Genome-wide identification of the AP2/ERF gene family in Rosa persica and expression profiling under abiotic stresses

Genome-wide identification of the AP2/ERF gene family in Rosa persica and expression profiling under abiotic stresses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification of the AP2/ERF gene family in Rosa persica and expression profiling under abiotic stresses Na Li, Yang Cui, Chenjie Zhang, Xiaolong Zhang, Ziguo Li, Chao Yu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7203695/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Background Rosa persica , the sole species in the subgenus Hulthemia of Rosaceae, thrives in the barren Gobi Desert, where its harsh environment has driven the evolution of exceptional stress-resistant traits, making it a valuable germplasm resource for studying extreme drought adaptation. With the recent completion of its telomere-to-telomere (T2T) genome assembly, an increasing number of stress-responsive gene families have been systematically characterized in R. persica . However, despite being one of the most critical transcription factor superfamilies involved in plant stress responses, the AP2/ERF superfamily has not yet been comprehensively identified or functionally analyzed in this extremophyte species, warranting in-depth investigation. Results Through comprehensive genome-wide analysis, we identified 114 AP2/ERF members in R. persica , which were classified into five subfamilies: AP2, DREB, ERF, RAV, and Soloist. The expression profiles of these AP2/ERF genes exhibited distinct tissue specificity. Notably, A005041.1 , A008193.1 , A013003.1 , and A014479.1 were exclusively expressed in roots, while A001403.1 and A026890.1 showed stem-specific expression. Seven genes ( A011282.1 , A010347.1 , A014504.1 , A025658.1 , A025595.1 , A002755.1 , and A024668.1 ) were simultaneously responsive to both drought and low temperature stress in R. persica . Among the ERF and DREB subfamilies, the abiotic stress-related genes A002909.1 , A014504.1 , A002755.1 , A010645.1 , and A009162.1 demonstrated transcriptional activation activity in yeast assays, whereas A005829.1 and A024668.1 lacked such activity. Conclusion This study provides the first systematic identification and characterization of the AP2/ERF gene family in R. persica , revealing key members involved in drought and cold stress responses. Our findings establish a theoretical foundation for elucidating the functional mechanisms of AP2/ERF genes in this extremophyte species and their roles in stress adaptation. Genome-wide analysis Rosa persica AP2/ERF gene family stress response Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Transcription factors precisely regulate gene expression by binding to specific cis-acting elements in target gene promoters, serving as master regulators in abiotic stress-responsive signaling networks [ 1 , 2 ]. The AP2/ER F family constitutes a unique class of plant-specific transcription factors that play essential roles in regulating plant growth/development and environmental stress responses [ 3 , 4 ]. Based on the Arabidopsis classification system [ 5 , 6 ], the AP2/ERF superfamily is categorized into five major subfamilies (AP2, DREB, ERF, RAV, and Soloist) according to their conserved domains, all of which contain at least one 60–70 amino acid AP2/ERF domain [ 7 ]. Specifically, the AP2 subfamily contains two AP2/ERF domains [ 8 ], while the RAV subfamily possesses one AP2/ERF domain along with an additional B3 domain [ 9 ]. The Soloist subfamily maintains a single AP2/ERF domain but displays low sequence homology with other subfamilies and lacks similar conserved motifs [ 10 ]. Both the ethylene-responsive ERF subfamily and the ethylene-independent DREB subfamily each contain one AP2/ERF domain [ 11 ]. In plants, the DREB and ERF subfamilies are of particular interest due to their crucial roles in enhancing tolerance to various abiotic stresses [ 12 ]. As plant-specific transcription factors, DREB proteins mediate responses to temperature- and water-related stresses by binding to dehydration-responsive elements (DRE/CRT; core sequence A/GCCGAC) in the promoter regions of target genes [ 13 ]. In contrast, the ERF subfamily preferentially recognizes ethylene-response elements (ERE) containing a GCC-box motif (AGCCGCC), which is predominantly found in promoters of ethylene-induced pathogenesis-related genes and certain abiotic stress-responsive genes [ 14 , 15 ]. Beyond regulating the biosynthesis of antioxidant metabolites to improve abiotic stress tolerance [ 11 ], ERF transcription factors also confer drought resistance by modulating the expression of anthocyanin biosynthesis genes (e.g., MdDFR , MdUF3GT , MdCHI , and MdCHS ) [ 16 ], thereby enhancing plant adaptation through multiple physiological pathways. The desert-adapted R. persica , the sole species in the subgenus Hulthemia of Rosaceae, thrives in arid desert and Gobi ecosystems, representing an exceptional biological resource for drought tolerance research [ 17 ]. With the recent completion of its telomere-to-telomere (T2T) genome assembly [ 18 ], multiple stress-responsive transcription factor families, including NAC [ 19 ], bZIP [ 20 ], MYB [ 21 ], and bHLH [ 22 ], have been systematically characterized. However, the RpAP2/ERF superfamily, despite its well-documented roles in abiotic stress responses across plant species, remains entirely uninvestigated in this extremophyte model. Indeed, beyond model plants such as Arabidopsis [ 5 ], rice [ 23 ], and maize [ 24 ], the regulatory roles of AP2/ERF transcription factors in abiotic stress responses have been extensively characterized across diverse ornamental species [ 25 , 26 , 27 ]. A comprehensive genome-wide identification of the AP2/ERF superfamily in R. persica is indispensable for deciphering its remarkable drought adaptation mechanisms. In this study, we performed systematic genome-wide identification and analysis of AP2/ERF transcription factors in R. persica , integrating these findings with our previous transcriptomic data to elucidate their responsive patterns under drought and low temperature stress. This work establishes a crucial foundation for future investigations into the functional mechanisms and regulatory networks of RpAP2/ERF genes during R. persica 's growth, development, and adaptation to environmental extremes. Results Identification and analysis of RpAP2/ERF genes A total of 114 RpAP2/ERF superfamily members were identified by combining of Hidden Markov Model(HMM) and BLAST search in the R. persica genome. The AP2/ERF protein sequences of R. persica exhibit considerable variation in characteristics (Table S1 ), with molecular weights ranging from 138 amino acids (aa) (A023126.1) to 836 aa (A015325.1). Specifically, 68 members consist of fewer than 300 aa, 37 members contain 301–500 aa, and 9 members comprise more than 501 aa. The protein molecular weights (MW) range from 15,727.41 Da (A023126.1) to 93,417.71 Da (A015325.1). Among them, 58 members have a MW less than 30,000 Da, and 40 members fall within the range of 30,000 Da to 50,000 Da. The predicted theoretical isoelectric point (PI) range from 4.55 (A009919.1) to 10.45 (A006323.1), with 82 proteins having an PI below 7 and 32 above 7. The instability index varies from 36.51 (A009919.1) to 78.31 (A002912.1), and the grand average of hydropathicity (GRAVY) ranges from 33.42 (A014479.1) to 78.65 (A025887.1). Prediction of subcellular location showed that RpAP2/ERF s mainly localized in the nucleus, 3 proteins were localized in the cytoplasm, 2 proteins were localized in the mitochondria, and 3 proteins were localized in the chloroplast. These results provided a theoretical basis for further studies on the function of RpAP2/ERF genes. Phylogenetic analysis of RpAP2/ERF protein To further understand the gene structure and evolutionary relationships of the RpAP2/ERF protein, we use the Arabidopsis AP2/ERF gene family as a reference, a phylogenetic tree of R. persica and Arabidopsis AP2/ERF protein sequences was constructed (Fig. 1 ). The AP2/ERF proteins of R. persica were classified into AP2, RAV, Soloist, DREB, and ERF subfamilies containing 15, 4, 1, 60 and 34 members, respectively. DREB subfamily was further split into six subgroups, from A1 to A6, containing 5, 6, 1, 12, 6, and 4 RpAP2/ERF proteins. The ERF subfamily was the largest among all subfamilies. It was further segregated into B1–6 subgroups, containing 7, 4, 21, 4, 5, and 19 RpAP2/ERF proteins, respectively. the Solosist subfamily is the smallest, consisting of only 1 member, indicating that RpAP2/ERF genes were distributed in different clades unevenly. Chromosomal location and colinearity analysis of RpAP2/ERF genes The chromosomal location analysis revealed that the 114 RpAP2/ERF genes were randomly distributed on the R. persica chromosome (Chr) (Fig. 2 ). Specifically, 25 RpAP2/ERFs were located on Chr2, and 22 on Chr7. However, the number of RpAP2/ERFs on Chr3 and Chr5 was the smallest, with only 10 RpAP2/ERF genes. As can be observed, there was no apparent correlation between chromosome length and RpAP2/ERF genes distribution. We further investigated the gene duplication events among RpAP2/ERF genes in the R. persica genome, as they play a crucial role in functional differentiation and gene expansion. In our study, ten pairs of tandem duplicated genes were revealed and distributed on the Chr 1, 5, 6 and 7, respectively (Fig. 2 ). Segmental duplication events revealed 28 AP2/ERF gene duplication pairs distributed across all chromosomes (Fig. 3 A). In conclusion, segmental and tandem duplication extensively contributes to the expansion of the RpAP2/ERF family. To further elucidate the phylogenetic mechanism of the AP2/ERF family, collinear analysis was performed on the AP2/ERF genes of Arabidopsis thaliana , and R. Chinese , as well as the AP2/ERF genes of R. persica (Fig. 3 B). The results suggested that there were 122 syntenic AP2/ERF gene pairs between R. persica and Arabidopsis, 138 between R. persica and R. Chinese , respectively. Structural and motifs analysis of the RpAP2/ERF genes The conserved motif analysis of the RpAP2/ERF protein was performed by MEME online sites. Twenty motifs were observed in 114 RpAP2/ERF protein sequences, with the number of motifs in RbeAP2/ERF proteins ranging from 2 to 7. Motif 1 was likely the most critical motif in RpAP2/ERF proteins, present in 97.37% (111) of them, followed by Motif 3, which was contained in 92.11% (105) of RpAP2/ERF proteins. Motifs 7, 13, 16, and 18 were exclusively present in the DREB subfamily, while Motifs 10, 12, 14, 15, and 17 were unique to the ERF subfamily. Motif 6 was specific to the AP2 family, and Motifs 11 and 20 were restricted to the RAV family. The Soloist family contains Motifs 19, 5, 4, 1, and 3. These results indicated that distinct subfamilies possess conserved motifs. The results of gene structural analysis showed that not all RpAP2/ERF genes had at least one intron, of which 61 out of 114 genes (53.51%) exhibiting no intron presence. The members within the same family possess the similar exon-intron structure. In the AP2 subfamily, RpAP2 genes generally contains a relatively large number of introns (5–10). For the DREB subfamily, most of RpDREB genes have 0–2 introns, except for A026649.1 containing 5 introns and A024668.1 containing 3. In the ERF subfamily, most of RpERF genes have 0–4 introns, while A025887.1 and A026368.1 contain 18 and 12 introns, respectively. In contrast, the RAV subfamily genes have no intron. In summary, the conserved motifs and gene structures indicate that RpAP2/ERF members of the same subfamily were highly conserved, which further verifies the reliability of phylogenetic tree clustering. Cis-regulatory elements analysis of the RpAP2/ERF genes To further understand the regulatory patterns and potential functions of the RpAP2/ERF genes, we analyzed the 2000bp upstream sequences from the start codons of the 114 RpAP2/ERF genes. These elements were divided into four main categories according to their putative functions, including light responsive (GA-motif, MRE, LAMP-element, etc.), phytohormone responsive (AuxRR-core, TGA-element, P-box, TATC-box, GARE-motif, CGTCA-motif, TGACG-motif, TCA-element and ABRE), abiotic and biotic stresses (ARE, LTR, TC-rich repeats, MBS, GC-motif) and plant growth and development (A-box, O2-site, HD-Zip 1, GCN4_motif, CAT-box, circadian, CCAAT-box, AT-rich element, MBSI and RY-element) (Fig. 5 ). These cis-acting elements are randomly distributed along the sequences of RpAP2/ERF genes. Among RpAP2/ERF members, 97.37% contain the anaerobic induction cis-acting regulatory element (ARE), representing the highest proportion. This is followed by the light-responsive cis-acting element (G-box), present in 95.61% of the members. A large number of members also contain abscisic acid-related cis-acting elements (ABRE), accounting for 92.98%. Among all cis-acting elements, the most abundant ones are G-box (594), ABRE (524), and ARE (383), followed by MeJA-responsive CGTCA-motif (357) and TGACG-motif (356). This indicates that RpERF/AP2 was not only involved in plant growth and development but also plays an important role in hormone signal transduction and stress responses. However, cis-acting elements such as TATC-box (36), AuxRR-core (19), and MBSI (10) have relatively low counts, suggesting that these regions regulate the expression of specific genes only under appropriate circumstances. Expression pattern analysis of RpAP2/ERF genes Based on the cis-acting element analysis of RpAP2/ERFs , these genes were likely involved in various abiotic stress responses. By integrating transcriptomic datasets from our previous studies, we analyzed the expression profiles of RpAP2/ERFs across different tissues. Furthermore, we characterized the organ-specific expression dynamics of AP2/ERF genes in R. persica , analyzing root and leaf tissues under drought stress, while examining root and stem segments under cold stress conditions. Transcriptomic analysis of five organs in R. persica (roots, stems, leaves, flowers, and fruits) revealed that most RpAP2/ERF genes were expressed across multiple tissues (Fig. 6 A). Overall, the RpAP2/ERF family displayed higher expression levels in roots, stems, and fruits compared to leaves and flowers. Root-specific expression was observed for four genes ( A005041.1 , A008193.1 , A013003.1 , A014479.1 ), while two genes ( A001403.1 , A026890.1 ) exhibited stem-specific expression. Only one gene was exclusively expressed in fruits ( A007190.1 ) and leaves ( A013214.1 ), respectively, suggesting these genes may have tissue-preferential functions. Moreover, we analyzed the expression patterns of 86 RpAP2/ERF genes using gene expression data from the drought transcriptome (Fig. 6 B). Transcriptomic analysis revealed distinct tissue-specific responses to drought stress among RpAP2/ERF family members. Six genes ( A002909.1 , A023874.1 , A002755.1 , A005638.1 , A010645.1 , A024668.1 ) exhibited leaf-specific drought responsiveness, while three genes ( A014162.1 , A025595.1 , A028433.1 ) showed root-specific activation. Notably, ten members ( A000782.1 , A011282.1 , A010347.1 , A026936.1 , A006323.1 , A025658.1 , A005829.1 , A014504.1 , A009162.1, A028597.1) demonstrated coordinated regulation in both leaves and roots. These findings clearly indicated that RpAP2/ERF genes play pivotal roles in R. persica 's drought adaptation, with some members functioning in tissue-specific manners while others orchestrating multi-tissue responses. We collected transcriptome data from roots and stems of wild R. persica in their natural habitat in Xinjiang from September to April, with average monthly temperatures remaining below 0°C during the coldest winter months (December to February). Our analysis revealed tissue-specific low temperature response patterns: ten genes ( A011282.1 , A008533.1 , A013004.1 , A010347.1 , A014162.1 , A025886.1 , A014504.1 , A014948.1 , A025658.1 , A023128.1 ) exhibited root-specific low temperature responsiveness, while nine stem-specific genes ( A005829.1 , A002912.1 , A002909.1 , A025595.1 , A009910.1 , A009919.1 , A002755.1 , A024668.1 , A026649.1 ) showed distinct activation patterns. Notably, only two genes ( A008425.1 , A024194.1 ) demonstrated coordinated low temperature response in both aerial and underground tissues (Fig. 6 C), suggesting most low temperature responsive genes in R. persica function in a tissue-specific manner. Transcriptional activation activity assay of RpAP2/ERF genes Under both drought and low temperature stress conditions, the significantly upregulated RpAP2/ERF genes exclusively belonged to the DREB and ERF subfamilies, consistent with the established paradigm that these two subfamilies are primary mediators of abiotic stress responses within the AP2/ERF superfamily [ 12 ]. To investigate the regulatory functions of AP2/ERF transcription factors, we selected seven drought- and low temperature-inducible genes ( A002909.1 , A005829.1 , A014504.1 , A002755.1 , A010645.1 , A024668.1 , A009162.1 ) (Fig. 7 ) from the DREB and ERF subfamilies for transcriptional activation analysis. Using a yeast system with stringent selection conditions (SD/-Trp-His-Ade medium), we observed that the positive control (pGBKT7-p53) and five candidate genes ( A002909 .1, A014504.1 , A002755.1 , A010645.1 , A009162.1 ) showed normal growth, indicating their transcriptional activation capability. In contrast, neither the empty vector (pGBKT7) nor two candidate genes ( A005829.1 and A024668.1 ) supported yeast growth under these selection conditions. These results demonstrate that among the seven tested transcription factors, five possess transcriptional activation activity in the yeast system, while A005829.1 and A024668.1 appear to lack this function. Protein-protein interaction network analysis of RpAP2/ERF genes To analyze the relationships and synergistic effects among RpAP2/ERF transcription factors, we constructed a protein-protein interaction (PPI) network using the STRING database with reference to homologous proteins in Arabidopsis thaliana (Fig. 8 ). The results revealed that A010347.1 , the homolog of AtAP2 , may serve as a central regulatory gene interacting with multiple proteins, particularly members of the DREB and ERF subfamilies. Additionally, A001403.1 (homolog of AtERF13 ), A010645.1 (homolog of AtDREB1B ), and A026930.1 (homolog of AtERF5 ) were identified as key hubs in the PPI network. These four genes likely play critical roles in mediating biological functions. Discussion As sessile organisms, plants inevitably face prolonged or transient abiotic stresses such as water deficit and temperature extremes [ 28 , 29 ]. Through evolutionary adaptation, they have developed sophisticated mechanisms to balance survival and growth under stressful conditions - a process fundamentally regulated by transcription factors at the molecular level [ 30 , 31 ]. Among these, the AP2/ERF family has been unequivocally established as a master regulator of abiotic stress responses [ 32 , 33 ]. This study presents the first comprehensive investigation of the AP2/ERF superfamily in R. persica , providing crucial insights into its remarkable stress adaptation strategies. Here we identified 114 AP2/ERF family genes in R. persica , which were classified into five subfamilies (AP2, DREB, ERF, RAV, and Soloist), with the ERF subfamily being the most abundant. This represents a common evolutionary pattern observed across diverse plant species - whether model plants like Arabidopsis thaliana (65/147 members) [ 34 ], Phaseolus vulgaris (95/180) [ 35 ] and Hordeum vulgare (54/121) [ 36 ], or ornamental species such as R. chinensis (68/135) [ 37 ], Juglans mandshurica (90/184) [ 39 ] and Rhododendron (53/120) [ 38 ] - where the ERF subfamily consistently constitutes the largest group. Some studies even suggest that the total size of AP2/ER F gene families was primarily determined by ERF subfamily expansion [ 33 ]. Analysis of the newly assembled T2T genome reveals that R. persica had a compact genome size of 364.44 Mb [ 18 ], smaller than that of R. chinensis (512 Mb) [ 41 ]. Correspondingly, R. persica contains fewer AP2/ERF genes (114) compared to R. chinensis (135) [ 37 ]. However, our comparative genomics analysis confirms that AP2/ERF family size does not directly correlate with genome size, as exemplified by Arabidopsis which maintains 147 AP2/ERF genes despite having a much smaller genome (125 Mb) [ 38 ]. This suggests that ecological adaptation pressures, rather than genome size constraints, primarily drive the expansion and diversification of this critical transcription factor family [ 42 ]. The phylogenetic tree clearly visualizes sequence relationships among AP2/ERF members. In our analysis, the AP2, RAV, and Soloist subfamilies each formed distinct monophyletic clades, while DREB and ERF subfamilies exhibited staggered distribution patterns across multiple subgroups. Notably, we observed a similar topological arrangement in the AP2/ERF phylogeny of R. chinensis [ 37 ] and Rhododendron [ 38 ]. From a fundamental classification perspective, phylogenetic trees are typically constructed based on gene structure as the primary criterion [ 43 ]. Both DREB and ERF subfamilies share a conserved feature of two tandemly arranged AP2 domains [ 44 ], representing a distinct architectural signature. This structural similarity has led some researchers to propose merging ERF and DREB into a single family [ 11 , 43 ]. Therefore, the observed staggered distribution of certain members between these two subfamilies likely reflects their shared evolutionary origin and structural conservation. Gene structure analysis provides important insights into gene function [ 44 ]. Our results demonstrate that members within the same subfamily share similar gene architectures, validating the reliability of our phylogenetic tree analysis. Comparative analysis of gene structures revealed distinct intron distribution patterns across the five AP2/ERF subfamilies. The AP2 and Soloist subfamilies consistently contained introns, with relatively higher intron numbers per gene. In contrast, all RAV subfamily members were completely intronless. The DREB and ERF subfamilies exhibited intermediate characteristics, with 52.94% and 65% of their members being intronless, respectively. These intron distribution patterns were consistent with previous reports in other plant species such as Prunus dulcis [ 45 ] and Saccharum spontaneum [ 42 ], suggesting evolutionary conservation of splicing mechanisms within the AP2/ERF superfamily. The cis-acting elements within promoter regions play pivotal roles in regulating gene expression [ 46 ], it associated with RpAP2/ERF genes primarily function in three major biological processes: hormonal regulation, stress responses, and light response [ 47 ]. Statistical analysis identified ABRE, ARE, and G-box as the most abundant cis-elements in these functional categories, respectively. Consistent with our current findings in the AP2/ERF family, comprehensive analysis of other major stress-responsive transcription factor families ( bHLH , NAC , and bZIP ) in R. persica demonstrates that a substantial proportion of members across these families contain ABRE (ABA-responsive element) cis-regulatory motifs [ 19 , 20 , 22 ]. The evolutionary conservation of ABRE enrichment among multiple TF families underscores the critical importance of ABA-mediated transcriptional reprogramming for R. persica 's adaptation to harsh desert environments. Expression analysis of R. persica AP2/ERF genes under drought and low temperature stress conditions revealed that the drought-responsive genes A011282.1 ( RpAt2g41710 ), A010347.1 ( RpAP2-3 ), A014504.1 ( RpERF115 ), A025658.1 ( RpERF118 ), A025595.1 ( RpRAP2-11 ), A002755.1 ( RpDREB2A ), and A024668.1 ( RpERF010 ) were simultaneously responsive to low temperature stress, demonstrating their dual regulatory roles in different abiotic stress responses. In studies investigating the low temperature stress response mechanisms of R. persica , MYC2 was identified as a key regulator of low temperature tolerance [ 48 ]. Yeast two-hybrid assays revealed that MYC2 physically interacts with multiple AP2/ERF family members, including RpDREB1B , RpERF114 , RpERF25 , and RpERF22 [ 48 ]. Notably, our drought-responsive expression profiling demonstrated that A010645.1 ( RpDREB1B ) also plays a significant role in drought stress adaptation, verified its dual functionality in mediating different stress responses. However, this stress response exhibits distinct tissue-specific patterns. Under drought conditions, A025595.1 ( RpRAP2-11 ) showed significant root-specific expression, whereas under low temperature stress it was exclusively upregulated in stems. Similarly, while A011282.1 ( RpAt2g41710 ), A010347.1 ( RpAP2-3 ), A014504.1 ( RpERF115 ), and A025658.1 ( RpERF118 ) were upregulated in both leaves and roots during drought exposure, these genes displayed root-specific induction under low temperature treatment. These observations demonstrate organ-specific partitioning of stress responses among RpAP2/ERF members. Among the seven genes co-regulated under both drought and low temperature stress conditions, two members ( A011282.1 and A010347.1 ) belong to the AP2 subfamily. While the AP2 subfamily was widely recognized for its primary roles in plant growth and developmental processes [ 49 – 51 ] such as floral development [ 52 ], leaf morphogenesis [ 53 ], and seed yield [ 54 ], emerging evidence suggests their involvement in abiotic stress responses as well [ 55 – 58 ]. Notably, studies on wheat disease resistance have identified that the AP2-10 transcription factor not only enhances disease resistance but is also significantly induced by PEG4000 and abscisic acid treatments [ 55 ]. These findings suggest that AP2 subfamily members may also play important functional roles in abiotic stress adaptation in R. persica . Conclusions This study systematically identified 114 AP2/ERF family members in R. persica and classified them into five distinct subfamilies based on their structural and sequence characteristics. We conducted comprehensive analyses of their fundamental physicochemical properties, phylogenetic relationships, gene structures, chromosomal distributions, and syntenic relationships. Furthermore, we investigated the expression patterns of these AP2/ERF members across different tissues and under abiotic stress conditions (drought and low temperature), identifying candidate genes responsive to these environmental challenges. Functional validation was performed for seven selected RpAP2/ERF transcription factors to confirm their transcriptional activation activity, and a protein-protein interaction network was constructed. Collectively, this work provides a comprehensive characterization of the RpAP2/ERF gene family, establishes a theoretical foundation for future functional studies of these genes and their protein interactions, and offers valuable insights for elucidating the stress resistance mechanisms in R. persica . Materials and methods Identification and physicochemical property analysis of RpAP2/ERF genes The phased and gap-free reference genome data of R. persica were derived from the previous research results of our team [ 18 ]. The amino acid sequences of AP2/ERF family in Arabidopsis thaliana were obtained from the Arabidopsis Information Resource (TAIR) ( https://www.arabidopsis.org ). AP2/ERF genes were screened using HMMER and BLASTP tools, and the Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov ) was employed for further analysis these genes. The AP2/ERF domain Hidden Markov Model (HMM) profile (PF00847) was downloaded from Pfam ( http://pfam.xfam.org/ ). The physicochemical characteristics of AP2/ERF genes, such as isoelectric point (PI) and molecular weight (MW), were analyzed using the Protein Parameter Calc tool in TBtools. Subcellular localization of AP2/ERF was predicted using the WoLFPSORT online platform ( https://wolfpsort.hgc.jp/ ). Phylogenetic analysis and chromosomal localization of RpAP2/ERF genes Multiple sequence alignment of AP2/ERF proteins from Arabidopsis thaliana and R. persica was performed using Clustal W. The alignment data was subsequently imported into MEGA11.0 software ( https://www.megasoftware.net/ ), and a phylogenetic tree was constructed using IQ-TREE software with the VT + F + R7 model and 1,000 bootstrap replicates. The phylogenetic tree was further modified with iTOL ( https://itol.embl.de ). The RpAP2/ERF genes were classified into five subfamilies, AP2, ERF, DREB, RAV and Soloist, depending on the number of AP2 domains and other features in sequence according to previous studies [ 34 ]. The chromosomal localization information of AP2/ERF family members was obtained from the R. persica genome annotation file. The results of gene chromosomal localization were visualized through the Gene Location Visualize from the GTF/GFF program of TBtools. Gene structure, conserved motif, colinearity analysis and cis-regulatory elements analysis of RpAP2/ERF genes The information of gene structures describing the exon and intron was extract from the gff file of genome sequence data. Visualization of the RpAP2/ERF gene structures using TBtools. The conserved motif analysis of RpAP2/ERF genes were determined using the MEME program ( http://meme-suite.org/ ) with the selected number of motifs set to 20. The resulting motifs were visualized using TBtools [ 59 ]. MCScanX and TBtools were employed to identify tandem duplicated genes of RpAP2/ERFs , and the inter-species collinearity of RpAP2/ERFs between R. persica and other plants. Finally, a 2000 bp genomic sequence was extracted of each RpAP2/ERF gene as the putative promoter region. The cis-acting elements in the RpAP2/ERF promoter regions were predicted by the PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) and the RpAP2/ERF cis-acting elements results were visualized by the Simple BioSequence Viewer program in TBtools. Expression profile analysis of RpAP2/ERF genes RNA-seq data of R. persica AP2/ERF superfamily genes were obtained from our previous research. Transcriptome data were used to analyze the expression patterns of RpAP2/ERF genes under drought stress, low temperature stress, and in various tissue structures. Normalized gene expression values expressed as FPKM (fragments per kilobase of exon per million fragments mapped) were transformed using log2 (FPKM + 1), we further used iTOL( https://itol.embl.de ) to construct the heatmap. The RpAP2/ERF protein interaction network was constructed by STRING ( https://cn.string-db.org/ ) with the reference organism of A. thaliana . Roots, stems, leaves, flowers, and fruits of R. persica were collected in Hutubi County, Xinjiang. Three-month-old seedlings of R. persica after sowing were subjected to drought treatment using the water restriction method. Samples were collected on day 0 (L0), day 7 (LD1), day 14 (LD2), and day 22 (LD3) after drought initiation, as well as on day 3 (LW1) and day 10 (LW2) after rewatering. Analysis of transcriptional activation activity For the transcriptional activation assay, the ORF of six genes ( A002909.1 , A005829.1 , A014504.1 , A002755.1 , A010645.1 , and A009162.1 ) were generated via recombination reactions and fused into the EcoRI and SalI sites of the GAL4 DNA-binding domain in the pGBKT7 vector. A024668.1 was fused into the PstI and BamHI sites of the same vector. The pGBKT7 (negative control), pGBKT7-p53 (positive control), pGBKT7- A002909.1 , pGBKT7- A005829.1 , pGBKT7- A014504.1 , pGBKT7- A002755.1 , pGBKT7- A010645.1 , pGBKT7- A009162.1 , and pGBKT7- A024668.1 plasmid was transformed into yeast Y2H and cultured on SD/-Trp medium. Transformed cells were verified using PCR followed by spotted on SD/-Trp and SD/-Trp-His-Ade media according to a gradient dilution method for 3 days. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Clinical trial number Not applicable Funding This work was supported by National Key Research and Development Project of China (2023YFD120010502), Fundamental Research Funds for the Central Universities (QNTD202503), National Natural Science Foundation of China, and Horizontal Project Commissioned by Enterprises and Institutions for Scientific and Technological Projects (2023-HXFW-428). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7203695","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":506512699,"identity":"3747b2f7-9b92-4e57-abc9-695b96e70a3a","order_by":0,"name":"Na Li","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Li","suffix":""},{"id":506512700,"identity":"8dd64e13-30ed-4fe1-afe2-fa8f1bb33aa7","order_by":1,"name":"Yang Cui","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Cui","suffix":""},{"id":506512701,"identity":"3cc32914-f705-4cae-ada6-3a5fb2949a08","order_by":2,"name":"Chenjie Zhang","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Chenjie","middleName":"","lastName":"Zhang","suffix":""},{"id":506512702,"identity":"59e2b69b-ed14-4568-9897-019acf644684","order_by":3,"name":"Xiaolong Zhang","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xiaolong","middleName":"","lastName":"Zhang","suffix":""},{"id":506512703,"identity":"56206704-ade6-4aaa-a0af-60c8f4b982dd","order_by":4,"name":"Ziguo Li","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Ziguo","middleName":"","lastName":"Li","suffix":""},{"id":506512704,"identity":"98b1d188-9dfd-4812-8039-d5d712df28bb","order_by":5,"name":"Chao Yu","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Yu","suffix":""},{"id":506512705,"identity":"4b097c06-6561-449b-b8e2-2ee764937d47","order_by":6,"name":"Huitang Pan","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Huitang","middleName":"","lastName":"Pan","suffix":""},{"id":506512706,"identity":"ce5664b5-d9da-42b5-8313-b55335cdf284","order_by":7,"name":"Yan Liu","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Liu","suffix":""},{"id":506512707,"identity":"ee225e7e-ca7e-4d57-88d1-fe46a6edbae8","order_by":8,"name":"Le Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACPgYGxgOMDWz8DOyNjQ8/EKOFDYhBWiQbeA43G0uQoIVBskEivU2AhygtEskHDnzcwSdhcPNhG4MEg52cbgNBLWkJB2eeYZMwuJ3Y9qCAIdnY7ABBLTkGh3nb2OqAWtoNJBgOJG4jSsvfNqAtNw+2SfAQrYURpOUGI7FaeJ4lHOwF+kXyTCIwkA2I8As/e/LBBz93HJPgO3784cMPFXZyBLVAwTEobUCcchCoIV7pKBgFo2AUjDwAAPkkQ6mty841AAAAAElFTkSuQmCC","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Le","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2025-07-24 09:08:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7203695/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7203695/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90125560,"identity":"8c42b106-3fb7-41e6-ac6a-c56f0b0094df","added_by":"auto","created_at":"2025-08-28 19:01:03","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9282296,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of \u003cem\u003eAP2/ERF\u003c/em\u003e transcription factors in \u003cem\u003eR. persica\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. The orange solid triangles on the tree branches represent bootstrap values, with larger triangles indicating higher bootstrap values. Different colors are used to distinguish different subfamilies.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/dcb1d54041d94b2b3b66140c.jpeg"},{"id":90125190,"identity":"2d24b013-012a-43b4-b565-395df9ae5e32","added_by":"auto","created_at":"2025-08-28 18:53:03","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3521466,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal localization of \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes. 114 \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes were mapped on the seven \u003cem\u003eR. persica\u003c/em\u003e chromosomes. The chromosome numbers are indicated on the left side of each white vertical bar. The tandem duplicated gene pairs are indicated within blue curve. The scale on the left is in megabases.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/d07b461a52d6ca3935d034c9.jpeg"},{"id":90125564,"identity":"47262edb-14b5-44d9-85f7-6f9bc83dff20","added_by":"auto","created_at":"2025-08-28 19:01:03","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46275225,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analysisof \u003cem\u003eAP2/ERF\u003c/em\u003e genes. A: Synteny analysis in \u003cem\u003eR. persica\u003c/em\u003e. The red lines indicate collinearity relationships among \u003cem\u003eRpAP2/ERF\u003c/em\u003egenes. Orange and yellow represent the level of gene distribution density on the chromosome. B: Synteny analysis between \u003cem\u003eR. persica\u003c/em\u003e and other plants. The blue lines highlight the syntenic \u003cem\u003eAP2/ERF\u003c/em\u003e gene pairs. At: \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, Rp: \u003cem\u003eR. persica\u003c/em\u003e, Rc: \u003cem\u003eR. Chinese\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/8912552bea701b0769e00c37.jpeg"},{"id":90125186,"identity":"55665f91-12e2-420d-9e88-60837d7e3d46","added_by":"auto","created_at":"2025-08-28 18:53:03","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2125945,"visible":true,"origin":"","legend":"\u003cp\u003eConserved motif and gene structure of \u003cem\u003eRpAP2/ERF\u003c/em\u003e gene family. A: Conserved motif\u003c/p\u003e\n\u003cp\u003edistributions of AP2/ERF proteins. B: Exon-intron distributions of \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/07c65067cb04ea7d90bc0169.jpeg"},{"id":90125201,"identity":"288eed06-5faf-44ec-824b-331fc0775b83","added_by":"auto","created_at":"2025-08-28 18:53:03","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28615976,"visible":true,"origin":"","legend":"\u003cp\u003eThe quantity information of cis-acting elements in \u003cem\u003eAP2/ERF\u003c/em\u003e promoters. The numbers in the figure represent the quantity of cis-acting elements; the greater the quantity, the redder the color of the squares.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/ed05b32d9d10f0aa2b16bb75.jpeg"},{"id":90125197,"identity":"7cd3b970-f5fa-40eb-a9cf-69b1ffacd27c","added_by":"auto","created_at":"2025-08-28 18:53:03","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":19284911,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap analysis of \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes expression patterns A Tissue-specific expression profiles across different organs. B Drought-responsive expression patterns in leaves (L0-LW2) and roots (R0-RW1). L0 and R0 represent controls, LD1-LD3/RD2-RD3 indicate drought treatment phases, and LW1-LW2/RW1 denote rewatering stages. C Low temperature stress responses in roots (G) and stems (J) from September to April (9-4)\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/5fb00ad5fd70c4ca397e3539.jpeg"},{"id":90125191,"identity":"492d09d0-2568-49a9-89c2-6b6d79ce46db","added_by":"auto","created_at":"2025-08-28 18:53:03","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3239264,"visible":true,"origin":"","legend":"\u003cp\u003eTransactivation activity analysis of seven RpAP2/ERF proteins. The negative and positive controls were pGBKT7 and pGBKT7-p53\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/bbe342ff4a56a33910f7ef10.jpeg"},{"id":90125195,"identity":"f03eff52-3c89-423a-bad3-d9ae61fb4f78","added_by":"auto","created_at":"2025-08-28 18:53:03","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1164805,"visible":true,"origin":"","legend":"\u003cp\u003ePartial protein-protein interaction network of RpAP2/ERF protein referring to orthologs in \u003cem\u003eArabidopsis\u003c/em\u003e. The red-labeled protein identifiers in the figure correspond to the Arabidopsis-homologous responsive RpAP2/ERF proteins.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/4874d11bd287c1c91009cc74.jpeg"},{"id":90125973,"identity":"585ffac6-ff6d-49b3-98f1-878c42b77b81","added_by":"auto","created_at":"2025-08-28 19:09:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":114527968,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/3dc96017-6896-4a35-a24b-d2aae25fa1cd.pdf"},{"id":90125559,"identity":"43a3b65c-0990-4a5f-a811-c480b3a855f8","added_by":"auto","created_at":"2025-08-28 19:01:03","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19604,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7203695/v1/216516cd8afe6ba7fe41576b.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification of the AP2/ERF gene family in Rosa persica and expression profiling under abiotic stresses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTranscription factors precisely regulate gene expression by binding to specific cis-acting elements in target gene promoters, serving as master regulators in abiotic stress-responsive signaling networks [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The \u003cem\u003eAP2/ER\u003c/em\u003eF family constitutes a unique class of plant-specific transcription factors that play essential roles in regulating plant growth/development and environmental stress responses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBased on the Arabidopsis classification system [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], the \u003cem\u003eAP2/ERF\u003c/em\u003e superfamily is categorized into five major subfamilies (AP2, DREB, ERF, RAV, and Soloist) according to their conserved domains, all of which contain at least one 60\u0026ndash;70 amino acid AP2/ERF domain [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Specifically, the AP2 subfamily contains two AP2/ERF domains [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], while the RAV subfamily possesses one AP2/ERF domain along with an additional B3 domain [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The Soloist subfamily maintains a single AP2/ERF domain but displays low sequence homology with other subfamilies and lacks similar conserved motifs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Both the ethylene-responsive ERF subfamily and the ethylene-independent DREB subfamily each contain one AP2/ERF domain [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn plants, the DREB and ERF subfamilies are of particular interest due to their crucial roles in enhancing tolerance to various abiotic stresses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As plant-specific transcription factors, DREB proteins mediate responses to temperature- and water-related stresses by binding to dehydration-responsive elements (DRE/CRT; core sequence A/GCCGAC) in the promoter regions of target genes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In contrast, the ERF subfamily preferentially recognizes ethylene-response elements (ERE) containing a GCC-box motif (AGCCGCC), which is predominantly found in promoters of ethylene-induced pathogenesis-related genes and certain abiotic stress-responsive genes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Beyond regulating the biosynthesis of antioxidant metabolites to improve abiotic stress tolerance [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], ERF transcription factors also confer drought resistance by modulating the expression of anthocyanin biosynthesis genes (e.g., \u003cem\u003eMdDFR\u003c/em\u003e, \u003cem\u003eMdUF3GT\u003c/em\u003e, \u003cem\u003eMdCHI\u003c/em\u003e, and \u003cem\u003eMdCHS\u003c/em\u003e) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], thereby enhancing plant adaptation through multiple physiological pathways.\u003c/p\u003e\u003cp\u003eThe desert-adapted \u003cem\u003eR. persica\u003c/em\u003e, the sole species in the subgenus Hulthemia of Rosaceae, thrives in arid desert and Gobi ecosystems, representing an exceptional biological resource for drought tolerance research [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. With the recent completion of its telomere-to-telomere (T2T) genome assembly [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], multiple stress-responsive transcription factor families, including \u003cem\u003eNAC\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], \u003cem\u003ebZIP\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], \u003cem\u003eMYB\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and \u003cem\u003ebHLH\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], have been systematically characterized. However, the \u003cem\u003eRpAP2/ERF\u003c/em\u003e superfamily, despite its well-documented roles in abiotic stress responses across plant species, remains entirely uninvestigated in this extremophyte model.\u003c/p\u003e\u003cp\u003eIndeed, beyond model plants such as Arabidopsis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], rice [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and maize [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], the regulatory roles of AP2/ERF transcription factors in abiotic stress responses have been extensively characterized across diverse ornamental species [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A comprehensive genome-wide identification of the \u003cem\u003eAP2/ERF\u003c/em\u003e superfamily in \u003cem\u003eR. persica\u003c/em\u003e is indispensable for deciphering its remarkable drought adaptation mechanisms. In this study, we performed systematic genome-wide identification and analysis of \u003cem\u003eAP2/ERF\u003c/em\u003e transcription factors in \u003cem\u003eR. persica\u003c/em\u003e, integrating these findings with our previous transcriptomic data to elucidate their responsive patterns under drought and low temperature stress. This work establishes a crucial foundation for future investigations into the functional mechanisms and regulatory networks of \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes during \u003cem\u003eR. persica\u003c/em\u003e's growth, development, and adaptation to environmental extremes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIdentification and analysis of\u003c/b\u003e \u003cb\u003eRpAP2/ERF\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 114 \u003cem\u003eRpAP2/ERF\u003c/em\u003e superfamily members were identified by combining of Hidden Markov Model(HMM) and BLAST search in the \u003cem\u003eR. persica\u003c/em\u003e genome. The AP2/ERF protein sequences of \u003cem\u003eR. persica\u003c/em\u003e exhibit considerable variation in characteristics (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), with molecular weights ranging from 138 amino acids (aa) (A023126.1) to 836 aa (A015325.1). Specifically, 68 members consist of fewer than 300 aa, 37 members contain 301\u0026ndash;500 aa, and 9 members comprise more than 501 aa. The protein molecular weights (MW) range from 15,727.41 Da (A023126.1) to 93,417.71 Da (A015325.1). Among them, 58 members have a MW less than 30,000 Da, and 40 members fall within the range of 30,000 Da to 50,000 Da. The predicted theoretical isoelectric point (PI) range from 4.55 (A009919.1) to 10.45 (A006323.1), with 82 proteins having an PI below 7 and 32 above 7. The instability index varies from 36.51 (A009919.1) to 78.31 (A002912.1), and the grand average of hydropathicity (GRAVY) ranges from 33.42 (A014479.1) to 78.65 (A025887.1). Prediction of subcellular location showed that \u003cem\u003eRpAP2/ERF\u003c/em\u003es mainly localized in the nucleus, 3 proteins were localized in the cytoplasm, 2 proteins were localized in the mitochondria, and 3 proteins were localized in the chloroplast. These results provided a theoretical basis for further studies on the function of \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analysis of RpAP2/ERF protein\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further understand the gene structure and evolutionary relationships of the RpAP2/ERF protein, we use the Arabidopsis \u003cem\u003eAP2/ERF\u003c/em\u003e gene family as a reference, a phylogenetic tree of \u003cem\u003eR. persica\u003c/em\u003e and Arabidopsis AP2/ERF protein sequences was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The AP2/ERF proteins of \u003cem\u003eR. persica\u003c/em\u003e were classified into AP2, RAV, Soloist, DREB, and ERF subfamilies containing 15, 4, 1, 60 and 34 members, respectively. DREB subfamily was further split into six subgroups, from A1 to A6, containing 5, 6, 1, 12, 6, and 4 RpAP2/ERF proteins. The ERF subfamily was the largest among all subfamilies. It was further segregated into B1\u0026ndash;6 subgroups, containing 7, 4, 21, 4, 5, and 19 RpAP2/ERF proteins, respectively. the Solosist subfamily is the smallest, consisting of only 1 member, indicating that \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes were distributed in different clades unevenly.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChromosomal location and colinearity analysis of\u003c/b\u003e \u003cb\u003eRpAP2/ERF\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chromosomal location analysis revealed that the 114 \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes were randomly distributed on the \u003cem\u003eR. persica\u003c/em\u003e chromosome (Chr) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Specifically, 25 \u003cem\u003eRpAP2/ERFs\u003c/em\u003e were located on Chr2, and 22 on Chr7. However, the number of \u003cem\u003eRpAP2/ERFs\u003c/em\u003e on Chr3 and Chr5 was the smallest, with only 10 RpAP2/ERF genes. As can be observed, there was no apparent correlation between chromosome length and \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes distribution.\u003c/p\u003e\u003cp\u003eWe further investigated the gene duplication events among \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes in the \u003cem\u003eR. persica\u003c/em\u003e genome, as they play a crucial role in functional differentiation and gene expansion. In our study, ten pairs of tandem duplicated genes were revealed and distributed on the Chr 1, 5, 6 and 7, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Segmental duplication events revealed 28 \u003cem\u003eAP2/ERF\u003c/em\u003e gene duplication pairs distributed across all chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In conclusion, segmental and tandem duplication extensively contributes to the expansion of the \u003cem\u003eRpAP2/ERF\u003c/em\u003e family.\u003c/p\u003e\u003cp\u003eTo further elucidate the phylogenetic mechanism of the \u003cem\u003eAP2/ERF\u003c/em\u003e family, collinear analysis was performed on the \u003cem\u003eAP2/ERF\u003c/em\u003e genes of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and \u003cem\u003eR. Chinese\u003c/em\u003e, as well as the \u003cem\u003eAP2/ERF\u003c/em\u003e genes of \u003cem\u003eR. persica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The results suggested that there were 122 syntenic \u003cem\u003eAP2/ERF\u003c/em\u003e gene pairs between \u003cem\u003eR. persica\u003c/em\u003e and Arabidopsis, 138 between \u003cem\u003eR. persica\u003c/em\u003e and \u003cem\u003eR. Chinese\u003c/em\u003e, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eStructural and motifs analysis of the\u003c/b\u003e \u003cb\u003eRpAP2/ERF\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe conserved motif analysis of the \u003cem\u003eRpAP2/ERF\u003c/em\u003e protein was performed by MEME online sites. Twenty motifs were observed in 114 \u003cem\u003eRpAP2/ERF\u003c/em\u003e protein sequences, with the number of motifs in RbeAP2/ERF proteins ranging from 2 to 7. Motif 1 was likely the most critical motif in RpAP2/ERF proteins, present in 97.37% (111) of them, followed by Motif 3, which was contained in 92.11% (105) of RpAP2/ERF proteins. Motifs 7, 13, 16, and 18 were exclusively present in the DREB subfamily, while Motifs 10, 12, 14, 15, and 17 were unique to the ERF subfamily. Motif 6 was specific to the AP2 family, and Motifs 11 and 20 were restricted to the RAV family. The Soloist family contains Motifs 19, 5, 4, 1, and 3. These results indicated that distinct subfamilies possess conserved motifs.\u003c/p\u003e\u003cp\u003eThe results of gene structural analysis showed that not all \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes had at least one intron, of which 61 out of 114 genes (53.51%) exhibiting no intron presence. The members within the same family possess the similar exon-intron structure. In the AP2 subfamily, \u003cem\u003eRpAP2\u003c/em\u003e genes generally contains a relatively large number of introns (5\u0026ndash;10). For the DREB subfamily, most of \u003cem\u003eRpDREB\u003c/em\u003e genes have 0\u0026ndash;2 introns, except for A026649.1 containing 5 introns and A024668.1 containing 3. In the ERF subfamily, most of \u003cem\u003eRpERF\u003c/em\u003e genes have 0\u0026ndash;4 introns, while A025887.1 and A026368.1 contain 18 and 12 introns, respectively. In contrast, the RAV subfamily genes have no intron. In summary, the conserved motifs and gene structures indicate that \u003cem\u003eRpAP2/ERF\u003c/em\u003e members of the same subfamily were highly conserved, which further verifies the reliability of phylogenetic tree clustering.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCis-regulatory elements analysis of the\u003c/b\u003e \u003cb\u003eRpAP2/ERF\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further understand the regulatory patterns and potential functions of the \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes, we analyzed the 2000bp upstream sequences from the start codons of the 114 \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes. These elements were divided into four main categories according to their putative functions, including light responsive (GA-motif, MRE, LAMP-element, etc.), phytohormone responsive (AuxRR-core, TGA-element, P-box, TATC-box, GARE-motif, CGTCA-motif, TGACG-motif, TCA-element and ABRE), abiotic and biotic stresses (ARE, LTR, TC-rich repeats, MBS, GC-motif) and plant growth and development (A-box, O2-site, HD-Zip 1, GCN4_motif, CAT-box, circadian, CCAAT-box, AT-rich element, MBSI and RY-element) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These cis-acting elements are randomly distributed along the sequences of \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes.\u003c/p\u003e\u003cp\u003eAmong \u003cem\u003eRpAP2/ERF\u003c/em\u003e members, 97.37% contain the anaerobic induction cis-acting regulatory element (ARE), representing the highest proportion. This is followed by the light-responsive cis-acting element (G-box), present in 95.61% of the members. A large number of members also contain abscisic acid-related cis-acting elements (ABRE), accounting for 92.98%. Among all cis-acting elements, the most abundant ones are G-box (594), ABRE (524), and ARE (383), followed by MeJA-responsive CGTCA-motif (357) and TGACG-motif (356). This indicates that \u003cem\u003eRpERF/AP2\u003c/em\u003e was not only involved in plant growth and development but also plays an important role in hormone signal transduction and stress responses. However, cis-acting elements such as TATC-box (36), AuxRR-core (19), and MBSI (10) have relatively low counts, suggesting that these regions regulate the expression of specific genes only under appropriate circumstances.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression pattern analysis of\u003c/b\u003e \u003cb\u003eRpAP2/ERF\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBased on the cis-acting element analysis of \u003cem\u003eRpAP2/ERFs\u003c/em\u003e, these genes were likely involved in various abiotic stress responses. By integrating transcriptomic datasets from our previous studies, we analyzed the expression profiles of \u003cem\u003eRpAP2/ERFs\u003c/em\u003e across different tissues. Furthermore, we characterized the organ-specific expression dynamics of \u003cem\u003eAP2/ERF\u003c/em\u003e genes in \u003cem\u003eR. persica\u003c/em\u003e, analyzing root and leaf tissues under drought stress, while examining root and stem segments under cold stress conditions.\u003c/p\u003e\u003cp\u003eTranscriptomic analysis of five organs in \u003cem\u003eR. persica\u003c/em\u003e (roots, stems, leaves, flowers, and fruits) revealed that most \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes were expressed across multiple tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Overall, the \u003cem\u003eRpAP2/ERF\u003c/em\u003e family displayed higher expression levels in roots, stems, and fruits compared to leaves and flowers. Root-specific expression was observed for four genes (\u003cem\u003eA005041.1\u003c/em\u003e, \u003cem\u003eA008193.1\u003c/em\u003e, \u003cem\u003eA013003.1\u003c/em\u003e, \u003cem\u003eA014479.1\u003c/em\u003e), while two genes (\u003cem\u003eA001403.1\u003c/em\u003e, \u003cem\u003eA026890.1\u003c/em\u003e) exhibited stem-specific expression. Only one gene was exclusively expressed in fruits (\u003cem\u003eA007190.1\u003c/em\u003e) and leaves (\u003cem\u003eA013214.1\u003c/em\u003e), respectively, suggesting these genes may have tissue-preferential functions.\u003c/p\u003e\u003cp\u003eMoreover, we analyzed the expression patterns of 86 \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes using gene expression data from the drought transcriptome (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Transcriptomic analysis revealed distinct tissue-specific responses to drought stress among \u003cem\u003eRpAP2/ERF\u003c/em\u003e family members. Six genes (\u003cem\u003eA002909.1\u003c/em\u003e, \u003cem\u003eA023874.1\u003c/em\u003e, \u003cem\u003eA002755.1\u003c/em\u003e, \u003cem\u003eA005638.1\u003c/em\u003e, \u003cem\u003eA010645.1\u003c/em\u003e, \u003cem\u003eA024668.1\u003c/em\u003e) exhibited leaf-specific drought responsiveness, while three genes (\u003cem\u003eA014162.1\u003c/em\u003e, \u003cem\u003eA025595.1\u003c/em\u003e, \u003cem\u003eA028433.1\u003c/em\u003e) showed root-specific activation. Notably, ten members (\u003cem\u003eA000782.1\u003c/em\u003e, \u003cem\u003eA011282.1\u003c/em\u003e, \u003cem\u003eA010347.1\u003c/em\u003e, \u003cem\u003eA026936.1\u003c/em\u003e, \u003cem\u003eA006323.1\u003c/em\u003e, \u003cem\u003eA025658.1\u003c/em\u003e, \u003cem\u003eA005829.1\u003c/em\u003e, \u003cem\u003eA014504.1\u003c/em\u003e, \u003cem\u003eA009162.1, A028597.1)\u003c/em\u003e demonstrated coordinated regulation in both leaves and roots. These findings clearly indicated that \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes play pivotal roles in \u003cem\u003eR. persica\u003c/em\u003e's drought adaptation, with some members functioning in tissue-specific manners while others orchestrating multi-tissue responses.\u003c/p\u003e\u003cp\u003eWe collected transcriptome data from roots and stems of wild \u003cem\u003eR. persica\u003c/em\u003e in their natural habitat in Xinjiang from September to April, with average monthly temperatures remaining below 0\u0026deg;C during the coldest winter months (December to February). Our analysis revealed tissue-specific low temperature response patterns: ten genes (\u003cem\u003eA011282.1\u003c/em\u003e, \u003cem\u003eA008533.1\u003c/em\u003e, \u003cem\u003eA013004.1\u003c/em\u003e, \u003cem\u003eA010347.1\u003c/em\u003e, \u003cem\u003eA014162.1\u003c/em\u003e, \u003cem\u003eA025886.1\u003c/em\u003e, \u003cem\u003eA014504.1\u003c/em\u003e, \u003cem\u003eA014948.1\u003c/em\u003e, \u003cem\u003eA025658.1\u003c/em\u003e, \u003cem\u003eA023128.1\u003c/em\u003e) exhibited root-specific low temperature responsiveness, while nine stem-specific genes (\u003cem\u003eA005829.1\u003c/em\u003e, \u003cem\u003eA002912.1\u003c/em\u003e, \u003cem\u003eA002909.1\u003c/em\u003e, \u003cem\u003eA025595.1\u003c/em\u003e, \u003cem\u003eA009910.1\u003c/em\u003e, \u003cem\u003eA009919.1\u003c/em\u003e, \u003cem\u003eA002755.1\u003c/em\u003e, \u003cem\u003eA024668.1\u003c/em\u003e, \u003cem\u003eA026649.1\u003c/em\u003e) showed distinct activation patterns. Notably, only two genes (\u003cem\u003eA008425.1\u003c/em\u003e, \u003cem\u003eA024194.1\u003c/em\u003e) demonstrated coordinated low temperature response in both aerial and underground tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), suggesting most low temperature responsive genes in \u003cem\u003eR. persica\u003c/em\u003e function in a tissue-specific manner.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptional activation activity assay of RpAP2/ERF genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eUnder both drought and low temperature stress conditions, the significantly upregulated \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes exclusively belonged to the DREB and ERF subfamilies, consistent with the established paradigm that these two subfamilies are primary mediators of abiotic stress responses within the \u003cem\u003eAP2/ERF\u003c/em\u003e superfamily [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To investigate the regulatory functions of \u003cem\u003eAP2/ERF\u003c/em\u003e transcription factors, we selected seven drought- and low temperature-inducible genes (\u003cem\u003eA002909.1\u003c/em\u003e, \u003cem\u003eA005829.1\u003c/em\u003e, \u003cem\u003eA014504.1\u003c/em\u003e, \u003cem\u003eA002755.1\u003c/em\u003e, \u003cem\u003eA010645.1\u003c/em\u003e, \u003cem\u003eA024668.1\u003c/em\u003e, \u003cem\u003eA009162.1\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) from the DREB and ERF subfamilies for transcriptional activation analysis.\u003c/p\u003e\u003cp\u003eUsing a yeast system with stringent selection conditions (SD/-Trp-His-Ade medium), we observed that the positive control (pGBKT7-p53) and five candidate genes (\u003cem\u003eA002909\u003c/em\u003e.1, \u003cem\u003eA014504.1\u003c/em\u003e, \u003cem\u003eA002755.1\u003c/em\u003e, \u003cem\u003eA010645.1\u003c/em\u003e, \u003cem\u003eA009162.1\u003c/em\u003e) showed normal growth, indicating their transcriptional activation capability. In contrast, neither the empty vector (pGBKT7) nor two candidate genes (\u003cem\u003eA005829.1\u003c/em\u003e and \u003cem\u003eA024668.1\u003c/em\u003e) supported yeast growth under these selection conditions. These results demonstrate that among the seven tested transcription factors, five possess transcriptional activation activity in the yeast system, while \u003cem\u003eA005829.1\u003c/em\u003e and \u003cem\u003eA024668.1\u003c/em\u003e appear to lack this function.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein-protein interaction network analysis of RpAP2/ERF genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo analyze the relationships and synergistic effects among \u003cem\u003eRpAP2/ERF\u003c/em\u003e transcription factors, we constructed a protein-protein interaction (PPI) network using the STRING database with reference to homologous proteins in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The results revealed that \u003cem\u003eA010347.1\u003c/em\u003e, the homolog of \u003cem\u003eAtAP2\u003c/em\u003e, may serve as a central regulatory gene interacting with multiple proteins, particularly members of the DREB and ERF subfamilies. Additionally, \u003cem\u003eA001403.1\u003c/em\u003e (homolog of \u003cem\u003eAtERF13\u003c/em\u003e), \u003cem\u003eA010645.1\u003c/em\u003e (homolog of \u003cem\u003eAtDREB1B\u003c/em\u003e), and \u003cem\u003eA026930.1\u003c/em\u003e (homolog of \u003cem\u003eAtERF5\u003c/em\u003e) were identified as key hubs in the PPI network. These four genes likely play critical roles in mediating biological functions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs sessile organisms, plants inevitably face prolonged or transient abiotic stresses such as water deficit and temperature extremes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Through evolutionary adaptation, they have developed sophisticated mechanisms to balance survival and growth under stressful conditions - a process fundamentally regulated by transcription factors at the molecular level [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Among these, the \u003cem\u003eAP2/ERF\u003c/em\u003e family has been unequivocally established as a master regulator of abiotic stress responses [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This study presents the first comprehensive investigation of the \u003cem\u003eAP2/ERF\u003c/em\u003e superfamily in \u003cem\u003eR. persica\u003c/em\u003e, providing crucial insights into its remarkable stress adaptation strategies.\u003c/p\u003e\u003cp\u003eHere we identified 114 \u003cem\u003eAP2/ERF\u003c/em\u003e family genes in \u003cem\u003eR. persica\u003c/em\u003e, which were classified into five subfamilies (AP2, DREB, ERF, RAV, and Soloist), with the ERF subfamily being the most abundant. This represents a common evolutionary pattern observed across diverse plant species - whether model plants like \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (65/147 members) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e (95/180) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and \u003cem\u003eHordeum vulgare\u003c/em\u003e (54/121) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], or ornamental species such as \u003cem\u003eR. chinensis\u003c/em\u003e (68/135) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], \u003cem\u003eJuglans mandshurica\u003c/em\u003e (90/184) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] and \u003cem\u003eRhododendron\u003c/em\u003e (53/120) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] - where the ERF subfamily consistently constitutes the largest group. Some studies even suggest that the total size of \u003cem\u003eAP2/ER\u003c/em\u003eF gene families was primarily determined by ERF subfamily expansion [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAnalysis of the newly assembled T2T genome reveals that \u003cem\u003eR. persica\u003c/em\u003e had a compact genome size of 364.44 Mb [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], smaller than that of \u003cem\u003eR. chinensis\u003c/em\u003e (512 Mb) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Correspondingly, \u003cem\u003eR. persica\u003c/em\u003e contains fewer \u003cem\u003eAP2/ERF\u003c/em\u003e genes (114) compared to \u003cem\u003eR. chinensis\u003c/em\u003e (135) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, our comparative genomics analysis confirms that \u003cem\u003eAP2/ERF\u003c/em\u003e family size does not directly correlate with genome size, as exemplified by Arabidopsis which maintains 147 \u003cem\u003eAP2/ERF\u003c/em\u003e genes despite having a much smaller genome (125 Mb) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This suggests that ecological adaptation pressures, rather than genome size constraints, primarily drive the expansion and diversification of this critical transcription factor family [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe phylogenetic tree clearly visualizes sequence relationships among \u003cem\u003eAP2/ERF\u003c/em\u003e members. In our analysis, the AP2, RAV, and Soloist subfamilies each formed distinct monophyletic clades, while DREB and ERF subfamilies exhibited staggered distribution patterns across multiple subgroups. Notably, we observed a similar topological arrangement in the \u003cem\u003eAP2/ERF\u003c/em\u003e phylogeny of \u003cem\u003eR. chinensis\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and \u003cem\u003eRhododendron\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. From a fundamental classification perspective, phylogenetic trees are typically constructed based on gene structure as the primary criterion [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Both DREB and ERF subfamilies share a conserved feature of two tandemly arranged AP2 domains [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], representing a distinct architectural signature. This structural similarity has led some researchers to propose merging ERF and DREB into a single family [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, the observed staggered distribution of certain members between these two subfamilies likely reflects their shared evolutionary origin and structural conservation.\u003c/p\u003e\u003cp\u003eGene structure analysis provides important insights into gene function [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Our results demonstrate that members within the same subfamily share similar gene architectures, validating the reliability of our phylogenetic tree analysis. Comparative analysis of gene structures revealed distinct intron distribution patterns across the five \u003cem\u003eAP2/ERF\u003c/em\u003e subfamilies. The AP2 and Soloist subfamilies consistently contained introns, with relatively higher intron numbers per gene. In contrast, all RAV subfamily members were completely intronless. The DREB and ERF subfamilies exhibited intermediate characteristics, with 52.94% and 65% of their members being intronless, respectively. These intron distribution patterns were consistent with previous reports in other plant species such as \u003cem\u003ePrunus dulcis\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] and \u003cem\u003eSaccharum spontaneum\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], suggesting evolutionary conservation of splicing mechanisms within the \u003cem\u003eAP2/ERF\u003c/em\u003e superfamily.\u003c/p\u003e\u003cp\u003eThe cis-acting elements within promoter regions play pivotal roles in regulating gene expression [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], it associated with \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes primarily function in three major biological processes: hormonal regulation, stress responses, and light response [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Statistical analysis identified ABRE, ARE, and G-box as the most abundant cis-elements in these functional categories, respectively. Consistent with our current findings in the \u003cem\u003eAP2/ERF\u003c/em\u003e family, comprehensive analysis of other major stress-responsive transcription factor families (\u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eNAC\u003c/em\u003e, and \u003cem\u003ebZIP\u003c/em\u003e) in \u003cem\u003eR. persica\u003c/em\u003e demonstrates that a substantial proportion of members across these families contain ABRE (ABA-responsive element) cis-regulatory motifs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The evolutionary conservation of ABRE enrichment among multiple TF families underscores the critical importance of ABA-mediated transcriptional reprogramming for \u003cem\u003eR. persica\u003c/em\u003e's adaptation to harsh desert environments.\u003c/p\u003e\u003cp\u003eExpression analysis of \u003cem\u003eR. persica AP2/ERF\u003c/em\u003e genes under drought and low temperature stress conditions revealed that the drought-responsive genes \u003cem\u003eA011282.1\u003c/em\u003e (\u003cem\u003eRpAt2g41710\u003c/em\u003e), \u003cem\u003eA010347.1\u003c/em\u003e (\u003cem\u003eRpAP2-3\u003c/em\u003e), \u003cem\u003eA014504.1\u003c/em\u003e (\u003cem\u003eRpERF115\u003c/em\u003e), \u003cem\u003eA025658.1\u003c/em\u003e (\u003cem\u003eRpERF118\u003c/em\u003e), \u003cem\u003eA025595.1\u003c/em\u003e (\u003cem\u003eRpRAP2-11\u003c/em\u003e), \u003cem\u003eA002755.1\u003c/em\u003e (\u003cem\u003eRpDREB2A\u003c/em\u003e), and \u003cem\u003eA024668.1\u003c/em\u003e (\u003cem\u003eRpERF010\u003c/em\u003e) were simultaneously responsive to low temperature stress, demonstrating their dual regulatory roles in different abiotic stress responses. In studies investigating the low temperature stress response mechanisms of \u003cem\u003eR. persica\u003c/em\u003e, \u003cem\u003eMYC2\u003c/em\u003e was identified as a key regulator of low temperature tolerance [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Yeast two-hybrid assays revealed that MYC2 physically interacts with multiple \u003cem\u003eAP2/ERF\u003c/em\u003e family members, including \u003cem\u003eRpDREB1B\u003c/em\u003e, \u003cem\u003eRpERF114\u003c/em\u003e, \u003cem\u003eRpERF25\u003c/em\u003e, and \u003cem\u003eRpERF22\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Notably, our drought-responsive expression profiling demonstrated that \u003cem\u003eA010645.1\u003c/em\u003e (\u003cem\u003eRpDREB1B\u003c/em\u003e) also plays a significant role in drought stress adaptation, verified its dual functionality in mediating different stress responses. However, this stress response exhibits distinct tissue-specific patterns. Under drought conditions, \u003cem\u003eA025595.1\u003c/em\u003e (\u003cem\u003eRpRAP2-11\u003c/em\u003e) showed significant root-specific expression, whereas under low temperature stress it was exclusively upregulated in stems. Similarly, while \u003cem\u003eA011282.1\u003c/em\u003e (\u003cem\u003eRpAt2g41710\u003c/em\u003e), \u003cem\u003eA010347.1\u003c/em\u003e (\u003cem\u003eRpAP2-3\u003c/em\u003e), \u003cem\u003eA014504.1\u003c/em\u003e (\u003cem\u003eRpERF115\u003c/em\u003e), and \u003cem\u003eA025658.1\u003c/em\u003e (\u003cem\u003eRpERF118\u003c/em\u003e) were upregulated in both leaves and roots during drought exposure, these genes displayed root-specific induction under low temperature treatment. These observations demonstrate organ-specific partitioning of stress responses among \u003cem\u003eRpAP2/ERF\u003c/em\u003e members.\u003c/p\u003e\u003cp\u003eAmong the seven genes co-regulated under both drought and low temperature stress conditions, two members (\u003cem\u003eA011282.1\u003c/em\u003e and \u003cem\u003eA010347.1\u003c/em\u003e) belong to the AP2 subfamily. While the AP2 subfamily was widely recognized for its primary roles in plant growth and developmental processes [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] such as floral development [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], leaf morphogenesis [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and seed yield [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], emerging evidence suggests their involvement in abiotic stress responses as well [\u003cspan additionalcitationids=\"CR56 CR57\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Notably, studies on wheat disease resistance have identified that the \u003cem\u003eAP2-10\u003c/em\u003e transcription factor not only enhances disease resistance but is also significantly induced by PEG4000 and abscisic acid treatments [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. These findings suggest that AP2 subfamily members may also play important functional roles in abiotic stress adaptation in \u003cem\u003eR. persica\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study systematically identified 114 \u003cem\u003eAP2/ERF\u003c/em\u003e family members in \u003cem\u003eR. persica\u003c/em\u003e and classified them into five distinct subfamilies based on their structural and sequence characteristics. We conducted comprehensive analyses of their fundamental physicochemical properties, phylogenetic relationships, gene structures, chromosomal distributions, and syntenic relationships. Furthermore, we investigated the expression patterns of these \u003cem\u003eAP2/ERF\u003c/em\u003e members across different tissues and under abiotic stress conditions (drought and low temperature), identifying candidate genes responsive to these environmental challenges. Functional validation was performed for seven selected \u003cem\u003eRpAP2/ERF\u003c/em\u003e transcription factors to confirm their transcriptional activation activity, and a protein-protein interaction network was constructed. Collectively, this work provides a comprehensive characterization of the \u003cem\u003eRpAP2/ERF\u003c/em\u003e gene family, establishes a theoretical foundation for future functional studies of these genes and their protein interactions, and offers valuable insights for elucidating the stress resistance mechanisms in \u003cem\u003eR. persica\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eIdentification and physicochemical property analysis of\u003c/b\u003e \u003cb\u003eRpAP2/ERF\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe phased and gap-free reference genome data of \u003cem\u003eR. persica\u003c/em\u003e were derived from the previous research results of our team [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The amino acid sequences of AP2/ERF family in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were obtained from the Arabidopsis Information Resource (TAIR) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). \u003cem\u003eAP2/ERF\u003c/em\u003e genes were screened using HMMER and BLASTP tools, and the Conserved Domain Database (CDD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed for further analysis these genes. The AP2/ERF domain Hidden Markov Model (HMM) profile (PF00847) was downloaded from Pfam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The physicochemical characteristics of \u003cem\u003eAP2/ERF\u003c/em\u003e genes, such as isoelectric point (PI) and molecular weight (MW), were analyzed using the Protein Parameter Calc tool in TBtools. Subcellular localization of AP2/ERF was predicted using the WoLFPSORT online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp/\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analysis and chromosomal localization of\u003c/b\u003e \u003cb\u003eRpAP2/ERF\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMultiple sequence alignment of AP2/ERF proteins from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eR. persica\u003c/em\u003e was performed using Clustal W. The alignment data was subsequently imported into MEGA11.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.megasoftware.net/\u003c/span\u003e\u003cspan address=\"https://www.megasoftware.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and a phylogenetic tree was constructed using IQ-TREE software with the VT\u0026thinsp;+\u0026thinsp;F\u0026thinsp;+\u0026thinsp;R7 model and 1,000 bootstrap replicates. The phylogenetic tree was further modified with iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de\u003c/span\u003e\u003cspan address=\"https://itol.embl.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes were classified into five subfamilies, AP2, ERF, DREB, RAV and Soloist, depending on the number of AP2 domains and other features in sequence according to previous studies [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The chromosomal localization information of \u003cem\u003eAP2/ERF\u003c/em\u003e family members was obtained from the \u003cem\u003eR. persica\u003c/em\u003e genome annotation file. The results of gene chromosomal localization were visualized through the Gene Location Visualize from the GTF/GFF program of TBtools.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene structure, conserved motif, colinearity analysis and cis-regulatory elements analysis of\u003c/b\u003e \u003cb\u003eRpAP2/ERF\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe information of gene structures describing the exon and intron was extract from the gff file of genome sequence data. Visualization of the \u003cem\u003eRpAP2/ERF\u003c/em\u003e gene structures using TBtools. The conserved motif analysis of \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes were determined using the MEME program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the selected number of motifs set to 20. The resulting motifs were visualized using TBtools [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. MCScanX and TBtools were employed to identify tandem duplicated genes of \u003cem\u003eRpAP2/ERFs\u003c/em\u003e, and the inter-species collinearity of \u003cem\u003eRpAP2/ERFs\u003c/em\u003e between \u003cem\u003eR. persica\u003c/em\u003e and other plants. Finally, a 2000 bp genomic sequence was extracted of each \u003cem\u003eRpAP2/ERF\u003c/em\u003e gene as the putative promoter region. The cis-acting elements in the \u003cem\u003eRpAP2/ERF\u003c/em\u003e promoter regions were predicted by the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the \u003cem\u003eRpAP2/ERF\u003c/em\u003e cis-acting elements results were visualized by the Simple BioSequence Viewer program in TBtools.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression profile analysis of\u003c/b\u003e \u003cb\u003eRpAP2/ERF\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRNA-seq data of \u003cem\u003eR. persica\u003c/em\u003e AP2/ERF superfamily genes were obtained from our previous research. Transcriptome data were used to analyze the expression patterns of \u003cem\u003eRpAP2/ERF\u003c/em\u003e genes under drought stress, low temperature stress, and in various tissue structures. Normalized gene expression values expressed as FPKM (fragments per kilobase of exon per million fragments mapped) were transformed using log2 (FPKM\u0026thinsp;+\u0026thinsp;1), we further used iTOL(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de\u003c/span\u003e\u003cspan address=\"https://itol.embl.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to construct the heatmap. The RpAP2/ERF protein interaction network was constructed by STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the reference organism of \u003cem\u003eA. thaliana\u003c/em\u003e. Roots, stems, leaves, flowers, and fruits of \u003cem\u003eR. persica\u003c/em\u003e were collected in Hutubi County, Xinjiang. Three-month-old seedlings of \u003cem\u003eR. persica\u003c/em\u003e after sowing were subjected to drought treatment using the water restriction method. Samples were collected on day 0 (L0), day 7 (LD1), day 14 (LD2), and day 22 (LD3) after drought initiation, as well as on day 3 (LW1) and day 10 (LW2) after rewatering.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAnalysis of transcriptional activation activity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor the transcriptional activation assay, the ORF of six genes (\u003cem\u003eA002909.1\u003c/em\u003e, \u003cem\u003eA005829.1\u003c/em\u003e, \u003cem\u003eA014504.1\u003c/em\u003e, \u003cem\u003eA002755.1\u003c/em\u003e, \u003cem\u003eA010645.1\u003c/em\u003e, and \u003cem\u003eA009162.1\u003c/em\u003e) were generated via recombination reactions and fused into the EcoRI and SalI sites of the GAL4 DNA-binding domain in the pGBKT7 vector. \u003cem\u003eA024668.1\u003c/em\u003e was fused into the PstI and BamHI sites of the same vector. The pGBKT7 (negative control), pGBKT7-p53 (positive control), pGBKT7- \u003cem\u003eA002909.1\u003c/em\u003e, pGBKT7- \u003cem\u003eA005829.1\u003c/em\u003e, pGBKT7- \u003cem\u003eA014504.1\u003c/em\u003e, pGBKT7- \u003cem\u003eA002755.1\u003c/em\u003e, pGBKT7- \u003cem\u003eA010645.1\u003c/em\u003e, pGBKT7- \u003cem\u003eA009162.1\u003c/em\u003e, and pGBKT7- \u003cem\u003eA024668.1\u003c/em\u003e plasmid was transformed into yeast Y2H and cultured on SD/-Trp medium. Transformed cells were verified using PCR followed by spotted on SD/-Trp and SD/-Trp-His-Ade media according to a gradient dilution method for 3 days.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eClinical trial number\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by National Key Research and Development Project of China (2023YFD120010502), Fundamental Research Funds for the Central Universities (QNTD202503), National Natural Science Foundation of China, and Horizontal Project Commissioned by Enterprises and Institutions for Scientific and Technological Projects (2023-HXFW-428).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eNa Li: Performed experiments, Analyze the data, Write manuscripts. Yang Cui: Investigation, Analyze the data. Chenjie Zhang, Xiaolong Zhang, Ziguo Li: Analyze the data. Chao Yu, Huitang Pan, Yan Liu: Provide ideas.Le Luo: Provide ideas, Design experiments, Writing-review \u0026amp; editing.All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEulgem T. Regulation of the Arabidopsis defense transcriptome. Trends Plant Sci. 2005, 10(2):71-78.\u003c/li\u003e\n\u003cli\u003eChinnusamy V, Schumaker K, Zhu JK. Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. J Exp Bot. 2004, 55(395):225\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eGong Z, Xiong L, Shi H, Yang S, Herrera-Estrella LR, Xu G, Chao DY, Li J, Wang PY, Qin F, et al. Plant abiotic stress response and nutrient use efficiency. Sci China Life Sci. 2020, 63(5):635\u0026ndash;74.\u003c/li\u003e\n\u003cli\u003eYu W, Yu Y, Wang C, Zhang Z, Xue Z. 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A high-quality genome sequence of \u003cem\u003eRosa chinensis\u003c/em\u003e to elucidate ornamental traits. Nat Plants. 2018, 4(7):473-484.\u003c/li\u003e\n\u003cli\u003eLi P, Chai Z, Lin P, Huang C, Huang G, Xu L, Deng Z, Zhang M, Zhang Y, Zhao X. Genome-wide identification and expression analysis of AP2/ERF transcription factors in sugarcane (\u003cem\u003eSaccharum spontaneum\u003c/em\u003e L.). BMC Genomics. 2020, 21(1):685.\u003c/li\u003e\n\u003cli\u003eMizoi J, Shinozaki K, Yamaguchi-Shinozaki K. AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta. 2012, 1819(2):86-96.\u003c/li\u003e\n\u003cli\u003eLiu Y, Cai L, Fan X, Zhang H, Chen M, Lin Y, Chen J, Xu K, Wu B. Genome-wide identification, evolutionary expansion and expression divergence of the AP2/ERF gene family in loquat (\u003cem\u003eEriobotrya japonica\u003c/em\u003e). Fruit Res. 2024, 4:11.\u003c/li\u003e\n\u003cli\u003eZhang D, Zeng B, He Y, Li J, Yu Z. Genome-wide identification and comparative analysis of the AP2/ERF gene family in \u003cem\u003ePrunus dulcis\u003c/em\u003e and \u003cem\u003ePrunus tenella\u003c/em\u003e: expression of \u003cem\u003ePdAP2/ERF\u003c/em\u003e genes under freezing stress during dormancy. BMC Genomics. 2025, 26(1):95.\u003c/li\u003e\n\u003cli\u003eZhao M, Li Y, Zhang X, You X, Yu H, Guo R, Zhao X. Genome-wide identification of \u003cem\u003eAP2/ERF\u003c/em\u003e superfamily genes in \u003cem\u003eJuglans mandshurica\u003c/em\u003e and expression analysis under cold stress. Int J Mol Sci. 2022, 23(23):19.\u003c/li\u003e\n\u003cli\u003eYan X, Huang W, Liu C, Hao X, Gao C, Deng M, Wen J. Genome-wide identification and expression analysis of the \u003cem\u003eAP2/ERF\u003c/em\u003e transcription factor gene family in hybrid tea rose under drought stress. Int J Mol Sci. 2024, 25(23):12849.\u003c/li\u003e\n\u003cli\u003eGeng L, Zhuang Y, Sui Y, Guo R, Luo L, Pan H, Zhang Q, Yu C. Molecular mechanism of response to low-temperature during the natural overwintering period of \u003cem\u003eRosa persica\u003c/em\u003e. Plant Cell Rep. 2025, 44(4):88.\u003c/li\u003e\n\u003cli\u003eKrizek B. \u003cem\u003eAINTEGUMENTA\u003c/em\u003e and \u003cem\u003eAINTEGUMENTA-LIKE6\u003c/em\u003e act redundantly to regulate Arabidopsis floral growth and patterning. Plant Physiol. 2009, 150(4):1916-1929.\u003c/li\u003e\n\u003cli\u003eJofuku KD, den Boer BG, Van Montagu M, Okamuro JK. Control of Arabidopsis flower and seed development by the homeotic gene\u003cem\u003e APETALA2\u003c/em\u003e. Plant Cell. 1994, 6(9):1211-1225.\u003c/li\u003e\n\u003cli\u003eOkamuro JK, Caster B, Villarroel R, Van Montagu M, Jofuku KD. The AP2 domain of \u003cem\u003eAPETALA2\u003c/em\u003e defines a large new family of DNA binding proteins in Arabidopsis. Proc. Natl. Acad. Sci. 1997, 94(13):7076-7081.\u003c/li\u003e\n\u003cli\u003eZhou L, Iqbal A, Yang M, Yang Y. Research Progress on Gene Regulation of Plant Floral Organogenesis. Genes-Basel. 2025, 16(1):17.\u003c/li\u003e\n\u003cli\u003eEl Ouakfaoui, Schnell J, Abdeen A, Colville A, Labbe H, Han S, Baum B, Laberge S, Miki B. Control of somatic embryogenesis and embryo development by AP2 transcription factors. Plant Mol Biol. 2010, 74(4-5):313-326.\u003c/li\u003e\n\u003cli\u003eJofuku KD, Omidyar PK, Gee Z, Okamuro JK. Control of seed mass and seed yield by the floral homeotic gene \u003cem\u003eAPETALA2\u003c/em\u003e. Proc. Natl. Acad. Sci. 2005, 102(8):3117-3122.\u003c/li\u003e\n\u003cli\u003eHu ZY, Wang XJ, Wei L, Wansee S, Rabbani Nasab HR, Chen L, Kang ZS, Wang JF. \u003cem\u003eTaAP2-10\u003c/em\u003e, an AP2/ERF transcription factor, contributes to wheat resistance against stripe rust. J Plant Physiol. 2023, 288:154078.\u003c/li\u003e\n\u003cli\u003eChang B, Qiu XY, Yang Y, Zhou WX, Jin B, Wang L. Genome-wide analyses of the \u003cem\u003eGbAP2\u003c/em\u003e subfamily reveal the function of \u003cem\u003eGbTOE1a\u003c/em\u003e in salt and drought stress tolerance in \u003cem\u003eGinkgo biloba\u003c/em\u003e. Plant Sci. 2024, 342:14.\u003c/li\u003e\n\u003cli\u003eZhou L, Cao H, Zeng X, Wu Q, Li Q, Martin JJJ, Fu D, Liu X, Li X, Li R et al. Oil palm AP2 subfamily gene \u003cem\u003eEgAP2.25\u003c/em\u003e improves salt stress tolerance in transgenic tobacco plants. Int J Mol Sci. 2024, 25(11):14.\u003c/li\u003e\n\u003cli\u003eChen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: An integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020, 13(8):1194-1202.\u003c/li\u003e\n\u003cli\u003eLiu JY, Bennett D, Demuth M, Burchard E, Artlip T, Dardick C, Liu Z. \u003cem\u003eeuAP2a\u003c/em\u003e, a key gene that regulates flowering time in peach (\u003cem\u003ePrunus persica\u003c/em\u003e) by modulating thermo-responsive transcription programming. Hortic Res-England. 2024, 11(5):14.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Genome-wide analysis, Rosa persica, AP2/ERF gene family, stress response","lastPublishedDoi":"10.21203/rs.3.rs-7203695/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7203695/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003eRosa persica\u003c/em\u003e, the sole species in the subgenus Hulthemia of Rosaceae, thrives in the barren Gobi Desert, where its harsh environment has driven the evolution of exceptional stress-resistant traits, making it a valuable germplasm resource for studying extreme drought adaptation. With the recent completion of its telomere-to-telomere (T2T) genome assembly, an increasing number of stress-responsive gene families have been systematically characterized in \u003cem\u003eR. persica\u003c/em\u003e. However, despite being one of the most critical transcription factor superfamilies involved in plant stress responses, the \u003cem\u003eAP2/ERF\u003c/em\u003e superfamily has not yet been comprehensively identified or functionally analyzed in this extremophyte species, warranting in-depth investigation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThrough comprehensive genome-wide analysis, we identified 114 \u003cem\u003eAP2/ERF\u003c/em\u003e members in \u003cem\u003eR. persica\u003c/em\u003e, which were classified into five subfamilies: AP2, DREB, ERF, RAV, and Soloist. The expression profiles of these \u003cem\u003eAP2/ERF\u003c/em\u003e genes exhibited distinct tissue specificity. Notably, \u003cem\u003eA005041.1\u003c/em\u003e, \u003cem\u003eA008193.1\u003c/em\u003e, \u003cem\u003eA013003.1\u003c/em\u003e, and \u003cem\u003eA014479.1\u003c/em\u003e were exclusively expressed in roots, while \u003cem\u003eA001403.1\u003c/em\u003e and \u003cem\u003eA026890.1\u003c/em\u003e showed stem-specific expression. Seven genes (\u003cem\u003eA011282.1\u003c/em\u003e, \u003cem\u003eA010347.1\u003c/em\u003e, \u003cem\u003eA014504.1\u003c/em\u003e, \u003cem\u003eA025658.1\u003c/em\u003e, \u003cem\u003eA025595.1\u003c/em\u003e, \u003cem\u003eA002755.1\u003c/em\u003e, and \u003cem\u003eA024668.1\u003c/em\u003e) were simultaneously responsive to both drought and low temperature stress in \u003cem\u003eR. persica\u003c/em\u003e. Among the ERF and DREB subfamilies, the abiotic stress-related genes \u003cem\u003eA002909.1\u003c/em\u003e, \u003cem\u003eA014504.1\u003c/em\u003e, \u003cem\u003eA002755.1\u003c/em\u003e, \u003cem\u003eA010645.1\u003c/em\u003e, and \u003cem\u003eA009162.1\u003c/em\u003e demonstrated transcriptional activation activity in yeast assays, whereas \u003cem\u003eA005829.1\u003c/em\u003e and \u003cem\u003eA024668.1\u003c/em\u003e lacked such activity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThis study provides the first systematic identification and characterization of the \u003cem\u003eAP2/ERF\u003c/em\u003e gene family in \u003cem\u003eR. persica\u003c/em\u003e, revealing key members involved in drought and cold stress responses. Our findings establish a theoretical foundation for elucidating the functional mechanisms of \u003cem\u003eAP2/ERF\u003c/em\u003e genes in this extremophyte species and their roles in stress adaptation.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification of the AP2/ERF gene family in Rosa persica and expression profiling under abiotic stresses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-28 18:52:58","doi":"10.21203/rs.3.rs-7203695/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-04T05:16:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256267047741786720743597136587320904498","date":"2026-03-24T06:09:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-20T08:27:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65004479494890168659736145704166491652","date":"2026-03-20T06:39:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51819765225541548578987624059030736050","date":"2026-03-19T12:20:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"152395060138989292094611149642787334312","date":"2025-09-05T15:22:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-02T17:15:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91407907908050235601301040074850195696","date":"2025-08-27T17:39:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211632786675711594668796882436929876278","date":"2025-08-22T11:21:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-20T09:14:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-19T10:13:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-04T05:00:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-04T05:00:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-07-24T09:06:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fb42ca90-92e5-4000-b9b1-b5756a86776c","owner":[],"postedDate":"August 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-08-28T18:52:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-28 18:52:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7203695","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7203695","identity":"rs-7203695","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}