Identification and multiple sequence alignment of CaERF proteins in pepper
A total of 142 ERF genes were obtained from the Capsicum annuum genome after excluding redundant sequences, the candidates containing an AP2 plus a B3 domain, and candidates containing more than two AP2 domains (Table S3). The 142 candidate genes were renamed consecutively according to the chromosomal positions (Table S3; Fig. S2). In addition, all identified ERF members encoded 44-672 residues. The molecular weight (Mw) of each CaERF protein ranged from 7.19 kDa to 74.91 kDa, and the theoretical pI varied from 4.24 to 11.10. Most of these proteins were unstable, and only fifteen CaERF proteins were stable (instability index<40) (Table S3).
Before phylogeny analysis was performed, multiple alignment analyses were performed using the amino acid sequences of the AP2 domains. The classification of all identified CaERFs is shown in Fig. 2, as described later. The alignment analyses indicated that the DREB subfamily possesses a specific WLG motif that is a completely conserved residue (Fig. 1A; Fig. S3), while more than 95% of members in the ERF subfamily had a WLGT motif for the ERF subfamily except for groups X and XI (Fig. 1B; Fig. S3). The DREB subfamily was completely conserved in V15 and E20, and more than 95% of the members of groups V to IX in the ERF subfamily contained A14 and D19 (Fig. 1AB; Fig. S3). The shaded residues shown for 37 DREB subfamily members indicate complete conservation in the AP2 domain (Fig. 1A; Fig. S3). However, the alignment revealed that the N-terminal regions of the AP2 domains in the ERF subfamily possessed a high homology, while those of the C-terminal regions showed very low conservation (Fig. 1B; Fig. S3). Moreover, groups X and XI possessed very low conservation in the 15th and 20th amino acids, and there was difficulty in in classifying these residues. Nevertheless, taking into account the topology of the tree in Fig. 2, groups X and XI were preliminarily classified as the ‘ERF-like subfamily’.
Phylogenetic analysis of the ERF family in four plant species
To clarify the phylogenetic relationships, an unrooted phylogenetic tree was constructed for all of the identified CaERF sequences based on their alignment with those in Arabidopsis by a neighbour-joining phylogenetic analysis. As shown in Fig. 2, based on the classification of AtERF in Nakano’s and Sakuma’s studies [15, 16], putative CaERF proteins were divided into two large subfamilies that corresponded to the DREB and ERF subfamilies (Fig. 2; Fig. S4). According to the cited studies [15] and taking into account the topology of the tree, the two subfamilies were further defined as 11 groups named group I to XI (Table 1; Fig. S4).
Notably, some differences existed in groups IX and X, which were then subdivided into IXa, IXb, Xa and Xb, because the members of groups IXb and Xb were only found in peppers. Additionally, the members of group XI were present only in pepper as well, whereas group V-Like (V-L) were absent in pepper (Table 1). These results indicated that the members of IXb, Xb and XI might be pepper-specific groups. To determine whether these three groups were specific to peppers, all CaERF genes were used to construct a neighbour-joining phylogenetic tree with those from tomato (137), rice (138) and Arabidopsis (Fig. S5). The topology of the phylogeny was mostly similar to that tree obtained when using only protein sequences from pepper and Arabidopsis (Fig. S4). The number of ERF proteins in each group is listed in Table 1. Groups IXb and Xb contained a significantly higher number of ERF TFs from peppers. In contrast, the members of ERF members in group XI included rice and tomato, and no significant differences were observed in other investigated species (Table 1). Therefore, groups IXb and Xb were designated as putative ‘pepper-specific groups’ (Fig. 2).
Table. 1. Summary of each group of ERF families in four plant species.
Subfamily
|
Group
|
Pepper
|
Arabidopsis
|
Rice
|
Tomato
|
DREB
|
37
|
57
|
54
|
43
|
|
I
|
5
|
10
|
9
|
7
|
|
II
|
8
|
15
|
9
|
7
|
|
III
|
5
|
23
|
13
|
7
|
|
IV
|
19
|
9
|
23
|
22
|
ERF
|
107
|
65
|
84
|
94
|
|
V
|
11
|
5
|
13
|
12
|
|
VI
|
5
|
8
|
2
|
4
|
|
VII
|
7
|
5
|
9
|
5
|
|
VIII
|
31
|
15
|
19
|
35
|
|
IXa
|
13
|
17
|
20
|
15
|
|
IXb
|
5
|
0
|
1
|
0
|
|
Xa
|
11
|
8
|
8
|
13
|
|
Xb
|
12
|
0
|
0
|
2
|
|
V-L
|
0
|
3
|
0
|
0
|
|
VI-L
|
2
|
4
|
1
|
4
|
|
XI
|
8
|
0
|
11
|
4
|
|
Total
|
142
|
122
|
138
|
137
|
To evaluate the biological functions of the CaERF protein of the groups, the functional characteristics of ERF from Arabidopsis, tomato and pepper were investigated in the literature. As shown in Table S4, the members of the same group possessed similar biological functions, and group VIII members were found to be likely involved in alkaloid biosynthesis. Because of the importance of capsaicinoids and capsorubin in pepper, the possibility of the Capsicum annuum genome (version 2.0) containing putative ERF homologs involved in secondary metabolites was investigated. A previous study demonstrated that Erf and Jerf in peppers were involved in the regulation of the pungency phenotype [31]. Erf and Jerf were mapped to CaERF53 and CaERF101 in the Capsicum annuum genome (version 2.0), respectively. (Table S5). Moreover, CaERF101 was identified as the putative orthologue of both CaPF1 and JERF1, and it was shown to be associated not only with the regulation of polyamine biosynthesis but also with ABA biosynthesis (Table S5). It was likely that the members of group VII which contained CaERF53 and CaERF101, were related to secondary metabolite biosynthesis.
Conserved motif analysis of CaERF
Conserved amino acid motifs represent functional areas maintained during the evolutionary process. The conserved motifs within the 142 CaERF sequences were analysed and compared using MEME. A total of 15 significantly conserved motifs (E-value < 10-32) possessing 11-41 residues were identified and named motif 1 to motif 15 (Table S6). Five conserved amino acid motifs, motif 1 to motif 5, were found to be located in the AP2 domain region, which were present in the majority of CaERF proteins and designated as “general motifs” (Fig. 3); however, both motif 2 and motif 5 were mainly shared within group VIII in the ERF subfamily (Fig. 3B). The remaining motifs (motif 6 to motif 15) were distributed outside of the AP2 domain and were classified as “specific motifs”. Motif 9 and motif 12 were primarily restricted to group IV in the DREB subfamily (Fig. 3A). Motif 10 and motif 11 were specifically contained in group VIII. Motifs 6 and 13 were found in group X, and motif 14 was in group V (Fig. 3B). Further, motif 15 was specifically present in group VII. Nevertheless, the same group of trees harboured similar motif patterns (Fig. 3).
Expression patterns of CaERFs in different developmental stages of pericarp and placenta
Expression patterns imply a biological function for genes. Capsorubin and capsaicinoids are specifically biosynthesized in pepper fruit, and their accumulation pattern is regulated through developmental stages. To gain further insight into the hypothetical roles of CaERFs during the capsorubin and capsaicinoids biosynthesis processes, the expression patterns of CaERFs and the genes involved in synthesis in the pericarp and placenta (including 6, 16, 25, 36, 38, 43, and 48 DPA stages) were investigated (Fig. 4). RNA-Seq raw data were retrieved from a public database [13] and all of the reads were remapped to the Capsicum annuum genome (version 2.0). The expression of the relevant capsorubin synthesis gene gradually increased at 36 DPA, and capsorubin itself primarily accumulated at this stage (Fig. 4A, cluster C). A total of 48 CaERF (33%) transcripts were expressed at a level that could not be detected. Based on similar expression patterns, the expression patterns of CaERF in the pericarp were hierarchically clustered, and divided into 10 clusters (Fig. 4B). The expression of members of cluster C9 and cluster C10 was in agreement with the transcriptional trend of relevant-capsorubin synthetic genes. Although the expression of members of cluster C10 gradually decreased after 43 DPA, the genes involved in the synthesis of capsorubin (i.e., CaPDS and CaLCYB) were also gradually expressed after this stage. This result indicated that these ERF TFs may regulate different genes involved in capsorubin biosynthesis. Thus, the members of cluster C9 and cluster C10 were candidates for the regulation of capsorubin biosynthesis.
The expression of genes involved in capsaicinoids synthesis tended to rapidly increase from 6 DPA to 25 DPA, and then they gradually decreased, which was consistent with abundant production of capsaicinoids at stages from 13 DPA to 25 DPA (Fig. 4C). A total of 38 CaERFs (26%) were expressed at a level that could not be detected in any of the developmental stages of the placenta. The placenta-expressed genes were hierarchically clustered based on similar expression patterns, yielding 10 clusters (Fig. 4D). Generally, CaERF in the same phylogenetic group revealed distinct expression. In the ten clusters, only the expression of members in cluster L3 and cluster L4 exhibited good agreement with the stages of abundant-capsaicinoids accumulation. However, the expression of cluster L3 members (CaERF85, CaERF101, CaERF65 and CaERF73) was high at 6 DPA, and then it was not detected at other stages, with the exception of CaERF116 and CaERF102. The transcript level of cluster L4 members (CaERF111, CaERF92, CaERF28, CaERF53, CaERF103, CaERF114, CaERF25 and CaERF139) increased from 6 DPA to 36 DPA, but levels slightly decreased at 25 DPA. A previous study demonstrated that CaERF53 and CaERF101 were related to capsaicinoids biosynthesis [31]. However, CaERF101 was included in cluster L5, and the members of this cluster exhibited increased transcript levels at 38 DPA. Therefore, the members of cluster L3 and cluster L4 were represent novel candidates for the regulation of capsaicinoids biosynthesis.
Additionally, the members of two putative ‘pepper-specific groups’ (IXb and Xb) were barely expressed during all of the developmental pericarp and placenta stages, with the exception of CaERF67, CaERF73, CaERF127, and CaERF129, which exhibited low expression in group IXb. The biological function of members of groups IXb and Xb might involve in capsorubin and capsaicinoids biosynthesis. Capsorubin and capsaicinoids are characteristically synthesized in pericarp and placental tissue, respectively. To further understand whether CaERFs are specifically expressed in different tissues, their expression patterns in the leaf, root, stem, pericarp and placenta were examined. The RNA-Seq raw data of leaves, roots and stems were not uploaded by Kim et al [13]; the RPKM values were published instead, and when they were mapped to the Capsicum annuum genome (version 1.5), the expression of CaERFs clearly exhibited no tissue specificity (Fig. S6).
Expression patterns of CaERFs in fruit pericarp and placenta in different developmental stages
To further determine whether the expression of ERF genes possessed a specific stage in the pericarp and tissue, ten CaERFs from the clusters of possible candidates associated with capsorubin and capsaicinoids biosynthesis that are highly expressed in the pericarp and placenta at different developmental stages, were selected for analysis with perform qRT-PCR experiments. As shown in Fig. 5A, the contents of β-carotene, zeaxanthin and capsorubin started to increasingly accumulate in pericarp tissue at the MG stage, whereas lutein content including the branch of the non-synthetic capsorubin was decreased. The expression of CaERF82, CaERF97, CaERF66, CaERF107 and CaERF101 in pericarp tissue not only maintained a good agreement with the tendency of carotenoids biosynthesis (β-carotene, zeaxanthin and capsorubin), but also it exhibited a lower level of transcription in other tissue (roots, flowers, stems, placentas, leaves and seeds) (Fig. 5B). Thus, it was likely that the members of cluster C9 and cluster C10 were involved in carotenoids biosynthesis.
Validation of capsaicinoids biosynthesis related ERF TFs
The capsaicin and dihydrocapsaicin content significantly increased in placental tissue from 10 DPA to 25 DPA, after which they increased slowly (Fig. 6A). The pattern of expression levels CaERF102, CaERF53, CaERF111 and CaERF92 in placental tissue were similar to the capsaicinoids biosynthesis patterns, while CaERF28 expression did not show a developmental stage-regulated pattern. With the exception of CaERF53, these genes were also highly expressed in certain tissues (Fig. 6B). Additionally, we aimed to obtain a preliminary understanding of whether capsaicinoids biosynthesis was regulated by CaERF genes in pepper to enable adaption to different temperatures. As shown in Fig. 6C, the capsaicin and dihydrocapsaicin content dramatically accumulated with increasing temperature but the capsaicin content at T25 was significantly higher than it was in T33. The expression of CaERF53, CaERF92 and CaERF28 was the highest in T25, which was consistent with the accumulated level of capsaicin, while the expression of CaERF102 and CaERF111 decreased with increasing temperature (Fig. 6D). Therefore, these results indicated that CaERF102, CaERF53, CaERF111 and CaERF92 might be associated with capsaicinoids biosynthesis in pepper, but they perform different functions response to temperature to control capsaicinoids biosynthesis.