The AP2/ERF superfamily is one of the largest groups of transcription factors in plants [1
]. It includes all genes coding for at least one APETALA2 (AP2) domain and can be further separated into the ETHYLENE RESPONSE FACTOR (ERF), the AP2, and the RAV families. The AP2 domain, which is responsible for DNA binding, was named after the Arabidopsis thaliana
APETALA2 protein identified by Jofuku et al
]. Subsequently, tobacco (Nicotiana tabacum
) transcription factors containing a motif related to the AP2 domain were also identified [3
]. Since these proteins are able to bind an ethylene responsive DNA element (AGCCGCC), they were classified as ERFs (Ethylene Responsive Factors) and their DNA binding domain was named after them [4
The subdivision of the AP2/ERF group into families is based on the number of AP2 domains present in the proteins, together with the presence of other DNA binding domains. The AP2 family contains proteins with a double, tandem repeated, AP2 domain [5
]. The ERF family genes code for transcription factors with a single AP2 domain. Lastly, the RAV family encodes proteins possessing a single AP2 domain plus an additional B3 domain, which is also present in other, non-ERF transcription factors [6
Two major efforts to define a nomenclature for ERF sequences have been published. Sakuma et al.
] divided the Arabidopsis
ERF family into two subfamilies based on the amino acid sequence of the DNA binding domain: the CBF/DREB subfamily (group A) and the ERF-like subfamily (group B), both further subdivided in six groups. Nakano et al
] on the other hand, proposed an innovative classification for Arabidopsis
ERFs, based on the amino acid sequence of the whole proteins, splitting the family into ten groups.
Despite the relatively high sequence conservation of the AP2/ERF domain, the number of DNA elements bound by different AP2/ERF transcription factors is extremely wide [[3
] and [10
The DREB group interacts with the core sequence CCGAC [11
], while the ERF group typically binds to an AGCCGCC sequence, called GCC box [3
In the AP2 family, a binding element gCAC(A/G)N(A/T)TcCC(a/g)ANG(c/t) has been reported for the protein AINTEGUMENTA (ANT) [12
], and the same sequence was shown to be bound by five other AP2 members [13
]. This DNA element is quite distinct from the consensus sequence CCGA/CC bound by the DREB/ERF group and, surprisingly, is not composed of two similar half sites, since the AP2 proteins contain a double AP2 domain [14
AP2/ERF transcription factors regulate a number of biological processes including development, reproduction, responses to hormones, adaptation to biotic and abiotic stresses [15
] and [19
After the release of the whole genomic sequences of several plant organisms, including Arabidopsis, rice (Oryza sativa)
and poplar (Populus tricocarpa
), the AP2/ERF transcription factor superfamily was analyzed, both to place each member in an organized nomenclature system, and to provide maps of their expression. To date, two full genome sequences of highly homozygous and heterozygous grapevine (Vitis vinifera
) Pinot noir genotypes have been carried out [[20
] and [22
]]. These two milestones provided a useful genomic platform to study this fruit crop. As AP2/ERF transcription factors are involved in flower development and tolerance to biotic and abiotic stresses, their superfamily represents one of the best pools to investigate, when searching for important grapevine traits. Since genome scale analyses of the transcriptional response to the development and environmental stimuli require a precise and complete annotation in order to provide reliable and exhaustive data, we decided to annotate the ERF family members and create a qRT-PCR platform that allows investigating their expression profile.
A recent study suggested that 132 genes encoding AP2/ERF proteins are present in the grapevine genome [23
]. However, a higher number of AP2/ERF genes is present in Arabidopsis and poplar genomes (147 and 202 sequences, respectively). We therefore re-screened the grapevine genome for AP2/ERF sequences, adopting two different strategies, in order to accurately identify AP2/ERF-like sequences in the Vitis vinifera
genome. In this study we provide a characterization of the grapevine AP2/ERF transcription factor superfamily and demonstrate that it is in fact composed of almost 149 genes. Moreover, using qRT-PCR platform encompassing the whole ERF/AP2 superfamily, we show how AP2/ERF-like genes are expressed in both vegetative and reproductive tissues at different developmental stages, and we infer roles and putative targets for some of these genes. Overall, our analysis suggests that AP2/ERF proteins play a strong role in ripening-related processes.