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Genome-wide identification and comparative analysis of the AP2/ERF gene family in Prunus dulcis and Prunus tenella: expression of PdAP2/ERF genes under freezing stress during dormancy

BMC Genomics volume 26, Article number: 95 (2025) Cite this article

Abstract

The AP2/ERF (APETALA2/ethylene responsive factor) transcription factor family, one of the largest in plants, plays a crucial role in regulating various biological processes, including plant growth and development, hormone signaling, and stress response. This study identified 114 and 116 AP2/ERF genes in the genomes of 'Wanfeng' almond (Prunus dulcis) and 'Yumin' wild dwarf almond (Prunus tenella), respectively. These genes were categorized into five subfamilies: AP2, DREB, ERF, RAV, and Soloist. The PdAP2/ERF and PtAP2/ERF members both demonstrated high conservation in protein motifs and gene structures. Members of both families were unevenly distributed across eight chromosomes, with 30 and 27 pairs of segmental duplications and 15 and 18 pairs of tandem repeated genes, respectively. The promoter regions of PdAP2/ERF and PtAP2/ERF family members contained numerous important cis-elements related to growth and development, hormone regulation, and stress response. Expression pattern analysis revealed that PdAP2/ERF family members exhibited responsive characteristics under freezing stress at different temperatures in perennial dormant branches. Quantitative fluorescence analysis indicated that PdAP2/ERF genes might be more intensely expressed in the phloem of perennial dormant branches of almond, with the opposite trend observed in the xylem. This study compared the characteristics of PdAP2/ERF and PtAP2/ERF gene family members and initially explored the expression patterns of PdAP2/ERF genes in the phloem and xylem of perennial dormant branches. The findings provide a theoretical foundation for future research on almond improvement and breeding, as well as the molecular mechanisms underlying resistance to freezing stress.

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Introduction

The external environment fluctuates during plant growth and development, often presenting adverse conditions such as biotic and abiotic stresses. These include extreme temperatures, drought, salinity, and exposure to pests and diseases, which can lead to plant damage or mortality [1]. Consequently, plants have evolved diverse mechanisms to mitigate the effects of unfavorable environmental changes. These adaptive strategies encompass metabolic regulation, osmoregulation, signal transduction, hormone regulation, gene expression modulation, and the activation of transcription factors [2, 3]. Transcription factors function as key regulators of gene expression within signaling networks, directly influencing the activation or suppression of target genes. This orchestration modulates the interactions between various gene signaling pathways [4]. The role of transcription factors in regulating plant stress responses has been extensively investigated, with numerous types identified, including WRKY [5], MYB [6], and bHLH [7].

The AP2/ERF transcription factor family represents one of the most extensive families of transcription factors in plants, playing a crucial role in plant responses to biotic and abiotic stresses [8, 9]. The defining characteristic of AP2/ERF transcription factors is the presence of the AP2 structural domain. This highly conserved DNA-binding structure typically comprises 60–70 amino acids and regulates target gene transcription by recognizing specific DNA sequences [10]. Based on variations in the number and sequence of AP2 structural domains, the AP2/ERF family can be categorized into five primary subfamilies: AP2, DREB, ERF, RAV, and Soloist [11]. The regulatory mechanisms of AP2/ERF transcription factors involve interactions with other transcription factors, co-regulators, and hormonal signals, enabling complex regulation of gene expression [12]. These factors not only activate target gene transcription but also function as transcriptional repressors, inhibiting specific gene expression and thus maintaining a balance between plant growth and survival under varying environmental conditions [13]. Given their significant roles in plant stress response and developmental regulation, AP2/ERF transcription factors have emerged as a focal point in contemporary agricultural research [14].

To date, the function of the AP2/ERF gene family has been extensively studied in numerous plants with significant findings. OsDREB2B regulates the expression of OsAP2-39 by binding to its promoter. Additionally, OsDREB2B interacts with OsWRKY21 to regulate gibberellin metabolism gene expression and inhibit gibberellin synthesis, resulting in decreased gibberellin content that negatively affects rice growth and development [15]. Overexpression of OsERF71 enhances rice tolerance to drought stress [16]. Transfection of the wheat TaERF-6-3A gene into Arabidopsis increased the sensitivity of transgenic Arabidopsis plants to drought and salt stress [17]. Overexpression of PtoER15 aids poplar in maintaining stem water potential, thereby improving drought tolerance [18]. EjCBF3, a homolog of PsAP2/ERF28, enhances cold tolerance in loquat by increasing antioxidant enzyme activity [19]. AtCBF2 regulates Arabidopsis thaliana response to cold stress through the ICE-CBF-COR response pathway, enabling cold sensors in the plasma membrane to perceive cold stress [20]. AP2/ERF genes are involved in plant hormone regulation and stress response. OsERF71 can regulate the accumulation of ethylene and abscisic acid, thereby increasing the amount of proline in rice and ultimately improving the ability of rice to resist drought stress [21]. The two genes ZmDREB39 and ZmDREB89 can regulate the accumulation of abscisic acid in maize, thereby improving the drought resistance of maize [22]. In Taxus x media, the expression levels of the three genes TmERF5, TmERF14, and TmERF36 were found to be increased after gibberellin treatment, thereby improving its cold resistance [23]. The OsERF096 gene can resist low temperature stress by regulating the accumulation of auxin in rice [24]. In Arabidopsis, cytokinin response factor 6 (CRF6) is highly expressed in the veins of mature leaves, promotes the synthesis of cytokinins, and is induced by abiotic stress [25]. AP2/ERF genes can improve the ability of plants to resist abiotic stress. OsEREBP1 can bind to the GCC-box element of the PR gene promoter, thereby improving the disease resistance of rice [26]. AtERF1 can regulate the production of ethylene and jasmonic acid, promote the accumulation of phytoalexins, and thus improve the disease resistance of Arabidopsis [27]. Arabidopsis can phosphorylate the two genes ERF6 and ERF72 through the MAPKs pathway, thereby enhancing its disease resistance to Botrytis cinerea [28]. Overexpression of the GmERF113 gene in soybean can promote the increased expression of GmPR1 and GmPR10-1, thereby enhancing resistance to soybean Phytophthora infection [29].

The AP2/ERF gene family usually has multiple copies in the plant genome, and these copies are mainly formed through gene duplication events, including whole genome duplication, segmental duplication, and tandem duplication [30]. For example, in Arabidopsis, members of the AP2/ERF gene family have expanded significantly through whole genome duplication events, forming multiple paralogous genes [31]. These duplicated genes may have undergone functional differentiation during evolution, thus giving plants a wider range of adaptability. In different plants, the duplication and evolution patterns of AP2/ERF genes show certain differences [32]. For example, in medicinal plants, the number and subfamily distribution of AP2/ERF gene family members vary from species to species, which may be closely related to the unique physiological characteristics and medicinal value of medicinal plants [32]. In addition, in plants such as wild strawberries, researchers have found through phylogenetic analysis and gene expression pattern studies that specific AP2/ERF genes play an important role in coping with adverse stresses such as low temperature, and these genes usually have duplication phenomena, further proving the importance of gene duplication in the process of plant adaptation to the environment [33]. In summary, the AP2/ERF gene duplication study not only reveals the evolutionary history of plant genomes, but also provides important clues for understanding the molecular mechanisms of plant growth, development and stress response.

Almond belongs to the genus Prunus of the Rosaceae family and is widely distributed globally. Wild dwarf almond, also belonging to the genus Prunus and family Rosaceae, is a wild almond species. It is a valuable relict of the Mediterranean Tertiary Miocene deciduous forest species in China, protected and recognized as a 'wild plant resource of China' and is often referred to as a 'living fossil of plants'. Although almond and wild dwarf almond are both distributed in Xinjiang, China, there are significant differences in their freezing resistance. The winter temperature in the wild dwarf almond distribution area is around -20℃, while the winter temperature in the almond cultivation area is within -10℃. However, in the almond cultivation area, short-term ultra-low temperature climate phenomena often occur in winter, and the temperature can reach around -25℃, causing a large area of almond trees to freeze and die. The AP2/ERF gene plays an important role in regulating plant response to low temperature stress. In this study, we analyzed the almond and wild dwarf almond genomes for AP2/ERF gene identification and conducted comparative analyses of protein physicochemical properties, phylogeny, conserved motif, gene structure, gene duplication, gene localization, promoter, and expression patterns. Additionally, we selected eight PdAP2/ERF genes for freeze-stress fluorescence quantitative expression analysis. This research provides a foundation for exploring the characteristics of almond and wild dwarf almond AP2/ERF family members. This study conducted bioinformatics analysis on the AP2/ERF family members of almond and wild dwarf almond, and compared the characteristics of the AP2/ERF family members between the two, providing a theoretical basis for subsequent almond molecular breeding.

Material methods

Identification of AP2/ERF family members

We identified AP2/ERF genes through the 'Wanfeng' almond genome (owned by our team) and the 'Yumin' wild dwarf almond genome [34]. The protein data of both genomes were compared against the AP2 domain Hidden Markov Model (PF00847), retaining genes with an e-value less than or equal to e−5 [35]. Additionally, 146 AP2/ERF protein sequences from A. thaliana, obtained from the Uniprot database, were used for BLAST comparison with the 'Wanfeng' almond and 'Yumin' wild dwarf almond genomic protein data. Genes with e-values less than or equal to e−5 were retained [36]. After combining the results of both comparisons and eliminating redundant sequences, 114 and 116 genes were identified in the 'Wanfeng' almond and 'Yumin' wild dwarf almond genomes, respectively. Furthermore, protein physicochemical properties were analyzed using ExPASy (http://web.expasy.org/protparam/), and protein subcellular localization was determined using WoLF PSORT II (https://www.genscript.com/wolf-psort.html?src=leftbar) [37, 38].

Phylogenetic tree construction and classification

Multiple sequence alignment of AP2/ERF protein sequences from three species—almond, wild dwarf almond, and Arabidopsis thaliana—was conducted using ClustalW within the MEGA 7 tool. Subsequently, a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with 1,000 bootstrap replications, employing the Poisson correction model and pairwise deletion [39]. The resulting phylogenetic tree was then categorized into five subgroups: AP2, DREB, ERF, RAV, and Soloist. Finally, the tree was aesthetically refined using the iTOL website [40].

Conservation of Motifs, Structural Domains and Gene Structures

The motif identification of PdAP2/ERF and PtAP2/ERF member protein sequences was conducted using the MEME tool (http://meme-suite.org/tools/meme) with a motif number of 10 and default parameters [41]. The NCBI Conserved Domain Database (CDD) online tool (https://www.ncbi.nlm.nih.gov/cdd/) was employed to search for the location and number of GATA structural domains in CmGATA family member protein sequences [42]. Exon and intron positions and numbers of PdAP2/ERF and PtAP2/ERF genes were analyzed using 'Wanfeng' almond and 'Yumin' wild dwarf almond genome information data. Subsequently, the phylogenetic tree, motif, structural domains, and gene structures of PdABC family members were clustered and mapped utilizing the TBtools software [43].

Gene localization, gene duplications, and Ka/Ks values

The chromosomal and positional distribution information of PdAP2/ERF and PtAP2/ERF genes on the genome was extracted and mapped using the TBtools software. Fragment duplication and tandem duplication gene pairs of PdAP2/ERF and PtAP2/ERF members were identified using the MCScanX tool. Chromosomal covariate distribution circles were then visualized using the Circos tool [44, 45]. Furthermore, AP2/ERF homologous gene pairs were identified in almond, wild dwarf almond, and Arabidopsis. Finally, the KaKs_Calculator tool was employed to calculate Ka (nonsynonymous substitution rate), Ks (synonymous substitution rate), and Ka/Ks values for all types of replicated genes [46].

Cis-acting element annotations

We extracted promoter sequences 2000 bp upstream of PdAP2/ERF and PtAP2/ERF members for cis-acting element annotation using the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [47]. The positional distribution of cis-acting elements was mapped according to three functionally relevant categories previously identified: abiotic and biotic stresses, phytohormone responsiveness, and plant growth and development. Additionally, we quantified all components for comparative analysis.

Freezing stress expression patterns

To analyze the gene expression patterns of PdAP2/ERF members, we utilized FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values derived from transcriptomes of annual dormant branches subjected to six freeze-stress treatments (-5, -10, -15, -20, -25, and -30 ℃). These transcriptomes were previously sequenced by our research team.

Plant material and fluorescence quantification

The annual branches of 'Wanfeng' almond served as the experimental material in this study. The specimens were obtained from the almond resource nursery of the state-owned Erlin factory in Shache County, Xinjiang, China (38°11′N, 77°12′E). In mid-January 2022, three 9-year-old 'Wanfeng' almond trees, exhibiting uniform growth and free from pests and diseases, were selected. Annual dormant branches of similar growth were uniformly chosen from the mid-crown in four directions: south, east, north, and west. The cuttings were sealed with paraffin wax. Dormant annual branches from one tree represented one biological replication, with three trees constituting three biological replications. These samples were subjected to freezing stress at six temperatures: -5, -10, -15, -20, -25, and -30 ℃, respectively, for 24 h. Subsequently, phloem and xylem tissues were extracted from the treated dormant annual branches and stored at -80 ℃ for future analysis.

Quantitative real-time fluorescence polymerase chain reaction (qRT-PCR) was employed to investigate the expression level changes of eight PdAP2/ERF genes in the ligamentous and xylem tissues of annual dormant branches of 'Wanfeng' almond under freezing stress. The experiment was conducted at six temperatures (-5, -10, -15, -20, -25, and -30 °C), with three biological replicates for each sample. The qRT-PCR primers for the selected PdAP2/ERF genes were designed using Primer Premier 5 [48]. We used Actin from a previously published article on almonds for this study [49]. The fluorescence quantification experimental procedure followed the protocol described in a previously published article by our team [49]. The relative expression of the target genes was calculated using the 2−ΔΔCT method (Table S1) [50].

Prediction of target genes and protein interactions

The protein sequences of PdAP2/ERF and PtAP2/ERF family members were uploaded to the STRING database (https://string-db.org/) for node comparisons. Subsequently, the relationship between PdAP2/ERF and PtAP2/ERF members was predicted based on Arabidopsis protein interactions [51].

The A. thaliana DREB1B (CBF) transcription factor binding map was obtained from the JASPAR Plantae database (https://jaspar.elixir.no/search?q=&collection=CORE&tax_group=plants) [52]. Subsequently, 2000 bp promoter sequences of all genes in the almond genome were extracted, and genes bound to DREB1B (CBF) transcription factors were identified using the motif FIMO (https://meme-suite.org/meme/) tool. Target gene structural domain prediction was then conducted based on the PFAM database. Finally, KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) enrichment analyses of target genes were performed using OmicShare Tools (https://www.omicshare.com/tools).

Results

Characterization of PdAP2/ERF and PtAP2/ERF members

We identified 114 and 116 AP2/ERF genes in the genomes of almond and wild dwarf almond, respectively, and renamed these genes according to their location and order on the chromosome. Almond genes were renamed PdAP2/ERF1 to PdAP2/ERF114, and wild dwarf almond genes were renamed PtAP2/ERF1 to PtAP2/ERF116. The physicochemical properties of the proteins differed significantly among the PdAP2/ERF members (Table S1). The amino acid count of PdAP2/ERF members ranged from 138 aa (PdAP2/ERF3) to 871 aa (PdAP2/ERF96), with 64 members having fewer than 299 aa, 39 members between 300 and 499 aa, and 11 members exceeding 500 aa. The protein molecular weights ranged from 15,131.69 Da (Dalton) (PdAP2/ERF3) to 95,101.09 Da (PdAP2/ERF96), with 50 members below 29,999 Da. PdAP2/ERF member proteins exhibited theoretical isoelectric points ranging from 4.54 to 9.99, with 80 member proteins below 7 and 34 above 7. The theoretical isoelectric point of PdAP2/ERF member proteins exhibited a range from 28.86 (PdAP2/ERF1) to 79.52 (PdAP2/ERF4), and the aliphatic index ranged from 43.76 (PdAP2/ERF11) to 77.04 (PdAP2/ERF26). All members were hydrophilic proteins, with most subcellularly localized primarily in the nucleus. Similarly, the physicochemical properties of the PtAP2/ERF members varied considerably (Table S2). The amino acid counts of the PtAP2/ERF members ranged from 138 aa (PtAP2-ERF2) to 1273 aa (PtAP2-ERF26), with 60 members below 299 aa, 36 between 300 and 499 aa, and 20 exceeding 500 aa. Protein molecular weights ranged from 15,259.86 Da (PtAP2/ERF2) to 140,697.38 Da (PtAP2/ERF26), with 52 members below 29,999 Da, 39 between 30,000 Da and 49,999 Da, and 25 above 50,000 Da. The theoretical isoelectric points of PtAP2/ERF member proteins ranged from 4.42 to 9.99, with 76 member proteins below 7 and 40 above 7. The instability index of PtAP2/ERF member proteins ranged from 33.89 (PtAP2/ERF42) to 84.39 (PtAP2/ERF3), and the aliphatic index ranged from 33.5 (PtAP2/ERF10) to 94.96 (PtAP2/ERF75). All members were hydrophilic proteins, predominantly localized in the nucleus. In conclusion, the PdAP2/ERF and PtAP2/ERF member proteins demonstrated a high degree of similarity in their physicochemical properties.

Phylogenetic analysis of PdAP2/ERF and PtAP2/ERF

We constructed neighbor-joining phylogenetic trees of AP2/ERF family members in three species: almond, wild dwarf almond, and A. thaliana. The classification followed the established categorization of A. thaliana AP2/ERF family members into five major subfamilies: AP2, RAV, Soloist, DREB (with six subgroups: A1, A2, A3, A4, A5, and A6), and ERF (with six subgroups: B1, B2, B3, B4, B5, and B6). The analysis revealed that both PdAP2/ERF and PtAP2/ERF have 20 members in the AP2 subfamily, 4 members in the RAV subfamily, and 1 member in the Soloist subfamily. In the DREB subfamily, PdAP2/ERF had 40 members while PtAP2/ERF had 39. The distribution of PdAP2/ERF (PtAP2/ERF) members in DREB subgroups was as follows: 14 (14) in A1, 6 (5) in A2, 1 (1) in A3, 7 (8) in A4, 7 (6) in A5, and 5 (5) in A6. In the ERF subfamily, PdAP2/ERF had 49 members and PtAP2/ERF had 52. The distribution of PdAP2/ERF (PtAP2/ERF) members in ERF subgroups was: 14 (16) in B1, 2 (3) in B2, 14 (14) in B3, 5 (5) in B4, 3 (3) in B5, and 11 (11) in B6. Notably, the number of PdAP2/ERF and PtAP2/ERF members in each subfamily or subgroup was comparable. In summary, the phylogenetic tree clustering results show that PdAP2/ERF and PtAP2/ERF family members have similar numbers in the same subfamily or group, and the AP2/ERF gene sequences of the two are highly conserved, which also indicates that almond and wild dwarf almond are less separated during the evolutionary process Fig. 1.

Fig. 1
figure 1

Neighbor-joining phylogenetic tree of almond, wild dwarf almond, and A. thaliana AP2/ERF family members. The red solid circles on the tree branches represent bootstrap values, with larger circles indicating higher bootstrap values. Different subfamilies or subgroups are denoted by distinct colored regions. The numbers in parentheses indicate the quantity of PtAP2/ERF genes in each group

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PdAP2/ERF and PtAP2/ERF members conserve motifs, structural domains, and gene structure

Using the MEME tool, we identified 10 motifs in the PdAP2/ERF and PtAP2/ERF member protein sequences, with motifs 1, 3, and 8 representing AP2 structural domains. We integrated the phylogenetic trees, motifs, and structural domains of PdAP2/ERF and PtAP2/ERF members to create a comprehensive map (Figure S1A). The analysis revealed (Figure S1B) that protein sequences of RAV subfamily members primarily contained motifs 1, 2, and 3. Soloist subfamily members predominantly included motifs 1, 2, 3, and 5. AP2 subfamily members mainly comprised motifs 1, 2, 3, 4, and 5. DREB subfamily members primarily contained motifs 1, 3, 5, 7, 9, and 10. ERF subfamily members principally included motifs 1, 2, 3, and 5. Furthermore, examination of conserved domains in PdAP2/ERF and PtAP2/ERF members showed that DREB, ERF, RAV, and Soloist subfamilies contained one AP2 domain (except for PtAP2/ERF103, which contained two), while AP2 subfamily members possessed two AP2 structural domains (except PtAP2/ERF45, which contained one) (Figure S1C). PdAP2/ERF and PtAP2/ERF members within the same subfamily exhibited high conservation of exons and introns (Figure S1D). In the RAV subfamily, PtAP2/ERF48 contained 4 exons and 1 intron, while the other 7 members contained only 1 exon. The Soloist subfamily genes contained 1 and 2 exons, and 0 and 1 introns, respectively. The number of exons in the AP2 subfamily ranged from 7 to 11, with introns ranging from 5 to 10. Among DREB subfamily members, PtAP2/ERF26 contained 22 exons and 21 introns, 17 members contained 2 exons and 1 intron, 1 member contained 3 exons and 2 introns, and 53 members contained only 1 exon. Four members of the ERF subfamily had between 7 and 10 exons and between 6 and 9 introns, while most other members had between 1 and 2 exons and between 0 and 1 intron. Figure S1E illustrates the shapes and colors of motifs, structural domains, and gene structures. PdAP2/ERF and PtAP2/ERF members demonstrated a high degree of conservation in motifs, structural domains, and gene structures. In summary, the conserved motifs, domains and gene structures all indicate that PdAP2/ERF and PtAP2/ERF members of the same subfamily are highly conserved, which further verifies the reliability of phylogenetic tree clustering and subfamily division results.

Gene localization and gene duplication analysis of PdAP2/ERF and PtAP2/ERF members

The 114 PdAP2/ERF genes exhibited an uneven distribution across eight chromosomes (Fig. 2A). Specifically, Superscaffold1, Superscaffold2, Superscaffold3, Superscaffold4, Superscaffold5, Superscaffold6, Superscaffold7, and Superscaffold8 contained 28, 14, 14, 4, 20, 16, 12, and 6 PdAP2/ERF genes, respectively. Similarly, 116 PtAP2/ERF genes demonstrated an uneven distribution across eight chromosomes (Fig. 2B). Chr1, Chr2, Chr3, Chr4, Chr5, Chr6, Chr7, and Chr8 harbored 26, 17, 14, 6, 17, 12, 5, and 19 PtAP2/ERF genes, respectively. Furthermore, both PdAP2/ERF and PtAP2/ERF members were predominantly localized within regions of high chromosomal gene density.

Fig. 2
figure 2

Distribution of chromosomal localization of PdAP2/ERF and PtAP2/ERF member genes. A: Distribution of localization on chromosomes of PdAP2/ERF family members. B: Distribution of localization on chromosomes of PtAP2/ERF family members. The left scale bar indicates chromosome length, with both Superscaffold and Chr representing chromosomes. Gene density for each chromosome was calculated using a genetic spacing of 100 kb. A gradient color scheme from blue (low gene density) to red (high gene density) is employed, with blank regions indicating genetic areas lacking gene distribution information. Green connecting lines denote tandemly repeated gene pairs

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To visualize the distribution of fragment duplication gene pairs, we mapped chromosome circles and compiled the data in an information table (Table S3). Our analysis revealed 30 fragment duplication gene pairs within the PdAP2/ERF family members (Fig. 3A and C), alongside 15 tandem duplication gene pairs (Fig. 2A). In the PtAP2/ERF family members, we identified 27 pairs of fragment duplication genes (Fig. 3B and D) and 18 pairs of tandem duplication genes (Fig. 2B).

Fig. 3
figure 3

Circle map illustrating the distribution of fragment replication genes. A Circular representation of PdAP2/ERF family member fragment replication gene pairs. B Tabular information on PdAP2/ERF family member fragment replication gene pairs. Red connecting lines indicate fragment replication gene pairs. C Circular representation of the distribution of fragment replication gene pairs among PtAP2/ERF family members. D Tabular information on fragment replication gene pairs of PtAP2/ERF family members. Red connectors denote segmental replication gene pairs

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We further characterized homologous gene pairs among almond, wild dwarf almond, and A. thaliana AP2/ERF family members (Fig. 4). The analysis identified 101 homologous gene pairs between almond and A. thaliana, 99 between wild dwarf almond and A. thaliana, and 140 between almond and wild dwarf almond. This result indicates that the PdAP2/ERF and PtAP2/ERF family members are highly conserved, and further suggests that almond and wild dwarf almond are less separated during evolution. The results revealed that the Ka/Ks values between these duplicated gene pairs and homologous gene pairs were predominantly less than 1, indicating that AP2/ERF genes were primarily subject to purifying selection during the evolutionary process. Additionally, three and four tandem duplicate gene pairs between PdAP2/ERF and PtAP2/ERF, respectively, had incalculable Ks values. Furthermore, five and eight tandem duplicate gene pairs between PdAP2/ERF and PtAP2/ERF, respectively, had indeterminable Ks values. Among the homologous genes, 15 pairs in almond and wild dwarf almond, 45 pairs in A. thaliana and almond, and 45 pairs in A. thaliana and wild dwarf almond had incalculable Ks values. These gene pairs with indeterminable Ks values were categorized as having high sequence divergence values.

Fig. 4
figure 4

Distribution of homologous gene pairs of almond, wild dwarf almond, and Arabidopsis thaliana AP2/ERF family members. Line segments represent chromosomes, and numbers correspond to specific chromosome designations

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Analysis of PdAP2/ERF and PtAP2/ERF member promoters

We identified 114 and 118 cis-acting elements in the PdAP2/ERF and PtAP2/ERF family members, respectively. These included numerous light-response-related cis-acting elements such as ACE, G-box, and MRE, in addition to a large number of TATA-box and CAAT-box cis-acting elements (Table S4 and Table S5). Three functional types of cis-acting elements were identified in both PdAP2/ERF and PtAP2/ERF family members: those related to abiotic and biotic stresses, phytohormone responsiveness, and plant growth and development. These types were consistent across both families and their distributions were mapped separately (Figure S2A and Figure S2B). The results revealed that cis-acting elements related to abiotic and biotic stresses included TC-rich repeats, LTR, ARE, GC-motif, MBS, and WUN-motif. Phytohormone-responsive elements encompassed TATC-box, TCA-element, SARE, ABRE, AuxRR-core, CGTCA-motif, TGACG-motif, P-box, GARE-motif, TGA-box, and AuxRE. Elements related to plant growth and development functions included eleven types: MSA-like, Unnamed_1, circadian, RY-element, O2-site, CAT-box, motif I, GCN4_motif, HD-Zip 1, AACA_motif, and MBSI. Furthermore, we quantified the occurrence of 30 types of cis-acting elements. It is noteworthy that these three types of cis-acting elements are exactly the same in PdAP2/ERF and PtAP2/ERF family members. The analysis revealed that five types of cis-acting elements—ABRE, ARE, CGTCA-motif, TGACG-motif, and MBS were present in more than 100 PdAP2/ERF and PtAP2/ERF family members (Figure S2C). Notably, while the number of ABRE elements differed substantially between the two families, the other four types showed similar frequencies. In summary, the results of cis-acting elements indicate that members of the AP2/ERF family are widely involved in the growth and development, stress response, and hormone regulation of both almond and wild dwarf almond.

Analysis of protein interactions between PdAP2/ERF and PtAP2/ERF family members

We conducted protein interaction predictions for the PdAP2/ERF and PtAP2/ERF family members using the STRING tool, referencing the A. thaliana database. The analysis revealed potential protein interactions among 31 members of the PdAP2/ERF family, with PdAP2/ERF92 identified as the predicted central gene (Figure S3A). Similarly, 25 members of the PtAP2/ERF family exhibited potential protein interactions, with PtAP2/ERF39 emerging as the predicted central gene (Figure S3B). Multiple potential interactions were observed between several members within both family interaction networks, such as PdAP2/ERF70 interacting with PdAP2/ERF92 and PdAP2/ERF100, and PtAP2/ERF110 interacting with PtAP2/ERF39 and PtAP2/ERF84. Furthermore, GO enrichment analysis of the PdAP2/ERF and PtAP2/ERF family member proteins, based on STRING tool predictions, indicated their primary distribution in the nucleus (GO:0005634) and involvement in processes including ethylene-activated signaling pathway (GO:0009873), hormone-mediated signaling pathway (GO:0009755), cellular response to chemical stimulus (GO:0070887), and DNA-binding transcription factor activity (GO:0003700) (Table S6, Table S7).

Predictive analysis of potential target genes of PdAP2/ERF family members

Based on the Arabidopsis DREB1B (CBF) transcription factor binding map (MA1669.2), we predicted 2980 potential target genes in the almond genome. This prediction included nine types of matching sequences: GCCGACAT, ACCGACAT, GCCGACAA, GCCGACGT, GCCGACAC, GCCGACTT, GCCGACCT, and GCCGACAG (Fig. 5A, Table S8). GO enrichment analysis of these potential target genes revealed that in the biological process category, most target gene proteins were enriched in cellular processes, metabolic processes, and cellular process functions (Fig. 5B). In the cellular component category, the majority of target gene proteins were enriched in cell, cell part, and organelle functions. For molecular function, most target gene proteins were enriched in catalytic activity, binding, and transcription regulator activity. Furthermore, we identified six ERF-type genes (PdAP2/ERF34, PdAP2/ERF86, PdAP2/ERF104, PdAP2/ERF107, PdAP2/ERF109, and PdAP2/ERF113) that were also among the potential target genes of DREB1B (CBF) (Fig. 5C).

Fig. 5
figure 5

PdAP2/ERF family member target genes. A DREB1B (CBF) transcription factor binding map. B Target gene GO-enriched functional distribution map. C DREB1B with six potential target genes map

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Analysis of the expression pattern of PdAP2/ERF family members

We excluded PdAP2/ERF genes with FPKM values below 1 in all six freezing stress (-5, -10, -15, -20, -25, and -30 °C) treated annual dormant branch samples, retaining 63 PdAP2/ERF members for expression pattern analysis (Fig. 6, Table S9). The results revealed that fifteen genes (PdAP2/ERF2, PdAP2/ERF7, PdAP2/ERF9, PdAP2/ERF12, PdAP2/ERF17, PdAP2/ERF21, PdAP2/ERF29, PdAP2/ERF52, PdAP2/ERF53, PdAP2/ERF77, PdAP2/ERF80, PdAP2/ERF84, PdAP2/ERF87, PdAP2/ERF97, and PdAP2/ERF98) were significantly expressed in the control samples. Ten genes (PdAP2/ERF3, PdAP2/ERF5, PdAP2/ERF16, PdAP2/ERF18, PdAP2/ERF26, PdAP2/ERF27, PdAP2/ERF29, PdAP2/ERF84, PdAP2/ERF89, and PdAP2/ERF108) were significantly expressed in -10 °C samples. Fourteen genes (PdAP2/ERF5, PdAP2/ERF26, PdAP2/ERF40, PdAP2/ERF61, PdAP2/ERF64, PdAP2/ERF65, PdAP2/ERF68, PdAP2/ERF71, PdAP2/ERF72, PdAP2/ERF75, PdAP2/ERF89, PdAP2/ERF91, PdAP2/ERF98, and PdAP2/ERF106) were significantly expressed in -15 °C samples. Eight genes (PdAP2/ERF3, PdAP2/ERF7, PdAP2/ERF15, PdAP2/ERF41, PdAP2/ERF61, PdAP2/ERF63, PdAP2/ERF73, and PdAP2/ERF92) were significantly expressed in -20 °C samples. Twenty-seven genes (PdAP2/ERF17, PdAP2/ERF20, PdAP2/ERF25, PdAP2/ERF28, PdAP2/ERF32, PdAP2/ERF36, PdAP2/ERF38, PdAP2/ERF39, PdAP2/ERF42, PdAP2/ERF45, PdAP2/ERF50, PdAP2/ERF51, PdAP2/ERF57, PdAP2/ERF58, PdAP2/ERF59, PdAP2/ERF62, PdAP2/ERF66, PdAP2/ERF70, PdAP2/ERF72, PdAP2/ERF76, PdAP2/ERF80, PdAP2/ERF86, PdAP2/ERF90, PdAP2/ERF93, PdAP2/ERF94, PdAP2/ERF104, and PdAP2/ERF105) were significantly expressed in -25 °C samples. Nineteen genes (PdAP2/ERF25, PdAP2/ERF36, PdAP2/ERF38, PdAP2/ERF39, PdAP2/ERF42, PdAP2/ERF51, PdAP2/ERF59, PdAP2/ERF60, PdAP2/ERF66, PdAP2/ERF70, PdAP2/ERF76, PdAP2/ERF77, PdAP2/ERF86, PdAP2/ERF90, PdAP2/ERF93, PdAP2/ERF104, PdAP2/ERF105, PdAP2/ERF106, and PdAP2/ERF108) were significantly expressed in the -30 °C samples. Notably, several genes such as PdAP2/ERF3, PdAP2/ERF25, and PdAP2/ERF42 were significantly expressed in multiple samples. In summary, members of the PdAP2/ERF family may play an important role in regulating the dormancy period of almonds to resist freezing stress.

Fig. 6
figure 6

Heatmap depicting the expression of PdAP2/ERF family members in freeze-stressed annual branches. The analysis was conducted using average row normalization, with blue representing low expression and red indicating high expression. Expression values exceeding 0.8 between genes and samples in the heatmap were considered significant

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Fluorescence quantitative expression analysis of PdAP2/ERF family members

We further investigated the changes in fluorescence quantitative expression levels of eight PdAP2/ERF genes in the phloem and xylem of 'Wanfeng' almond after six freezing stress treatments at temperatures of -5, -10, -15, -20, -25, and -30 °C (Figure S4). The results revealed that PdAP2/ERF5, PdAP2/ERF40, PdAP2/ERF64, PdAP2/ERF86, PdAP2/ERF104, and PdAP2/ERF109 were up-regulated in the phloem under various freezing stress temperatures. Notably, PdAP2/ERF86 and PdAP2/ERF104 exhibited the most significant expression at -15, -25, and -30 °C, with levels more than tenfold higher than the control (Fig. 7A). Conversely, the expression levels of PdAP2/ERF18 and PdAP2/ERF113 appeared to be suppressed by freezing stress. In the xylem, the expression levels of PdAP2/ERF5 and PdAP2/ERF104 were elevated under different freezing stress treatments, while the other six PdAP2/ERF genes showed varied responses, either decreasing or increasing under different freezing stress temperatures (Fig. 7B). This result suggests that PdAP2/ERF family members may comprehensively resist the effects of freezing stress by regulating almond phloem and xylem.

Fig. 7
figure 7

Fluorescence quantitative expression levels of eight PdAP2/ERF genes in 'Wanfeng' almond annual dormant branches under six temperature gradients of freezing stress. A phloem. B xylem. The horizontal coordinates A, B, C, D, E, and F represent -5, -10, -15, -20, -25, and -30 °C, respectively

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Discussions

AP2/ERF transcription factors comprise a large family of proteins extensively involved in plant growth and development, signal transduction, hormone regulation, and stress response [8, 9]. To date, the AP2/ERF family has been identified and analyzed in numerous plant species, including A. thaliana (147) [53], Oryza sativa (164) [53], Fagopyum tataricum (134) [54], Triticum aestivum (320) [55], Glycine max (301) [56], Prunus persica (131) [57], Prunus mume (116) [58], and Juglans mandshurica (184) [59], among others. Recently, researchers in Europe and the United States have sequenced and assembled the genomes of three almond varieties: 'Texas' [60], 'Nonpareil' [61], and 'Lauranne' [62]. Concurrently, Chinese researchers have accomplished the same for the Xinjiang native cultivar 'Wanfeng' almond and wild dwarf almond. Consequently, investigating the AP2/ERF gene family based on the genomes of Xinjiang native cultivated 'Wanfeng' almond and wild dwarf almond aligns more closely with the genetic characteristics of native almond. Therefore, by studying the differences between the genomes of 'Wanfeng' almond and wild dwarf almond and exploring the regulatory genes of their respective characteristic traits, a better approach can be provided for the breeding of native almonds in China. In this study, we identified 114 and 116 AP2/ERF genes in the almond and wild dwarf almond genomes, respectively, designating them as PdAP2/ERF and PtAP2/ERF. The analysis revealed significant variability in amino acid number, protein molecular weight, theoretical isoelectric point, instability index, and aliphatic index among members of the PdAP2/ERF and PtAP2/ERF families. All proteins were found to be hydrophilic, with most member proteins subcellularly localized in the nucleus.

In A. thaliana, AP2/ERF family members were classified into five subfamilies: AP2, RAV, Soloist, DREB, and ERF, based on their structural domains. The DREB subfamily was further categorized into six subgroups (A1-A6), and the ERF subfamily into six subgroups (B1-B6), as observed in A. thaliana [53]. Consequently, subsequent studies on AP2/ERF family members in related plants have adopted this five-subfamily classification [56, 63]. We constructed neighbor-joining phylogenetic trees for almond, wild dwarf almond, and A. thaliana AP2/ERF family members. The distribution of PdAP2/ERF members across the five subfamilies was: AP2 20 (17.54%), RAV 4 (3.51%), Soloist 1 (0.88%), DREB 40 (35.88%), and ERF 49 (42.98%). Similarly, PtAP2/ERF members were distributed as: AP2 20 (17.24%), RAV 4 (3.45%), Soloist 1 (0.86%), DREB 39 (33.62%), and ERF 52 (44.83%). The highly similar distribution of members within the same subfamily for both species indicates that almond and wild dwarf almond AP2/ERF family members are highly conserved in gene sequences, suggesting limited divergence in their evolutionary trajectories. Gene function is closely related to conserved motif and gene structure in protein sequences [64]. The protein sequences of PdAP2/ERF and PtAP2/ERF members within the same subfamily exhibited highly conserved motif types. Most members of the DREB, ERF, RAV, and Soloist subfamilies contained only one exon, whereas the number of exons in AP2 subfamily members ranged from seven to eleven. This finding aligns with analyses of conserved motif and exon–intron numbers in AP2/ERF subfamily members of species such as P. persica [57], P. mume [58], and Pennisetum glaucum [65].

Both PdAP2/ERF and PtAP2/ERF family members were unevenly distributed on eight chromosomes, with no significant correlation between chromosome length and gene count. This distribution pattern aligned with AP2/ERF members in other species [57, 66]. Gene duplication plays a crucial role in the expansion and evolution of gene family members, contributing to species' environmental adaptation and maintenance of normal life processes [65]. The extent of gene duplication in AP2/ERF family members varies across species. For instance, 38 pairs of fragment duplication genes were identified in Medicago truncatula [67], while Zingiber officinale exhibited 4 tandem duplicate pairs and 15 pairs of fragment duplication genes [68]. In our study, we identified 30 and 27 pairs of fragment duplication genes and 15 and 18 pairs of tandem duplication genes in PdAP2/ERF and PtAP2/ERF family members, respectively. These findings suggest that gene duplication may contribute to the amplification of PdAP2/ERF and PtAP2/ERF family members. Furthermore, we identified 101, 99, and 140 pairs of AP2/ERF homologous genes between almond and A. thaliana, wild dwarf almond and A. thaliana, and almond and wild dwarf almond, respectively. AP2/ERF members between almond and wild dwarf almond demonstrated high conservation, with Ka/Ks values less than 1, indicating purifying selection. However, a substantial number of AP2/ERF homologous genes between almond and A. thaliana, and between wild dwarf almond and A. thaliana, could not be calculated for the Ks value, falling into the sequence divergence value category. This result suggests significant differential variations in AP2/ERF gene sequences between arborvitae and herbaceous plants.

Cis-acting elements in the promoter region play a crucial role in regulating gene expression [69]. Numerous cis-acting elements have been identified in the promoter regions of the PdAP2/ERF and PtAP2/ERF family members, encompassing various important elements for hormone regulation, growth and development, and stress response. These cis-acting elements indicate that AP2/ERF genes are extensively involved in multiple response processes in almond and wild dwarf almond. Transcription factors often regulate various response processes holistically through protein interactions [70]. Within the PdAP2/ERF family, 31 members exhibited potential protein interactions with each other, while 25 members of the PtAP2/ERF family demonstrated potential protein interactions among themselves. These PdAP2/ERF and PtAP2/ERF members play significant roles in the ethylene-activated signaling pathway, hormone-mediated signaling pathway, and DNA-binding transcription factor activity, among others. CBF (C-repeat binding transcription factor/dehydrate responsive element binding factor) is a key gene in plant response to freezing stress [71]. Consequently, we predicted 2980 potential target genes in the almond genome based on the DREB1B (CBF) transcription factor binding MA1669.2. This result can reveal the regulatory network of CBF transcription factors in almond and has reference significance for understanding the growth, development and stress adaptation mechanism of almond.

Investigating gene expression during tissue development and under stress conditions is crucial for understanding the molecular mechanisms underlying biological development [72]. We examined the expression profiles of PdAP2/ERF family members in six freezing stress-treated annual fruiting shoots of 'Wanfeng' almond. Following the principle that genes were considered expressed when the FPKM value exceeded 1, 51 PdAP2/ERF genes were found to be potentially inactive in the six freezing-stress-treated annual fruiting shoots, possibly due to dormancy. Conversely, 63 PdAP2/ERF genes exhibited significant expression in annual fruiting shoots after any of the six freezing stress treatments, suggesting they may play diverse functional roles during almond dormancy. Notably, 27 PdAP2/ERF genes showed significant expression at -25 ℃. Currently, the role of AP2/ERF in regulating low-temperature stress resistance has been verified in several species. For instance, AmCBF2 (Avicennia marina) [73], BpERF13 (Glycine max) [74], TdSHN1 (Triticum durum) [75], and ZjDREB1.4 (Zoysia japonica) [76] have been shown to enhance plant freezing tolerance. We investigated the fluorescence quantitative expression level changes of eight PdAP2/ERF genes in the phloem and xylem of annual dormant branches of 'Wanfeng' almond after six temperature freezing stress treatments at -5, -10, -15, -20, -25, and -30 °C. The results revealed that most PdAP2/ERF genes exhibited higher expression levels in the phloem compared to relatively lower levels in the xylem. This finding suggests that the phloem of dormant annual almond branches may play a significant role in the response to freezing stress.

Conclusion

In this study, we identified 114 and 116 AP2/ERF genes from the 'Wanfeng' almond genome and the 'Yumin' wild dwarf almond genome, respectively. (1) According to the classification of Arabidopsis AP2/ERF family members, PdAP2/ERF and PtAP2/ERF members were divided into five subfamilies: AP2, RAV, Soloist, DREB and ERF. The results of protein physicochemical properties, protein conserved motifs and gene structure showed that members of the same subfamily of PdAP2/ERF and PtAP2/ERF were highly conserved, while members of different subfamilies were poorly conserved. (2) Gene duplication results showed that there were 30 pairs of segmental duplication genes and 15 pairs of tandem duplication genes in the PdAP2/ERF family members, and 27 pairs of segmental duplication genes and 18 pairs of tandem duplication genes in the PtAP2/ERF family members. Homologous genes showed that PdAP2/ERF and PtAP2/ERF members were highly conserved, and the Ka/Ks values of these homologous gene pairs were all less than 1, indicating that AP2/ERF genes were mainly subject to purifying selection during evolution. (3) The results of cis-acting elements showed that AP2/ERF genes were widely involved in the growth and development, stress response and hormone regulation of almond and wild dwarf almond, and the expression pattern showed that PdAP2/ERF family members may be involved in the almond dormancy period to resist freezing stress. (4) Fluorescence quantitative results showed that the expression levels of 8 PdAP2/ERF genes in the phloem and xylem of annual dormant branches of almond were different, indicating that both may play a role in resisting freezing stress. The results of this study may help to study the characteristics of AP2/ERF genes between almond and wild dwarf almond species, and provide data reference for subsequent almond frost resistance research.

Data availability

The 'Wanfeng' almond reference genome data used in this study has been uploaded to the NCBI database with login number GCA_040581505.1. You can access them at the following website https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_040581505.1/. The whole genome sequence data of 'Yumin' wild almond is stored in the genome warehouse of the Chinese Academy of Sciences Beijing Institute of Genomics/National Genome Data Center of China National Biological Information Center, with the login number of GWHCBGA00000000, and can be accessed publicly at the following website https://ngdc.cncb.ac.cn/gwh. The dataset generated and analyzed in this study can be obtained from the corresponding author upon reasonable request. The original sequence data of the transcriptome corresponding to the six freezing stresses of 'Wanfeng' almond used in this research has been saved in the genome sequence file (genomics, proteomics and bioinformatics 2021) of the National Genome Data Center (Nucleic Acid Research 2024) of the China National Bioinformatics Center/Chinese Academy of Sciences Beijing Genome Research Institute (GSA: CRA007323), which can be accessed publicly at the following website https://ngdc.cncb.ac.cn/gsa.

The whole genome sequence data of 'Yumin' wild almond is stored in the genome warehouse of the Chinese Academy of Sciences Beijing Institute of Genomics/National Genome Data Center of China National Biological Information Center, with the login number of GWHCBGA00000000, and can be accessed publicly at the following website https://ngdc.cncb.ac.cn/gwh. The dataset generated and analyzed in this study can be obtained from the corresponding author upon reasonable request.

The original sequence data of the transcriptome corresponding to the six freezing stresses of 'Wanfeng' almond used in this research has been saved in the genome sequence file (genomics, proteomics and bioinformatics 2021) of the National Genome Data Center (Nucleic Acid Research 2024) of the China National Bioinformatics Center/Chinese Academy of Sciences Beijing Genome Research Institute (GSA: CRA007323), which can be accessed publicly at the following website https://ngdc.cncb.ac.cn/gsa.

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Acknowledgements

All authors express their gratitude to Dr. Han Zhao from the Research Institute of Non-Timber Forestry, Chinese Academy of Forestry for providing the genome data of Prunus tenella for use in this study.

Funding

1.The key research and development project of Xinjiang Uyghur Autonomous Region, (Grant Number 2023B02026).

2.Xinjiang Uygur Autonomous Region Youth Fund Project (Grant Number 2024D01B35).

3.The key research and development project of Xinjiang Uyghur Autonomous Region, (Grant Number 2024B02018).

4. Xinjiang Agricultural University High-level Talent Scientific Research Cultivation Program (Grant Number 2524GCCRC)

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Authors and Affiliations

  1. College of Horticulture, Xinjiang Agricultural University, Urumqi, 830052, China

    Dongdong Zhang, Bin Zeng, Yawen He & Zhenfan Yu

  2. Forestry and Landscape Architecture College, Xinjiang Agricultural University, Urumqi, 830052, China

    Jiangui Li

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  1. Dongdong Zhang
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  2. Bin Zeng
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  4. Jiangui Li
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Contributions

D.D.Z. and Z.F.Y. designed the study, conducted analyses, and wrote and edited the manuscript. Y.W.H. prepared figures. B.Z. and J.G.L. participated in data curation, investigation, methodology development, and played crucial roles in reviewing and editing the manuscript.

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Correspondence to Zhenfan Yu.

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This plant materials don’t include any species at risk of extinction. We declare that all the experimental plants were collected with permission from local authorities of agricultural department and the plant materials in the experiment. We comply with relevant institutional, national, and international guidelines and legislation for plant study.

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Supplementary Information

Supplementary Material 1. 

12864_2025_11275_MOESM2_ESM.pdf

Supplementary Material 2: Figure S1. Phylogenetic tree, conserved motif, structural domain, and gene structure combination diagrams of PdAP2/ERF and PtAP2/ERF family members. A: NJ phylogenetic tree of PdAP2/ERF and PtAP2/ERF members. Different colored regions represent the five subfamilies: AP2, RAV, Soloist, DREB, and ERF, respectively. B: Conserved motif. C: AP2 structural domains. D: Gene structures. E: Diagrams of different element types.

12864_2025_11275_MOESM3_ESM.pdf

Supplementary Material 3: Figure S2. Distribution location and frequency statistics of three functional cis-acting elements related to Abiotic and biotic stresses, Phytohormone responsiveness, and Plant growth and development. A: Distribution location of the three types of functional cis-acting elements in the PdAP2/ERF family members. B: Distribution location of the three types of functional cis-acting elements in the PtAP2/ERF family members. C: Frequency statistics of 30 types of cis-acting elements in PdAP2/ERF and PtAP2/ERF family members. Red font indicates cis-acting elements with a frequency exceeding 100.

12864_2025_11275_MOESM4_ESM.pdf

Supplementary Material 4. Figure S3. Protein interaction network diagram. a: PdAP2/ERF family members protein interaction network diagram. b: PtAP2/ERF family members protein interaction network diagram. Red circles represent hub genes.

12864_2025_11275_MOESM5_ESM.png

Supplementary Material 5. Figure S4. Quantitative Real-time polymerase chain reaction. (A) Amplification Curve. (B) Melting curve.

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Zhang, D., Zeng, B., He, Y. et al. Genome-wide identification and comparative analysis of the AP2/ERF gene family in Prunus dulcis and Prunus tenella: expression of PdAP2/ERF genes under freezing stress during dormancy. BMC Genomics 26, 95 (2025). https://doi.org/10.1186/s12864-025-11275-9

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