Genome-wide identification of the oat (Avena sativa L.) R2R3-MYB gene family and expression analysis under different abiotic stresses

Abstract

Background

Oat (Avena sativa L.), a member of the Avena genus in the Poaceae family, exhibits strong adaptability to harsh environments, including nutrient-poor soils, salinity, cold, and drought. The R2R3-MYB (v-myb avian myeloblastosis viral oncogene homologue) transcription factors form one of the most extensive transcription factor families in plants, contributing significantly to growth regulation and responses to abiotic stresses. However, the AsR2R3-MYB gene family has not yet been systematically characterized in A. sativa, and its expression patterns and potential functions under drought, cold, or salt stress remain unexplored. (2) Methods: This study identified AsR2R3-MYB genes from the A. sativa genome and examined their chromosomal distribution, gene architecture, phylogenetic relationships, protein-protein interaction networks, and promoter cis-elements. Additionally, expression profiles across various tissues and in response to different abiotic stresses were validated using qRT-PCR. (3) Results: A total of 237 AsR2R3-MYB family members were identified in this study. Based on phylogenetic analysis with Arabidopsis R2R3-MYB proteins, the 237 AsR2R3-MYBs were categorized into 29 subfamilies (A1–A29). Among these, the A1 subfamily contained the largest number of members. Protein–protein interaction analysis revealed that 45 AsMYB proteins were involved in interaction networks, with AsMYB201 exhibiting the highest number of interacting partners. Twenty AsMYB genes were expressed in all tissues with significant differences in expression levels across different organs. All 20 AsMYB genes responded to salt, drought, and cold stresses, with AsMYB204 and AsMYB107 showing significant upregulation under multiple abiotic stresses, identifying them as key candidate genes for further functional validation. (4) Conclusions: These results establish a basis for future investigation into the roles of A. sativa AsMYB genes in abiotic stress responses and offer important insights to support molecular breeding efforts aimed at enhancing stress resilience in A. sativa.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08723-3.

Introduction

Oats (Avena sativa L.) is an annual grass crop in the Poaceae family that is notable for its tolerance to poor soil, salinity, drought, and cold, as well as its broad adaptability and high forage yield, making it an important source of quality fodder for herbivorous livestock that plays a key role in global agricultural production [1, 2]. Current0ly, there are 30 known species within the genus Avena, with common cultivated A. sativa planted widely, mainly in the temperate regions of Europe, North America, and Asia above latitude 40 N. Based on whether the A. sativa grains are covered with husks, they can be categorized into hulled or hull-less A. sativa [3]. The cultivation of forage A. sativa helps alleviate forage shortages in winter and spring, thereby contributing significantly to the conservation of grassland ecosystems and the advancement of sustainable grassland livestock production and ecological balance [4].

Transcription factors generally comprise several key functional domains, such as segments involved in DNA binding, gene activation, nuclear import, and protein interactions. They play central roles in regulating gene expression at the transcriptional level [5]. The MYB family of transcription factors belongs to the class of trans-acting proteins encoded by distinct regulatory genes; thus, MYB transcription factors are often referred to as MYB proteins [6]. MYB proteins contain an evolutionarily conserved MYB-type DNA-binding domain composed of one to four imperfect repeats (R1–R4), each approximately 52 amino acids long [7, 8]. The second and third helices form a helix–turn–helix (HTH) motif that recognizes the major groove of the DNA double helix [9]. Based on repeat composition, MYB transcription factors are classified into four types: 1R, 2R (R2R3), 3R (R1R2R3), and 4R-MYB [10], among which 1R-MYB and R2R3-MYB are the most common in plants [11]. The MYB gene family was first identified in the avian myeloblastosis virus (v-MYB) and later in animal cells (c-MYB), both sharing a conserved MYB-binding motif [12, 13]. In plants, the R2R3-MYB subfamily defined by the presence of R2 and R3 repeats is the most widespread. It is hypothesized that R2R3-MYB genes originated through deletion of the R1 repeat from ancestral R1R2R3-MYB genes or duplication of R1-containing variants [14, 15].

During their development, plants are constantly exposed to various abiotic and biotic stresses and must rely on internal mechanisms to cope with these challenges. Through evolution, plants have developed sophisticated regulatory systems to adapt to changing environments, among which MYB transcription factors play crucial roles. As regulatory proteins, MYB transcription factors modulate gene expression to regulate plant growth and stress responses [16]. The first plant MYB transcription factor, ZmMYBC1, was identified in Zea mays. Among them, the R2R3-MYB subfamily is the most extensively studied and represents the largest MYB group in plants, playing key roles in growth regulation, cell differentiation, environmental stress response, and primary and secondary metabolism [17, 18]. In plants, the MYB gene family is deeply involved in regulating responses to various abiotic stresses. For example, overexpression of MwMYB4 from Agropyron mongolicum reduced leaf bleaching and enhanced cold tolerance in Arabidopsis thaliana [19]. Ectopic expression of OsMYB4 in A. thaliana also markedly improved cold resistance [20]. Transgenic Hordeum vulgare lines overexpressing HvMYB1 exhibited enhanced drought tolerance [21]. SiMYB16 from Setaria italica improved salt resistance in Oryza sativa by regulating lignin and suberin biosynthesis [22], and overexpression of GhMYB4 in A. thaliana enhanced tolerance to both salinity and drought [23]. Although numerous MYB transcription factors have been functionally characterized in various plant species, the molecular mechanisms underlying stress tolerance in oat remain poorly understood. A previous genome-wide analysis of the MYB transcription factor family in A. sativa identified 268 MYB members and revealed that the MYB1R subfamily plays an important role in drought stress response and reactive oxygen species (ROS) homeostasis regulation [24]. However, a comprehensive analysis focusing specifically on the R2R3-MYB subfamily and its expression patterns under multiple abiotic stresses in oat remains limited. Therefore, systematic identification and characterization of the AsR2R3-MYB gene family is essential to better understand their roles in stress responses and to provide valuable genetic resources for breeding stress-resilient oat cultivars.

In this study, R2R3-MYB gene family members in oat were detected and systematically analyzed through bioinformatics approaches. Expression patterns under various abiotic stresses were examined using quantitative real-time PCR (qRT-PCR). The results provide valuable understanding of the functional significance of R2R3-MYB genes in oat and lay the groundwork for genetically modifying new oat cultivars with enhanced stress tolerance.

Materials and methods

Genome-wide identification of R2R3-MYB genes in oat

The complete A. sativa genome was sourced from the EnsemblPlants repository, and the A. thaliana R2R3-MYB amino acid sequences were extracted via the TAIR platform (https://www.arabidopsis.org/). The Hidden Markov Model (HMM) profile corresponding to the MYB domain (PF00249) was obtained through Pfam (https://pfam.xfam.org/, accessed 6 January 2024), then aligned to the A. sativa genome using A. thaliana R2R3-MYB protein sequences as references, applying an e-value threshold of 10⁻⁵. The resulting gene set was curated manually to remove redundant and truncated entries. For each initially detected candidate, the PF000249 conserved domain present in candidate R2R3-MYB peptides was validated via CDD at NCBI (https://www.ncbi.nlm.nih.gov/), and visual inspection was carried out using TBtools to discard repetitive or truncated polypeptides lacking the MYB domain. This yielded a finalized candidate set for the A. sativa R2R3-MYB gene group. Full-length polypeptides encompassing the MYB domain were renamed as AsMYB proteins. Molecular mass and pI values for AsMYB proteins were computed using the ExPASy server (https://web.expasy.org/compute_pi/), and subcellular localization was predicted via WoLF PSORT (https://wolfpsort.hgc.jp/).

Evolutionary analysis of the R2R3-MYB gene family in A. sativa

In order to explore the evolutionary connections within the R2R3-MYB gene group of A. sativa, a corresponding phylogenetic tree was generated through MEGA 7.0, using R2R3-MYB family protein from A. thaliana and A. sativa. The neighbor-joining (NJ) method was applied with the following parameters: Poisson model, pairwise deletion, and 1,000 bootstrap replications. The phylogenetic tree was further visualized using iTOL.

Analysis of the AsMYB gene structure and conserved protein motifs

The genome annotation (GFF format) of A. sativa was acquired via Ensembl Plants (https://plants.ensembl.org/index.html) to investigate the gene architecture of the R2R3-MYB family. Conserved sequence motifs in AsR2R3-MYB proteins were detected using the MEME software package (http://meme-suite.org/tools/meme), with a cap of 10 motifs per sequence. Intron–exon configurations and motif localization were visualized in TBtools (V2.310). Genomic coordinates of R2R3-MYB members were pinpointed and plotted using TBtools as well. Additionally, 2,000 bp upstream regions from each AsR2R3-MYB gene’s initiation codon were extracted to identify cis-acting elements within promoter segments via the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Prediction outcomes were subjected to statistical evaluation in Excel and rendered through TBtools.

Collinearity analysis and gene duplication patterns

To further investigate the R2R3-MYB gene repertoire in A. sativa, tandem and segmental duplication events involving AsMYB loci were detected through the MCScanX algorithm. Visualization of the results was performed by constructing high resolution Circos plots via TBtools. Moreover, syntenic associations between A. sativa and three additional species (A. thaliana, Triticum aestivum, and Zea mays) were explored using the One Step MCScanX-Super Fast function within TBtools. The resulting collinearity patterns were presented through Dual Synteny Plot output.

Gene regulatory network analysis of AsMYB

Protein-protein interaction (PPI) prediction was conducted for A. sativa R2R3-MYB proteins using the STRING database (https://string-db.org/), with A. thaliana as a reference species owing to its well-characterized genome. A confidence threshold of 0.700 was applied to filter interaction scores. Based on documented associations among MYB proteins in A. thaliana, potential interactions among AsMYB proteins were inferred. The resulting PPI network was further processed via Cytoscape (version 3.7.2) (http://www.cytoscape.org/), using a lower cutoff of 0.4 for interaction confidence. In the interaction map, proteins were denoted as nodes, while predicted links between them were shown as edges. Final PPI network diagrams were optimized using Adobe Illustrator 2024.

Plant materials and treatments

The A. sativa cultivar used in this experiment was new strain Zhongxuyan No. 2, sourced from the forage germplasm collection and breeding unit at the Institute of Animal Science, Chinese Academy of Agricultural Sciences. Uniform, well-developed seeds were chosen and germinated in culture boxes for 7 d before being transferred into seedling containers containing a 1:1 blend of vermiculite and nutrient substrate. Plants were cultivated under controlled environmental settings of 25 °C/18°C (day/night), 65%-75% relative humidity, and a photosynthetic photon flux density (PPFD) of 200–250 µmol·m^-2·s^-1. When the seedlings reached the three-leaf stage, thinning was performed to retain uniform and vigorous plants. After an additional 14 d of growth, stress treatments were applied. A. sativa seedlings were treated with cold (4°C), 20% PEG6000, or 150 mM NaCl for cold, drought, or salt stress treatments, respectively, with three replicates for each treatment. A. sativa leaf and root tissues were collected at different time points during stress (0, 2, 4, 8, 12, and 24 h) for expression pattern analysis of target genes under stresses. Germinated seeds were sown into soil, and potted plants were placed in the growth chamber under the same conditions. Different A. sativa tissue parts (stem, leaf, spike, and stem nodes) at the wax ripening stage were sampled, quickly frozen in liquid nitrogen for convenient total RNA extraction, and then stored in a -80°C freezer.

Gene expression analysis by qRT-PCR and statistical analyses

Total RNA was isolated from A. sativa tissues using the Plant Total RNA Kit (Simgene, Hangzhou, China), and first-strand cDNA was generated using the Simgene cDNA Synthesis Kit (Hangzhou, China). To gain a more detailed understanding of R2R3-MYB gene expression patterns, 20 AsMYB genes from distinct subgroups were analyzed across various tissues and under drought, salinity, and cold stress through quantitative RT-PCR (qRT-PCR). The qRT-PCR experiments were conducted on a LightCycler^® 96 system with TB Green^® Premix Ex Taq™ II (TAKARA, Beijing, China). Primers specific to AsMYB were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi), and their sequences are listed in Table S1. The β-actin gene was used as an internal reference for normalization, and its primer sequences are also provided in Table S1. PCR amplifications were carried out using the TIANGEN Fluorescent Quantification Kit (FP206-02) with a final reaction volume of 20 µL, consisting of 5 µL cDNA, 1.6 µL primer mix, 3.4 µL ddH₂O, and 10 µL of 2× SuperReal PreMix Plus. The thermal cycling protocol involved an initial step of 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Each treatment condition included three independent biological replicates. Transcript abundance was calculated through the 2^-ΔΔCT method. Heatmaps reflecting expression changes of the 20 AsMYB genes under various tissues and abiotic stresses were visualized using TBtools. Statistical analysis of qRT-PCR data was performed using one way ANOVA followed by Tukey’s post-hoc test in Originpro 2025b. Differences were considered statistically significant at p < 0.05.

Results

Identification of R2R3-MYB family genes in the A. sativa genome

A total of 237 R2R3-MYB genes were identified in the A. sativa genome through HMM profiling and conserved domain analysis. Detailed information on these AsR2R3-MYB family members is provided in Table S1. The encoded proteins showed considerable variation in length, ranging from 104 amino acids (AsMYB123) to 1064 amino acids (AsMYB75). Their predicted molecular weights spanned from 11,317.79 Da (AsMYB123) to 116,891.42 Da (AsMYB68), while theoretical isoelectric points (pI) ranged between 4.60 (AsMYB135, AsMYB210) and 10.76 (AsMYB60). Subcellular localization analysis predicted that most MYB proteins are primarily located in the nucleus (Table S2).

Based on A. sativa genome information, the distribution of 237 MYB genes across 21 chromosomes was analysed. The results (Fig. 1) showed that the 237 A. sativa MYB genes were unevenly distributed across the 21 chromosomes, with the greatest number of AsMYB genes located on chromosome 6 A (20 genes). Chromosome 4D contained 17 AsMYB genes; chromosome 1 A had 16 AsMYB genes; chromosomes 1D, 3 C, and 6D each had 14 AsMYB genes; chromosomes 2 C, 2D, and 5 A each had 13 AsMYB genes; chromosome 6 C had 12 AsMYB genes; chromosome 4 C had 11 AsMYB genes; chromosomes 3D and 5 C each had 10 AsMYB genes; chromosome 5D had 9 AsMYB genes; chromosomes 2 A, 7 A, and 7D each had 8 AsMYB genes; chromosome 3 A had 6 AsMYB genes; chromosome 7 C had 5 AsMYB genes; and chromosome 1 C only had 2 AsMYB genes. These genes were named AsMYB1-AsMYB237 based on their chromosomal positions.

Fig. 1.

Chromosomal locations of AsMYB genes

Phylogenetic analysis of A. sativa R2R3-MYB proteins

To better clarify the evolutionary associations among AsMYB proteins, we applied the NJ method in MEGA software in order to generate a phylogenetic tree encompassing the 237 MYB protein sequences from A. sativa and 126 R2R3-MYB sequences from A. thaliana (Fig. 2). Based on phylogenetic tree bootstrap support values exceeding 50%, the R2R3-MYB gene family was classified into 29 subfamilies (named A1-A29). With the 237 AsMYB members distributed among 23 of these subfamilies. Within these subfamilies, the A1 subfamily (48 members), A8 subfamily (30 members), A9 subfamily (29 members) and A28 subfamily (30 members) were the four largest subfamilies, which together constituted over 37% of all identified R2R3-MYB gene members. The A5 subfamily contained only 2 R2R3-MYB members and made up the smallest subfamily. Additionally, AsMYB members were not distributed in the A5, A15, and A24 subfamilies, while the A11, A12, A13, A20, A21, A22, A23, and A26 subfamilies consisted exclusively of R2R3-MYB members. In the A1, A3, A4, A6, A27 and A29 subfamilies, the number of AsMYB members in A. sativa was approximately two to three times higher than that in the corresponding subfamilies of A. thaliana. The findings suggest the existence of shared ancestral origins between the A. sativa and A. thaliana MYB gene families, as well as specific amplifications and diversifications that occurred during the evolutionary process after their separation.

Fig. 2.

Phylogenetic tree of the AsR2R3-MYB genes in A. sativa and A. thaliana

Conserved gene structure and motif composition of AsR2R3-MYB

Through MEME suite analysis of 237 AsMYB proteins, a total of 10 motifs were identified, with the sequence lengths of these motifs ranging from a minimum of 11 amino acids (Motif4) to a maximum of 50 amino acids (Motif5, Motif6, Motif7, Motif8, Motif9, and Motif10). Some motifs were conserved across most AsMYB members, while others were unique to a few AsMYB members. For example, 223 AsMYB members possessed Motif1 and Motif4, whereas only 17 AsMYB members had Motif5 and Motif10. Most AsMYB members contained Motif1, Motif2, Motif3, and Motif4, but only 17, 17, 18, 18, 30, 20, and 38 AsMYB members respectively contain Motif5, Motif6, Motif7, Motif8, Motif9, and Motif10, respectively. AsMYB166, AsMYB167, AsMYB58, AsMYB190, AsMYB225, AsMYB226, AsMYB171, and AsMYB185 contained only Motif3 (Figure 3).

Fig. 3.

Conserved motifs and gene structure of R2R3-MYB family genes

Promoter cis-acting elements of the A. sativa AsR2R3-MYB gene family

To better understand the functional characteristics of the MYB gene family in oat, we analyzed the cis-acting elements within the 2,000 bp upstream promoter regions of 237 AsMYB genes. A total of 37 distinct types of cis-elements were identified, which were classified into five functional categories: core transcriptional elements, hormone-responsive elements, light-responsive elements, abiotic stress-responsive elements, and development-related elements. Among these, core transcriptional elements, such as core promoter elements and common promoter/enhancer elements were the most abundant, accounting for approximately 58% of all elements (Figure S1). Hormone responsive elements made up about 19%, with MeJA and ABA responsive elements being the most prevalent. Auxin-responsive elements (e.g., TGA-element) and gibberellin-responsive elements (e.g., GARE-motif) were also present. Light-responsive elements accounted for about 13%, including GT1-motif, TCT-motif, and MRE. Abiotic stress-related elements comprised roughly 7%, including MBS (drought-responsive), LTR (low-temperature responsive), and TC-rich repeats (defense and stress responsive). Although development-related elements, such as GCN4_motif involved in endosperm expression and CAT-box related to meristem activity were less frequent, their presence suggests that some AsMYB genes may exhibit tissue or stage-specific expression. Overall, These findings indicate that AsMYB genes are regulated by diverse signaling pathways, such as hormone signaling, light response, and defense mechanisms, and may play crucial roles in oat tolerance to drought, salinity, and cold stresses. (Figure 4;Table S3).

Fig. 4.

Cis-acting elements of R2R3-MYB gene family members in A. sativa

Gene duplication events and collinearity analysis of R2R3-MYB gene family members in A. sativa

Gene duplication plays an essential role in generating novel genes and biological functions, with segmental and tandem duplications recognized as factors contributing significantly to gene family growth. In order to investigate the evolutionary dynamics of A. sativa R2R3-MYB genes, MCScanX software was used to detect whole-genome duplication events among gene family components, as well as to generate Circos plots. It was found that 80 homologous gene pairs were present among the 237 genes showing segmental duplication. For example, a segmental duplication event occurred between AsMYB189 on chromosome 5 A and AsMYB1 on chromosome 5D, leading to many homologies between AsMYB genes on different chromosomes and increasing the possibilities for evolution. Segmental duplication might be the main mechanism by which the AsR2R3-MYB gene family has evolved and expanded (Figure 5).

Fig. 5.

AsR2R3-MYB gene family repeat events. The gray lines indicate collinearity among all genes in A. sativa, while the red lines highlight collinear relationships specifically within the AsR2R3-MYB gene family

Furthermore, to explore possible evolutionary patterns within the MYB gene family across various crops, we investigated the collinearity between A. sativa MYB genes and those of four representative plant species: A. thaliana, Z. mays, O. sativa, and T. aestivum. Our findings revealed a substantial number of collinear genes between A. sativa and each of these species. Specifically, 4,246 A. sativa genes were collinear with those of A. thaliana, accounting for 4.61% of the analyzed genes (4,246 of 92,089 genes), with 29 of the 237 AsMYB genes showing collinearity. A total of 44,559 A. sativa genes were collinear with those of O. sativa, representing 41.72% of the analyzed genes (44,559 of 106,803 genes), including 105 of the 237 AsMYB genes. Similarly, 44,559 A. sativa genes were collinear with those of Z. mays, accounting for 43.05% of the analyzed genes (44,559 of 103,496 genes), with 108 AsMYB genes involved. In addition, 73,413 A. sativa genes were collinear with those of T. aestivum, representing 42.68% of the analyzed genes (73,413 of 172,006 genes), among which 111 AsMYB genes were identified. The degree of collinearity of A. sativa MYB genes with O. sativa, Z. mays, and T. aestivum notably exceeded that with A. thaliana, suggesting a relatively conserved distribution of MYB genes among these three Poaceae species (Figure 6).

Fig. 6.

Homology Analysis of R2R3-MYB Gene Family Members in A. sativa, A. thaliana, O. sativa, and T. Aestivum

Protein-protein interaction network prediction of R2R3-MYB gene family members in A. sativa

Protein-protein interactions are essential for elucidating the functional mechanisms and regulatory activities of transcription factors. In order to investigate potential interactions within the oat R2R3-MYB gene family, predicted interaction networks were generated using the STRING online database, with the A. thaliana protein database serving as a comparative reference. The results showed that the regulatory network contained 45 nodes (representing AsMYB proteins) and 140 edges (representing predicted protein-protein interactions). The PPI network was further optimized using the degree algorithm in Cytoscape. In the network diagram, the color of the 45 nodes transitions from dark to light, indicating a decreasing degree score. Among them, AsMYB201 exhibited the highest degree, interacting with 12 other AsMYB proteins. These findings imply that AsMYB201 could serve an important function in the AsR2R3-MYB protein family, functionally associated with other proteins and involved in various physiological and biochemical processes in plants.

Fig. 7.

A. sativa R2R3-MYB protein interaction network

Expression pattern analysis of AsMYB genes in different A. sativa tissues

A total of 20 genes originating from different subfamilies of the A. sativa R2R3-MYB family were selected for analysis. These genes exhibited diverse structural domains of proteins, along with profiles of cis-regulatory elements. To examine how they are expressed in various tissues, a heatmap was generated for displaying the expression distribution of the MYB genes across different A. sativa tissues (Fig. 8A and Table S4). It was observed that all 20 AsR2R3-MYB genes from various subgroups presented distinct expression trends across organs, indicating functional differentiation among gene members in specific tissues. A majority of AsMYB genes, including AsMYB169, AsMYB165, and AsMYB163, showed stronger expression in the internodes and leaves compared to the stems and spikes. In contrast, AsMYB161 was more abundantly expressed in spikes and stems. Two members of the A28 subgroup, AsMYB69 and AsMYB134, exhibited elevated transcript levels in internodes relative to other tissues. AsMYB158 and AsMYB36, belonging to the A10 subgroup, were predominantly expressed in leaf tissues. Overall, most AsMYB genes showed high expression in leaves and stem nodes.

Fig. 8.

A Heatmap showing the expression of 20 AsMYB genes in various oat tissues (stem, leaf, spike, and stem nodes). B-D Expression patterns of the same genes under drought (B), salt (C), and cold (D) stress at 0, 2, 4, 8, 12, and 24 h. Color scale represents the relative expression level (log₂-transformed FPKM values), where red indicates high expression and blue indicates low expression

Expression analysis of the R2R3-MYB gene family in A. sativa under different abiotic stress treatments

The expression patterns of 20 AsR2R3-MYB genes under cold, drought, and high-salinity stresses were analyzed using qRT-PCR to explore the potential functions of different gene members (Table S5). As shown in Fig. 8B, the 20 AsMYB genes responded to the three stress treatments to varying degrees. Specifically, AsMYB122 and AsMYB135 exhibited significant upregulated expression 2 h after PEG treatment, while AsMYB230, AsMYB134, AsMYB204, and AsMYB107 showed significantly upregulated expression 12 h after PEG treatment, at which point the genes reaching peak expression levels. Under PEG treatment, the expression levels of AsMYB237, AsMYB100, AsMYB12, AsMYB165, and AsMYB163 were significantly downregulated, suggesting that these genes may be involved in oat responses to drought stress. Cluster analysis based on expression profiles further revealed that several AsMYB genes exhibited similar expression patterns under PEG treatment. For instance, AsMYB230 and AsMYB134 showed comparable expression trends, with transcript levels significantly increasing at 8 h and reaching a peak at 12 h. These results indicate that some AsMYB genes may play important roles in the drought stress response in A. sativa (Fig. 8B). NaCl stress significantly upregulated the expression of AsMYB134, AsMYB163, AsMYB204, AsMYB107, AsMYB158, AsMYB36, and AsMYB12, with their transcript levels reaching peak values at 12 h after treatment. The expression levels of AsMYB107 and AsMYB204 were significantly increased at multiple time points following NaCl treatment. Cluster analysis further revealed that AsMYB12 and AsMYB188 exhibited similar expression patterns at different time points after NaCl treatment. These results suggest that some AsMYB genes may be involved in the response of A. sativa to salt stress (Fig. 8C). Cold stress (4 °C) significantly increased the expression levels of AsMYB36, AsMYB230, AsMYB100, AsMYB82, AsMYB134, and AsMYB135 after 2 h of treatment, reaching peak levels at this time point. In contrast, AsMYB161, AsMYB122, AsMYB237, AsMYB169, AsMYB81, and AsMYB69 were downregulated at different cold stress time points, whereas the remaining 14 AsMYB genes showed upregulated expression during the treatment period. Among them, AsMYB163 exhibited the greatest increase in expression. Most genes reached their peak expression levels after 8 h of cold stress (Fig. 8D). Cluster analysis revealed that AsMYB12 and AsMYB188 exhibited similar expression responses to cold treatment. In addition, AsMYB97 and AsMYB158 showed significant expression changes during cold stress, although their peak expression occurred at different time points. These results suggest that these genes may play important roles in the response of A. sativa to cold stress.

Discussion

Members of the R2R3-MYB gene family are broadly present throughout eukaryotic organisms [25]. As genome sequencing technologies have advanced rapidly, comprehensive analyses on a whole-genome scale of the R2R3-MYB gene family have been performed across a wide range of plant species. To date, 126 members have been identified in A. thaliana, 174 in Nicotiana tabacum, 244 in Glycine max, 121 in Solanum lycopersicum, 157 in Z. mays, and 110 in O. sativa [26–31]. The quantity of identified MYB genes varies among different plant species, as does the size of their genomes, which are 125 Mb, 4.5 Gb, 1.03 Gb, 828 Mb, 2300 Mb, and 430 Mb, respectively. In this study, 237 R2R3-MYB genes were identified in A. sativa, a greater number than that in A. thaliana, N. tabacum, S. lycopersicum, Z. mays, and O. sativa, but fewer than that in soybean. A previous genome-wide study identified 268 MYB transcription factor genes in A. sativa, including members of the MYB1R, MYB2R, and MYB3R subfamilies [24]. In contrast, the present study focused specifically on the R2R3-MYB subgroup of the MYB family. The difference in the number of MYB genes reported in the two studies results from differences in gene identification criteria, genome annotation versions, and the classification scope of MYB subfamilies. In addition, the previous study mainly investigated the role of MYB1R genes in regulating ROS homeostasis under drought stress, whereas our study systematically analyzed the evolutionary characteristics of the R2R3-MYB gene family and their expression responses under drought, salt, and cold stresses. These findings provide complementary insights into the potential roles of MYB transcription factors in abiotic stress responses in A. sativa. The N. tabacum genome is much larger than the soybean genome, but tobacco has fewer R2R3-MYB genes compared to G. max. A comparative analysis of genomic size and how many R2R3-MYB genes have been detected across different plant species indicates that R2R3-MYB gene abundance does not show a strict correlation with genome size. Furthermore, AsR2R3-MYB proteins contained 10 shared motifs, and genes grouped within the same subfamily exhibited comparable motif types and numbers, indicating that both the evolutionary conservation and variation within the A. sativa R2R3-MYB gene family are related to these conserved motifs and the regulatory functions of different subfamilies. Like most plant species, most AsR2R3-MYB genes are composed of three exons and two introns. Interestingly, AsR2R3-MYB genes within the same subfamily display conserved exon intron organization, with the majority containing two or fewer introns a structural feature commonly observed in R2R3-MYB genes across various plant species [32]. To classify the A. sativa AsR2R3-MYB proteins, a phylogenetic tree was constructed based on 126 A. thaliana R2R3-MYB proteins, thereby dividing them into 29 subfamilies. AsR2R3-MYB genes with similar motifs were classified into the same branch based on homology alignment. The sequence structures of R2R3-MYB proteins from different subfamilies are often related to their biological functions. Most AtR2R3-MYB proteins in subfamilies A1, A10, and A28 are associated with abiotic stress [33–35]. Subfamily A17 plays an important role in anthocyanin biosynthesis, members of subfamily A14 are related to the regulation of the jasmonic acid pathway in response to plant injury, and members of subfamily A6 play a significant role in the biosynthesis of flavonoids [36–38]. Although the functions of nearly all AsR2R3-MYB proteins are unclear, homologous genes with close evolutionary branches may have similar functions. Moreover, three subfamilies (A5, A15, and A24) contained only A. thaliana members, suggesting that A. sativa lacks MYB genes that perform the functions of these A. thaliana members, this result is similar to findings reported by Aoyagi et al. [39].

Cis-regulatory elements within promoter regions are essential for the regulation of gene expression [40]. Variations in these elements among gene promoters may contribute to the differential expression profiles observed in A. sativa [41]. In this study, 37 cis-acting elements were identified in the promoter region of AsR2R3-MYB genes, 16 of which were related to cell development, 7 of which were plant hormone-responsive elements, 9 of which were light-responsive elements, and 5 of which were involved in the defence and stress responses. These results suggest that AsR2R3-MYB gene family members may participate in regulating multiple hormone signaling pathways and stress responses, enabling plants to swiftly adapt to both biotic and abiotic challenges and improving their resilience under adverse environmental conditions. Comparable roles have been reported in other species; for instance, in A. thaliana, AtMYB30 binds to the promoter of AOX1a, enhancing its expression to maintain cellular redox balance and thereby increasing salt tolerance [42]. Moreover, salt stress can alter the methylation of the promoter region of OsMYB91 in O. sativa, thereby increasing the expression of OsMYB91 under salt stress conditions [43]. These results demonstrate that the ability of plants to adapt to environmental stresses relies heavily on sophisticated mechanisms of gene expression regulation. The diversity and complexity of cis-acting elements provide plants with a highly flexible and dynamic gene expression regulatory network, thus enabling them to respond quickly and effectively to various environmental stresses. This molecular-level adaptation and regulation endows plants with the ability to survive and grow under various abiotic stresses, such as salt, drought, and cold stresses. This regulatory capability may not only help stabilize grain yield but may also maintain forage biomass production under adverse environmental conditions, which could be of potential significance for oats as a crop used for both grain and forage [44].

Salt, drought, and cold stress severely impact plant development and growth. For A. sativa, these stresses can reduce both grain yield and total forage biomass, thereby affecting its dual economic and agricultural value [45]. To cope with these adverse environments, plants have acquired sophisticated molecular strategies. Among them, MYB-type transcriptional regulators are essential for coordinating plant responses to a wide range of environmental cues, both from biotic and abiotic origins [46, 47]. In this research, we analyzed the transcriptional behavior of 20 AsR2R3-MYB genes derived from different R2R3-MYB lineages exposed to salt, low temperature, and drought stress. The results indicate that most of these genes are likely associated with adaptation to non-biological stresses. Specifically, under water-deficit conditions, seven AsR2R3-MYB genes showed increased expression levels, implying their potential role in promoting molecular pathways that contribute to enhanced drought tolerance in A. sativa. Studies have demonstrated that the homologous gene OsMYBAS1 of AsMYB122 in O. sativa functions in the regulatory pathway associated with drought stress in rice seedlings. Under drought conditions, the OsMYB48 gene, homologous to AsMYB135, has been reported to contribute to enhanced drought tolerance. Elevated expression of OsMYB48 results in increased levels of LEA proteins and proline, reduces transpirational water loss in transgenic lines, and consequently strengthens drought resilience in rice [48]. Therefore, such genes present promising candidates for modulating drought stress responses in A. sativa. In Cajanus cajan, the expression of CcMYB107 was markedly elevated by drought exposure, and its overexpression promoted the activation of drought-responsive genes, thereby improving the plant’s drought tolerance [49]. Additionally, overexpression of OsMYB2 has been shown to raise intracellular proline and soluble sugar contents and increase antioxidant enzyme activities, while decreasing levels of hydrogen peroxide and malondialdehyde, collectively enhancing drought resistance [50]. Under saline conditions, the expression of AsMYB100, AsMYB163, AsMYB12, AsMYB158, AsMYB204, and AsMYB107 was significantly elevated, indicating a potential role for these six genes in positively modulating salt tolerance in A. sativa. Previous studies have shown that overexpressing A. thaliana AtMYB12 can improve plant salt tolerance Salvia miltiorrhiza Bunge [51]. Furthermore, overexpression of MsMYB206 in Medicago sativa significantly enhanced the salt tolerance of the transgenic lines, indicating that R2R3-MYB genes play a positive regulatory role in plant responses to salt and drought stress [52]. Under cold stress conditions, the expression of eight AsMYB family genes was inhibited, indicating that these genes might inhibit the molecular regulatory pathways related to cold adaptability in A. sativa. Previous research has shown that the O. sativa OsMYB30 transcription factor reduces cold tolerance by interacting with JAZ proteins to suppress the expression of β-amylase [53]. Under cold stress, overexpression of SlMYB41 in S. lycopersicum reduced cold tolerance, suggesting that SlMYB41 plays a role in regulating cold resistance in S. lycopersicum [54]. Under cold stress, AsMYB genes in A. sativa exhibited two primary expression patterns: “upregulated-downregulated” and “downregulated-upregulated.” These observations suggest that AsMYB gene expression in response to low-temperature conditions is regulated by a complex signaling network. Nonetheless, the precise roles of AsR2R3-MYB genes in conferring cold tolerance remain unclear. AsMYB36 and AsMYB158, both belonging to the A10 subgroup, display similar expression profiles under cold and salt stress, indicating potential functional redundancy driven by evolutionary conservation. Nonetheless, functional divergence was also observed within the same subfamily, AsMYB69 was significantly repressed under cold stress, whereas AsMYB134 exhibited a marked upregulation. The A7 subfamily gene AsMYB204 and the A9 subfamily gene AsMYB107 showed significantly upregulated expression under salt, drought, and cold stresses, suggesting that AsMYB genes play important roles in the stress response of A. sativa. The strong induction of these two genes is consistent with the presence of multiple stress and hormone-responsive cis-acting elements in their promoter regions, such as ABRE (abscisic acid-responsive element), DRE (dehydration-responsive element), and LTR (low-temperature-responsive element). Similar expression patterns have also been reported in other plant species, such as TaMYBL1 in wheat and AtMYB15 in A. thaliana, where these homologous genes enhance tolerance to abiotic stresses by activating downstream stress-related pathways [55, 56]. Therefore, AsMYB107 and AsMYB204 may represent important candidate genes of the AsMYB gene family involved in abiotic stress responses. Further functional characterization of these genes will be valuable for elucidating the molecular and genetic mechanisms underlying stress tolerance and for facilitating molecular-assisted breeding of stress-resilient A. sativa cultivars.

Conclusions

A genome-wide survey was conducted to identify the AsR2R3-MYB gene family in oat, resulting in the detection of 237 AsR2R3-MYB genes. These genes were categorized into 29 subfamilies based on phylogenetic analysis with Arabidopsis R2R3-MYB proteins, with the A1 subfamily comprising the largest number of members (48), while the A5 subfamily contained only two. Among the encoded AsMYB proteins, 45 were predicted to participate in protein–protein interactions, with AsMYB201 displaying the most interacting partners. Transcript profiling of 20 selected AsR2R3-MYB genes revealed ubiquitous expression across all tissues, though expression intensities differed notably among organs. All examined genes were responsive to salt, drought, and cold stresses. Notably, AsMYB204 (A7 subgroup) and AsMYB107 (A9 subgroup) exhibited marked upregulation, suggesting that they may play important roles in abiotic stress adaptation. This expression pattern is consistent with the presence of multiple stress-related cis-acting elements in their promoter regions, indicating that these elements may facilitate transcriptional activation under abiotic stress conditions. This work comprehensively cataloged the AsR2R3-MYB gene repertoire and annotated their coding sequences, offering theoretical support and practical insights for future investigations into oat gene function and for breeding stress-resilient germplasm based on this gene lineage.

Authors’ contributions

X Z designed and conducted the experiments, analyzed the data, and wrote the manuscript. S G was involved in conception and investigation. X Z and W Y collected and identified plant materials. D Y, W Y, Q J, and Z Z provided analytical tools and analyzed data. Corresponding authors L M and X W was responsible for funding acquisition, supervision, and writing-review & editing.

Funding

This work was supported by the earmarked fund for CARS (CARS-34) and the Agricultural Science and Technology Innovation Program (ASTIP-IAS10).

Data availability

The genomic data supporting the results of this study are available from the databases cited in the manuscript. The download links and accession numbers are provided in the relevant sections of the main text.

Declarations

Ethics approval and consent to participate

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Not applicable.

Competing interests

The authors declare no competing interests.