The results are consistent with those observed by Lin et al. [26
], who reported that RAP2.4 is involved in light, ethylene and ABA-independent drought tolerance but not in ABA response. However, similar to RAP2.4L OX lines, RAP2.4 OX lines were glucose-sensitive and both RAP2.4 and RAP2.4L OX lines were salt-sensitive (Figure ). Additionally, single or double knockout lines of RAP2.4 and RAP2.4L grew faster than wild type plants (Additional file 3
), suggesting their role in seedling growth control.
It is not known whether other CEBFs are involved in ABA response. Another important question to be addressed is the mechanism of their function, if they are involved in ABA response. CE1 constitutes an ABA response complex with the G-box type ABRE and functions in combination with ABRE. Therefore, CEBFs are expected to interact with the transcription factors ABFs/AREBs, which mediate ABA response in seedlings via the G-box type ABRE. In the case of DREB2C, which binds another coupling element DRE, its physical interaction with ABFs/AREBs has been demonstrated [37
]. It would be worthwhile to determine whether CEBFs can physically interact with ABFs/AREBs. As described before, several CEBFs mediate plant defense response. Thus, our results raise an interesting possibility that CE1 may be a converging point of ABA and defense responses.