Genome-Wide Analysis of the Almond AP2/ERF Superfamily and Its Functional Prediction during Dormancy in Response to Freezing Stress

Abstract

The AP2/ERF transcription factor family is one of the largest transcription factor families in plants and plays an important role in regulating plant growth and development and the response to biotic and abiotic stresses. However, there is no report on the AP2/ERF gene family in almond (Prunus dulcis). In this study, a total of 136 PdAP2/ERF genes were identified from the almond genome, and their protein physicochemical properties were analyzed. The PdAP2/ERF members were divided into five subgroups: AP2, RAV, ERF, DREB, and Soloist. The PdAP2/ERF members in each subgroup had conserved motif types and exon/intron numbers. PdAP2/ERFS members are distributed on eight chromosomes, with 22 pairs of segmental duplications and 28 pairs of tandem duplications. We further explored the colinear relationship between almond and Arabidopsis thaliana, Oryza sativa, Malus domestica, and Prunus persicaAP2/ERF genes and their evolution. The results of cis-acting elements showed that PdAP2/ERF members are widely involved in various processes, such as growth and development, hormone regulation, and stress response. The results based on transcriptome expression patterns showed that PdAP2/ERF genes had significant tissue-specific expression characteristics and were involved in the response of annual dormant branches of almond to low-temperature freezing stress. In addition, the fluorescence quantitative relative expression results of 13 representative PdAP2/ERF genes in four tissues of 'Wanfeng' almond and under six low-temperature freezing treatments of annual dormant branches were consistent with the transcriptome results. It is worth noting that the fluorescence quantitative expression level showed that the PdERF24 gene was extremely significant at -30 °C, suggesting that this gene may play an important role in the response of almond dormancy to ultralow temperature freezing stress. Finally, we identified 7424 and 6971 target genes based on AP2 and ERF/DREB DNA-binding sites, respectively. The GO and KEGG enrichment results showed that these target genes play important roles in protein function and multiple pathways. In summary, we conducted bioinformatics and expression pattern studies on PdAP2/ERF genes, including 13 PdAP2/ERF genes, and performed fluorescence quantitative analysis of annual dormant shoots under different low-temperature freezing stress treatments to understand the tolerance of almond dormancy to freezing stress and suggest future improvements.

Keywords: AP2/ERF gene family; Prunus dulcis; evolutionary analyses; expression patterns; freezing stress.

Figure 1

Phylogenetic tree of AP2/ERF family members in almond and Arabidopsis. Each color block represents a group. The area between the two green segments represents the ERF subgroup; the area between two purple segments represents DREB subgroup.

Figure 2

Phylogenetic clustering of PdAP2/ERF members based on motifs and gene structures. (A) Neighbor-joining phylogenetic tree of members of the PdAP2/ERF family. Different colored areas represent different groups. (B) Conserved protein motifs. (C) AP2 domains. (D) Gene structures. (E) Motif LOGO. The ruler at the bottom of B and C figures represents the length of amino acids. The ruler at the bottom of D figure represents the number of nucleotides (base pair: bp).

Figure 3

Chromosome localization of almond PdAP2/ERF family members. The green lines show tandem duplicated genes. The scale on the left shows the chromosome length information, and the chromosome names are listed on the right. The blue regions in the chromosome segments represent low gene density, and the red regions represent high gene density.

Figure 4

Interchromosomal relationships of the PdAP2/ERF genes in the almond genome. The red lines link the segmentally duplicated PdAP2/ERF gene pairs.

Figure 5

Synteny analysis of AP2/ERF family members in almond compared to those from four other plant species. (A) Prunus dulcis vs. Arabidopsis thaliana, (B) Prunus dulcis vs. Oryza sativa, (C) Prunus dulcis vs. Malus domestica, and (D) Prunus dulcis vs. Prunus persica. Each horizontal line represents a chromosome, and the red lines represent collinear genes. Eight chromosomes of Prunus dulcis are represented by red segments. Five chromosomes of Arabidopsis thaliana are represented by green segments. Twelve chromosomes of Oryza sativa are represented by yellow segments. Seventeen chromosomes of Malus domestica are represented by light pink segments. Eight chromosomes of Prunus persica are represented by pink fragments. The number represents the corresponding chromosome name.

Figure 6

Cis-acting elements in the promoter regions of PdAP2/ERF family members. Each color square represents a cis-element type.

Figure 7

Heatmap of the expression patterns of PdAP2/ERF gene family members. (A): Heatmap of expression patterns in different tissues. (B): Expression heatmap of annual dormant branches under freezing stress at different temperatures. The ROW normalization method was used to draw the heatmap. Red squares indicate upregulation of expression, black squares indicate no expression, and green squares indicate downregulated expression.

Figure 8

Fluorescence quantitative relative expression of 13 representative PdAP2/ERF genes in leaves, branches, flesh, and cores of ‘Wanfeng’ almond. The ordinate represents the relative expression level of genes, and the abscissa represents leaves, branches, flesh, and cores from left to right.

Figure 9

Fluorescence quantitative relative expression of 13 representative PdAP2/ERF genes under six temperature gradient freezing stress treatments in the annual dormant branches of ‘Wanfeng’ almond. CK, T1, T2, T3, T4, and T5 represent −5, −10, −15, −20, −25, and −30 °C, respectively. The ordinate represents the relative expression level of genes, and the abscissa represents CK, T1, T2, T3, T4, and T5 from left to right.

Figure 10

Protein–protein interaction network of PdAP2/ERF members. Red gene names represent central genes. The black dashed line represents the interaction between different proteins.

Figure 11

Top 20 significantly enriched GO terms for PdAP2/ERF-interacting proteins. (A): Molecular Function. (B): Biological Process.

Figure 12

The top 20 significantly enriched GO terms for target genes. (A): Top 20 significantly enriched GO terms for AP2 target genes. (B): Top 20 significantly enriched GO terms for ERF/DREB target genes.