The AP2/ERF superfamily is one of the largest TF families in the plant kingdom, and it has been successfully identified and investigated in many plant species of sequenced genomes [47–49]. Although the AP2/ERF superfamily in peppers was reported by Jin et al , they indicated that CaAP2/ERFs might be involved in the response to P. capsici in peppers. Capsorubin and capsaicinoids are unique to Capsicum spp., and they possess high economic and nutritional values. This study put more emphasis on demonstrating the relationship between Capsicum-specific secondary metabolites and the ERF family (the largest branch of the AP2/ERF superfamily). study of the Capsicum genome contributes to understanding the structure of gene families and predicting their biological functions. In this study, a total of 142 non-redundant ERF genes were identified from the Capsicum annuum genome. The ERF family in Arabidopsis (122) , watermelon (120) , rice (143) , Chinese cabbage (248) , cauliflower (146)  and Bryum argenteum (75)  were successfully identified and investigated. These results indicated that the number of ERF genes in different plants was distinct. Additionally, alignment analyses showed that the members of the ERF and DREB subfamilies possessed a specific WLG motif, as observed in the report of Cui et al . The distinction between the ERF and DREB subfamilies is that they can interact with the different motifs. The ERF subfamily typically binds to the GCC-box in the promoter regions, whereas the DREB subfamily is characterized by dehydration-responsive element binding factor containing a core motif of CCGAC [29, 54] According to Nakano and Sakuma’s study [15, 16], this DNA-binding specificity is mainly determined by the 14th and 19th amino acids in the AP2 domain (V14 and E19 for the DREB subfamily but A14 and D19 for the ERF subfamily); however, the DREB subfamily is completely conserved at V15 and E20, and the ERF subfamily is highly conserved at A14 and D19 (Fig. 2).
All CaERF members were used to construct a phylogenetic tree with matched proteins from tomato, rice and Arabidopsis. The classification of the tree was defined and annotated based on the proposed by Nakano et al. , and it ultimately defined 11 groups. This result was similar to that of Jin’s study in peppers , no matter the topology or classification of the tree. However, in this study, both groups X and IX were subdivided, and a new group XI was identified. Group XI showed a very low conservation of certain amino acids, which resulted in difficult classification. They group was classified as the ‘ERF-like subfamily’. It was likely that many gene signature motifs underwent divergent evolution after duplication from a common ancestor. Moreover, groups IXb and Xb were regarded as putative ‘pepper-specific groups’ (Fig. 3), and we cannot completely rule out the possibility that the members of putative “pepper-specific groups” were related to capsorubin and capsaicinoids biosynthesis. However, the members of these TFs were rarely expressed both in the pericarp and placenta throughout different developmental stages. Therefore, it seems that these ‘pepper-specific groups’ are not the master regulators of capsorubin and capsaicinoids biosynthesis.
Numerous studies have indicated that the members of a group in large families of plant TFs generally possess similar conserved amino acid motifs or domains, such as MYB, WAKY, and NAC [55–57]. In most cases, similar amino acid motifs are likely to share a similar function. Motifs 1 to 5, which are mainly located in the AP2 domain region were defined as “general motifs”, (Fig. 4B). Motifs 6 to 15 distributed outside the AP2 domain and were designated as “specific motifs” (Fig. 4B); they are potentially related to nuclear localization and transcription regulation . Some reports suggested that the D(I/V)QAA sequences were regarded as the basic characteristics for the DREB family in cauliflower [48, 59], whereas motif 8 contained these conserved sequences, and it was primarily restricted to groups VI and X of the ERF family (Fig. 4). It was likely because TFs have occurred divergent evolution in different species. Indeed, groups VI and X in the phylogenetic tree were near the branch of the DREB family (Fig. 3).
In some cases, the same phylogenetic subgroup had a similar transcript level , implying that members of the same phylogenetic subgroup might perform similar functions. SlERF6 was involved in the regulation of carotenoids biosynthesis and fruit ripening in tomato (Table S4) , which was located in group VII (Fig. S4). However, in this study, the genes of cluster C9 and cluster C10 were from different groups (except for CaERF101, which was in group VII), and they were regarded as candidates for the regulation of capsorubin biosynthesis. Because their expression patterns exhibited good agreement with the transcriptional level of the capsorubin synthesis gene (Fig. 5AB), and the members of this two pericarp highly expressed cluster (CaERF82, CaERF97, CaERF66, CaERF107 and CaERF101) maintained good agreement with the increase in carotenoids biosynthesis (β-carotene, zeaxanthin and capsorubin) in pericarp tissue (Fig. 6B). These results indicated that the genes of the same phylogenetic subgroup exhibited distinct expression patterns, which is consistent with the observation from a previous study [60, 61]. Moreover, previous studies have demonstrated that CaERF101 is involved in multiple secondary metabolic pathway and phytohormone, such as pungent capsaicinoids, polyamine and ABA biosynthesis [31, 42, 44]. Thus, it is likely that CaERF101 also regulates secondary metabolic pathways in the ripening pericarp, and the members of cluster C9 and cluster C10 are involved in carotenoids biosynthesis.
Erf and Jerf in the pepper have been proposed to be involved in accumulation of pungency , and they were mapped to CaERF53 and CaERF101, respectively, in this study. CaERF101 was identified as the putative orthologue of both CaPF1 and JERF1 in other reports, and it was shown to be associated with polyamine and ABA biosynthesis (Table S5) [42–44]. It is likely that the members of the group containing CaERF53 and CaERF101 (VII) regulates capsaicinoids or secondary metabolite biosynthesis. However, the members of cluster L3 (CaERF102, CaERF85, CaERF101, CaERF65, CaERF73 and CaERF116) and cluster L4 (CaERF111, CaERF92, CaERF28, CaERF53, CaERF103, CaERF114, CaERF25 and CaERF139) were candidates for the regulation of capsaicinoids biosynthesis in placental tissue, and only CaERF53 came from group VII. However, CaERF101 was placed into cluster L5. The expression of these members rapidly increased at 38 DPA, which was not similar to the stages of abundant-capsaicinoids accumulation (Fig. 5C and 5D). These results implied that CaERF101 may perform multiple functions in addition to capsaicinoids biosynthesis. Moreover, the expression of four CaERFs CaERF102, CaERF53, CaERF111 and CaERF92 showed a positive correlation with the level of capsaicinoids biosynthesis (Fig. 7AB). In addition, capsaicinoids biosynthesis is regulated by environmental factors. ERF TF transcription is influenced by different temperatures, and ERF TFs have been shown to enhance plant tolerance to stress by being partially responsible for increasing certain metabolites [45, 62]. For example, overexpression of DREB1A can cause accumulation of monosaccharides, disaccharides, trisaccharides, and sugar alcohols to improve the tolerance to freezing and dehydration stress in transgenic plants . In this study, the placenta significantly accumulated capsaicin and dihydrocapsaicin content following the higher temperature treatment. The expression of CaERF53 and CaERF92 increased, but that of CaERF102 and CaERF111 decreased with increasing temperature (Fig. 7C). Therefore, it may be that the members of cluster L3 and cluster L4 are related to temperature mediated capsaicinoids biosynthesis. However, CaERF111, CaERF92, CaERF102 and CaERF111 might play different roles in the regulation of capsaicinoids biosynthesis when exposed to different temperatures.