Phylogenomic Analysis and Functional Characterization of the APETALA2/Ethylene-Responsive Factor Transcription Factor Across Solanaceae

Abstract

The AP2/ERF family constitutes one of the largest groups of transcription factors in the Solanaceae. AP2/ERF contributes to various plant biological processes, including growth, development, and responses to various stresses. The origins and functional diversification of AP2/ERF within the Solanaceae family remain poorly understood, primarily because of the complex interactions between whole-genome duplications (WGDs) and tandem duplications. In this study, a total of 1282 AP2/ERF proteins are identified from 7 Solanaceae genomes. The amplification of AP2/ERF genes was driven not only by WGDs but also by the presence of clusters of tandem duplicated genes. The conservation of synteny across different chromosomes provides compelling evidence for the impact of the WGD event on the distribution pattern of AP2/ERF genes. Distinct expression patterns suggest that the multiple copies of AP2/ERF genes evolved in different functional directions, catalyzing the diversification of roles among the duplicated genes, which was of great significance for the adaptability of Solanaceae. Gene silencing and overexpression assays suggest that ERF-1 members' role in regulating the timing of floral initiation in C. annuum. Our findings provide insights into the genomic origins, duplication events, and function divergence of the Solanaceae AP2/ERF.

Keywords: AP2/ERF; Solanaceae; flowering time; gene duplications; phylogenomics; synteny.

Figure 1

Phylogenetic tree and distribution of the AP2/ERF family in Solanaceae. (A) The maximum-likelihood gene tree was constructed for the AP2/ERF gene family, and the syntenic relationships between these genes were analyzed. Terminal branches represent nine categories, with each connecting line positioned within the circular gene tree indicating significant synteny conservation between gene pairs within subclades. (B) The copy numbers of various AP2/ERF categories across plant species. The number represented the level of enrichment among gene family members. The species names were displayed on the outer ring, accompanied by their corresponding abbreviations as documented in Supplementary Table S1. The identification of AP2/ERF categories was displayed on the inner ring of the figure. The various species and groups are visually distinguished by different colors.

Figure 2

Phylogenetic profiling and synteny relationships of the AP2/ERF homologous genes in Solanaceae. (A) Phylogenomic profile of AP2/ERF syntelogs (syntenic homologous genes) across Solanaceae genomes. Species names are listed at the top of (A) as detailed in Supplementary Table S1. The corresponding phylogenetic tree of species is shown on the top side. Cluster IDs are indicated at the right of the figure. Columns represent syntelog clusters, and the color scale indicates the number of nodes (genes grouped in that cluster) per species. (B,C) Synteny network of the AP2/ERF gene family in Solanaceae species. Clusters from (A) can be divided into large-conserved clusters and lineage-specific clusters. The size of each node corresponds to the number of edges it has (node degree).

Figure 3

Microsynteny relationships of homologous genomic segments for two gene clusters in Solanaceae species. (A) Microsynteny relationships of conserved CBF homologous segments in Solanaceae species. (B) Microsynteny relationships of APE/ERF tandem gene clusters belonging to the ERF-1 subfamily in Solanaceae species. Syntenic relationships at the AP2/ERF loci are shown in Solanaceae species, including Solanum lycopersicum, Capsicum annuum, Capsicum chinense, Capsicum baccatum, Solanum tuberosum, Petunia axillaris, and Ipomoea nil. Gray curves connected the identified syntenic genes, while the annotated genes are represented by rectangles. Genes located on the forward strand are shown in blue and genes located on the reverse strand are shown in green. AP2/ERF genes were denoted by green arrows and connected by red lines.

Figure 4

Expression patterns of AP2/ERF genes in Solanum lycopersicum. Expression profiles of candidate genes in tomato (M82) fruit are presented. (A) Expression patterns of AP2/ERF genes in tomato pericarp. (B) Expression patterns of AP2/ERF genes in tomato septum. (C) Expression patterns of AP2/ERF genes in tomato locular tissue. (D) Expression patterns of AP2/ERF genes in tomato placenta. (E) Expression patterns of AP2/ERF genes in tomato columella. (F) Expression patterns of AP2/ERF genes in tomato seed. The figure shows plots comparing expression levels among nine gene subfamilies, with the central line denoting the mean value. The data have been standardized. A color scale indicating expression levels is displayed on the right. Expression levels are normalized using a log2 scale. The differently colored top box denotes the diverse gene groups to which they belong. DPA, days post anthesis; MG, mature green; Br, breaker; Pk, pink; LR, light red; RR, red ripe.

Figure 5

Expression patterns of AP2/ERF genes in Capsicum annuum. This figure illustrates the expression profiles of candidate genes within pepper tissues from the cultivar Zunla. Differences in expression among nine gene subfamilies are depicted, with the central horizontal line marking the mean expression level. The color scale representing the expression values for (A,B) is located at the top right. The color scale for (C–E) is positioned at the bottom right. The expression levels are normalized using log2.

Figure 6

Phenotypes of flowering time in AP2/ERF-silenced pepper and AP2/ERF-overexpressed Arabidopsis. The phenotype and flowering time of gene-silenced plants are presented in (A,B), while those of gene-overexpressed Arabidopsis are shown in (C,D). Error bars indicate standard deviations (SDs) from three replicates. Data are presented as means ± SD. WT, wide type Arabidopsis; OE, Arabidopsis transgenic lines. Different uppercase letters indicate statistically significant differences from the WT or TRV2:00 group at p ≤ 0.0001, as determined by Tukey’s HSD. Scale bars = 2.5 cm.