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. 2026 Jan 30;26:373. doi: 10.1186/s12870-026-08181-x

Genome-wide identification of the AP2/ERF gene family and its alternative splicing to reveal their expression profiles in different tissues of Rosa roxburghii

Yani Chen 1,4,#, Xueqi Li 1,4,#, Shize Li 1,3,4, Xinyue Li 1,4, Guangqi Gong 1,4, Tingting Jing 2,✉, Yanlin An 1,4,✉
PMCID: PMC12930606  PMID: 41612186

Abstract

Background

The AP2/ERF is one of the largest gene families in plants, which plays an important role in plant growth and development and resistance to stresses. However, the quantity, evolutionary characteristics and expression characteristics in different tissues of this gene family in Rosa roxburghii are still unknown.

Results

In this study, 106 members of the AP2/ERF gene family were identified in the genome of Rosa roxburghii. These members are unevenly distributed on seven chromosomes, and most of them are predicted to be located in the nucleus. Phylogenetic analysis reveals that all the family members are classified into four subfamilies, DREB, ERF, AP2 and RAV with their numbers being 31, 57, 14 and 4 respectively. Analysis of cis-acting elements, conserved motifs, and gene structure shows that a large number of cis-acting elements related to hormones and stress are found. Furthermore, the conserved motif 1 is present in all family members, and except for the RAV subfamily, each subfamily contains unique motifs. However, the gene structure of AP2 subfamily is obviously different from the other three subfamilies, which contains more specific motifs and cds. Segmental duplication and synteny analysis reveal that the number of AP2/ERF varies greatly among Rosaceae, and segmental duplication is one of the important factors for the expansion of the AP2/ERF family in Rosa roxburghii. Furthermore, based on transcriptome data and RT-qPCR, the specific expression characteristics of AP2/ERF genes and their alternative splicing in eight different tissues of Rosa roxburghii were presented and verified.

Conclusions

In conclusion, this study comprehensively identified and analyzed the classification, evolution, and expression characteristics of the AP2/ERF family genes in Rosa roxburghii, and provided a reference for further molecular breeding of Rosa roxburghii.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08181-x.

Keywords: Rosa Roxburghii tratt, AP2/ERF gene family, Gene expression, Alternative splicing, RT-qPCR

Introduction

Rosa roxburghii (2n = 2x = 14) is a perennial plant of the Rosaceae family mainly distributed in southwest China [1]. The fruits of Rosa roxburghii contain many substances beneficial to the human body such as vitamins, amino acids, superoxide dismutase, flavonoids and polysaccharides [2, 3]. As more and more studies have proved that the fruits of Rosa roxburghii have effects such as antioxidation, enhancing immunity, protecting nerves and anti-cancer, its planting area and consumption continue to expand, and it has become an important way for farmers in southwest China to increase their incomes [4]. However, due to the lack of available reference genome data, previous studies paid less attention to the functional genes and genetic regulatory mechanisms of growth and development of Rosa roxburghii. It was not until 2023 that the first high-quality reference genome of Rosa roxburghii was released. Zong et al. [1] comprehensively utilized multiple sequencing technologies to assemble a Rosa roxburghii genome with a size of 504 Mb and identified 40,020 protein-coding genes, all of which were mainly anchored to 7 chromosomes. Subsequently, Yang et al. [5] assembled the genome of ‘Guinong 5’ Rosa roxburghii, obtained a genome with a size of 531 Mb, containing 45,226 coding genes, and found a key enzyme GDH related to L-ascorbic acid biosynthesis.

The release of above genomes provides important support for in-depth research on the functional genes and molecular breeding of Rosa roxburghii. For example, Yin et al. [6] identified 41 calmodulin-like proteins (CMLs) in Rosa roxburghii genome, and found that the overexpression of RrCML13 in fruits and roots significantly promoted the transcription of RrGGP2 and the accumulation of AsA, and further confirmed the interaction between RrCML13 and RrGGP2 proteins; Qin et al. [7] identified 38 members of HD-Zip gene family in the genome of ‘Guinong 5’ Rosa roxburghii, and these genes were divided into 4 subfamilies and proved to be involved in regulating the development of trichomes. However, compared with other plants, many genes or gene families that may have important functions in Rosa roxburghii need to be further identified [8–13].

The AP2/ERF is a superfamily of transcription factors widely present in plants. Based on the number of domains and binding sequences of AP2/ERF transcription factors, they can be divided into 5 subfamilies: AP2, ERF, DREB, RAV, and Soloist [14, 15]. The AP2 subfamily contains two highly conserved and tandemly repeated AP2/ERF domains. The ERF and DREB subfamilies each contain only one AP2/ERF domain, with the difference lying in the amino acid residues of the conserved sequence in this domain: the 14th and 19th positions of the ERF subfamily are alanine and aspartic acid, respectively, while those of the DREB subfamily are valine and glutamic acid, respectively. The RAV subfamily contains one AP2/ERF domain and one B3 domain, where the AP2 domain is located at the N-terminus and the B3 domain at the C-terminus [16]. The Soloist subfamily also contains one AP2/ERF domain, but its amino acid motifs and gene structure differ significantly from those of transcription factors in other subfamilies. In addition, the number of such transcription factors is small, and there have been few studies on the Soloist subfamily transcription factors so far [17–20].

The protein members of this family are closely related to plant growth and stress response. Studies in tomato have shown that ERF2 plays a key role in multiple signaling pathways such as SA, JA and ROS. It may directly or indirectly regulate the expression of Pto, PR1b1 and PR-P2 and enhance tomato resistance to Stemphylium lycopersici [21]; In Tritipyrum, a gene TtERF_B2-50 that is highly expressed in the roots, stems, and leaves and is highly sensitive to salt stress has been identified [22]; Xing et al. [23] identified 163 AP2/ERF genes in ginger and found that they played an important role in regulating the development of ginger rhizome and flower; Liu et al. [24] identified nine candidate CsAP2/ERFs genes related to the spring bud break of tea plants; while studies in Chinese prickly ash show that ZbERF13.2, ZbRAP2-12, and ZbERF2.1 showed high levels of expression in the early stages of fruit development, ZbRAP2-4 and ZbERF3.1 were significantly expressed at the fruit coloring stage, ZbERF16 was significantly expressed at fruit ripening and the expression level increased as the fruit continued to develop. Moreover, SlAP2a has been confirmed to be a negative regulator of tomato fruit ripening; interestingly, LeERF2 promotes tomato fruit ripening by guiding ethylene synthesis [25, 26]. These results indicate that the AP2/ERF gene family in plants plays important roles in regulating organ development and stress resistance. Currently, Rosa roxburghii faces various stresses such as extreme high temperature, drought, and diseases during its vigorous growth period. Revealing the quantitative characteristics of the AP2/ERF family in Rosa roxburghii and their expression profiles during fruit development is of great significance for Rosa roxburghii breeding.

In this study, we first conducted a comprehensive identification of the members of the AP2/ERF gene family in the Rosa roxburghii genome and revealed their distribution information on chromosomes and physicochemical characteristics such as isoelectric points and amino acid numbers. Meantime, based on the phylogenetic tree, the members of the AP2/ERF gene family in Rosa roxburghii were classified, and the conserved motifs, cis-acting elements, and gene structures were analyzed. Moreover, gene duplication and synteny events were revealed. In addition, transcriptome analysis and RT-qPCR revealed the expression characteristics of the AP2/ERF gene family in eight tissues of Rosa roxburghii, and verified their alternative splicing. This study provides new insights into the role of AP2/ERF in the tissue development of Rosa roxburghii and will help to deeply reveal the diverse expression characteristics and molecular functions of different AP2/ERF family members.

Materials and methods

Plant materials

Eight Rosa roxburghii samples including FL1 (Flower 1, flower bud), FL2 (Flower 2, flower), LF (Leaf, mature leaf), FR1 (Fruit 1, fruits that have just started to develop after flowering), FR2 (Fruit 2, fruits that continue to develop after the F1 stage), FR3 (Fruit 3, fruits in the rapid-growth period), FR4 (Fruit 4, pre-maturity stage of fruits), and FR5 (Fruit 5, fruits at the mature stage) were collected from Renhuai City, Guizhou Province (E:106.38、N:27.85). Samples were collected from May to August, 2023, and the collected samples were immediately transported to the laboratory with dry ice and placed in the refrigerator at -80℃. All samples contained three biological repeats. Voucher specimens for all plant species were deposited in the herbarium of Moutai Institute under the accession numbers PE-2024-001. The specimens were identified by Xiaoqin Zhang. The plant sample collection complied with relevant institutional, national, and international guidelines and legislation. Permission was obtained from the land owner prior to sample collection [27].

Identification of the AP2/ERF family in Rosa roxburghii

Before identifying the AP2/ERF gene family, the relevant sequences of Rosa roxburghii genome were downloaded from the CNGB Nucleotide Sequence Archive database (https://db.cngb.org/cnsa/, accession CNP0004212) [1, 27]. Subsequently, the Hidden Markov Model (HMM) profile of the AP2/ERF family (PF00847) was downloaded from the InterPro database (https://www.ebi.ac.uk/interpro/download/Pfam/) [22]. Then, the HMMER software (v3.3.2, http://hmmer.org/download.html) was used to conduct a preliminary identification of the members of the AP2/ERF gene family in the Rosa roxburghii genome (the e-value was set to 1e-10), and the NCBI Batch CD-Search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and PlantTFDB (Plant Transcription Factor Database, https://planttfdb.gao-lab.org/) were used to further verify all genes [8, 28]. Meanwhile, the members of the AP2/ERF family were classified by combining protein domains with phylogenetic trees [29].

Chromosome distribution and sequence analysis

The location information of each AP2/ERF gene family member is obtained from the gff file matched with the genome, and then used the MG2C online software (http://mg2c.iask.in/mg2c_v2.1/) to visualize its chromosomal distribution characteristics [10]. The ExPasy website (https://web.expasy.org/) was used to predict the molecular weight (MW), number of amino acids and isoelectric point (pI) of all AP2/ERF proteins [30]. The subcellular localization results were predicted by the WoLF PSORT (https://wolfpsort.hgc.jp/) website [6]. At the same time, after extracting the DNA sequence of the upstream 2000 bp before the promoter of the gene family members, these sequences were submitted to the Plant CARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for analysis of cis-regulatory elements [9, 31]. In addition, the conserved motifs and gene structures of the members of the AP2/ERF gene family were identified and visualized using TBtools under the default parameters [32].

Phylogenetic tree construction, gene duplication and synteny analysis

To construct a reliable phylogenetic tree, we first downloaded the protein sequences of all AP2/ERF family members of Arabidopsis thaliana 10 from TAIR (https://www.arabidopsis.org/) and then constructed an NJ phylogenetic tree (the bootstrap replicates value was set to 1000) using MEGA 11 (https://www.megasoftware.net/) [18]. Further, the genomes and related annotation files of Arabidopsis thaliana, peach tree (CN14, https://www.rosaceae.org/Analysis/13087664), plum tree (Zhongli No.6, https://www.rosaceae.org/Analysis/9019655) and apple (GDDH13, https://iris.angers.inra.fr/gddh13/the-apple-genome-downloads.html) were downloaded. To determine the types of gene duplication and reveal the evolutionary characteristics of the gene family, gene duplication and synteny analysis were performed using TBtools software (v2.3, https://github.com/CJ-Chen/TBtools-II/releases) under default parameters [13, 33, 34]. In addition, the Ka/Ks values of the members of the AP2/ERF family are also calculated using TBtools software, and the relevant results were drawn through its built-in “Advanced Circos” or “Dual Systeny Plot for MCScanX” plugins. Meanwhile, the protein-protein interaction network was constructed using the STRING online tool (https://cn.string-db.org/).

Expression pattern analysis and alternative splicing identification

The transcriptome data of eight tissues (FL1, FL2, LF, FR1, FR2, FR3, FR4, and FR5) of Rosa roxburghii were downloaded from the China National Center for Bioinformation/Beijing Institute of Genomics (GSA: CRA017453, https://ngdc.cncb.ac.cn/gsa). Raw transcriptome sequencing data filtering and gene expression level analysis were performed with reference to our previous studies [27]. Based on the gene expression levels, the series test of cluster was performed using the Omicshare (https://www.omicshare.com/) online toolset to explain the expression patterns of the AP2/ERF family in Rosa roxburghii. To identify alternative splicing variants of the AP2/ERF gene family, all new transcripts were retained when calculating gene expression levels. Then, the generated gtf file was imported into IGV software (v 2.19.5, https://igv.org/doc/desktop/#DownloadPage/) to screen the splicing variants of the AP2/ERF gene family, and genes with a transcript number greater than 2 were considered as candidate alternatively spliced genes. To increase the reliability of the results, the remaining genes and their corresponding transcripts are treated as alternatively spliced after the transcripts with TPM values less than 1 in each set of samples are filtered out [35].

RNA extraction and real-time quantitative PCR (RT-qPCR) analysis

Total RNA was extracted using the Total RNA Purification Kit (cat DP441, Tiangen, China). First-strand cDNA synthesis was performed using the PrimeScript RT Reagent Kit (cat 6110 A, TaKaRa, Japan) according to the manufacturer’s instructions [36]. The primers used were designed with Primer 5 and synthesized by Sangon Biotech (Shanghai, China). Quantitative real-time PCR was performed on a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, USA) using TB Green Premix Ex Taq™ II (TaKaRa, Japan). The RT-qPCR was performed using 10 µl of PCR product, with cycling conditions referring to our previous studies, and each biological replicate contained three technical replicates [27]. And the β-actin gene was used as an internal reference gene [37]. Detailed primer sequence information was listed in Table S1.

Statistical analysis

The expression levels of AP2/ERF gene family members in different tissues of Rosa roxburghii were analyzed by one-way ANOVA Tukey’s test using SPSS 22.0 software (https://www.ibm.com/cn-zh/products/spss). The significance level was set to 0.05.

Results

Genome-wide identification of the AP2/ERF gene family

In the genome of Rosa roxburghii, a total of 106 members of the AP2/ERF gene family were successfully identified through a series of methods (Table S2). All these family members are unevenly distributed on 7 chromosomes (Fig. 1). Among them, chromosome 6 has the fewest number and chromosome 1 has the largest number, with 9 and 27 respectively. They vary widely in amino acid counts, ranging from 119 aa (Rr103810) to 830 aa (Rr600028), while MW ranges from 13.29 kDa to 90.59 kDa (Table S3). The pI value varies between 4.53 (Rr100626) and 9.95 (Rr302608). Subcellular localization prediction revealed that 79 AP2/ERF family members were localized in the nucleus, 21 were localized in the chloroplast, 3 were localized in the mitochondrion (Rr104292, Rr104069 and Rr500891), 1 was localized in the plasma (Rr106153), and 2 was localized in the vacuolar (Rr103537, Rr303797). The differences in the above characteristics suggest that there may be functional diversity.

Fig. 1.

Fig. 1

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Distribution of the AP2/ERF genes in the Rosa roxburghii genome. The left axis shows the length of each chromosome, and it was estimated in mega base (Mb)

Protein domain, phylogenetic analysis and classification of AP2/ERF family

Before constructing the phylogenetic tree and classifying the gene family, we first identified and visualized the protein domains of all members of the AP2/ERF family. The results showed that except for 14 identified family members which contain two AP2 domains, all other members contain only one AP2 domain. Additionally, B3 domains were identified in four protein sequences. Both of these domains are typical structural features of the AP2/ERF family. Furthermore, some members also contain structural features such as SOBP and XET_C (Fig. 2).

Fig. 2.

Fig. 2

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Protein sequence domain analysis of the AP2/ERF gene family

To further investigate the evolutionary relationships and classification characteristics among members of the AP2/ERF family in Rosa roxburghii, a phylogenetic tree was constructed based on the NJ method. Then, based on the similarity to the protein sequences of Arabidopsis thaliana, all AP2/ERF family members of Rosa roxburghii were divided into four subfamilies: DREB, ERF, AP2, and RAV, with their numbers being 31, 57, 14, and 4 respectively (Fig. 3). However, no members of the Soloist subfamily were found in Rosa roxburghii [38, 39].

Fig. 3.

Fig. 3

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Construction and analysis of the phylogenetic tree. An neighbor-joining phylogenetic tree was constructed based on the AP2/ERF protein sequences in the genomes of Rosa roxburghii Tratt and Arabidopsis thaliana. The branches of blue, cyan, red and green represent ERF, DREB, AP2 and RAV subfamilies respectively

Motif, gene structure and cis-acting regulatory elements analysis of the AP2/ERF gene family

Conserved motif, gene structure, and cis-acting elements located in the promoter region can affect gene expression levels and functional differentiation [40]. As shown in Fig. 3, we first performed conserved motif analysis on the protein sequences of all Rosa roxburghii AP2/ERF families and found a total of 10 different types of motifs. Among them, motif 1 is highly conserved and shared by all members of the AP2/ERF gene family. motif 9 only exists in the DREB subfamily; Motifs 3, 4, and 7 only exist in the AP2 subfamily; motifs 6 and 8 only exist in the ERF subfamily. The RAV subfamily is the most special, as it contains only one motif 1 (Fig. 4A). Overall, the number of motif per AP2/ERF protein ranged from 1 to 7 (Rr501814, Rr501818), and the AP2 subfamily has the largest average number of motifs. Meantime, the 2000 bp upstream sequence of the start codon was used as the promoter region of the gene for cis-acting element analysis. In addition to the large number of light-responsive elements, the cis-acting elements related to hormonal response and stress response are mainly shown in Fig. 4B. Among these elements located in the promoter region, there are a large number of elements related to SA, ABA, GA and MeJA responses, indicating that these AP2/ERF family members may play an important role in regulating the growth and development of Rosa roxburghii. In previous studies, it has been proven that AP2/ERF has multiple stress resistance functions [22, 41]. In the promoter region of the above genes, some cis-acting elements related to low temperature and mixed stress have also been found, which may endow them with the function of participating in stress responses. Further gene structure analysis showed that most of the AP2/ERF gene families contained 1–4 cds, but the AP2 subfamily was significantly different from the DREB, RAV and ERF subfamilies, and many of its members contained more than 7 cds (such as Rr601642, Rr205264) (Fig. 4C). These results provide an important basis for further understanding the function of AP2/ERF gene family in Rosa roxburghii.

Fig. 4.

Fig. 4

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Motif, gene structure and cis-acting regulatory elements analysis of the AP2/ERF gene family. A Protein motifs of AP2/ERF genes. Ten conservative motifs are shown in the figure. B Cis-acting regulatory elements analysis of the AP2/ERF genes. Sequences of the 2000 bp above the start codon were used to identify cis-acting elements. C Gene structure of AP2/ERF genes. In different subgraphs, the abscissa represents the length of the gene sequence, with the unit being bp

Segmental duplication genes and synteny analysis of AP2/ERF gene family

Segmental duplication events are one of the important driving forces for genome expansion [8]. Here, 15 pairs of segmental duplication genes were identified in the AP2/ERF gene family of Rosa roxburghii (Table S4). As shown in Fig. 5A, among these gene family members, only the Rr100358 gene forms two gene pairs distributed on different chromosomes (Rr100358 vs. Rr104292, Rr100358 vs. Rr204331). At the same time, the Ka/Ks analysis results show that all Ka/Ks ratios are less than 1, indicating that these genes have experienced purifying selection or negative selection during the evolutionary process (Table S5). To further investigate the evolutionary characteristics of the AP2/ERF gene family in Rosaceae plants, collinearity analysis of Rosa roxburghii vs. Arabidopsis, Rosa roxburghii vs. Prunus persica, Rosa roxburghii vs. Prunus salicina, and Rosa roxburghii vs. Malus pumila was also performed (Fig. 5B). A total of 85, 113, 110 and 215 syntenic gene pairs were identified between the four genome pairs mentioned above. Many AP2/ERF family members of Rosa roxburghii have multiple corresponding relationships with peach trees, plums and apples. In addition, it is obvious that the members of AP2/ERF gene family in apple have expanded significantly, which indicates that Rosa roxburghii has a closer genetic relationship with peach trees and plum trees.

Fig. 5.

Fig. 5

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Segmental duplication and synteny analysis. A Circle plot showing collinearity of AP2/ERF gene family in Rosa roxburghii Tratt. Collinearity genes were highlighted with red curved lines. B Synteny analysis of AP2/ERF genes from Arabidopsis, Prunus persica, Prunus salicina and Malus pumila

Analysis of gene expression patterns and protein-protein interactions

In order to investigate the functional characteristics of AP2/ERF in different tissues of Rosa roxburghii, we first performed expression level analysis of all gene family members based on transcriptome. After excluding the unexpressed genes, the overall expression characteristics are shown in the form of a heat map in Fig. 6A and Table S6. The results showed that the expression of many AP2/ERF genes was tissue-specific. At the same time, we hypothesized 8 gene expression patterns (from profile 0 to profile 7). The dynamic analysis results prove that profile 0 and profile 6 are significantly enriched, indicating that these two expression trend characteristics have broader representativeness. In addition, the interaction relationship among members of the AP2/ERF gene family was predicted. The results showed that there may be interactions among many members of the AP2/ERF gene family, Rr601642 and Rr305139, Rr702533 and Rr701339 are the potential core of these interactions, indicating the diversification of its functions (Fig. 6B, C). Meanwhile, correlation analysis based on gene expression levels also demonstrates that members of the AP2/ERF gene family have extensive correlations in Rosa roxburghii (Fig. 6D).

Fig. 6.

Fig. 6

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Transcriptome-based expression level analysis and protein interaction of AP2/ERF gene family. A Heatmap of the expression levels of the AP2/ERF gene family. B Protein-protein interaction network among members of the AP2/ERF gene family. C Analysis of the expression trend of AP2/ERF genes, where the red profile indicates that P value < 0.05. D Correlation analysis of expression levels among AP2/ERF gene family members

To further reveal the expression characteristics of the AP2/ERF gene family in different tissues of Rosa roxburghii, we analyzed in detail the significant differences of highly expressed genes. As shown in Fig. 7, the expression levels of many genes in different tissues of Rosa roxburghii were significantly different. For example, the expression levels of genes such as Rr101000, Rr205567, Rr500891 and Rr703591 in leaves were significantly higher than those in other tissues; while the expression levels of Rr103537, Rr205389, Rr306181, Rr500954, Rr601984 and Rr704562 were significantly higher in FL2. Meantime, some genes such as Rr100626, Rr106153, Rr204002, Rr203451, Rr300733, Rr400515, Rr203451, etc. show stage-wise high expression in fruits, indicating that these genes may be involved in regulating the development of Rosa roxburghii fruits.

Fig. 7.

Fig. 7

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The expression characteristics of AP2/ERF gene family in eight tissues of Rosa roxburghii were analyzed based on transcriptome sequencing. Different letters above the bars represent significant differences at p < 0.05 by Tukey’s test

Identification of alternative splicing isoforms of the AP2/ERF family

Alternative splicing is an important mechanism for increasing the complexity of the transcriptome and proteome. Through splicing the gene transcription products in different ways, multiple mRNA isoforms can be generated, and then proteins with different functions can be translated [35, 40]. Many studies have indicated that alternative splicing events can regulate the synthesis of plant secondary metabolites and the growth and development processes of plants [42, 43]. Among the members of the AP2/ERF family in Rosa roxburghii, two genes have undergone alternative splicing. For Rr305193, intron retention events occurred, while for the two splicing isoforms of Rr701339, intron retention and exon skipping occurred respectively (Fig. 8). Meanwhile, transcriptome analysis shows that the expression trends among different alternative splicing isoforms of the same gene are highly consistent. Detailed sequencing results have been listed in Figure S1.

Fig. 8.

Fig. 8

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Alternative splicing events identified in the AP2/ERF gene family

RT-qPCR verified the expression level of AP2/ERF gene family

To further validate the accuracy of the expression levels obtained based on transcriptome data, 8 genes with higher expression levels were selected for RT-qPCR analysis (Table S7). As shown in Fig. 9, the expression characteristics of these genes are highly consistent with the transcriptome results. For example, genes Rr205389, Rr403648, and Rr704562 are all highly expressed in FL2. This result implies that the expression level obtained based on transcriptome data has high reliability. In addition, because quantitative primers for alternative splicing can only be designed at splicing sites (exon-exon junction sites), it is difficult to find suitable specific primers.

Fig. 9.

Fig. 9

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Expression of AP2/ERF genes analyzed by RT-qPCR in different tissues of Rosa roxburghii. Different letters above the bars represent significant differences at p < 0.05

Discussion

The AP2/ERF is one of the largest gene families in plants. Studies in many plants such as Arabidopsis and rice have proven that its family members have the functions of regulating plant morphogenesis, stress responses, hormone signal transduction, and metabolite biosynthesis [38]. In this study, we identified 106 members of the AP2/ERF gene family from the genome of Rosa roxburghii, which are unevenly distributed across 7 chromosomes. These family members were divided into four subfamilies: DREB, ERF, AP2, and RAV, with the numbers being 31, 57, 14, and 4 respectively (Figs. 1, 2 and 3). However, the number in Rosa roxburghii is significantly less than that in Arabidopsis (143), maize (214), and wheat (576), and it is also fewer than that in plants of the Rosaceae family such as apple (259), Pyrus pyrifolia (234), and peach (131) [18, 44–46]. The reason for such results may be, on the one hand, that we adopted more stringent identification criteria (all identified members must pass the triple verification of HMM and CDD/PlantTFDB); on the other hand, it may be closely related to the quality of genome annotation. Meanwhile, it can be clearly seen from the phylogenetic tree that no Soloist subfamily was identified in Rosa roxburghii. This result is similar to the identification results in pear and peach trees, reflecting that the Soloist may have been lost in many plants of the Rosaceae family [44, 45, 47].

The function and expression level of genes are multiply influenced by gene structure, cis-acting elements and conserved motifs. For example, the insertion of a 181 bp transposon in the promoter region of CsMYB75 promotes a large accumulation of anthocyanins and is a determinant of the color change of tea leaves from green to purple [48]. Meanwhile, the W-box and G-box elements play an important role in the premature senescence of the flag leaf of rice [49]. For the four subfamilies of AP2/ERF in Rosa roxburghii, each subfamily has unique motifs identified. For example, motif 9 is only found in the DREB subfamily, motif 6 and motif 8 are only identified in the ERF subfamily, while motif 3, 4, and 7 are only found in the AP2 subfamily (Fig. 4). Meanwhile, factors such as differences in the number and types of cis-acting elements on the promoter, as well as significant variations in gene structure, have collectively shaped the functional roles and expression profiles of AP2/ERF family members in Rosa roxburghii. However, compared with apple and sand pear (Pyrus pyrifolia), the AP2/ERF family in Rosa roxburghii and peach has not undergone significant expansion (Fig. 5). Furthermore, Ka/Ks analysis revealed that the AP2/ERF family in Rosa roxburghii has obviously experienced strong negative selection. This may be due to the fact that Rosa roxburghii has long been in a wild state and has undergone little artificial domestication [50].

Additionally, although the AP2/ERF has been proven to have multiple functional roles in plants, its expression characteristics in different tissues of Rosa roxburghii have not been revealed yet [51, 52]. Therefore, we first analyzed the expression levels of the AP2/ERF gene family in eight tissues including the flower, leaf and fruit of Rosa roxburghii through transcriptome data. The results indicated that the expression levels of 44 family members were not detected in any of the 8 tissues, which might be caused by abnormal genome annotation, the requirement of specific conditions for expression activation, or loss of gene function; in contrast, the other 69 genes displayed tissue-specific expression characteristics. Abundant correlations, protein-protein interaction relationships, as well as diverse functional roles, result in only 43 out of these 69 genes being classified into eight expression profiles. (Fig. 6). Genes whose expression levels are not detected may be activated only in specific environments or tissues, or may have lost their functions during the evolutionary process. Many studies have demonstrated that alternative splicing events can affect the expression levels of genes and endow them with different functions. Approximately 40%~60% of intron-containing genes in plants undergo alternative splicing events [53, 54]. In the AP2/ERF gene family of Rosa roxburghii, only two genes (Rr701339 and Rr305193) were identified to have undergone alternative splicing events, and this number is far below the whole genome level (Fig. 8) [40, 42, 43]. However, it should be noted that some alternative splicing events occur only under specific stress conditions [55]. In addition, similar expression levels were exhibited among different spliceosomes of two genes. Finally, in order to verify the actual expression levels of the AP2/ERF genes in Rosa roxburghii, the expression levels of 8 genes were verified by RT-qPCR (Fig. 9). The results are highly consistent with the transcriptome results, once again demonstrating the reliability of the data in this study.

Overall, the above findings provide insight into the potential functional role of the AP2/ERF gene in Rosa roxburghii. The comprehensive analysis was helpful to screen candidate AP2/ERF genes, and lay a foundation for further functional identification and genetic improvement of agronomic traits in Rosa roxburghii. However, these findings lack more reliable functional verification; although the sequencing of alternative splicing transcripts has been completed, their expression levels have not been analyzed quantitatively. In the future research, more key functional genes should be further explored to reveal their relationship with stress resistance and fruit development of Rosa roxburghii, so as to promote the breeding research of Rosa roxburghii.

Conclusions

In this study, we conducted a comprehensive identification of the AP2/ERF family in Rosa roxburghii, and revealed the expression levels of 106 AP2/ERF family members in eight different tissues of Rosa roxburghii based on transcriptome and RT-qPCR, as well as analyzed the alternative splicing events that occurred in them. At present, research on molecular breeding of Rosa roxburghii is still in its infancy, and these research results can provide important clues for further in-depth studies on AP2/ERF. In the future, a variety of functional verifications will be carried out to demonstrate the specific functional roles of these genes in Rosa roxburghii, thereby promoting research on the functional genome of Rosa roxburghii.

Supplementary Information

Supplementary Material 1. (131.6KB, pdf)
Supplementary Material 2. (95.5KB, xls)
Supplementary Material 3. (32.5KB, xls)
Supplementary Material 4. (20KB, xls)
Supplementary Material 5. (20.5KB, xls)
Supplementary Material 6. (32.5KB, xls)
Supplementary Material 7. (34.1KB, xlsx)

Acknowledgements

We would like to express our gratitude to Teacher Xiaoqin Zhang for the assistance provided during the sample collection process.

Authors’ contributions

Y.A. and T.J.: Conceived and designed the research project. Y.C. and X.L.: Writing original draft, sample collection, and DNA and RNA extraction, visualization and data curation. S.L., X.L. and G.G.: Gene family identification, transcriptome analysis, and RT-qPCR validation. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Guizhou Province (ZK[2023]453); research Foundation for Scientific Scholars of Moutai Institute (mygccrc[2022]074, mygccrc[2022]077, mygccrc [2022]083, mygccrc [2022]097)；Guizhou Engineering Research Center for Specialty Food Resources (KY(2020)022); Science and Technology Innovation Team of Moutai Institute (MTXYTD202502).

Data availability

The raw sequence data reported in this paper were downloaded from the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA017453, https://ngdc.cncb.ac.cn/gsa).

Declarations

Ethics approval and consent to participate

Not applicable. The sampling of plant material was performed in compliance with institutional guidelines. The research conducted in this study required neither approval from an ethics committee, nor involved any human or animal subjects.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yani Chen and Xueqi Li contributed equally to this work.

Contributor Information

Tingting Jing, Email: jtt0127@163.com.

Yanlin An, Email: 17855124165@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (131.6KB, pdf)
Supplementary Material 2. (95.5KB, xls)
Supplementary Material 3. (32.5KB, xls)
Supplementary Material 4. (20KB, xls)
Supplementary Material 5. (20.5KB, xls)
Supplementary Material 6. (32.5KB, xls)
Supplementary Material 7. (34.1KB, xlsx)

Data Availability Statement

The raw sequence data reported in this paper were downloaded from the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA017453, https://ngdc.cncb.ac.cn/gsa).

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