Characterization of the AP2/ERF Transcription Factor Family and Expression Profiling of DREB Subfamily under Cold and Osmotic Stresses in Ammopiptanthus nanus

Abstract

APETALA2/ethylene-responsive factor (AP2/ERF) is one of the largest transcription factor (TF) families in plants, which play important roles in regulating plant growth, development, and response to environmental stresses. Ammopiptanthus nanus, an unusual evergreen broad-leaved shrub in the arid region in the northern temperate zone, demonstrates a strong tolerance to low temperature and drought stresses, and AP2/ERF transcription factors may contribute to the stress tolerance of A. nanus. In the current study, 174 AP2/ERF family members were identified from the A. nanus genome, and they were divided into five subfamilies, including 92 ERF members, 55 dehydration-responsive element binding (DREB) members, 24 AP2 members, 2 RAV members, and 1 Soloist member. Compared with the other leguminous plants, A. nanus has more members of the DREB subfamily and the B1 group of the ERF subfamily, and gene expansion in the AP2/ERF family is primarily driven by tandem and segmental duplications. Promoter analysis showed that many stress-related cis-acting elements existed in promoter regions of the DREB genes, implying that MYB, ICE1, and WRKY transcription factors regulate the expression of DREB genes in A. nanus. Expression profiling revealed that the majority of DREB members were responsive to osmotic and cold stresses, and several DREB genes such as EVM0023336.1 and EVM0013392.1 were highly induced by cold stress, which may play important roles in cold response in A. nanus. This study provided important data for understanding the evolution and functions of AP2/ERF and DREB transcription factors in A. nanus.

Keywords: AP2/ERF; Ammopiptanthus nanus; cold stress; gene family; osmotic stress; transcription factor.

Figure 1

The number of APETALA2/ethylene-responsive factor (AP2/ERF) family members in Ammopiptanthus nanus and some other plant species.

Figure 2

Phylogenetic analysis of AP2/ERF superfamily members in A. nanus.

Figure 3

Phylogenetic analysis of dehydration-responsive element binding (DREB) subfamily members in A. nanus and Arabidopsis thaliana.

Figure 4

Phylogenetic analysis of ERF subfamily members in A. nanus and A. thaliana.

Figure 5

Distribution of the AP2/ERF family genes on the A. nanus chromosomes. The black vertical bars to the right of the gene names indicate tandem duplication.

Figure 6

Representative motif compositions and gene structures of the AP2/ERF family members in A. nanus. One or two members from Soloist, RAV, and AP2 subfamilies and one member from each ERF and DREB subfamilies were used to show the typical motif compositions and gene structures of members in the subfamily or the group. (A) Conserved motif analysis of the AnAP2/ERF family transcription factors. All motifs were identified using the MEME database. (B) Gene structure of AnAP2/ERF family members. Exons and introns are represented by yellow boxes and black lines, respectively, and the untranslated region (UTR) is shown in green.

Figure 7

Distribution of segmentally duplicated AnAP2/ERF genes on A. nanus chromosomes. Paralogous pairs in the DREB subfamily are shown in blue, those of the ERF subfamily are shown in red, those of the AP2 subfamily are shown in orange, and those of the RAV subfamily are shown in green.

Figure 8

Ks and Ka/Ks value distributions of the AP2/ERF family genes in the genomes of A. nanus and Glycine max. Distribution of Ks and Ka/Ks values were calculated from the paralogous gene pairs in the A. nanus genome (A and B), and the orthologous gene pairs between the A. nanus and G. max genomes (C and D). Four orthologous gene pairs with Ks > 2.5 were not used for preparing the above figure.

Figure 9

Cis-acting element prediction in the promoter region of the DREB subfamily genes in A. nanus. The color of the square depicts the quantity of the predicted cis-acting elements in the promoter region.

Figure 10

Expression profiles of the DREB subfamily genes calculated using RNA-seq datasets. Transcript per million TPM values of all AnDREB subfamily genes in A. nanus leaves under normal conditions (A); fold change values of all AnDREB subfamily genes exposed to delayed short-term osmotic stress or cold stress (7d) in A. nanus leaves (B). The color scale represents low expression with blue and high expression with red.

Figure 11

Expression profiles of 32 DREB subfamily genes under 3 h, 6 h, 12 h, and 24 h of osmotic and cold treatment in A. nanus leaves. Gene expression levels were quantified through quantitative reverse transcriptase PCR (qRT-PCR) analysis and the experimental values were normalized using eIF1 as the reference gene.