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Article

Genome-Wide Identification and Expression Profiling of AP2/ERF Transcription Factor Genes in Prunus armeniaca L.

by
Yanguang He
1,2,†,
Lin Wang
1,†,
Nan Jiang
1,†,
Donglin Zhang
3,
Xiaodan Shi
4,
Tana Wuyun
1 and
Huimin Liu
1,*
1
Research Institute of Non-Timber Forestry, Chinese Academy of Forestry, Zhengzhou 450003, China
2
College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China
3
Department of Horticulture, University of Georgia, Athens, GA 30602, USA
4
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2025, 16(8), 1353; https://doi.org/10.3390/f16081353
Submission received: 7 July 2025 / Revised: 5 August 2025 / Accepted: 18 August 2025 / Published: 20 August 2025
(This article belongs to the Special Issue Forest Tree Breeding: Genomics and Molecular Biology)
Browse Figures
Versions Notes

Abstract

The APETALA2/Ethylene Responsive Factor (AP2/ERF transcription factor) family plays pivotal roles in plant growth, stress responses, and metabolic regulation. Here, we identified 118 AP2/ERF family members in the apricot (Prunus armeniaca L.) genome, which were classified into four major subfamilies (AP2, DREB, ERF, and RAV) and Soloists (few unclassified factors), through phylogenetic analysis. The ERF subfamily exhibited the largest expansion (55 members), driven predominantly by 10 tandem and 14 segmental duplication events. Gene structures and conserved motifs exhibited similar patterns within each subfamily. Chromosomal distribution was uneven, with chromosome 1 harboring the highest gene density. PaWRI1 was specifically expressed in apricot kernel and positively correlated with oil accumulation. A total of 47 lipid-related genes were predicted as potential targets of PaWRI1 through correlation analysis, which covers the entire three-stage process of plant oil synthesis. These results advance our understanding of how core AP2/ERF transcription factors modulate oil accumulation pathways in apricot, offering potential targets for metabolic engineering.

1. Introduction

Apricot (Prunus armeniaca L.), an important member of the Rosaceae family [1], is one of the most commercially valuable fruit crops worldwide. Its fruits are not only prized for their unique flavor, but the kernels are also rich in high-value oils (40%–60% oil content) and various bioactive compounds, including oleic acid, linoleic acid, tocopherols, and phenolic substances, which have wide applications in the food, pharmaceutical, and cosmetic industries [2,3,4].
Transcription factors (TFs) are DNA-binding proteins that specifically recognize and bind to cis-acting elements in the promoter regions of target genes, thereby regulating transcriptional activity [5]. The APETALA2/Ethylene Responsive Factor (AP2/ERF) family, a major group of plant TFs, contains a conserved AP2/ERF domain that recognizes and binds to specific cis-elements such as the GCC-box and DRE/CRT motifs in target gene promoters [6,7,8,9]. Based on structural features and phylogenetic relationships, this family can be divided into four major subfamilies: (1) the AP2 subfamily, characterized by dual AP2 domains, primarily regulates plant growth and development; (2) the ERF subfamily, containing a single AP2 domain, is involved in stress responses and hormone signaling; (3) the Related to ABI3/VP1 (RAV) subfamily, which possesses both AP2 and B3 domains, modulates diverse biological processes; and (4) the dehydration-responsive element binding protein (DREB) subfamily, containing only one AP2 domain, regulates genes involved in hormone and stress responses [6,7,8,10]. At the molecular level, the typical AP2 domain consists of 60 amino acids, forming a characteristic three-dimensional structure with three DNA-binding β-sheets and an α-helix mediating protein–protein interactions [7,11,12]. Notably, the distribution of this family varies significantly across organisms: AP2 and ERF subfamilies are plant-specific, whereas RAV members are also found in animals [10,13]. During plant evolution, the AP2/ERF family has undergone significant expansion (e.g., 147 members in Arabidopsis thaliana (L.) Heynh., 163 in rice (Oryza sativa L.)), with gene duplication contributing to functional diversification [14,15,16]. Although AP2/ERF TFs have been extensively studied in model plants such as Arabidopsis and rice, research in Rosaceae Prunus species remains uneven—most studies have focused on peach (Prunus persica (L.) Batsch) [17,18,19].
The AP2/ERF transcription factors play important roles in many biological and physiological processes, including spikelet and floral organ development [6,20], fruit ripening and seed development [21,22], root initiation and development [23], stem growth and development [24], leaf size and development [25], and biotic and abiotic stress [26,27]. In apricot studies, flowers, fruits, and seeds constitute primary research targets owing to their centrality in reproduction and agricultural value. However, most of the related studies were conducted on model plants, including Arabidopsis [28], rice [29], maize (Zea mays L.) [30], and Brassica napus L. [31] fewer studies were report on other plant [21], especially Rosaceae, which underpins global fruit production and drives critical advances in pomology, genomics, and perennial crop breeding. Significantly, the regulatory functions of AP2/ERF transcription factors in apricot fruit maturation and seed development await characterization.
In this study, we performed a genome-wide systematic analysis of the AP2/ERF TF family in apricot. Through bioinformatics approaches, we identified 118 AP2/ERF members and classified them into four major subfamilies: AP2 (20), RAV (5), DREB (37), and ERF (55), and Soloist (1 unclassified member). Comprehensive analyses of gene structure, conserved motifs, chromosomal localization, duplication events, and syntenic relationships were conducted using multiple bioinformatics tools. Notably, cis-element analysis revealed that AP2/ERF members are enriched with hormone-responsive (e.g., ABRE) and stress-related elements. Particularly, PaERF35 and PaDREB5 contain both endosperm-specific and seed-preferential regulatory motifs, suggesting their potential roles in seed development in apricot. To systematically investigate expression patterns, transcriptomic data from apricot flowers, flower buds, leaves, and five kernel (K1–K5) and eight flesh (F1–F8) developmental stages were analyzed. Five AP2/ERF genes that harbor endosperm expression or seed-specific regulation elements were specifically expressed in apricot kernels, indicating their potential functions in kernel development. Through correlation analysis, 47 lipid-related genes were predicted as the potential targets of PaWRI1. This study provides preliminary insights into the potential molecular mechanisms by which the AP2/ERF transcription factor family (with particular emphasis on the WRI1 homolog PaAP2-5) regulates oil biosynthesis in apricot kernels. These findings lay a foundation for further investigations into the molecular mechanism of AP2/ERF genes in regulating apricot oil biosynthesis.

2. Materials and Methods

2.1. Genome-Wide Identification of AP2/ERF Transcription Factors in Apricot

To identify AP2/ERF proteins, we employed a computational pipeline using the AP2 DNA-binding domain HMM profile (PF00847) from InterPro. This profile was used to scan the annotated proteome of P. armeniaca cv. ‘Sungold’ (Genome assembly v1.0, 4 June 2021); accessed via https://www.rosaceae.org/Analysis/10254125 on 8 August 2024 using HMMER software (v3.4) with default parameters.
For comparative analysis, 146 AP2/ERF protein sequences from A. thaliana were retrieved from the TAIR database (https://www.arabidopsis.org/, accessed on 12 August 2024). The sequences served as BLASTP (BLAST-2.12.0+) queries against a local P. armeniaca protein database. Candidate genes identified by HMMER and BLASTP underwent NCBI CDD validation for AP2 domain confirmation, with verified sequences designated as final AP2/ERF family members.
Key physicochemical properties (molecular weight, pI, instability index, aliphatic index, GRAVY) of the identified AP2 TFs were predicted using Expasy ProtParam (https://web.expasy.org/protparam/, accessed on 15 August 2024).

2.2. Phylogenetic and Evolutionary Analysis

Protein sequences were aligned using MUSCLE (v5.1, default parameters) following standard protocols [32,33]. Phylogenetic reconstruction employed two complementary methods: maximum likelihood (TreeBeST module in MEGA7, 1000 bootstraps) and Bayesian inference in IQ-TREE (v2.0.4) [34]. Resultant phylogenies were visualized and annotated via iTOL (https://itol.embl.de/, accessed on 17 August 2025) [35,36]. In total, 115 AP2/ERF protein sequences from peach were retrieved from the JGI Phytozome 14 (https://phytozome-next.jgi.doe.gov/, accessed on 2 August 2025).

2.3. Structural Characterization of AP2/ERF Genes

Gene structure analysis was conducted through a comprehensive examination of exon–intron organization using TBtools -II (v2.100) [37]. Conserved motifs were identified via MEME (https://meme-suite.org/meme/, accessed on 17 August 2025, 20 maximum motifs, width 6–200 aa). Domain architecture was verified using the NCBI Batch CD-Search tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 17 August 2025) [37,38], with all structural features visualized through TBtools.

2.4. Chromosomal Localization and Synteny Analysis of AP2/ERF Gene Family

Chromosomal localization of the PaAP2/ERF gene family was visualized using TBtools, with genomic positional information extracted from the annotated genome assembly of P. armeniaca. Gene duplication events and synteny analysis among apricot, Arabidopsis, and peach were analyzed using One StepMCScanX-SuperFast module and visualized in TBtools [33].

2.5. Analysis of Regulatory Elements and Functional Annotation

Promoter regions (2 kb upstream of AP2/ERF genes) were analyzed for cis-regulatory elements using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 17 August 2025) with standard parameters [39]. Functional annotation was performed through eggNOG-mapper (http://eggnog-mapper.embl.de/, accessed on 17 August 2025), followed by Gene Ontology enrichment analysis using TBtools ORA (Over-Representation Analysis) module [40,41].

2.6. Transcriptional Profiling and Experimental Validation

AP2/ERF expression patterns were analyzed using RNA-seq data (https://www.cncb.ac.cn/, accessed on 17 August 2025, PRJCA001987), with FPKM values calculated across multiple tissues (young leaves, flower buds, flowers) and developmental stages (kernels: K1–K5; flesh: F1–F8). Hisat2-aligned reads (default parameters) were mapped to the ‘Sungold’ reference genome [42]. Gene expression levels were quantified as read counts with htseq-count, followed by FPKM calculation in R according to the formula: FPKM = (total exon fragments)/(mapped reads in millions × exon length in kilobases) [43]. Normalized FPKM values were used to generate heatmaps, with hierarchical clustering performed via the “complete” linkage method.
To validate the RNA-seq expression data, RT-qPCR analysis was performed. Kernel samples were collected from nine-year-old P. armeniaca cv. ‘Shenyangdashuaifu’ trees cultivated at the Yuanyang Long-Term Experimental Base of the Non-Timber Forest Research Institute, Chinese Academy of Forestry (Xinxiang, China). Samples from three healthy trees were harvested at 50 (17 April), 60, 70, 80, and 90 days after flowering (DAF) in spring, immediately frozen in liquid nitrogen for 10 min, and stored at −80 °C. For each tree, 30 kernels were collected from the east, south, west, and north canopy directions. Total RNA extraction and RT-qPCR were conducted using a CFX96 Touch Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA) [44]. The primer sequences are provided in Table S1, using UBQ as the reference gene. Expression levels were determined via the 2−ΔΔCt method with three biological replicates per gene [45].

2.7. Oil Content Quantification

Kernels used for oil content detection were the same samples used for RT-qPCR. Total oil content was determined using a nuclear magnetic resonance (NMR) analyzer (PQ001-SFC-Solid Fat Content Analyzer Benchtop NMR, Niumag, Suzhou, China) with three biological replicates. The oil calibration was constructed according to the manufacturer’s instruction using pure apricot kernel oil, which was obtained following Soxhlet extraction of 30 g freeze-dried mature kernel samples as previously described [46].

2.8. Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 8:
(1)
Differences in gene expression (RT-qPCR) and oil content across sample groups were assessed using one-way ANOVA.
(2)
Pearson correlation analyses were performed to evaluate the concordance between RNA-seq and RT-qPCR data, and the relationships between gene expression patterns and oil accumulation profiles.

2.9. Targets Prediction of PaWRI1

To identify potential lipid biosynthesis targets of PaWRI1 in apricot kernels, we employed a dual-criterion approach: (1) Cis-element screening: Promoters must contain the AW-box motif CnTnG7[CG] (where n = any nucleotide); (2) Expression correlation: Pearson correlation coefficient between PaWRI1 and target genes ≥ 0.9. Target genes satisfying both criteria were functionally annotated via KEGG pathway analysis. Corresponding Arabidopsis orthologs were identified through BLASTP alignment (E-value < 1 × 10−5). Key candidate genes with putative roles in lipid metabolism are highlighted in Table S2.

3. Results

3.1. Identification of AP2/ERF Family Members in Apricot

Through HMM (Hidden Markov Model) screening and BLASTP analysis, followed by verification using NCBI’s CDD, we identified 118 members of the AP2/ERF gene family in apricot (Table S2). The encoded proteins exhibited considerable diversity in their physicochemical properties: amino acid lengths spanning 167–837 residues, molecular weight between 14.08 and 91.52 kDa, and isoelectric points (pI) from 4.63 to 10.61. Protein stability analysis revealed instability indices of 31.69–82.43, aliphatic indices of 32.45–78.93, and hydropathicity scores (GRAVY) ranging from −1.107 to −0.337 (Table S3).

3.2. Phylogenetic Analysis of the PaAP2/ERF Family

To elucidate the evolutionary relationships among apricot AP2/ERF genes, we performed comparative phylogenetic analysis using 118 apricot, 146 Arabidopsis, and 115 peach AP2/ERF protein sequences through multiple sequence alignment followed by maximum likelihood (ML) tree construction (Figure 1). The ML-based phylogenetic classification revealed that apricot AP2/ERF superfamily members segregate into five distinct groups: four major subfamilies, ERF (55 members), DREB (37), AP2 (20), and RAV (5), along with one unclassified member (Soloist) (Figure 1, Table S4). Notably, the ERF subfamily represents the most abundant group, while RAV constitutes the smallest subset.

3.3. Gene Structure, Conserved Motif, and Domain Analysis of the AP2/ERF Family in Apricot

To investigate the genomic architecture and phylogenetic characteristics of apricot AP2/ERF genes, we analyzed their exon-intron organization (Figure 2A). The analysis revealed considerable structural variation, with exon numbers ranging from 1 to 10 across all members, while DREB subfamily genes exhibited a more conserved structure containing only 1–2 exons. Members of the ERF and RAV subfamily contain 1–3 exons. The AP2 subfamily members contained significantly more exons (ranging from 4 to 10) than other subfamilies, exhibiting the most diverse gene structures among the four subfamilies. Consistent with the phylogenetic analysis, AP2 subfamily members with similar exon numbers exhibit a closer evolutionary relationship, such as PaAP2-10 (6) and PaAP2-14 (4).
Using the MEME suite in TBtools, 20 conserved motifs were identified in AP2/ERF protein sequences, with widths ranging from 6 to 200 bp (Figure 2B). Certain motifs, such as motifs 1 and 5, appeared across all subfamilies. Members within the same subfamily displayed similar motif compositions. For example, all DREB members contained adjacent motif 1 and motif 2, while the RAV subfamily uniquely possessed motif 13. Most AP2 subfamily members harbored motifs 1, 4, 6, and 8. The ERF subfamily showed the most complex motif organization, though all its members shared a conserved arrangement of motifs 1, 2, and 3. In contrast, the Soloist subfamily, represented by a single member, exclusively contained motifs 1 and 18.
NCBI Batch CD Search revealed key conserved domains in the apricot AP2/ERF protein sequences (Figure 2C). While all members possessed AP2 or AP2 superfamily domains, their number and type varied by subfamily. Most DREB and ERF members had a single AP2 domain, with one gene from each subfamily containing an AP2 superfamily domain. The AP2 subfamily primarily featured two domains—either two AP2 domains or one AP2 domain paired with an AP2 superfamily domain. Five AP2 members had only one domain of either type. Despite having the fewest members, the RAV subfamily exhibited the highest domain diversity, with five distinct types. Each RAV member contained one AP2 domain, while the remaining four domains were unique to this subfamily, including the rare Bfil_C_EcoRII_N_B3 and PLN02501 superfamily domains, each appearing only once.

3.4. Chromosomal Localization and Syntenic Relationships of the PaAP2/ERF Family

To visualize the distribution of AP2/ERF genes on apricot chromosomes, the Gene Density Profile in TBtools was employed. The 118 genes were mapped across all eight chromosomes (Figure 3), showing variations in gene counts per chromosome and an uneven distribution within individual chromosomes. Chromosome 1 contained the highest number (24), including 10 ERF, 9 DREB, 4 AP2, and 1 RAV subfamily members. In contrast, chromosome 8 had the fewest genes (7), with six belonging to the ERF subfamily and clustered near the chromosome end.
To investigate the evolutionary dynamics of AP2/ERF transcription factors in P. armeniaca, we employed MCScanX to analyze duplication events, identifying 25 segmental duplication pairs and 17 tandem duplication clusters (Figure 4A, Table S5).
Comparative synteny analysis with A. thaliana and peach revealed 110 and 147 syntenic gene pairs, respectively (Figure 4B, Table S6). The results showed distinct evolutionary patterns: 29 apricot AP2/ERF genes had one-to-one synteny with Arabidopsis homologs, while 31 genes exhibited one-to-multiple relationships, including 17 (one-to-two), 10 (one-to-three), 3 (one-to-four), and 1 (one-to-five). Similarly, synteny analysis with peach identified 50, 30, 11, and 1 apricot genes with one-to-one, one-to-two, one-to-three, and one-to-four relationships, respectively.

3.5. Analysis of Cis-Regulatory Elements and Functional Annotation of the PaAP2/ERF Family

To investigate the potential functions of AP2/ERF genes in apricot, we analyzed the 2 kb promoter regions upstream of each AP2/ERF gene using PlantCARE (Figure 2D). The analysis revealed that AP2/ERF gene promoters are enriched with diverse hormone- and stress-responsive cis-elements, demonstrating their extensive regulatory roles in plant growth and environmental adaptation. The cis-regulatory elements were functionally classified into hormone-responsive elements (including gibberellin and abscisic acid responsive motifs) mediating signal transduction, stress-responsive elements (such as low temperature and drought inducible motifs) involved in stress adaptation, and tissue-specific elements (e.g., seed and endosperm preferential motifs) regulating organ development. Promoter analysis revealed that the ABRE (abscisic acid-responsive element) was widely distributed across the AP2/ERF family, being present in 47 out of 55 ERF subfamily members, 33 out of 37 DREBs, 18 out of 20 AP2s, and all RAVs. In total, 25 and 18 AP2/ERF genes contain endosperm expression and seed-specific regulation elements, respectively. Notably, PaERF35 and PaDREB5 harbor both endosperm-specific expression and seed-specific regulatory cis-elements.
Comprehensive gene ontology (GO) enrichment analysis of AP2/ERF transcription factors in apricot revealed their multifaceted regulatory roles in plant growth and stress adaptation (Table S7). At the molecular level, the AP2/ERF family exhibited significant enrichment in DNA-binding transcription factor activity and sequence-specific DNA binding. Biological process annotation demonstrated their involvement in four major functional categories: developmental processes including post-embryonic organ morphogenesis and root system development; stress responses to abiotic stimuli, drought, and pathogen defense; metabolic regulation of primary metabolism and lipid biosynthesis; and hormone signaling pathways mediated by ethylene and jasmonate (Figure 5). Cellular component analysis revealed their predominant nuclear localization.

3.6. Expression Patterns of AP2/ERF Genes Across Tissues and Their Roles in Apricot Oil Accumulation

Among the tested samples, 89 AP2/ERF genes were expressed (FPKM > 1) in at least one tissue (Figure 6A). Tissue-specific expression was observed for 3 genes in flesh, 2 in flower/flower bud, and 10 each in young leaf and kernel. The majority of these genes exhibited distinct expression patterns across different tissues.
Most of the AP2 subfamily indicated a higher expression level in the kernel than in other samples, including PaAP2-1, PaAP2-5, PaAP2-13, PaAP2-16, and PaAP2-20, which presented higher expression activity at the last two developmental stages of kernel. Among all AP2 genes, PaAP2-5 (WRI1), PaAP2-13, and PaAP2-19 were specifically expressed in the kernel, whereas PaAP2-2 was specifically expressed in the flower or flower bud. Most of the RAV subfamily genes represented very low expression levels, except PaRAV1, the expression level of which is much higher in young leaves than that of any other RAV members’ expression in other samples. Most of the ERF subfamily represented a higher expression level in leaf and flesh than in other samples. Among all members of the AP2/ERF family, PaERF25 exhibits the highest expression level across all detected tissues and developmental stages, with an average FPKM value of 713. PaERF38, PaERF43, PaERF44, and PaERF53 were specifically expressed in young leaves. PaERF39 was specifically expressed in the flower and flower bud. Interestingly, PaERF26, PaERF42, and PaERF51 were specifically expressed in the kernel and had a different expression pattern, i.e., PaERF42 expressed more in the early development stage of the apricot kernel, whereas the other two in the later stage, indicating that they might play different roles during apricot kernel development. Similar to the ERF subfamily, genes in the DREB subfamily also showcased high expression activity in flesh and young leaves. PaDREB8, PaDREB11, and PaDREB36 were specifically expressed in flesh and had a higher expression level in the later, middle, and early develop pmental stages of flesh, respectively, indicating that they might play different roles during apricot flesh development. PaDREB5, PaDREB9, PaDREB20, PaDREB21, PaDREB24, and PaDREB37 were specifically expressed in young leaves, while PaDREB12 and PaDREB32 were specifically expressed in the kernel.
Since WRI1 was confirmed to bind AW-box and play pivotal roles in plant lipid biosynthesis [47], we mainly focus on the further analysis of PaWRI1. In apricot, the oil accumulated mainly from 50 DAF to 90 DAF (mature stage), indicating S-shaped growth (Figure S1). The expression pattern of PaWRI1 was positively correlated with oil accumulation, with a high Pearson correlation coefficient (0.7). To identify potential target genes of PaWRI1 involved in lipid biosynthesis in apricot kernel, we conduct correlation analysis between PaWRI1 and genes involved in lipid metabolism based on FPKMs, and AW-box scanning in the promoter sequences of lipid genes. In total, 47 lipid metabolism-related genes were identified with AW-box and co-expressed with PaWRI1 with a high Pearson correlation coefficient (>0.9), including genes involved in glycolysis, de novo FA synthesis, and TAG assembly (Table S2). To identify other AP2/ERF genes that might be co-expressed with PaWRI1 in the same network during kernel development, we conducted expression correlation analysis based on FPKMs. As a result, 14 AP2/ERF genes were co-expressed with PaWRI1 with a Pearson coefficient > 0.9, among which PaERF26, PaRAV3, PaDREB12, and PaAP2-13 were kernel-specifically expressed (Table S8).
To validate the RNA-seq expression profiles, we performed RT-qPCR analysis on selected genes (PaAP2-5, PaAP2-10, PaAP2-20, PaDREB4, PaDREB6, and PaDREB12) across different developmental stages (Figure 6B–G). The RT-qPCR results showed strong consistency with RNA-seq data, with significant positive correlations (Pearson’s r > 0.83, p < 0.05) between the two datasets. This concordance confirms the reliability of our transcriptomic analysis.

4. Discussion

The AP2/ERF transcription factor family represents one of the most important regulatory gene families in plants, playing crucial roles in various biological processes ranging from development to stress responses [48,49,50,51]. Our comprehensive genome-wide analysis identified 118 AP2/ERF genes in apricot, expanding our understanding of this gene family in Rosaceae species. The identified AP2/ERF proteins exhibited considerable variation in their physicochemical properties (molecular weights: 14.08–91.52 kDa; pI: 4.63–10.61; GRAVY: −1.107 to −0.337), suggesting extensive functional diversification within this family. The molecular diversity likely underlies the functional plasticity of AP2/ERF proteins in regulating various aspects of apricot growth and environmental adaptation.
According to phylogenetic analysis, apricot AP2/ERF transcription factors were classified into four distinct subfamilies (AP2, DREB, ERF, and RAV), with ERF being the largest (55 members) and RAV the smallest (5 members). This classification is consistent with reports in other plant species [52], which confirms the evolutionary conservation of AP2/ERF gene family organization. Compared to Arabidopsis, the total number of AP2 genes and the number of genes in each subfamily are more similar between apricot and peach (Table S4). This result indicates that the evolutionary trajectories of the AP2 gene family are strongly constrained by phylogenetic context, reflecting shared genomic and selective pressures between closely related species.
Notably, structural analysis showed that AP2 subfamily members possessed the most complex gene architectures (4–10 exons), while the other three subfamilies generally contained fewer exons (1–3). This situation was also found in other plants [52]. Research has indicated that evolutionary processes can lead to the progressive loss of introns [53,54]. In light of this theory, it is plausible that the AP2 subfamily gave rise to other subfamilies during evolution in plants. This structural diversity, particularly the alternative splicing potential in AP2/ERF subfamily genes [48], likely contributes to functional specialization among subfamilies. Domain architecture analysis further supported this notion—while most DREB/ERF members contained a single AP2 domain, AP2 subfamily proteins typically possessed two AP2 domains. Intriguingly, the small RAV subfamily exhibited the greatest domain diversity, which may reflect an evolutionary strategy where functional versatility compensates for limited gene numbers [55].
Chromosomal distribution analysis revealed an uneven distribution of AP2/ERF genes across the apricot genome, with chromosome 1 harboring the highest number (24 genes) and chromosome 8 the fewest (7 genes). This non-random distribution pattern suggests potential hotspots for gene family expansion [56,57,58].
The evolutionary expansion of gene families constitutes a fundamental mechanism for environmental adaptation in plants [59,60,61]. Our study reveals that the ERF subfamily within the AP2/ERF transcription factor family of apricot has undergone a distinctive expansion, characterized by frequent segmental and tandem duplication events. Genomic analyses identified 14 segmental duplications (out of 25 total) and 10 tandem duplications (out of 17 total) within the ERF subfamily. Notably, genes forming physical clusters exhibit complex expression patterns. For instance, PaERF1, PaERF2, and PaERF3 display divergent expression profiles, while the tandemly duplicated PaDREB genes show functional redundancy: PaDREB20, PaDREB21, and PaDREB24 were specifically expressed in young leaves, whereas PaDREB22 and PaDREB23 remain transcriptionally silent in all test samples. These results demonstrate that tandem duplication provides both genetic redundancy for developmental robustness and functional diversity for specialized environmental responses.
Comparative synteny analysis elucidates evolutionary connections among species while revealing how genomic architecture shapes biological function [62]. Our analysis identified 110 syntenic AP2/ERF gene pairs between apricot and Arabidopsis, and 147 pairs between apricot and peach (Figure 4B and Table S6), demonstrating conserved evolutionary trajectories of this superfamily across these species. Notably, 29 apricot AP2/ERF genes exhibited one-to-one synteny with Arabidopsis homologs, while 31 genes showed one-to-multiple relationships—including 17 (1:2), 10 (1:3), 3 (1:4), and 1 (1:5) gene pairs (Figure 4B). This asymmetry implies differential expansion rates following species divergence [62], with Arabidopsis undergoing more frequent gene duplications than apricot—a pattern potentially linked to the shorter life cycle of the former. Given that evolution typically preserves ortholog function [39], these syntenic relationships establish a robust framework for functional annotation of apricot AP2/ERF genes based on characterized Arabidopsis orthologs. The increased synteny conservation (including predominant 1:1 orthologs) in the apricot–peach comparison reflects their closer phylogenetic affinity relative to apricot–Arabidopsis pairs.
Cis-regulatory elements in promoters are key modulators of metabolic gene expression [39]. Numerous characterized cis-elements participate in hormonal signaling and stress-responsive gene regulation [63]. Comprehensive cis-element analysis of apricot AP2/ERF promoters demonstrated significant enrichment of diverse regulatory elements, including hormone-responsive motifs (auxin, GA, and ABA), stress adaptation elements, and tissue-specific regulators. Notably, five AP2/ERF genes that harbor endosperm expression or seed-specific regulation elements were specifically expressed in apricot kernels, indicating their potential functions in kernel development. Emerging evidence shows tissue-specific promoters mediate stronger gene expression than the constitutive 35S promoter [30,64]. Recent studies have demonstrated that the seed-specific PsOLE1 promoter from Prunus sibirica L. exhibits stronger transcriptional activity than the 35S constitutive promoter during late seed maturation stages [64]. Selecting optimal promoters to regulate lipid-related genes is critical in oil metabolic engineering. Employing the FUS3 promoter to control AtWRI1 expression represents an effective strategy for enhancing seed oil production by prolonging the accumulation phase during mid-seed development [65]. These findings suggest that future applications utilizing seed-specific promoters to drive expression of the key lipid biosynthesis genes and regulators could enable precise spatial-temporal control of oil accumulation specifically in developing apricot kernels. Importantly, ABA-responsive elements (ABREs) were overrepresented (89%) in DREB subfamily promoters, correlating with their stress-induced expression patterns [66]. Furthermore, auxin- and GA-responsive elements in flesh-specific AP2/ERFs (PaDREB8, PaDREB11, and PaDREB36) exhibited developmental stage-matched expression profiles during fruit development, recapitulating conserved hormonal regulation mechanisms observed in peach [67,68,69]. These findings collectively establish the molecular basis for AP2/ERF-mediated regulation of specialized metabolic and developmental processes in apricot.
The temporospatial characteristics of gene expression reflect their functions in different tissues and different developmental stages of plants. The functional diversification of AP2/ERF genes in apricot is evident from their varied expression patterns and predicted regulatory roles. The RAV subfamily genes represent very low expression levels, except PaRAV1, which had a much higher expression level in young leaves than in other samples. Previous study found that overexpression of RAV1 in Arabidopsis delays flowering, causes rosette leaf development to slow, and induces leaf senescence [70,71]. Thus, we speculate that PaRAV1 may play an important role in apricot leaves. Most of the AP2 subfamily had a higher expression level in the kernel than in other tissues, which is consistent with the important roles of AP2 in seed development and yield of Arabidopsis [22]. Most of the ERF and DREB subfamilies were found to have a higher expression level in leaf and flesh than in other samples. A previous study reported that PsERF1 played important roles in plum (Prunus salicina L.) fruit ripening [21]. Thus, we speculated that those ERFs with high transcriptional activity in flesh might be related to fruit development and the ripening of apricot. By targeting DRE/CRT motifs in promoter regions, DREB proteins modulate the expression of stress-responsive genes to play a pivotal role in ABA-mediated pathways, dehydration tolerance, and chilling stress responses [48]. More stress-related experiments should be performed to illustrate DREBs’ roles in apricot.
WRI1, as a member of the AP2 subfamily, was reported to play a pivotal role in plant oil biosynthesis in direct or indirect ways [72,73]. In apricot, the oil accumulated mainly from 50 DAF (when the kernel coat size reached the maximum) to 90 DAF (mature stage), along with seed maturation and dehydration (Figure S1). The expression of PaWRI1 was specifically activated in the kernel and positively correlated with apricot oil accumulation, indicating its potential role in lipid biosynthesis and kernel development in apricot. PsWRI1 was confirmed to orchestrate the regulation of carbon partitioning for oleic acid accumulation in P. sibirica kernel, based on transcriptome analysis and a transient co-transformation experiment [74]. These authors found that PsWRI1 could specifically up-regulate the promoter activity of stearoyl-ACP desaturase (SAD), phosphoenolpyruvate carboxykinase (PEPCK), and biotin synthase (BS). In this study, we predicted 47 target lipid-related genes of PaWRI1 on a whole-genomic scale, spanning the entire three-stage process of plant oil synthesis. These 47 target lipid-related genes include key genes, such as diacylglycerol acyltransferase (DGAT), Stearoyl-ACP Desaturase (SAD), and oleosin, and confirmed Ababidopsis genes, such as pyruvate kinase beta subunit 1 (PKP-β1), biotin carboxyl carrier protein (BCCP2; encoding a subunit of the ACCase), and BIOTIN ATTACHMENT DOMAIN-CONTAINING (BADC) [75]. Enhancing vegetable oil production by manipulating transcription factors has shown greater efficacy compared to strategies centered on individual enzymes [63,64,65]. This represents a strategic advantage for engineered lipid biosynthesis: once a stable genetic transformation system for apricot is established, we can employ gene editing technology to upregulate PaWRI1 expression, thereby increasing the kernel oil content of apricot. What’s more, four AP2/ERF genes specifically expressed in kernels were co-expressed with PaWRI1, among which PaDREB12 is the homolog of AtABI4. In Arabidopsis, abi4 mutants decreased sensitivity to ABA inhibition of germination and altered seed-specific gene expression [28]. These results indicated their potential synergistic role in the apricot kernel developmental network. Further experiments will be needed to verify our results and establish the WRI1-mediated oil biosynthesis and kernel development network in the apricot kernel.

5. Conclusions

In this study, we identified 118 putative AP2/ERF transcription factors in apricot, which were divided into four distinct subfamilies (20 AP2s, 37 DREBs, 55 ERFs, and 5 RAVs) and one Soloist. The clustered PaAP2/ERFs exhibited comparable gene architectures and conserved motif patterns, reinforcing the phylogenetic associations observed. The PaAP2/ERF genes displayed uneven chromosomal distribution, with expansion driven by both segmental (25 events) and tandem (17 events) duplications. In total, 25 AP2/ERF genes were tissue-specifically expressed in apricot. Based on co-expression analysis and AW-box scanning, 47 lipid-related genes were predicted as the potential targets of PaWRI1, which is pivotal in plant oil biosynthesis regulation. This study delivers the first chromosome-scale genomic annotation of the PaAP2/ERF family, establishing a foundation for elucidating the functional roles of critical PaAP2/ERF members in apricot lipid metabolism.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16081353/s1. Table S1: Primers used in this study. Table S2: Correlation between the expression level of PaWRI1 and lipid-related genes. Table S3: Characteristics of apricot (Prunus armeniaca L.) AP2/ERF transcription factor superfamily. Table S4: Summary of the AP2/ERF family in apricot, Arabidopsis thaliana (L.) Heynh., and peach (Prunus persica (L.) Batsch). Table S5: Duplication events of apricot AP2/ERF genes. Table S6: Synteny gene pairs of apricot vs Arabidopsis and peach. Table S7: GO enrichment of AP2/ERF transcription factors. Table S8: Identification of co-expressed AP2/ERF genes with PaWRI1. Figure S1: Oil content of apricot kernels at different developmental stages. K1, K2, K3, K4, and K5 refer to 50, 60, 70, 80, and 90 days after flowering.

Author Contributions

Conceptualization, H.L.; methodology, N.J.; software, Y.H. and X.S.; validation, Y.H.; formal analysis, L.W. and N.J.; investigation, H.L.; resources, H.L.; writing—original draft preparation, Y.H., N.J., and L.W.; writing—review and editing, N.J., L.W., T.W., and H.L.; visualization, D.Z.; project administration, T.W. and D.Z.; funding acquisition, H.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the central non-profit research institution of the Chinese Academy of Forestry, grant number CAFYBB2021MA008, and the National Natural Science Foundation of China, grant number 32101562.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The evolutionary relationships among AP2/ERF family members were reconstructed through maximum likelihood (ML) analysis with IQ-TREE software (v2.0.4), with graphical representation generated using iTOL. Color-coded clusters distinguish the five phylogenetic groups, including four major subfamilies and Soloist members.
Figure 1. The evolutionary relationships among AP2/ERF family members were reconstructed through maximum likelihood (ML) analysis with IQ-TREE software (v2.0.4), with graphical representation generated using iTOL. Color-coded clusters distinguish the five phylogenetic groups, including four major subfamilies and Soloist members.
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Figure 2. Structural characterization of apricot (Prunus armeniaca L.) AP2/ERF genes. (A) Gene architecture showing exon (yellow boxes), intron (black lines), and UTR (green boxes) organization. Gene labels are color-coded according to phylogenetic classification (four subfamilies plus Soloist). (B) Protein motif composition, with 20 distinct motifs represented by colored segments. (C) Domain architecture, illustrating six conserved domains through color-coded annotations. (D) Predicted cis-element in apricot AP2/ERF genes. The 2.0 kb promoter sequences were subjected to PlantCARE for cis-element analysis, with visualization implemented in TBtools. In the resulting schematic, eighteen distinct color-coded segments along the promoter lines represent eighteen functional categories of cis-regulatory elements.
Figure 2. Structural characterization of apricot (Prunus armeniaca L.) AP2/ERF genes. (A) Gene architecture showing exon (yellow boxes), intron (black lines), and UTR (green boxes) organization. Gene labels are color-coded according to phylogenetic classification (four subfamilies plus Soloist). (B) Protein motif composition, with 20 distinct motifs represented by colored segments. (C) Domain architecture, illustrating six conserved domains through color-coded annotations. (D) Predicted cis-element in apricot AP2/ERF genes. The 2.0 kb promoter sequences were subjected to PlantCARE for cis-element analysis, with visualization implemented in TBtools. In the resulting schematic, eighteen distinct color-coded segments along the promoter lines represent eighteen functional categories of cis-regulatory elements.
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Figure 3. The chromosomal distribution of AP2/ERF genes across all eight apricot chromosomes was visualized using TBtools’ Gene Location Visualize (GTF/GFF) and Gene Density Profile. Gene names are indicated on the right side of each chromosome.
Figure 3. The chromosomal distribution of AP2/ERF genes across all eight apricot chromosomes was visualized using TBtools’ Gene Location Visualize (GTF/GFF) and Gene Density Profile. Gene names are indicated on the right side of each chromosome.
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Figure 4. Comparative synteny analysis of AP2/ERF transcription factors in apricot. (A) Intra-genomic synteny of AP2/ERF genes. (B) Inter-genomic synteny among apricot, Arabidopsis thaliana (L.) Heynh., and peach (Prunus persica (L.) Batsch). MCScanX was used for duplication detection and TBtools for visualization, with gray and red lines representing segmental duplications and syntenic AP2/ERF pairs, respectively.
Figure 4. Comparative synteny analysis of AP2/ERF transcription factors in apricot. (A) Intra-genomic synteny of AP2/ERF genes. (B) Inter-genomic synteny among apricot, Arabidopsis thaliana (L.) Heynh., and peach (Prunus persica (L.) Batsch). MCScanX was used for duplication detection and TBtools for visualization, with gray and red lines representing segmental duplications and syntenic AP2/ERF pairs, respectively.
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Figure 5. Functional annotation of apricot AP2/ERF proteins was performed through Gene Ontology (GO) classification. The eggNOG-mapper tool was employed for gene function annotation, followed by GO enrichment analysis conducted with TBtools. The color-coded bars represent distinct GO categories: biological process (blue), cellular component (orange), and molecular function (green).
Figure 5. Functional annotation of apricot AP2/ERF proteins was performed through Gene Ontology (GO) classification. The eggNOG-mapper tool was employed for gene function annotation, followed by GO enrichment analysis conducted with TBtools. The color-coded bars represent distinct GO categories: biological process (blue), cellular component (orange), and molecular function (green).
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Figure 6. Expression profiles of apricot AP2/ERF genes in kernel, fruit, flower, flower bud, and young leaf tissues. (A) The heatmap illustrates Z-score-transformed FPKM values. Cluster analysis was performed based on expression trends and displayed in the circular map, grouping genes with similar patterns. K1–K5: Developing kernels at sequential stages; F1–F8: Fruit flesh samples representing progressive developmental phases; FL: Mature flower; FB: Flower bud. Data visualization was performed using R with normalized FPKM values. Flower, leaf, kernel, and fruit icons adjacent to gene names denote the tissue-specific expression of corresponding genes. (B–G) RT-qPCR validation of the expression levels of AP2/ERF genes in the different developmental stages of apricot kernels (Blue bars and left Y-axis). Error bars represent the means ± SE (n = 3). The FPKM values of the corresponding genes are also shown (orange lines and Y-axis on the right).
Figure 6. Expression profiles of apricot AP2/ERF genes in kernel, fruit, flower, flower bud, and young leaf tissues. (A) The heatmap illustrates Z-score-transformed FPKM values. Cluster analysis was performed based on expression trends and displayed in the circular map, grouping genes with similar patterns. K1–K5: Developing kernels at sequential stages; F1–F8: Fruit flesh samples representing progressive developmental phases; FL: Mature flower; FB: Flower bud. Data visualization was performed using R with normalized FPKM values. Flower, leaf, kernel, and fruit icons adjacent to gene names denote the tissue-specific expression of corresponding genes. (B–G) RT-qPCR validation of the expression levels of AP2/ERF genes in the different developmental stages of apricot kernels (Blue bars and left Y-axis). Error bars represent the means ± SE (n = 3). The FPKM values of the corresponding genes are also shown (orange lines and Y-axis on the right).
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MDPI and ACS Style

He, Y.; Wang, L.; Jiang, N.; Zhang, D.; Shi, X.; Wuyun, T.; Liu, H. Genome-Wide Identification and Expression Profiling of AP2/ERF Transcription Factor Genes in Prunus armeniaca L. Forests 2025, 16, 1353. https://doi.org/10.3390/f16081353

AMA Style

He Y, Wang L, Jiang N, Zhang D, Shi X, Wuyun T, Liu H. Genome-Wide Identification and Expression Profiling of AP2/ERF Transcription Factor Genes in Prunus armeniaca L. Forests. 2025; 16(8):1353. https://doi.org/10.3390/f16081353

Chicago/Turabian Style

He, Yanguang, Lin Wang, Nan Jiang, Donglin Zhang, Xiaodan Shi, Tana Wuyun, and Huimin Liu. 2025. "Genome-Wide Identification and Expression Profiling of AP2/ERF Transcription Factor Genes in Prunus armeniaca L." Forests 16, no. 8: 1353. https://doi.org/10.3390/f16081353

APA Style

He, Y., Wang, L., Jiang, N., Zhang, D., Shi, X., Wuyun, T., & Liu, H. (2025). Genome-Wide Identification and Expression Profiling of AP2/ERF Transcription Factor Genes in Prunus armeniaca L. Forests, 16(8), 1353. https://doi.org/10.3390/f16081353

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