APETALA2/ethylene responsive factors (AP2/ERF) are unique regulators in the plant kingdom and are involved in the whole life activity processes such as development, ripening, and biotic and abiotic stresses. In tomato (Solanum lycopersicum), there are 140 AP2/ERF genes; however, their functionality remains poorly understood. In this work, the 14th and 19th amino acid differences in the AP2 domain were used to distinguish DREB and ERF subfamily members. Even when the AP2 domain of 68 ERF proteins from 20 plant species and motifs in tomato DREB and ERF proteins were compared, the binding ability of DREB and ERF proteins with DRE/CRT and/or GCC boxes remained unknown. During fruit development and ripening, the expressions of 13 DREB and 19 ERF subfamily genes showed some regular changes, and the promoters of most genes had ARF, DRE/CRT, and/or GCC boxes. This suggests that these genes directly or indirectly respond to IAA and/or ethylene (ET) signals during fruit development and ripening. Moreover, some of these may feedback regulate IAA or ET biosynthesis. In addition, 16 EAR motif-containing ERF genes in tomato were expressed in many organs and their total transcripts per million (TPM) values exceeded those of other ERF genes in most organs. To determine whether the EAR motif in EAR motif-containing ERF proteins has repression function, their EAR motifs were retained or deleted in a yeast one-hybrid (YIH) assay. The results indicate that most of EAR motif-containing ERF proteins lost repression activity after deleting the EAR motif. Moreover, some of these were expressed during ripening. Thus, these EAR motif-containing ERF proteins play vital roles in balancing the regulatory functions of other ERF proteins by completing the DRE/CRT and/or GCC box sites of target genes to ensure normal growth and development in tomato.

Background: APETALA2/ethylene responsive factors (AP2/ERFs) are unique regulators found in the plant kingdom that are involved in all life activity processes, including flowering, fruit ripening, floral meristem growth, and defense responses. In tomato (Solanum lycopersicum), there are 60 DREB and 80 ERF subfamily members, however, their functionality remains poorly understood.Results: In this work, the AP2 domain conserved amino acid sequences of 68 ERF proteins from 20 plant species were compared and a Multiple Em for Motif Elicitation (MEME) analysis was conducted. Results revealed that the 9th amino acid of the AP2 domain exhibited marked characteristics during the selection of DRE/CRT and/or GCC boxes as protein binding sites. Moreover, motifs near the AP2 domain may be involved in protein binding to DNA, whereas motifs far away from the AP2 domain may function as a part of the transactivation domain. Furthermore, we compared the expression levels of all ERF genes in 30 tomato organs and under biotic and abiotic stresses. Results indicated that most of 17 ERF and DREB repressor genes were highly expressed in almost all tomato organs and under some biotic and abiotic stress. The transcripts per million (TPM) value ratios of all repressor genes exceeded that of all activator genes in 16 tomato organs. Thus, it can be inferred that these repressor genes play vital roles in balancing the regulatory functions of activator genes and activator genes may also conversely compete with repressor genes to ensure normal growth, development, and defense responses in tomato.Conclusions: This work uncovered the potential functions of all ERF and DREB genes that regulate tomato growth, development, and defense responses, and considers the binding ability of the AP2 domain unique sequences with DRE/CRT and GCC boxes, as well as the relationship of unique motifs with the transactivation domain. These findings will expand upon our understanding of the functions of ERF and DREB genes in tomato.