The AP2/ERF transcription factors superfamily is one of the largest groups of transcription factors in plants, which includes at least one APETALA2 (AP2) domain. According to the number of AP2 domains, and the presence of other DNA binding domains, AP2/ERF can be divided into the ERF, AP2, RAV and Soloist families. The ERF family encodes proteins with a single AP2 domain, while the AP2 gene family codes for transcription factors with two AP2 domains [1–3]. With the exception of a single AP2 domain, however, there is one additional B3 domain in the RAV gene family. The B3 domain is a DNA-binding domain conserved in other plant specific transcription factors .
To date, two major schemes have been applied to define the ERF family nomenclature. According to DNA binding domain protein sequences, the ERF family was divided into two major subfamilies, the ERF and DREB subfamilies. ERF and DREB were divided into six groups in Arabidopsis. Alternatively, based on AP2/ERF domain amino acid sequences, Arabidopsis and rice ERF families were divided into 12 and 15 respective groups . Similarly, 10 groups were identified in the grape and cucumber ERF family [1, 4].
Despite high sequence conservation in the AP2/ERF domain, each family exhibits different DNA elements. Generally, the ERF subfamily binds to an AGCCGCC sequence, i.e. the GCC box , while the DREB subfamily typically interacts with a CCGAC core sequence . The AP2 family, even with the presence of two AP2 domains, does not bind to the CCGA/CC sequence as in DREB/ERF subfamilies, but binds to the GCAC(A/G)N(A/T)TCCC(A/G)ANG(C/T) element [8, 9]. AP2 family genes are regulated by microRNA (miR172), and can be divided into AP2 and ANT groups [10, 11]. RAV family binds to the CAACA and CACCTG sequence. Such as pepper CARAV1 can recognize and bind to these motifs, and activate the yeast reporter gene .
A variety of AP2/ERF transcription factors have been successfully identified and investigated in some plants, including Arabidopsis, rice [2, 13], grape , poplar (Populus tricocarpa) , wheat (Triticum aestivum) , cucumbers , barley (Hordeum vulgare) , and soybean (Glycine max) . The AP2/ERF transcription factors regulate diverse biological processes in plant function and development, such as hormones, reproduction, cell proliferation, abiotic and biotic stress responses [18, 19].
Commonly, the DREB subfamily is used as viable candidate to enhance crop abiotic stress tolerance. The DREB subfamily exhibits different response patterns under environmental stress, including low-temperature (AtCBF1) , heat (ZmDREB2A, AtDREB1A) [21, 22], osmotic (CkDREB) , drought (OsDREB1) [24, 25], and water-deficit and high-salt stress (CaDREBLP1) . The DREB transcription factors activate multiple dehydration/cold-regulated genes by interacting with DRE/CRT elements (A/GCCGAC), which are present in the RD/COR gene promoters . In addition, several DREB subfamily genes are reportedly positive and negative mediators of ABA and sugar responses, primarily during germination and early seedling stages .
ERF transcription factors are also involved in signal pathways during environmental stress or pathogen and disease-related stimuli. ERF transcription factors directly regulate pathogenesis-related (PR) gene expression by binding DNA with the GCC-box (GCCGCC), such as PR1 to PR5 [6, 28, 29]. ERF transcription factors play an important role in plant development, as well as tolerance to biotic and abiotic stress. ERF transcription factor overexpression has been reported in rice [30, 31], tomato, and tobacco [32, 33], leading to drought and salt tolerant improvements in transgenic plants. Signal molecules, including JA, salicylic acid (SA), ethylene (ET), and abscisic acid (ABA) regulate several important defense-signaling pathways. ERF transcription factors potentially play a role in abiotic and biotic stress in plants, such as drought (SHN1, SHN2 and SHN3), salt (AP37), freezing (TaERF1) [34–36], hypoxic stress (SNORKEL1, SNORKEL2, RAP2.2, AtERF73 and HRE1) [1, 37, 38], cell dedifferentiation (WIND1) , metabolite biosynthesis (LeERF-1, Nud), and trait development (ORC1, ERN and EFD)[40–44]. Most of these ERF transcription factors improve abiotic tolerance in crops without causing undesirable growth phenotypes . However CRL5, an AP2 subfamily in rice, promoted crown root initiation in response to ABA . Moreover, CRL5 affected sepal abscission (BnAP2), plant height (NsAP2), and leaf shape in Brassica napus, water lily, and maize [46–48]. The RAV family was shown to mediate plant defense during abiotic and biotic stress. CaRAV1 overexpression increased tolerance to high salinity and osmotic stress in Arabidopsis, and the B. napus RAV-1-HY15 gene was induced by cold, NaCl, and PEG treatments [49, 50]. These observations emphasize the importance of identifying all AP2/ERF superfamily genes to interpret the mechanisms underlying stress signal transmission, and ultimately manipulate AP2/ERF protein regulation to improve crop stress resistance. ERF-mediated plant defense responses can be better understood by elucidating the signaling pathways involved in defense response regulation.
Chinese cabbage, a member of the genus Brassica, is an important leaf vegetable crop grown worldwide. The Chinese cabbage genome (Chiifu-401-42) was recently sequenced and assembled. Data indicated B. rapa ssp. pekinensis exhibits a close relationship with A. thaliana, and experienced a whole genome triplication since its divergence from Arabidopsis 13 to 17 Mya [51, 52]. The release of the entire Chinese cabbage genome sequence, as well as others, including Arabidopsis, potato, and tomato, provided us an opportunity for comparative genome research on AP2/ERF transcription factors. Characterization of AP2/ERF superfamily genes in B. rapa ssp. pekinensis can serve to clarify the molecular mechanisms responsible for abiotic and biotic stress responses, such as cold, heat, salt, or disease resistance. Subsequently, Brassica varieties with increased tolerance to many adverse environments can be developed using transgenic technology. A recent study reported 62 AP2/ERF superfamily genes using expressed sequence tags (ESTs) in Chinese cabbage . In this study, we systematically and comprehensively describe the AP2/ERF transcription factors in B. rapa ssp. pekinensis through a comparative genome analysis. The objectives of our study were as follows: (i) identify and characterize the AP2/ERF transcription factors in the B. rapa ssp. pekinensis genome; (ii) analyze AP2/ERF transcription factor phylogenetic relationships and orthologous genes between the B. rapa ssp. pekinensis and A. thaliana genome; and (iii) construct AP2/ERF transcription factor interaction networks, and analyze AP2/ERF transcription factor expression patterns through comparative genomics. ESTs were applied in AP2/ERF transcription factor expression analyses.