Genome-Wide Identification and Stress-Responsive Expression Analysis of the Actin-Depolymerizing Factor (ADF) Gene Family in Avena sativa L

Genome-Wide Identification and Stress-Responsive Expression Analysis of the Actin-Depolymerizing Factor (ADF) Gene Family in Avena sativa L | 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 and Stress-Responsive Expression Analysis of the Actin-Depolymerizing Factor (ADF) Gene Family in Avena sativa L Deying Wang, Tao Liu, Wenhui Tian, Hong Chen, Guoqing Wang, Bo Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8235971/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Feb, 2026 Read the published version in BMC Plant Biology → Version 1 posted 14 You are reading this latest preprint version Abstract Background Oat ( Avena sativa L.) is an important cereal crop globally, valued for both its grain and forage. Actin depolymerization factors (ADFs) are highly conserved eukaryotic proteins that facilitate remodeling of the actin cytoskeleton. Accumulating evidence indicates that ADFs play crucial roles in plant growth, development, and stress responses. Nevertheless, the AsADFs family has not yet been identified in oat, an important cereal with high abiotic stress tolerance. Results In this study, a total of 18 ADF genes ( AsADF ) were identified from the oat reference genome (Sang.v1.1) and mapped to 12 different chromosomes. Based on the phylogenetic analysis, these genes were classified into four groups, which was confirmed by their structure and the distribution of conserved motifs in the encoded proteins. Collinearity analysis demonstrated strong relationships between oat and wheat ADFs . The promoters of most AsADFs family contain cis-elements associated with growth, development, and stress responses, suggesting their potential involvement in these biological processes. Subcellular localization prediction indicated that ADFs are mainly located in the cytoplasm, and this localization is consistent with their role in cytoskeletal maintenance. Analysis of qRT-PCR results indicated that most AsADFs exhibited differential expression patterns under circadian rhythm and responded to various abiotic stresses. Among them, the expression levels of AsADF9 , 13 , and 14 from group B showed significant changes under all four abiotic stress conditions, suggesting their important roles in abiotic stress resistance. Conclusions This study revealed that AsADF genes play crucial roles in oat's response to various abiotic stresses. Notably, the expression of AsADF9 , 13 and 14 was strongly induced under stress conditions, highlighting them as key candidates mediating oat's stress response. In summary, the present study established a theoretical foundation for analyzing the molecular mechanism of stress resistance in oat and provides valuable insights for molecular breeding to enhance stress resistance. actin-depolymerizing factor oat circadian rhythm abiotic stresses expression analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background The actin-based microfilament cytoskeleton is essential for numerous cellular processes, providing mechanical support and motive force for the cell, and playing critical roles in plant growth, development, and stress responses [ 1 – 3 ]. Actin exists in two forms: globular actin (G-actin) and filamentous actin (F-actin) [ 4 ]. The polymerization of G-actin into F-actin enables a wide range of biological functions [ 5 , 6 ]. The remodeling of F-actin is driven by a diverse set of actin-binding proteins (ABPs), which in turn regulate various cellular and physiological functions [ 7 , 8 ]. Among these, the actin depolymerizing factor (ADF) represents a crucial ABP that severs and depolymerizes F-actin [ 9 , 10 ]. The number of ADF family genes varies considerably among different organisms. Most non-plant organisms have only one or two ADF/cofilin genes. In contrast, plants typically possess an expanded ADF family, which enables them to fulfill diverse physiological functions [ 5 , 11 ]. Given the important functions of the actin cytoskeleton in plant development and stress responses, ADF proteins are regulators of cellular processes [ 5 , 9 ]. Consequently, genome-wide identification and functional characterization of the ADF family have been performed in numerous plant species (Table S1 ). The ADF gene family plays crucial roles in plant growth and development, and the functional characterization of ADFs has been conducted in various plant species. For instance, in Arabidopsis thaliana , the atadf9 mutant displays reduced lateral branches and diminished callus-forming capacity [ 12 ]. AtADF1 and AtADF4 promote hypocotyl elongation by interacting with 14-3-3 λ proteins [ 13 , 14 ]. AtADF7 and AtADF10 regulate pollen tube growth by modulating cytosolic pH [ 15 ]. Beyond A. thaliana , studies in cotton have shown that suppressing GhADF1 expression enhances both fiber elongation and secondary cell wall deposition [ 16 ]. In maize, ZmADF1 is pollen-specific and negatively regulates pollen quantity, viability, germination, and seed set [ 17 ]. Beyond growth, ADFs are crucial for stress responses. The atadf5 mutant exhibits reduced drought survival due to impaired stomatal closure [ 18 ]. AtADF1 responds to salt stress via its association with MYB73 [ 19 ], while AtADF4 confers osmotic stress tolerance [ 14 ]. GhADF6 is implicated in the response to Verticillium dahliae [ 20 ]. Heterologous expression of GmADF13 in Arabidopsis and soybean hairy roots enhances drought tolerance [ 21 ]. Overexpression of OsADF3 also improves drought tolerance in transgenic A. thaliana seedlings [ 22 ]. ZmADF1 plays a critical role in regulating growth under high-temperature stress [ 11 ]. In wheat, TaADF3 negatively regulates Puccinia striiformis (PST) resistance, likely by modulating ROS homeostasis [ 23 ]. In contrast, TaADF4 and TaADF7 are shown to confer resistance to PST by remodeling the actin cytoskeleton [ 24 , 25 ]. Furthermore, heterologous overexpression of TaADF16 is shown to enhance cold tolerance in transgenic A. thaliana by boosting ROS scavenging capacity [ 26 ]. As the world's seventh most widely grown cereal crop, oat ( Avena sativa L.) is a valuable cultivated dual-purpose species [ 27 – 29 ]. This health-promoting cereal has garnered increasing attention due to its rich content of dietary fiber, phytochemicals, and essential nutrients [ 30 , 31 ]. Concurrently, as a crucial forage crop, oat provides high-quality feed for livestock. This helps alleviate pressure on conventional feed grains and natural grasslands, thereby facilitating the restoration of degraded pastures and ecological conservation [ 32 , 33 ]. Oats not only possess significant economic value but are also considered a valuable gene pool due to their tolerance to stress conditions. Oat contains numerous stress-resistance genes yet to be discovered, which can provide vital resources for crop stress-resistance improvement. Stable yield is crucial for oat production. Despite oat’s considerable ability to tolerate environmental stresses, their growth and development are still affected by various stresses, such as extreme temperatures, cold, and drought, leading to substantial yield losses [ 34 – 36 ]. In this study, 18 AsADF genes were identified from the oat Sang reference genome (Sang.v1.1) and a comprehensive analysis was conducted. Initially, we assessed their fundamental physicochemical properties and chromosomal locations. To elucidate their evolutionary relationships, we constructed a phylogenetic tree and analyzed the protein structures. Furthermore, cis-elements within the promoter regions were predicted, along with their subcellular localization. Finally, to investigate their potential regulatory mechanisms and functions, we examined their expression patterns under circadian rhythms and their expression level changes under abiotic stress. This study establishes a foundation for screening key candidate genes within the ADF family in oats and provides valuable genetic resources for molecular breeding aimed at stress resistance. Results Identification of ADF genes in oat Through a comprehensive analysis of the ADF-H domain (PF00241) annotation and the CD-Search tool from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/cdd/), a total of 18 putative ADF genes were identified in the oat genome. These genes were located on 12 different chromosomes and were designated AsADF1 to AsADF18 based on their respective gene IDs in the Sang reference genome (Table 1, Fig. 1). Analysis of the basic physicochemical properties of AsADF proteins revealed substantial variation among members. Their amino acid lengths ranged from 139 aa (AsADF4, 5, 6, 7, and 17) to 152 aa (AsADF1). The molecular weight (MW) varied between 15.75 kDa (AsADF13) and 17.30 kDa (AsADF1), and the theoretical isoelectric point (pI) spanned from 4.43 (AsADF13) to 8.73 (AsADF2 and AsADF6). The instability index (InI) ranged from 31.61 (AsADF6) to 47.73 (AsADF17), while the aliphatic index (AI) values between 61.08 (AsADF6) and 85.65 (AsADF14) suggest relatively high thermal stability. Finally, the grand average of hydropathicity (GRAVY) scores ranged from -0.037 (AsADF17) to -0.567 (AsADF4), indicating that all these proteins are hydrophilic (Table 1). Subcellular localization predictions indicated that the ADF proteins are mainly localized in the cytoplasm, followed by the chloroplast, nucleus, extracellular space, mitochondria, and peroxisomes. This distribution implies potential functional roles for ADF proteins within these organelles (Fig. S1). Table 1 Physicochemical properties of AsADF proteins in oats Sequence ID Name Protein Length MW(kDa) pI InI AI GRAVY AVESA.00010b.r2.1AG0061340.1 AsADF1 152 17307.39 5.22 45.64 74.47 -0.493 AVESA.00010b.r2.1DG0136740.1 AsADF2 143 16402.91 8.73 43.25 74.34 -0.3 AVESA.00010b.r2.1DG0179840.1 AsADF3 150 17278.35 5.1 44.51 72.13 -0.541 AVESA.00010b.r2.2AG0200070.1 AsADF4 139 16043.09 5.4 36.85 63.24 -0.567 AVESA.00010b.r2.2CG0312780.1 AsADF5 139 15755.91 5.55 43.77 61.8 -0.439 AVESA.00010b.r2.2DG0370090.1 AsADF6 139 15885.01 5.25 31.61 61.08 -0.488 AVESA.00010b.r2.3DG0568620.1 AsADF7 139 15931.18 5.52 42.74 77.91 -0.376 AVESA.00010b.r2.4AG0610360.1 AsADF8 145 16778.06 5.94 42.93 72.69 -0.44 AVESA.00010b.r2.4AG0615680.1 AsADF9 147 15789.71 4.64 39.33 83.67 -0.059 AVESA.00010b.r2.4AG0615690.1 AsADF10 142 16055.08 5.46 44.36 74.23 -0.484 AVESA.00010b.r2.4CG1327640.1 AsADF11 143 16416.94 8.73 45.53 74.34 -0.3 AVESA.00010b.r2.4DG0753700.1 AsADF12 145 16720.02 6.31 42.93 72.69 -0.419 AVESA.00010b.r2.4DG0759170.1 AsADF13 142 15754.6 4.44 38.69 87.18 -0.238 AVESA.00010b.r2.4DG0759210.1 AsADF14 147 15837.76 4.6 39.09 85.65 -0.037 AVESA.00010b.r2.4DG0759220.1 AsADF15 142 16085.11 5.46 44.95 74.23 -0.487 AVESA.00010b.r2.5AG0799410.1 AsADF16 143 16402.91 8.73 43.25 74.34 -0.3 AVESA.00010b.r2.6AG1032230.1 AsADF17 139 16013.33 5.56 47.73 70.79 -0.384 AVESA.00010b.r2.7CG0705670.1 AsADF18 145 16746 5.94 43.45 74.69 -0.424 Chromosomal location and collinearity analysis of AsADFs A total of 18 AsADFs were identified and distributed across 12 of the 20 chromosomes, with one to four genes per chromosome (Fig. 1). Chromosome 4D contained the highest number ( AsADF12 , 13 , 14, and 15 ), followed by chromosome 4A ( AsADF8 , 9, and 10 ), and chromosome 1D ( AsADF2 and 3 ). The remaining chromosomes (1A, 2A, 2C, 2D, 3D, 4C, 5A, 6A, and 7C) each contained only one AsADF gene (Fig. 1). Gene duplication is a widespread phenomenon that plays a crucial role in plant evolution [37]. To identify gene duplication events within the AsADF family, genomic collinearity segments in the oat genome were systematically analyzed using TBtools, and the results were visualized in Circos plots (Fig. 2). The results showed 13 pairs of tandem duplication events in the oat genome (Fig. 2). They suggest an evolutionary relationship between these AsADF members, and that these genes may have similar functions. To further investigate the phylogenetic relationships between the oat ADF family and the ADF families of other plants, a collinearity analysis was conducted between oat and four other species: A. thaliana, maize, rice, and wheat. The analysis revealed 3, 28, 17, and 44 orthologous gene pairs between oat and A. thaliana , maize, rice, and wheat, respectively. Notably, the ADF genes exhibited the highest degree of evolutionary conservation between oat and wheat (Fig. 3). Phylogenetic analysis of the AsADFs To further elucidate the phylogenetic relationships of oat ADF proteins, we collected ADF family genes from several identified species, including the model plant A. thaliana ; the Poaceae species rice, maize, and wheat; and soybean (previously reported by our lab), and constructed a phylogenetic tree using the neighbor-joining (NJ) method in MEGA X with the ADF family of oat (Table S2, Fig. 4). The results showed that the ADF proteins were grouped into four groups, and the number in each group was different. With the exception of Group B, which contained only four Poaceae ADFs, the other three groups comprised ADFs from all six species. Notably, AsADFs and OsADFs within each subgroup showed a close evolutionary relationship. Among the oat ADFs, Group C contained the smallest number (3), while Groups A, B, and D each contained 4 members (Fig. 4). Characteristics of the AsADFs protein sequences The protein sequence identity among the AsADFs was greater than 58%, with the lowest identity observed between AsADF3 and AsADF14. Notably, the protein sequences of AsADF2/11/16 and AsADF8/18 were identical (Fig. S2). Multiple sequence alignment revealed that all AsADF proteins contain the conserved ADF-H domain and actin-binding regions (Fig. S3). Based on amino acid sequence prediction, the secondary structure of AsADF proteins consists mainly of three α-helices, six β-sheets, and four β-turns. This structural composition suggests that AsADF family proteins possess actin-binding functions, which is consistent with the known role of ADFs in binding to F-actin (Fig. S3). Furthermore, 3D structural models of AsADFs predicted by SWISS-MODEL show that proteins within the same group share high structural similarity, implying potential functional conservation (Fig. S4). Conserved motifs and gene structures of the AsADFs Among the nine conserved motifs identified, Motifs 1-4 constitute the core ADF domain, indicating their conservation and functional importance within the oat ADF family (Fig. 5B; Fig. S5). Notably, several motifs exhibited specific distributions: Motif 9 was unique to AsADF1 ; Motif 8 was exclusively present in AsADF9 and AsADF14 ; Motif 6 was specific to Group C; and Motifs 5 and 7 were found only in Group D (Fig. 5B). Structural analysis of AsADF genes revealed that members of the same group generally share similar exon-intron organizations (Fig. 5A, C). Within the AsADF gene family, all genes contained three exons, except for AsADF4 and certain members of Group B (excluding AsADF10 ) (Fig. 5C). In these three exon genes, the second exon is the longest, flanked by shorter exons at the 5' and 3' ends, with the 3' exon being the shortest (Fig. 5C). Furthermore, size variation among the AsADF genes appears to be primarily due to differences in intron length. Functional analysis of cis-elements in the promoter region of AsADFs To further understand the functional roles of the AsADF gene family, we performed an analysis of the 2,000 bp promoter regions using the online software PlantCARE. The results showed that the cis-elements in the AsADF promoters could be classified into four major categories based on their predicted functions: stress response, photoperiod responsiveness, phytohormone response, and growth/development (Fig. 6; Fig. S6). Notably, the promoters of the AsADF family genes contained 11 low-temperature response and 21 drought responsive elements, suggesting their important roles in environmental stress adaptation. Furthermore, we identified 23 photoperiod-related and 9 hormone-related elements, with abscisic acid (ABA) and methyl jasmonate (MeJA) response elements being the most abundant (Fig. 6; Fig. S6). These findings suggest that the AsADF family is crucial not only for growth and development but also for responding to abiotic stresses in oat. Analysis of differential expression under circadian rhythm To further investigate the potential roles of AsADF genes in oat growth, we analyzed their circadian expression rhythms by qRT-PCR. In all qRT-PCR analyses, a gene was considered to respond to a treatment if it showed a minimum twofold difference compared to the control with a statistical significance of P < 0.01. The results showed that AsADF genes in the same group had similar rhythmic expression patterns (Fig. 7). Specifically, in Group A, the expression levels of four genes (excluding AsADF17 ) exhibited a circadian rhythm, being up-regulated after sunrise (8:00), peaking at noon (12:00), and subsequently down-regulated. Conversely, Group C genes displayed an opposite pattern, with the highest expression at 8:00 that decreased throughout the day and began to rise again around 24:00. Within Group D, AsADF8 , 12 , and 18 showed the same expression pattern as Group C, with levels down-regulated during the day and up-regulated at night to a peak at 24:00, but AsADF1 , AsADF3 and all Group B genes did not exhibit significant circadian rhythmicity (Fig. 7). In summary, the expression of most AsADF genes exhibits diurnal fluctuations, suggesting their involvement in photoperiod-related growth and development processes. Transcription patterns of AsADFs in response to abiotic stress To further understand the potential functions of AsADFs in response to abiotic stresses, we collected leaves and roots from oat seedlings at the three-leaf stage following exposure to cold, heat, drought, and salt treatments. The relative expression levels of the AsADF genes were then examined by qRT-PCR (Fig. 8). The results indicated that most AsADF genes were responsive to various abiotic stresses to differing degrees. Notably, the expression of AsADF4 , 5 and 7 of Group A, AsADF9 , 13 , and 14 of Group B, AsADF8 , 12 and 18 of Group D responded to all four stress treatments, suggesting their potential essential functions in oat's stress response. The expression levels of AsADF9 , 13 and 14 were significantly up-regulated under all four stresses; those of AsADF8 , 12 and 18 were up-regulated by heat, salt, and drought stress, while under cold stress, their expression was initially up-regulated, peaked at 3 h and subsequently down-regulated; The relative expression of AsADF4 , 5 , and 7 was down-regulated under heat and salt stress, but under cold stress, their expression was initially down-regulated, up-regulated at 12 h, and then down-regulated again, drought stress caused little overall change, although a transient spike in expression was notably observed at 9 h. Under cold stress treatment, the expression levels of the 13 AsADF genes, except for AsADF1 , 3 , 10 , 15 , and 17 , were up-regulated or down-regulated to varying degrees; whereas under drought stress, the expression levels of all AsADF genes were up-regulated, among which the expression levels of AsADF9 , 13 and 14 were up-regulated by more than a 100-fold (Fig. 8). Discussion ADFs, recognized as one of the key members among ABPs, are involved in many plant growth and development processes [ 5 , 9 ]. Their crucial role in plant stress responses has been well-established [ 5 ]. The ADF gene family has been identified in a variety of plant species through genome-wide analyses. The number of ADF genes varies, with 11 in A. thaliana , 11 in rice, 14 in poplar, 11 in tomato, 27 in banana, 13 in maize, 9 in common bean, 10 in pigeon pea, 25 in wheat, 18 in soybean, 18 in melon, 18 in Chinese cabbage, and 9 in alfalfa [ 11 , 22 , 26 , 38 – 47 ]. However, the functions of the ADF family genes in oats remain unclear. Therefore, we identified ADF genes in oat and conducted a comprehensive analysis of their phylogenetic relationships, sequence characteristics, and expression patterns. In this study, we identified 18 ADF genes (Fig. 1 , Table 1 ), which were located on 12 chromosomes (Fig. 1 ). Previous studies have suggested that ADF genes in plants likely evolved from a common ancestor [ 41 ]. A comparative analysis of the characterized ADF families in Poaceae reveals that this number of ADF genes is greater than that in rice (11) and maize (13) but less than that in wheat (25). This variation in gene copy number among different plant species is potentially driven by gene duplication events [ 11 , 22 , 26 , 48 ]. Although oats and wheat are both important allopolyploid cereals with complex and large genomes, we found that oats possess a smaller number of ADF genes than wheat [ 26 ]. We speculate that this difference from the relatively late initiation of systematic research on oats compared to wheat, resulting in a less complete genome assembly and annotation. Evolutionary analysis indicated that oat ADF genes were classified into four groups (Fig. 4 ). This grouping pattern is consistent with that reported in A. thaliana , rice, tomato, maize, melon, soybean, Chinese cabbage, and alfalfa [ 11 , 38 , 40 , 44 – 47 ]. Phylogenetic analysis revealed that many oat ADF genes were closely clustered with previously reported ADF genes known to play critical roles in stress responses, suggesting potential functional conservation. For instance, AsADF9 , AsADF13 , and AsADF14 cluster within the same clade as OsADF3 and TaADF16 . Based on the reported roles of these genes in drought and cold stress tolerance, we speculate that the oat ADFs in this clade may exhibit a conserved biological function in response to these abiotic stresses [ 22 , 26 ](Fig. 4 ). Furthermore, the exon-intron structures of these genes were largely conserved compared to previous reports (Fig. 5 C). Analysis of the oat ADF protein motifs revealed that these proteins are highly conserved (Fig. 5 B). All AsADF proteins contain the ADF-H domain, which is composed of motifs 1 to 4. Additionally, we identified some motifs specific to certain genes, which may be linked to functional specialization among them (Fig. 5 B; Fig. S5). Given that gene expression is largely regulated by cis-elements in promoter sequences, analyzing these elements helps elucidate the regulatory mechanisms of ADF genes and infer their potential biological functions [ 49 , 50 ]. Previous studies have indicated that phytohormonal signals and environmental stresses are key regulators of ADF gene expression [ 17 , 26 ]. Various phytohormone and stress-responsive elements, including LTR (low-temperature response), MBS (drought stress response), ABRE (ABA response), and TGACG-/CGTAC-motifs (MeJA response), were identified in the promoters of oat ADF genes through cis-element analysis (Fig. 6 ; Fig. S6). Notably, light-responsive elements were also identified in all AsADF gene promoters, further supporting their potential roles in regulating oat growth, development, and stress responses. Light is fundamental to plant life, and its perception governs key aspects of plant growth and development [ 51 – 53 ]. The expression of numerous genes involved in these processes is under circadian rhythm regulation, which in turn affects the growth and development of plants [ 54 ]. Given the presence of multiple light-responsive elements identified in the promoters of oat ADF genes, we speculate that they might function in photoperiodic regulation (Fig. 6 ; Fig. S6). To investigate whether there was differential circadian expression of the AsADF genes, we conducted qRT-PCR analysis (Fig. 7 ). Our results indicated that the expression of most AsADF genes followed a circadian rhythm (Fig. 7 ). Genes in Group A (except AsADF17 ) exhibited higher expression during the day than at night, with their transcript levels peaking at noon coinciding with the highest temperature of the day, suggesting a potential role in sensing ambient temperature fluctuations. Conversely, the Group C genes showed higher expression during the night. Phylogenetic relationships showed these genes are in the same clade as AtADF5 , which has been reported to regulate drought tolerance by modulating stomatal aperture. Based on this, we infer that the Group C AsADFs may perform a similar function in drought stress response [ 18 ](Fig. 4 ; Fig. 7 ). In summary, AsADF genes likely participate in various growth and developmental pathways by responding to photoperiodic rhythms. During growth and development, plants are usually exposed to various environmental stresses, such as soil salinity, drought, and extreme temperatures [ 55 ]. As an important nutrient-rich grain and forage crop, oat holds significant value in stable yields under stress conditions [ 27 – 29 ]. Plants require optimal temperatures for growth and development, and temperatures beyond their physiological optimum ranges can result in heat or cold stress [ 56 ]. With the intensification of global warming in recent years, the fluctuations in spring temperatures have significantly increased the risk of cereal crops being exposed to late spring coldness (LSC) [ 57 , 58 ]. LSC causes severe damage or even plant death. As oats are mainly cultivated in cold high-altitude regions, they are highly vulnerable to LSC, often leading to significant yield losses. Moreover, oat growth requires cool climatic conditions, and this trait limits its cultivation in warmer regions and constrains the supply of high-quality oats to meet consumer demand [ 59 , 60 ]. Besides temperature stress, salt and drought stress also negatively affect oat growth and productivity [ 61 , 62 ]. Soil salinization is a major environmental challenge to global agricultural production [ 34 , 63 ], and drought conditions are common in many oat-growing regions. Although oats are moderately stress tolerant, their yield remains limited by salt and drought [ 34 , 35 , 64 ]. Therefore, identifying key genes associated with abiotic stress tolerance is crucial for oat's stable yields. The remodeling of the actin cytoskeleton serves as a critical target for signaling pathways triggered by diverse developmental and environmental stimuli [ 8 , 65 ]. Plant ADFs function through cytoskeletal remodeling, and studies have reported that ADF genes in various Poaceae species participate in abiotic stress responses. For instance, heterologous expression of OsADF3 enhances drought tolerance in A. thaliana [ 22 ]. In addition, TaADF16 plays an important role in low temperature tolerance [ 26 ]. Based on the significant role of the ADF gene family in abiotic stress responses, it is necessary to analyze its function in oats. Leaves are the primary site for temperature stress perception in plants, while roots are a major organ for sensing salt and drought stress. Therefore, we used qRT-PCR to analyze the expression patterns of AsADF genes under these stresses [ 66 , 67 ]. As shown in Fig. 8 , all AsADF genes were responsive to drought stress. Notably, Group B members AsADF9 , AsADF13 , and AsADF14 exhibited significant up-regulation under all four abiotic stress treatments, suggesting their important role in stress response of oats (Fig. 8 ). These ADF genes are potential candidates for abiotic stress tolerance in oats and could be further verified as key genes. Conclusions This study identified 18 ADF genes unevenly distributed across 12 chromosomes in the oat genome. Phylogenetic analysis classified these genes into four groups, with members of each group exhibiting conserved gene structures and motif compositions. Expression analysis revealed that most ADF genes showed circadian rhythms, suggesting roles in plant growth regulation. Furthermore, multiple ADF genes responded to various abiotic stresses, particularly drought, indicating their potential involvement in stress adaptation. Notably, AsADF9 , AsADF13 , and AsADF14 from Group B were significantly up-regulated under four stress conditions, indicating that they are promising candidates for further functional characterization. These results provide valuable insights into the functions of the ADF family and offer new genetic resources for the development of stress-tolerant oat varieties. Materials and methods Identification of the ADF genes in the genome of oat The whole genome and GFF3 of oat (Sang.v1.1) and other plants ( A. thaliana , rice, maize, and wheat) were obtained from the Ensembl Plants database ( https://plants.ensembl.org/ ). A collection of experimentally validated ADF family sequences from published studies served as query sequences for homology-based screening via the BLAST Compare Two Seqs function in TBtools [ 68 ]. The hidden Markov model (HMM) profile of the ADF-H domain (PF00241) was obtained from the Pfam database ( http://pfam.xfam.org/ ) and domain architecture analysis was conducted using the Simple HMM Search module within TBtools with an E-value cutoff of 1e-5. The presence of the ADF-H domain in putative ADF proteins was verified using the batch CD-search tool available through the NCBI Conserved Domain Database (CDD) ( https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi ). A consensus strategy integrating sequence similarity and structural domain conservation yielded the final candidate set of oat ADF family members. Physicochemical characteristics and localization to chromosomes The physicochemical properties of the identified AsADF proteins, including amino acid length, molecular weight (MW), isoelectric point (pI), instability index (II), aliphatic index (AI), and grand average of hydropathicity (GRAVY), were predicted using the ProtParam tool on the ExPASy server [ 69 ]( https://web.expasy.org/protparam/ ). The physical positions of the AsADF genes were determined from the genome annotation file, and the genes were mapped to their chromosomes. The genomic distribution was visualized using the "Gene Location Visualize from GTF/GFF" function in TBtools, based on the Sang reference genome. Analysis of the gene structure and identification of the conserved motif in the protein Structural analysis of AsADF genes was performed using the oat genome annotation file (GFF3 format) obtained from the Ensembl Plants database. A phylogenetic tree of AsADF proteins was constructed with MEGA X software via the NJ method, with 1000 bootstrap replicates. Conserved motifs in the AsADF proteins were identified using the MEME (Multiple Expectation Maximization for Motif Elicitation) suite, with the maximum number of motifs set to 9, and were visualized using the "Gene Structure View" function in TBtools. Exon-intron structures were visualized using the "Visualize Gene Structure" function in TBtools. Furthermore, conserved domains in AsADF proteins were predicted using the Batch CD-Search tool on the NCBI platform and visualized with the "Visualize NCBI CDD Domain Pattern" function in TBtools. Analysis of protein sequence characteristics, phylogeny, and collinearity Multiple sequence alignment of ADF family proteins was performed using MEGA X software, and the resulting alignment was exported in FASTA format. For visualization, the tertiary structure file of the ADF protein (1f7s.1.pdb) was obtained from SWISS-MODEL and used to annotate the alignment results using the online tool ESPript 3.0. The protein sequences of the 18 AsADF genes were submitted to the SWISS-MODEL online server [ 70 ] ( https://swissmodel.expasy.org/ ) for homology modeling. The template (1f7s.1.A) with the highest Global Model Quality Estimation (GMQE) score in the PDB database was selected to ensure high structural consistency across the AsADF protein family.The phylogenetic tree of oat, wheat, rice, and maize ADF proteins was constructed using the NJ method in MEGA X software with 1000 bootstrap replicates. The resulting tree was then visualized and annotated using the iTOL online platform ( https://itol.embl.de/ ) to enhanced graphical representation. Gene collinearity analysis was carried out among A. thaliana , wheat, rice, and maize. The One-Step MCScanX-Super Fast plugin in TBtools was employed for the collinearity analysis and visualization. Cis-elements analysis and subcellular localization Putative cis-acting elements within the 2,000 bp promoter region upstream of the AsADF genes were identified using the PlantCARE database ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). The predicted cis-acting elements were visualized using TBtools. Subcellular localization was predicted using WoLF PSORT ( https://wolfpsort.hgc.jp/ ). The results were subsequently visualized and comparatively analyzed with TBtools to facilitate biological interpretation. Plant materials and stress treatments materials of oat seedlings The oat cultivar Keyan No.4, used in this study, was developed and stored at the Key Laboratory of Adaptation and Evolution of Plateau Biota (AEPB) of the Northwest Institute of Plateau Biology, Chinese Academy of Sciences. The oat seeds were disinfected using 75% ethanol for 5 min, followed by treatment with 3% H 2 O 2 for 1 min, and then washed 5 times with sterile water before being germinated for 5 days in a growth chamber. The germinated seeds were then cultivated in a modified Hoagland solution (NS10205-500 mL, Coolaber) under a 14-h light/10-h dark cycle, with temperatures at 23°C during the day and 18°C at night for 15 days. Subsequently, seedlings were exposed to various stress conditions: heat (40°C), cold (4°C), salt (150 mM NaCl), and drought (simulated with 20% w/v PEG-6000), for durations of 0, 1, 3, 6, 12, and 24 hours. Untreated Hoagland's solution was used as the control. For circadian rhythm analysis of AsADFs , seedlings were grown until the three-leaf stage, and samples were collected every 4 hours. Following collection, roots and leaves were immediately frozen in liquid nitrogen and stored at -80°C for subsequent RNA extraction. RNA extraction, cDNA preparation, and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from plant samples using FastPure Universal Plant Total RNA Isolation Kit (RC411-01, Vazyme) according to the manufacturer's instructions. And then reverse-transcribed into cDNA with HiScriptIII RT SuperMix for qPCR (+ gDNA wiper) (R323-01, Vazyme). The qRT-PCR analysis was performed using ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme Biotech), and the protocol was 95℃ for 30 s followed by 34 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. Relative quantification of gene expression was performed based on the 2 −ΔΔCt method [ 71 ], with AsEIF4A used as the internal reference gene. The sequences of all primers used in this study are listed in Supplementary Table S3. Data analysis Data were analyzed using SPSS 22 (SPSS Inc., USA) and GraphPad Prism 8 (GraphPad Software Inc., USA). Continuous variables are expressed as mean ± standard deviation (SD). Differences between the control and treatment groups at various time points were evaluated using t-tests. For comparisons among multiple treatment conditions, one-way ANOVA was applied. A p-value of less than 0.05 was considered statistically significant. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This work was supported by National Major Agricultural Science and Technology Project (NK20220402), National Natural Science Foundation of China (U24A20427), and the Qinghai Provincial Key Laboratory of Crop Molecular Breeding (2023-1-1). Author Contribution D.W. and T.L. conceived and designed the research. H.C. and B.Z. guided the experiment. D.W. and G.W. conducted the experiments. D.W. and B.Z. wrote the manuscript. W.T. H.C. and G.W. provided technical assistance. T.L. and W.T. critically reviewed and revised the manuscript. All authors have read and agreed to the published version of the manuscript. Acknowledgements Not applicable. Data Availability The genome sequences and annotation files of oat, *A. thaliana* , maize, rice, and wheat were downloaded from the Ensembl Plants database (https://plants.ensembl.org/index.html). References Bamburg JR, McGough A, Ono S. 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17:09:13","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4233370,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/82a784eae0632c9ac5a31b6b.jpeg"},{"id":98546432,"identity":"118b613d-c3bb-4b4f-8895-90ec9036c66d","added_by":"auto","created_at":"2025-12-18 19:11:39","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14498,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/706fc457610d92ead62dacf8.png"},{"id":98626267,"identity":"fe02c9c6-20c8-45c4-9463-49f91747e6c5","added_by":"auto","created_at":"2025-12-19 17:09:39","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75467,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/248cfb1c8220b632e61fe42c.png"},{"id":98546444,"identity":"d23438d0-6b96-4001-9729-36c7b2dfc552","added_by":"auto","created_at":"2025-12-18 19:11:39","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67053,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/7924796051b108c946e928d0.png"},{"id":98546434,"identity":"ad60c332-05e2-4186-8199-9f643332ffbe","added_by":"auto","created_at":"2025-12-18 19:11:39","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":110010,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/145c7bcfddc73d0fe8be5cd2.png"},{"id":98546437,"identity":"d60bad5b-412f-4463-98ed-d68155b15f64","added_by":"auto","created_at":"2025-12-18 19:11:39","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20596,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/e9d6074d46f0fec33a832428.png"},{"id":98546442,"identity":"91211dc2-e36a-4180-b5d3-89c810bd141c","added_by":"auto","created_at":"2025-12-18 19:11:39","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":77666,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/d9017d933f93d8ab150a2155.png"},{"id":98625704,"identity":"6d01c148-5604-41b8-9280-02342646b41a","added_by":"auto","created_at":"2025-12-19 17:09:17","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119360,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/8c397aae63d029ac466cf1fe.png"},{"id":98626063,"identity":"8d0722b7-c41f-432d-bffd-6d437aa2459c","added_by":"auto","created_at":"2025-12-19 17:09:29","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":99161,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/6c731452cf6700f7a26540f1.png"},{"id":98546445,"identity":"eca1aab7-4260-48c1-ba46-f41f97508e0f","added_by":"auto","created_at":"2025-12-18 19:11:40","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":171304,"visible":true,"origin":"","legend":"","description":"","filename":"501f83b7429f48a28640a555f9eb7dc61structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/8e8f6f177d2524e582ce24b4.xml"},{"id":98546447,"identity":"d686109e-1d6f-4f24-a760-0d465869c9d8","added_by":"auto","created_at":"2025-12-18 19:11:40","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":188594,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/bafa01e7c1c45695bafb2a49.html"},{"id":98546420,"identity":"69018fa9-deeb-4a03-927a-349c9718377f","added_by":"auto","created_at":"2025-12-18 19:11:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99971,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal locations of the\u003cem\u003e ADF \u003c/em\u003egene family.\u003c/p\u003e\n\u003cp\u003eGreen bars represent the twelve chromosomes with \u003cem\u003eAsADF \u003c/em\u003egenes. The scale indicates the chromosome length, and the black lines indicate the position of each \u003cem\u003eAsADF\u003c/em\u003e gene.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/0cfb935ff4b10608109ffdb4.png"},{"id":98626406,"identity":"c73f8ade-9001-4114-a67d-ad2841673403","added_by":"auto","created_at":"2025-12-19 17:09:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":725416,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome localization of \u003cem\u003eAsADF\u003c/em\u003e duplicated genes in oat.\u003c/p\u003e\n\u003cp\u003eThe green boxes represent different chromosomes.\u003c/p\u003e\n\u003cp\u003eThe red lines represent the segmentally duplicated genes.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/67f8d367d193f8b5cb97ce41.png"},{"id":98546419,"identity":"95002a5d-efb5-4382-b052-e5ced5ac01be","added_by":"auto","created_at":"2025-12-18 19:11:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1042588,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analyses of the \u003cem\u003eADF \u003c/em\u003egenes between oat and four representa-tive species.\u003c/p\u003e\n\u003cp\u003eThe collinear blocks within \u003cem\u003eA. sativa\u003c/em\u003e and other specie genomes were displayed by the gray lines.\u003c/p\u003e\n\u003cp\u003eThe syntenic\u003cem\u003e ADF \u003c/em\u003egene pairs between oat and other species were highlighted with the red lines.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/f62e132382eba18e4032f2c7.png"},{"id":98546421,"identity":"6d2421ab-7604-44cd-a6e0-22fd8ae72793","added_by":"auto","created_at":"2025-12-18 19:11:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":953164,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of ADF family.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree is divided into four groups (A, B, C, and D).\u003c/p\u003e\n\u003cp\u003eThe background is shaded in yellow, green, pink, and purple for Group A, B, C, and D, respectively.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/9a41418ef814ef725187ff3b.png"},{"id":98625915,"identity":"8a7cf2fe-b891-4c3c-8c0e-1d83e778b1db","added_by":"auto","created_at":"2025-12-19 17:09:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":195197,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis, conserved motifs, and gene structure of ADF proteins in oat.\u003c/p\u003e\n\u003cp\u003e(A) Phylogenetic tree of the oat ADF family constructed by the neighbor-joining method using MEGA software. (B) Distribution of conserved motifs in AsADF proteins. Different motifs are represented by numbered colored boxes. (C) Exon-intron structure of \u003cem\u003eAsADF\u003c/em\u003egenes. Yellow boxes and black lines represent exons and introns, respectively.\u003c/p\u003e\n\u003cp\u003eThe scale at the bottom applies to both panels (B) and (C).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/bffd3de1b3253090455ed78e.png"},{"id":98627552,"identity":"a5b79aa9-54f7-488a-936a-e750d660b194","added_by":"auto","created_at":"2025-12-19 17:10:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":665378,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction of promoter cis-elements.\u003c/p\u003e\n\u003cp\u003eThe color scale from white to red indicates an increasing number of predicted elements.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/32cdb513e22efed3e6fa593d.png"},{"id":98625984,"identity":"9f605cf7-4144-4406-ba80-57507e5bad2a","added_by":"auto","created_at":"2025-12-19 17:09:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":421583,"visible":true,"origin":"","legend":"\u003cp\u003eCircadian expression patterns of the \u003cem\u003eAsADF\u003c/em\u003e gene family.\u003c/p\u003e\n\u003cp\u003eRelative expression levels were determined by qRT-PCR using \u003cem\u003eAsEIF4A\u003c/em\u003e as an internal reference and calculated via the 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/36c54108c2c6d312a81d776a.png"},{"id":98626592,"identity":"70feed2a-cdde-4102-8b2b-23d33fb4d523","added_by":"auto","created_at":"2025-12-19 17:09:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":667348,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of 18 \u003cem\u003eAsADF\u003c/em\u003e genes under cold, heat, salt, and drought stress in oat.\u003c/p\u003e\n\u003cp\u003eRelative expression levels were determined by qRT-PCR using \u003cem\u003eAsEIF4A\u003c/em\u003e as an internal reference and calculated via the 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e method. Lowercase letters indicate statistically significant differences (P \u0026lt; 0.01) among time points within each treatment as determined by Tukey's test.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/03d09cd9486a385067f5f309.png"},{"id":102785901,"identity":"c7435d36-e970-4b3c-8b7d-e0af2038a769","added_by":"auto","created_at":"2026-02-16 16:10:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6232460,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/08c710c1-d805-484e-b7fa-dc4d9b431bb5.pdf"},{"id":98546448,"identity":"23b06ee9-64b8-4e62-9e25-7ffbd09d64ac","added_by":"auto","created_at":"2025-12-18 19:11:40","extension":"rar","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":30941192,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.rar","url":"https://assets-eu.researchsquare.com/files/rs-8235971/v1/402fbfaf855d1c506f6a9175.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Wide Identification and Stress-Responsive Expression Analysis of the Actin-Depolymerizing Factor (ADF) Gene Family in Avena sativa L","fulltext":[{"header":"Background","content":"\u003cp\u003eThe actin-based microfilament cytoskeleton is essential for numerous cellular processes, providing mechanical support and motive force for the cell, and playing critical roles in plant growth, development, and stress responses [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Actin exists in two forms: globular actin (G-actin) and filamentous actin (F-actin) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The polymerization of G-actin into F-actin enables a wide range of biological functions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The remodeling of F-actin is driven by a diverse set of actin-binding proteins (ABPs), which in turn regulate various cellular and physiological functions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Among these, the actin depolymerizing factor (ADF) represents a crucial ABP that severs and depolymerizes F-actin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe number of \u003cem\u003eADF\u003c/em\u003e family genes varies considerably among different organisms. Most non-plant organisms have only one or two ADF/cofilin genes. In contrast, plants typically possess an expanded ADF family, which enables them to fulfill diverse physiological functions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Given the important functions of the actin cytoskeleton in plant development and stress responses, ADF proteins are regulators of cellular processes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Consequently, genome-wide identification and functional characterization of the \u003cem\u003eADF\u003c/em\u003e family have been performed in numerous plant species (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eADF\u003c/em\u003e gene family plays crucial roles in plant growth and development, and the functional characterization of \u003cem\u003eADFs\u003c/em\u003e has been conducted in various plant species. For instance, in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the \u003cem\u003eatadf9\u003c/em\u003e mutant displays reduced lateral branches and diminished callus-forming capacity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eAtADF1\u003c/em\u003e and \u003cem\u003eAtADF4\u003c/em\u003e promote hypocotyl elongation by interacting with 14-3-3 λ proteins [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eAtADF7\u003c/em\u003e and \u003cem\u003eAtADF10\u003c/em\u003e regulate pollen tube growth by modulating cytosolic pH [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Beyond A. \u003cem\u003ethaliana\u003c/em\u003e, studies in cotton have shown that suppressing \u003cem\u003eGhADF1\u003c/em\u003e expression enhances both fiber elongation and secondary cell wall deposition [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In maize, \u003cem\u003eZmADF1\u003c/em\u003e is pollen-specific and negatively regulates pollen quantity, viability, germination, and seed set [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Beyond growth, \u003cem\u003eADFs\u003c/em\u003e are crucial for stress responses. The \u003cem\u003eatadf5\u003c/em\u003e mutant exhibits reduced drought survival due to impaired stomatal closure [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003eAtADF1\u003c/em\u003e responds to salt stress via its association with MYB73 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], while \u003cem\u003eAtADF4\u003c/em\u003e confers osmotic stress tolerance [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. \u003cem\u003eGhADF6\u003c/em\u003e is implicated in the response to \u003cem\u003eVerticillium dahliae\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Heterologous expression of \u003cem\u003eGmADF13\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e and soybean hairy roots enhances drought tolerance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Overexpression of \u003cem\u003eOsADF3\u003c/em\u003e also improves drought tolerance in transgenic \u003cem\u003eA. thaliana\u003c/em\u003e seedlings [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. \u003cem\u003eZmADF1\u003c/em\u003e plays a critical role in regulating growth under high-temperature stress [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In wheat, \u003cem\u003eTaADF3\u003c/em\u003e negatively regulates \u003cem\u003ePuccinia striiformis\u003c/em\u003e (PST) resistance, likely by modulating ROS homeostasis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In contrast, \u003cem\u003eTaADF4\u003c/em\u003e and \u003cem\u003eTaADF7\u003c/em\u003e are shown to confer resistance to PST by remodeling the actin cytoskeleton [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, heterologous overexpression of \u003cem\u003eTaADF16\u003c/em\u003e is shown to enhance cold tolerance in transgenic \u003cem\u003eA. thaliana\u003c/em\u003e by boosting ROS scavenging capacity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs the world's seventh most widely grown cereal crop, oat (\u003cem\u003eAvena sativa\u003c/em\u003e L.) is a valuable cultivated dual-purpose species [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This health-promoting cereal has garnered increasing attention due to its rich content of dietary fiber, phytochemicals, and essential nutrients [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Concurrently, as a crucial forage crop, oat provides high-quality feed for livestock. This helps alleviate pressure on conventional feed grains and natural grasslands, thereby facilitating the restoration of degraded pastures and ecological conservation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Oats not only possess significant economic value but are also considered a valuable gene pool due to their tolerance to stress conditions. Oat contains numerous stress-resistance genes yet to be discovered, which can provide vital resources for crop stress-resistance improvement. Stable yield is crucial for oat production. Despite oat\u0026rsquo;s considerable ability to tolerate environmental stresses, their growth and development are still affected by various stresses, such as extreme temperatures, cold, and drought, leading to substantial yield losses [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, 18 \u003cem\u003eAsADF\u003c/em\u003e genes were identified from the oat Sang reference genome (Sang.v1.1) and a comprehensive analysis was conducted. Initially, we assessed their fundamental physicochemical properties and chromosomal locations. To elucidate their evolutionary relationships, we constructed a phylogenetic tree and analyzed the protein structures. Furthermore, cis-elements within the promoter regions were predicted, along with their subcellular localization. Finally, to investigate their potential regulatory mechanisms and functions, we examined their expression patterns under circadian rhythms and their expression level changes under abiotic stress. This study establishes a foundation for screening key candidate genes within the \u003cem\u003eADF\u003c/em\u003e family in oats and provides valuable genetic resources for molecular breeding aimed at stress resistance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification of \u003cem\u003eADF\u003c/em\u003e genes in oat\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough a comprehensive analysis of the ADF-H domain (PF00241) annotation and the CD-Search tool from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/cdd/), a total of 18 putative \u003cem\u003eADF\u003c/em\u003e genes were identified in the oat genome. These genes were located on 12 different chromosomes and were designated AsADF1 to AsADF18 based on their respective gene IDs in the Sang reference genome (Table 1, Fig. 1).\u003c/p\u003e\n\u003cp\u003eAnalysis of the basic physicochemical properties of AsADF proteins revealed substantial variation among members. Their amino acid lengths ranged from 139 aa (AsADF4, 5, 6, 7, and 17) to 152 aa (AsADF1). The molecular weight (MW) varied between 15.75 kDa (AsADF13) and 17.30 kDa (AsADF1), and the theoretical isoelectric point (pI) spanned from 4.43 (AsADF13) to 8.73 (AsADF2 and AsADF6). The instability index (InI) ranged from 31.61 (AsADF6) to 47.73 (AsADF17), while the aliphatic index (AI) values between 61.08 (AsADF6) and 85.65 (AsADF14) suggest relatively high thermal stability. Finally, the grand average of hydropathicity (GRAVY) scores ranged from -0.037 (AsADF17) to -0.567 (AsADF4), indicating that all these proteins are hydrophilic (Table 1).\u003c/p\u003e\n\u003cp\u003eSubcellular localization predictions indicated that the ADF proteins are mainly localized in the cytoplasm, followed by the chloroplast, nucleus, extracellular space, mitochondria, and peroxisomes. This distribution implies potential functional roles for ADF proteins within these organelles (Fig. S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Physicochemical properties of AsADF proteins in oats\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"543\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eSequence ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eName\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eProtein Length\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMW(kDa)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003epI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eInI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eGRAVY\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.1AG0061340.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e152\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17307.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.493\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.1DG0136740.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16402.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.1DG0179840.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e17278.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e44.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e72.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.541\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.2AG0200070.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16043.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e36.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e63.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.567\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.2CG0312780.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15755.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e61.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.439\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.2DG0370090.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15885.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e31.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e61.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.488\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.3DG0568620.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15931.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e77.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.376\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.4AG0610360.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16778.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e72.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.4AG0615680.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15789.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e39.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e83.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.059\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.4AG0615690.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16055.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e44.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.484\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.4CG1327640.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16416.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.4DG0753700.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16720.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e42.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e72.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.419\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.4DG0759170.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15754.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e87.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.238\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.4DG0759210.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e15837.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e39.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e85.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.4DG0759220.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16085.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e44.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.487\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.5AG0799410.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16402.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e8.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.6AG1032230.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16013.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e47.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e70.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.384\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eAVESA.00010b.r2.7CG0705670.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsADF18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e145\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e16746\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e43.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e74.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-0.424\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eChromosomal location and collinearity analysis of \u003cem\u003eAsADFs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 18 \u003cem\u003eAsADFs\u003c/em\u003e were identified and distributed across 12 of the 20 chromosomes, with one to four genes per chromosome (Fig. 1). Chromosome 4D contained the highest number (\u003cem\u003eAsADF12\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, \u003cem\u003e14,\u003c/em\u003e and \u003cem\u003e15\u003c/em\u003e), followed by chromosome 4A (\u003cem\u003eAsADF8\u003c/em\u003e,\u003cem\u003e\u0026nbsp;9,\u003c/em\u003e and \u003cem\u003e10\u003c/em\u003e), and chromosome 1D (\u003cem\u003eAsADF2\u003c/em\u003e and \u003cem\u003e3\u003c/em\u003e). The remaining chromosomes (1A, 2A, 2C, 2D, 3D, 4C, 5A, 6A, and 7C) each contained only one \u003cem\u003eAsADF\u003c/em\u003e gene\u0026nbsp;(Fig. 1).\u003c/p\u003e\n\u003cp\u003eGene duplication is a widespread phenomenon that plays a crucial role in plant evolution\u0026nbsp;[37]. To identify gene duplication events within the \u003cem\u003eAsADF\u003c/em\u003e family, genomic collinearity segments in the oat genome were systematically analyzed using TBtools, and the results were visualized in Circos plots (Fig. 2). The results showed 13 pairs of tandem duplication events in the oat genome (Fig. 2). They suggest an evolutionary relationship between these \u003cem\u003eAsADF\u003c/em\u003e members, and that these genes may have similar functions.\u003c/p\u003e\n\u003cp\u003eTo further investigate the phylogenetic relationships between the oat \u003cem\u003eADF\u003c/em\u003e family and the \u003cem\u003eADF\u0026nbsp;\u003c/em\u003efamilies of other plants, a collinearity analysis was conducted between oat and four other species: \u003cem\u003eA. thaliana,\u003c/em\u003e maize, rice, and wheat. The analysis revealed 3, 28, 17, and 44 orthologous gene pairs between oat and \u003cem\u003eA. thaliana\u003c/em\u003e, maize, rice, and wheat, respectively. Notably, the \u003cem\u003eADF\u003c/em\u003e genes exhibited the highest degree of evolutionary conservation between oat and wheat (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of the AsADFs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further elucidate the phylogenetic relationships of oat ADF proteins, we collected \u003cem\u003eADF\u0026nbsp;\u003c/em\u003efamily genes from several identified species, including the model plant \u003cem\u003eA. thaliana\u003c/em\u003e; the Poaceae species rice, maize, and wheat; and soybean (previously reported by our lab), and constructed a phylogenetic tree using the neighbor-joining (NJ) method in MEGA X with the ADF family of oat (Table S2, Fig. 4). The results showed that the ADF proteins were grouped into four groups, and the number in each group was different. With the exception of Group B, which contained only four Poaceae ADFs, the other three groups comprised ADFs from all six species. Notably, AsADFs and OsADFs within each subgroup showed a close evolutionary relationship. Among the oat ADFs, Group C contained the smallest number (3), while Groups A, B, and D each contained 4 members (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacteristics of the AsADFs protein sequences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein sequence identity among the AsADFs was greater than 58%, with the lowest identity observed between AsADF3 and AsADF14. Notably, the protein sequences of AsADF2/11/16 and AsADF8/18 were identical (Fig. S2). Multiple sequence alignment revealed that all AsADF proteins contain the conserved ADF-H domain and actin-binding regions (Fig. S3). Based on amino acid sequence prediction, the secondary structure of AsADF proteins consists mainly of three α-helices, six β-sheets, and four β-turns. This structural composition suggests that AsADF family proteins possess actin-binding functions, which is consistent with the known role of ADFs in binding to F-actin (Fig. S3). Furthermore, 3D structural models of AsADFs predicted by SWISS-MODEL show that proteins within the same group share high structural similarity, implying potential functional conservation (Fig. S4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConserved motifs and gene structures of the \u003cem\u003eAsADFs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the nine conserved motifs identified, Motifs 1-4 constitute the core ADF domain, indicating their conservation and functional importance within the oat \u003cem\u003eADF\u003c/em\u003e family (Fig. 5B; Fig. S5). Notably, several motifs exhibited specific distributions: Motif 9 was unique to \u003cem\u003eAsADF1\u003c/em\u003e; Motif 8 was exclusively present in \u003cem\u003eAsADF9\u003c/em\u003e and \u003cem\u003eAsADF14\u003c/em\u003e; Motif 6 was specific to Group C; and Motifs 5 and 7 were found only in Group D (Fig. 5B).\u003c/p\u003e\n\u003cp\u003eStructural analysis of \u003cem\u003eAsADF\u0026nbsp;\u003c/em\u003egenes revealed that members of the same group generally share similar exon-intron organizations (Fig. 5A, C). Within the \u003cem\u003eAsADF\u003c/em\u003e gene family, all genes contained three exons, except for \u003cem\u003eAsADF4\u003c/em\u003e and certain members of Group B (excluding \u003cem\u003eAsADF10\u003c/em\u003e) (Fig. 5C). In these three exon genes, the second exon is the longest, flanked by shorter exons at the 5' and 3' ends, with the 3' exon being the shortest (Fig. 5C). Furthermore, size variation among the \u003cem\u003eAsADF\u003c/em\u003e genes appears to be primarily due to differences in intron length.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional analysis of cis-elements in the promoter region of \u003cem\u003eAsADFs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further understand the functional roles of the \u003cem\u003eAsADF\u003c/em\u003e gene family, we performed an analysis of the 2,000 bp promoter regions using the online software PlantCARE. The results showed that the cis-elements in the \u003cem\u003eAsADF\u003c/em\u003e promoters could be classified into four major categories based on their predicted functions: stress response, photoperiod responsiveness, phytohormone response, and growth/development (Fig. 6; Fig. S6). Notably, the promoters of the \u003cem\u003eAsADF\u003c/em\u003e family genes contained 11 low-temperature response and 21 drought responsive elements, suggesting their important roles in environmental stress adaptation. Furthermore, we identified 23 photoperiod-related and 9 hormone-related elements, with abscisic acid (ABA) and methyl jasmonate (MeJA) response elements being the most abundant (Fig. 6; Fig. S6). These findings suggest that the \u003cem\u003eAsADF\u003c/em\u003e family is crucial not only for growth and development but also for responding to abiotic stresses in oat.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of differential expression under circadian rhythm\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the potential roles of \u003cem\u003eAsADF\u003c/em\u003e genes in oat growth, we analyzed their circadian expression rhythms by qRT-PCR. In all qRT-PCR analyses, a gene was considered to respond to a treatment if it showed a minimum twofold difference compared to the control with a statistical significance of P \u0026lt; 0.01. The results showed that \u003cem\u003eAsADF\u003c/em\u003e genes in the same group had similar rhythmic expression patterns (Fig. 7). Specifically, in Group A, the expression levels of four genes (excluding \u003cem\u003eAsADF17\u003c/em\u003e) exhibited a circadian rhythm, being up-regulated after sunrise (8:00), peaking at noon (12:00), and subsequently down-regulated. Conversely, Group C genes displayed an opposite pattern, with the highest expression at 8:00 that decreased throughout the day and began to rise again around 24:00. Within Group D, \u003cem\u003eAsADF8\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e, and \u003cem\u003e18\u003c/em\u003e showed the same expression pattern as Group C, with levels down-regulated during the day and up-regulated at night to a peak at 24:00, but \u003cem\u003eAsADF1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;AsADF3\u003c/em\u003e and all Group B genes did not exhibit significant circadian rhythmicity (Fig. 7). In summary, the expression of most\u003cem\u003e\u0026nbsp;AsADF\u003c/em\u003e genes exhibits diurnal fluctuations, suggesting their involvement in photoperiod-related growth and development processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscription patterns of \u003cem\u003eAsADFs\u003c/em\u003e in response to abiotic stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further understand the potential functions of \u003cem\u003eAsADFs\u003c/em\u003e in response to abiotic stresses, we collected leaves and roots from oat seedlings at the three-leaf stage following exposure to cold, heat, drought, and salt treatments. The relative expression levels of the \u003cem\u003eAsADF\u003c/em\u003e genes were then examined by qRT-PCR (Fig. 8). The results indicated that most \u003cem\u003eAsADF\u003c/em\u003e genes were responsive to various abiotic stresses to differing degrees. Notably, the expression of \u003cem\u003eAsADF4\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e and \u003cem\u003e7\u003c/em\u003e of Group A, \u003cem\u003eAsADF9\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, and \u003cem\u003e14\u0026nbsp;\u003c/em\u003eof Group B, \u003cem\u003eAsADF8\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e and \u003cem\u003e18\u003c/em\u003e of Group D responded to all four stress treatments, suggesting their potential essential functions in oat's stress response. The expression levels of \u003cem\u003eAsADF9\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e and \u003cem\u003e14\u0026nbsp;\u003c/em\u003ewere significantly up-regulated under all four stresses; those of \u003cem\u003eAsADF8\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e and \u003cem\u003e18\u003c/em\u003e were up-regulated by heat, salt, and drought stress, while under cold stress, their expression was initially up-regulated, peaked at 3 h and subsequently down-regulated; The relative expression of \u003cem\u003eAsADF4\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e, and \u003cem\u003e7\u003c/em\u003e was down-regulated under heat and salt stress, but under cold stress, their expression was initially down-regulated, up-regulated at 12 h, and then down-regulated again, drought stress caused little overall change, although a transient spike in expression was notably observed at 9 h. Under cold stress treatment, the expression levels of the 13 \u003cem\u003eAsADF\u003c/em\u003e genes, except for \u003cem\u003eAsADF1\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e, and \u003cem\u003e17\u003c/em\u003e, were up-regulated or down-regulated to varying degrees; whereas under drought stress, the expression levels of all \u003cem\u003eAsADF\u003c/em\u003e genes were up-regulated, among which the expression levels of \u003cem\u003eAsADF9\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e and \u003cem\u003e14\u003c/em\u003e were up-regulated by more than a 100-fold (Fig. 8).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eADFs, recognized as one of the key members among ABPs, are involved in many plant growth and development processes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Their crucial role in plant stress responses has been well-established [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The \u003cem\u003eADF\u003c/em\u003e gene family has been identified in a variety of plant species through genome-wide analyses. The number of \u003cem\u003eADF\u003c/em\u003e genes varies, with 11 in \u003cem\u003eA. thaliana\u003c/em\u003e, 11 in rice, 14 in poplar, 11 in tomato, 27 in banana, 13 in maize, 9 in common bean, 10 in pigeon pea, 25 in wheat, 18 in soybean, 18 in melon, 18 in Chinese cabbage, and 9 in alfalfa [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39 CR40 CR41 CR42 CR43 CR44 CR45 CR46\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, the functions of the \u003cem\u003eADF\u003c/em\u003e family genes in oats remain unclear. Therefore, we identified \u003cem\u003eADF\u003c/em\u003e genes in oat and conducted a comprehensive analysis of their phylogenetic relationships, sequence characteristics, and expression patterns.\u003c/p\u003e \u003cp\u003eIn this study, we identified 18 \u003cem\u003eADF\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which were located on 12 chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Previous studies have suggested that \u003cem\u003eADF\u003c/em\u003e genes in plants likely evolved from a common ancestor [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A comparative analysis of the characterized \u003cem\u003eADF\u003c/em\u003e families in Poaceae reveals that this number of ADF genes is greater than that in rice (11) and maize (13) but less than that in wheat (25). This variation in gene copy number among different plant species is potentially driven by gene duplication events [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Although oats and wheat are both important allopolyploid cereals with complex and large genomes, we found that oats possess a smaller number of \u003cem\u003eADF\u003c/em\u003e genes than wheat [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We speculate that this difference from the relatively late initiation of systematic research on oats compared to wheat, resulting in a less complete genome assembly and annotation.\u003c/p\u003e \u003cp\u003eEvolutionary analysis indicated that oat \u003cem\u003eADF\u003c/em\u003e genes were classified into four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This grouping pattern is consistent with that reported in \u003cem\u003eA. thaliana\u003c/em\u003e, rice, tomato, maize, melon, soybean, Chinese cabbage, and alfalfa [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Phylogenetic analysis revealed that many oat \u003cem\u003eADF\u003c/em\u003e genes were closely clustered with previously reported \u003cem\u003eADF\u003c/em\u003e genes known to play critical roles in stress responses, suggesting potential functional conservation. For instance, \u003cem\u003eAsADF9\u003c/em\u003e, \u003cem\u003eAsADF13\u003c/em\u003e, and \u003cem\u003eAsADF14\u003c/em\u003e cluster within the same clade as \u003cem\u003eOsADF3\u003c/em\u003e and \u003cem\u003eTaADF16\u003c/em\u003e. Based on the reported roles of these genes in drought and cold stress tolerance, we speculate that the oat \u003cem\u003eADFs\u003c/em\u003e in this clade may exhibit a conserved biological function in response to these abiotic stresses [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e](Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Furthermore, the exon-intron structures of these genes were largely conserved compared to previous reports (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Analysis of the oat ADF protein motifs revealed that these proteins are highly conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). All AsADF proteins contain the ADF-H domain, which is composed of motifs 1 to 4. Additionally, we identified some motifs specific to certain genes, which may be linked to functional specialization among them (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Fig. S5).\u003c/p\u003e \u003cp\u003eGiven that gene expression is largely regulated by cis-elements in promoter sequences, analyzing these elements helps elucidate the regulatory mechanisms of \u003cem\u003eADF\u003c/em\u003e genes and infer their potential biological functions [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Previous studies have indicated that phytohormonal signals and environmental stresses are key regulators of \u003cem\u003eADF\u003c/em\u003e gene expression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Various phytohormone and stress-responsive elements, including LTR (low-temperature response), MBS (drought stress response), ABRE (ABA response), and TGACG-/CGTAC-motifs (MeJA response), were identified in the promoters of oat \u003cem\u003eADF\u003c/em\u003e genes through cis-element analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Fig. S6). Notably, light-responsive elements were also identified in all \u003cem\u003eAsADF\u003c/em\u003e gene promoters, further supporting their potential roles in regulating oat growth, development, and stress responses.\u003c/p\u003e \u003cp\u003eLight is fundamental to plant life, and its perception governs key aspects of plant growth and development [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The expression of numerous genes involved in these processes is under circadian rhythm regulation, which in turn affects the growth and development of plants [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Given the presence of multiple light-responsive elements identified in the promoters of oat \u003cem\u003eADF\u003c/em\u003e genes, we speculate that they might function in photoperiodic regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Fig. S6). To investigate whether there was differential circadian expression of the \u003cem\u003eAsADF\u003c/em\u003e genes, we conducted qRT-PCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Our results indicated that the expression of most \u003cem\u003eAsADF\u003c/em\u003e genes followed a circadian rhythm (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Genes in Group A (except \u003cem\u003eAsADF17\u003c/em\u003e) exhibited higher expression during the day than at night, with their transcript levels peaking at noon coinciding with the highest temperature of the day, suggesting a potential role in sensing ambient temperature fluctuations. Conversely, the Group C genes showed higher expression during the night. Phylogenetic relationships showed these genes are in the same clade as \u003cem\u003eAtADF5\u003c/em\u003e, which has been reported to regulate drought tolerance by modulating stomatal aperture. Based on this, we infer that the Group C \u003cem\u003eAsADFs\u003c/em\u003e may perform a similar function in drought stress response [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e](Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In summary, \u003cem\u003eAsADF\u003c/em\u003e genes likely participate in various growth and developmental pathways by responding to photoperiodic rhythms.\u003c/p\u003e \u003cp\u003eDuring growth and development, plants are usually exposed to various environmental stresses, such as soil salinity, drought, and extreme temperatures [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. As an important nutrient-rich grain and forage crop, oat holds significant value in stable yields under stress conditions [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Plants require optimal temperatures for growth and development, and temperatures beyond their physiological optimum ranges can result in heat or cold stress [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. With the intensification of global warming in recent years, the fluctuations in spring temperatures have significantly increased the risk of cereal crops being exposed to late spring coldness (LSC) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. LSC causes severe damage or even plant death. As oats are mainly cultivated in cold high-altitude regions, they are highly vulnerable to LSC, often leading to significant yield losses. Moreover, oat growth requires cool climatic conditions, and this trait limits its cultivation in warmer regions and constrains the supply of high-quality oats to meet consumer demand [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Besides temperature stress, salt and drought stress also negatively affect oat growth and productivity [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Soil salinization is a major environmental challenge to global agricultural production [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and drought conditions are common in many oat-growing regions. Although oats are moderately stress tolerant, their yield remains limited by salt and drought [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Therefore, identifying key genes associated with abiotic stress tolerance is crucial for oat's stable yields. The remodeling of the actin cytoskeleton serves as a critical target for signaling pathways triggered by diverse developmental and environmental stimuli [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Plant \u003cem\u003eADFs\u003c/em\u003e function through cytoskeletal remodeling, and studies have reported that \u003cem\u003eADF\u003c/em\u003e genes in various Poaceae species participate in abiotic stress responses. For instance, heterologous expression of \u003cem\u003eOsADF3\u003c/em\u003e enhances drought tolerance in \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, \u003cem\u003eTaADF16\u003c/em\u003e plays an important role in low temperature tolerance [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Based on the significant role of the \u003cem\u003eADF\u003c/em\u003e gene family in abiotic stress responses, it is necessary to analyze its function in oats. Leaves are the primary site for temperature stress perception in plants, while roots are a major organ for sensing salt and drought stress. Therefore, we used qRT-PCR to analyze the expression patterns of \u003cem\u003eAsADF\u003c/em\u003e genes under these stresses [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, all \u003cem\u003eAsADF\u003c/em\u003e genes were responsive to drought stress. Notably, Group B members \u003cem\u003eAsADF9\u003c/em\u003e, \u003cem\u003eAsADF13\u003c/em\u003e, and \u003cem\u003eAsADF14\u003c/em\u003e exhibited significant up-regulation under all four abiotic stress treatments, suggesting their important role in stress response of oats (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These \u003cem\u003eADF\u003c/em\u003e genes are potential candidates for abiotic stress tolerance in oats and could be further verified as key genes.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study identified 18 \u003cem\u003eADF\u003c/em\u003e genes unevenly distributed across 12 chromosomes in the oat genome. Phylogenetic analysis classified these genes into four groups, with members of each group exhibiting conserved gene structures and motif compositions. Expression analysis revealed that most \u003cem\u003eADF\u003c/em\u003e genes showed circadian rhythms, suggesting roles in plant growth regulation. Furthermore, multiple \u003cem\u003eADF\u003c/em\u003e genes responded to various abiotic stresses, particularly drought, indicating their potential involvement in stress adaptation. Notably, \u003cem\u003eAsADF9\u003c/em\u003e, \u003cem\u003eAsADF13\u003c/em\u003e, and \u003cem\u003eAsADF14\u003c/em\u003e from Group B were significantly up-regulated under four stress conditions, indicating that they are promising candidates for further functional characterization. These results provide valuable insights into the functions of the \u003cem\u003eADF\u003c/em\u003e family and offer new genetic resources for the development of stress-tolerant oat varieties.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eIdentification of the\u003c/b\u003e \u003cb\u003eADF\u003c/b\u003e \u003cb\u003egenes in the genome of oat\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe whole genome and GFF3 of oat (Sang.v1.1) and other plants (\u003cem\u003eA. thaliana\u003c/em\u003e, rice, maize, and wheat) were obtained from the Ensembl Plants database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org/\u003c/span\u003e\u003cspan address=\"https://plants.ensembl.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA collection of experimentally validated \u003cem\u003eADF\u003c/em\u003e family sequences from published studies served as query sequences for homology-based screening via the BLAST Compare Two Seqs function in TBtools [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The hidden Markov model (HMM) profile of the ADF-H domain (PF00241) was obtained from the Pfam database (\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) and domain architecture analysis was conducted using the Simple HMM Search module within TBtools with an E-value cutoff of 1e-5.\u003c/p\u003e \u003cp\u003eThe presence of the ADF-H domain in putative ADF proteins was verified using the batch CD-search tool available through the NCBI Conserved Domain Database (CDD) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA consensus strategy integrating sequence similarity and structural domain conservation yielded the final candidate set of oat \u003cem\u003eADF\u003c/em\u003e family members.\u003c/p\u003e\n\u003ch3\u003ePhysicochemical characteristics and localization to chromosomes\u003c/h3\u003e\n\u003cp\u003eThe physicochemical properties of the identified AsADF proteins, including amino acid length, molecular weight (MW), isoelectric point (pI), instability index (II), aliphatic index (AI), and grand average of hydropathicity (GRAVY), were predicted using the ProtParam tool on the ExPASy server [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e](\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe physical positions of the \u003cem\u003eAsADF\u003c/em\u003e genes were determined from the genome annotation file, and the genes were mapped to their chromosomes. The genomic distribution was visualized using the \"Gene Location Visualize from GTF/GFF\" function in TBtools, based on the Sang reference genome.\u003c/p\u003e\n\u003ch3\u003eAnalysis of the gene structure and identification of the conserved motif in the protein\u003c/h3\u003e\n\u003cp\u003eStructural analysis of \u003cem\u003eAsADF\u003c/em\u003e genes was performed using the oat genome annotation file (GFF3 format) obtained from the Ensembl Plants database. A phylogenetic tree of AsADF proteins was constructed with MEGA X software via the NJ method, with 1000 bootstrap replicates. Conserved motifs in the AsADF proteins were identified using the MEME (Multiple Expectation Maximization for Motif Elicitation) suite, with the maximum number of motifs set to 9, and were visualized using the \"Gene Structure View\" function in TBtools. Exon-intron structures were visualized using the \"Visualize Gene Structure\" function in TBtools. Furthermore, conserved domains in AsADF proteins were predicted using the Batch CD-Search tool on the NCBI platform and visualized with the \"Visualize NCBI CDD Domain Pattern\" function in TBtools.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of protein sequence characteristics, phylogeny, and collinearity\u003c/h2\u003e \u003cp\u003eMultiple sequence alignment of ADF family proteins was performed using MEGA X software, and the resulting alignment was exported in FASTA format. For visualization, the tertiary structure file of the ADF protein (1f7s.1.pdb) was obtained from SWISS-MODEL and used to annotate the alignment results using the online tool ESPript 3.0. The protein sequences of the 18 \u003cem\u003eAsADF\u003c/em\u003e genes were submitted to the SWISS-MODEL online server [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for homology modeling. The template (1f7s.1.A) with the highest Global Model Quality Estimation (GMQE) score in the PDB database was selected to ensure high structural consistency across the AsADF protein family.The phylogenetic tree of oat, wheat, rice, and maize ADF proteins was constructed using the NJ method in MEGA X software with 1000 bootstrap replicates. The resulting tree was then visualized and annotated using the iTOL online platform (\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 enhanced graphical representation.\u003c/p\u003e \u003cp\u003eGene collinearity analysis was carried out among \u003cem\u003eA. thaliana\u003c/em\u003e, wheat, rice, and maize. The One-Step MCScanX-Super Fast plugin in TBtools was employed for the collinearity analysis and visualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCis-elements analysis and subcellular localization\u003c/h2\u003e \u003cp\u003ePutative cis-acting elements within the 2,000 bp promoter region upstream of the \u003cem\u003eAsADF\u003c/em\u003e genes were identified using the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The predicted cis-acting elements were visualized using TBtools.\u003c/p\u003e \u003cp\u003eSubcellular localization was predicted using WoLF PSORT (\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). The results were subsequently visualized and comparatively analyzed with TBtools to facilitate biological interpretation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and stress treatments materials of oat seedlings\u003c/h2\u003e \u003cp\u003eThe oat cultivar Keyan No.4, used in this study, was developed and stored at the Key Laboratory of Adaptation and Evolution of Plateau Biota (AEPB) of the Northwest Institute of Plateau Biology, Chinese Academy of Sciences. The oat seeds were disinfected using 75% ethanol for 5 min, followed by treatment with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 1 min, and then washed 5 times with sterile water before being germinated for 5 days in a growth chamber.\u003c/p\u003e \u003cp\u003eThe germinated seeds were then cultivated in a modified Hoagland solution (NS10205-500 mL, Coolaber) under a 14-h light/10-h dark cycle, with temperatures at 23\u0026deg;C during the day and 18\u0026deg;C at night for 15 days. Subsequently, seedlings were exposed to various stress conditions: heat (40\u0026deg;C), cold (4\u0026deg;C), salt (150 mM NaCl), and drought (simulated with 20% w/v PEG-6000), for durations of 0, 1, 3, 6, 12, and 24 hours. Untreated Hoagland's solution was used as the control. For circadian rhythm analysis of \u003cem\u003eAsADFs\u003c/em\u003e, seedlings were grown until the three-leaf stage, and samples were collected every 4 hours. Following collection, roots and leaves were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C for subsequent RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction, cDNA preparation, and quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003e Total RNA was extracted from plant samples using FastPure Universal Plant Total RNA Isolation Kit (RC411-01, Vazyme) according to the manufacturer's instructions. And then reverse-transcribed into cDNA with HiScriptIII RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (R323-01, Vazyme). The qRT-PCR analysis was performed using ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme Biotech), and the protocol was 95℃ for 30 s followed by 34 cycles of 95 ℃ for 10 s and 60 ℃ for 30 s. Relative quantification of gene expression was performed based on the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], with \u003cem\u003eAsEIF4A\u003c/em\u003e used as the internal reference gene. The sequences of all primers used in this study are listed in Supplementary Table S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS 22 (SPSS Inc., USA) and GraphPad Prism 8 (GraphPad Software Inc., USA). Continuous variables are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between the control and treatment groups at various time points were evaluated using t-tests. For comparisons among multiple treatment conditions, one-way ANOVA was applied. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by National Major Agricultural Science and Technology Project (NK20220402), National Natural Science Foundation of China (U24A20427), and the Qinghai Provincial Key Laboratory of Crop Molecular Breeding (2023-1-1).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eD.W. and T.L. conceived and designed the research. H.C. and B.Z. guided the experiment. D.W. and G.W. conducted the experiments. D.W. and B.Z. wrote the manuscript. W.T. H.C. and G.W. provided technical assistance. T.L. and W.T. critically reviewed and revised the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe genome sequences and annotation files of oat, *A. thaliana* , maize, rice, and wheat were downloaded from the Ensembl Plants database (https://plants.ensembl.org/index.html).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBamburg JR, McGough A, Ono S. Putting a new twist on actin: ADF/cofilins modulate actin dynamics. Trends Cell Biol. 1999;9(9):364\u0026ndash;70.\u003c/li\u003e\n\u003cli\u003ePollard TD, Cooper JA. Actin, a central player in cell shape and movement. 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Methods. 2010;50(4):227\u0026ndash;30.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"actin-depolymerizing factor, oat, circadian rhythm, abiotic stresses, expression analysis","lastPublishedDoi":"10.21203/rs.3.rs-8235971/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8235971/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOat (\u003cem\u003eAvena sativa\u003c/em\u003e L.) is an important cereal crop globally, valued for both its grain and forage. Actin depolymerization factors (ADFs) are highly conserved eukaryotic proteins that facilitate remodeling of the actin cytoskeleton. Accumulating evidence indicates that \u003cem\u003eADFs\u003c/em\u003e play crucial roles in plant growth, development, and stress responses. Nevertheless, the \u003cem\u003eAsADFs\u003c/em\u003e family has not yet been identified in oat, an important cereal with high abiotic stress tolerance.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, a total of 18 \u003cem\u003eADF\u003c/em\u003e genes (\u003cem\u003eAsADF\u003c/em\u003e) were identified from the oat reference genome (Sang.v1.1) and mapped to 12 different chromosomes. Based on the phylogenetic analysis, these genes were classified into four groups, which was confirmed by their structure and the distribution of conserved motifs in the encoded proteins. Collinearity analysis demonstrated strong relationships between oat and wheat \u003cem\u003eADFs\u003c/em\u003e. The promoters of most \u003cem\u003eAsADFs\u003c/em\u003e family contain cis-elements associated with growth, development, and stress responses, suggesting their potential involvement in these biological processes. Subcellular localization prediction indicated that ADFs are mainly located in the cytoplasm, and this localization is consistent with their role in cytoskeletal maintenance. Analysis of qRT-PCR results indicated that most \u003cem\u003eAsADFs\u003c/em\u003e exhibited differential expression patterns under circadian rhythm and responded to various abiotic stresses. Among them, the expression levels of \u003cem\u003eAsADF9\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, and \u003cem\u003e14\u003c/em\u003e from group B showed significant changes under all four abiotic stress conditions, suggesting their important roles in abiotic stress resistance.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study revealed that \u003cem\u003eAsADF\u003c/em\u003e genes play crucial roles in oat's response to various abiotic stresses. Notably, the expression of \u003cem\u003eAsADF9\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e and \u003cem\u003e14\u003c/em\u003e was strongly induced under stress conditions, highlighting them as key candidates mediating oat's stress response. In summary, the present study established a theoretical foundation for analyzing the molecular mechanism of stress resistance in oat and provides valuable insights for molecular breeding to enhance stress resistance.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Identification and Stress-Responsive Expression Analysis of the Actin-Depolymerizing Factor (ADF) Gene Family in Avena sativa L","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 19:11:31","doi":"10.21203/rs.3.rs-8235971/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-02T06:45:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-02T06:36:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-31T08:11:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-30T16:07:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-27T15:04:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293523920476184845556457246210404488810","date":"2025-12-12T17:56:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120262359071378900458337081907448322462","date":"2025-12-12T00:10:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45643424648113882730758106123044246841","date":"2025-12-11T18:26:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328873961495825211038874358581907186008","date":"2025-12-11T15:57:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-11T15:45:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-03T07:17:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-01T07:53:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-01T07:50:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-11-29T09:19:57+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":"7b29d285-77db-4a3c-aaf8-6dace2d83916","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:08:29+00:00","versionOfRecord":{"articleIdentity":"rs-8235971","link":"https://doi.org/10.1186/s12870-026-08329-9","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2026-02-09 15:57:43","publishedOnDateReadable":"February 9th, 2026"},"versionCreatedAt":"2025-12-18 19:11:31","video":"","vorDoi":"10.1186/s12870-026-08329-9","vorDoiUrl":"https://doi.org/10.1186/s12870-026-08329-9","workflowStages":[]},"version":"v1","identity":"rs-8235971","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8235971","identity":"rs-8235971","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}