Origination, Expansion, Evolutionary Trajectory, and Expression Bias of AP2/ERF Superfamily in Brassica napus

Abstract

The AP2/ERF superfamily, one of the most important transcription factor families, plays crucial roles in response to biotic and abiotic stresses. So far, a comprehensive evolutionary inference of its origination and expansion has not been available. Here, we identified 515 AP2/ERF genes in B. napus, a neo-tetraploid forming ~7500 years ago, and found that 82.14% of them were duplicated in the tetraploidization. A prominent subgenome bias was revealed in gene expression, tissue-specific, and gene conversion. Moreover, a large-scale analysis across plants and alga suggested that this superfamily could have been originated from AP2 family, expanding to form other families (ERF, and RAV). This process was accompanied by duplicating and/or alternative deleting AP2 domain, intragenic domain sequence conversion, and/or by acquiring other domains, resulting in copy number variations, alternatively contributing to functional innovation. We found that significant positive selection occurred at certain critical nodes during the evolution of land plants, possibly responding to changing environment. In conclusion, the present research revealed origination, functional innovation, and evolutionary trajectory of the AP2/ERF superfamily, contributing to understanding their roles in plant stress tolerance.

Keywords: AP2/ERF superfamily; B. napus; RNA-seq; polyploid; positive selection; stress tolerance.

Figure 1

The number and classification of AP2/ERF genes in B. napus and other 14 species used in this study. Information regarding genome duplication or triplication was obtained from the Plant Genome Duplication Database (PGDD). The phenotypic picture for each species was obtained from the Phytozome.

Figure 2

The orthologous, paralogous, and cluster analyses of the AP2/ERF genes. (A) The number of orthologous genes identified between B. napus and other 14 species. The ratio indicated that the one gene in species (A. thaliana, B. oleracea, or B. rapa) has the one or more orthologous genes with B. napus. (B) The number of paralogous genes identified in B. napus and other 14 species. The ratio indicated that the one gene has the one or more orthologous genes in each species. (C) The Venn diagram shows the number of common and specific clusters and AP2/ERF genes in five Green alga species. The first number in the brackets represents the number of cluster, and the second number represents the number of genes. (D) The Venn diagram shows the number of common and specific clusters and AP2/ERF genes in eight angiosperms species. (E) The Venn diagram shows the number of common and specific clusters and AP2/ERF genes in B. napus and other 14 species used in this study. The abbreviations represent the species as follows: Bna, B. napus; Bra, B. rapa; Bol, B. oleracea; Ath, A. thaliana; Ptr, P. trichocarpa; Vvi, V. vinifera; Osa, O. sativa; Atr, A. trichopoda; Smo, S. moellendorffii; Ppa, P. patens; Cre, C. reinhardtii; Vca, V. carteri; Csu, C. subellipsoidea C-169; Mpu, M. pusilla RCC299; Olu, O. lucimarinus.

Figure 3

The three-dimensional histogram of the duplicated type number for AP2/ERF genes and all genes in the whole genome of B. napus and other 14 species.

Figure 4

The identification of collinearity block and Ks analyses between B. napus and other species. (A) The summary of collinearity blocks and AP2/ERF genes number between B. napus and other related species. (B) The circle plot of Ks values for all syntenic superfamily AP2/ERF genes in B. napus. (C) The density of Ks values for syntenic AP2/ERF gene pairs of each species. (D) The density of Ks values for syntenic AP2/ERF gene pairs between B. napus and other related species.

Figure 5

The phylogenetic relationship and positive selection analyses of AP2/ERF superfamily. (A) Phylogenetic tree constructed using neighbor-joining method by MEGA6, using the AP2 domain sequences of AP2/ERF genes in B. napus and Arabidopsis. The numbers are bootstrap values based on 1000 iterations. Only bootstrap values with >50% supports are indicated. (B) The positive selection analyses among DREB subfamily (I), ERF subfamily (VII), and AP2 families in representative species. The ω on the clades is dn/ds value under M8 of codeml, which represent the results of positive selection analyses.

Figure 6

The phylogenetic relationship and evolutionary trajectories of AP2 family in plants. (A) Phylogenetic tree constructed using neighbor-joining method by MEGA6, using AP2 domain sequences of AP2 family genes in higher (Green branches) and lower (Red branches) plants. (B) The major evolutionary trajectories of AP2 family. The R1 to R7 represent the AP2 domains from 5′ to 3′ of the AP2 family genes. The blue X indicated the AP2 domain was lost in the genes.

Figure 7

The expression analyses for AP2/ERF genes in B. napus. The expression values were calculated by RPKM (Reads Per Kilobase per Million mapped reads). (A) The circle plot of the expression values for all AP2/ERF genes in B. napus. The red and green lines indicated the AP2/ERF gene expression for three replicates in leaf and root, respectively. (B) The Heat map representation and hierarchical clustering of AP2 genes in root and leaf. The expression values were log2 transformed.

Figure 8

The expression comparative analyses for AP2/ERF genes in B. napus. (A) The comparative of AP2/ERF gene expression between leaf and root in AA-subgenome. (B) The comparative of AP2/ERF gene expression between leaf and root in CC-subgenome. (C) The comparative of AP2/ERF gene expression between AA-subgenome and CC-subgenome in leaf. (D) The comparative of AP2/ERF gene expression between AA-subgenome and CC-subgenome in root. ^*P < 0.05; ^**P < 0.01.