Genome-Wide Identification of PP2C Gene Family in Oat (Avena sativa L.) and Its Functional Analyses in Response to ABA and Abiotic Stresses

Abstract

Plant protein phosphatase 2C (PP2C) represents the largest and most functionally diverse group of protein phosphatases in plants, playing pivotal roles in regulating metabolic processes, hormone signaling, stress responses, and growth regulation. Despite its significance, a comprehensive genome-wide analysis of the PP2C gene family in oat (Avena sativa L.) has remained unexplored. Leveraging the recently published oat genome, we identified 194 AsaPP2C genes, which were unevenly distributed across all 21 chromosomes. A phylogenetic analysis of PP2C classified these genes into 13 distinct subfamilies (A-L), with conserved motif compositions and exon-intron structures within each subfamily, suggesting evolutionary functional specialization. Notably, a promoter analysis revealed an abundance of stress-responsive cis-regulatory elements (e.g., MYB, MYC, ARE, and MBS), implicating AsaPP2Cs in hormones and biotic stress adaptation. To elucidate their stress-responsive roles, we analyzed transcriptomic data and identified seven differentially expressed AsaPP2C (Asa_chr6Dg00217, Asa_chr6Ag01950, Asa_chr3Ag01998, Asa_chr5Ag00079, Asa_chr4Cg03270, Asa_chr6Cg02197, and Asa_chr7Dg02992) genes, which were validated via qRT-PCR. Intriguingly, these genes exhibited dynamic expression patterns under varying stress conditions, with their transcriptional responses being both time-dependent and stress-dependent, highlighting their regulatory roles in oat stress adaptation. Collectively, this study provides the first comprehensive genomic and functional characterization of the PP2C family in oat, offering valuable insights into their evolutionary diversification and functional specialization.

1. Introduction

Plants in natural ecosystems endure simultaneous abiotic stresses (e.g., drought, extreme temperatures, salinity, nutrient deficits, heavy metals) and biotic threats (e.g., pathogens, insects), requiring integrated physiological and molecular adaptations for survival [1,2]. These stressors not only impair growth and developmental processes but also significantly diminish agricultural land productivity, often resulting in substantial yield losses or even complete crop failure [3]. To combat these multifaceted stresses, plants have evolved sophisticated and interconnected regulatory networks capable of precisely perceiving environmental cues and initiating appropriate adaptive responses through complex signal transduction cascades [4]. Among these mechanisms, protein post-translational modifications (PTMs) serve as a crucial regulatory layer, dynamically modulating protein functions by altering enzymatic activity, protein-protein interactions, subcellular localization, and stability [5]. Notably, reversible modifications, such as phosphorylation, ubiquitination, and sumoylation, enable the rapid reprogramming of cellular processes, allowing plants to mount efficient stress responses while maintaining metabolic homeostasis. This sophisticated regulatory plasticity highlights the evolutionary of plants in responding to ever-changing environmental pressures [6,7].

PTMs represent one of the most sophisticated and dynamic regulatory strategies in cellular signaling, with protein phosphorylation and dephosphorylation constituting the predominant reversible mechanisms that govern protein function [8]. This exquisite balance is maintained through the opposing actions of protein kinases (PKs) and protein phosphatases (PPs), which collectively orchestrate vital physiological processes through the precise, spatiotemporal control of protein activity [9]. Protein kinases catalyze the transfer of phosphate groups from ATP/GTP to specific serine (Ser), threonine (Thr), or tyrosine (Tyr) residues on target proteins, thereby modulating their conformation, interactions, and functional states [10]. This phosphorylation cascade serves as a molecular switch that activates or inhibits downstream signaling pathways. A quintessential example is the SnRK1 kinase in Arabidopsis, which phosphorylates the transcription factor bZIP63 to promote seed storage mobilization and seedling establishment by activating the cyPPDK promoter, a critical metabolic adaptation during energy deprivation [11]. Conversely, protein phosphatases counter-balance kinase activity by hydrolytically removing phosphate groups, thereby resetting signaling circuits and maintaining cellular homeostasis [12]. The type 1 protein phosphatase TOPP exemplifies this regulatory logic by dephosphorylating ATG13a to facilitate the formation of the ATG1a-ATG13a autophagy initiation complex, a key mechanism for nutrient recycling during stress [13]. Among phosphatases, the protein phosphatase type 2C (PP2C) family plays a pivotal role in stress responses by negatively regulating SnRK2 kinases to modulate ABA signaling and fine-tune plant stress adaptation [14].

PP2C represents the most abundant and functionally diverse family of protein phosphatases in plants, serving as master regulators of both abiotic and biotic stress responses [15]. These enzymes orchestrate adaptive responses through ABA-dependent and ABA-independent pathways, fine-tuning cellular signaling to enhance plant resilience [16]. Under drought and saline-alkali stress, PP2Cs modulate stomatal closure and ion homeostasis, while during low-temperature stress, they activate OST1 kinase to bolster cold tolerance [16]. Notably, in Arabidopsis, over 50% of PP2C genes exhibit dynamic regulation in response to ABA, salt, cold, and heat, underscoring their pivotal role in environmental adaptation [14]. Among PP2C subfamilies, subfamily A stands out for its universal stress responsiveness. For instance, all 10 OsPP2C genes in rice (Oryza sativa L.) are upregulated under ABA and salt stress, highlighting their conserved function in stress signaling [17]. This regulatory pattern extends across diverse species, including alfalfa [12], maize [18], oilseed rape [19], strawberry [20], and tomato [21], where PP2Cs are consistently induced by drought, salinity, extreme temperatures, and nutrient deficiencies, enabling tailored stress responses. Beyond abiotic stress, PP2Cs also contribute to plant immunity, modulating defense pathways against pests and pathogens. Given their multifaceted roles, deciphering the molecular mechanisms of PP2Cs in stress regulation is critical for developing stress-tolerant crops [20].

With the rapid advancement of genome sequencing technologies, the PP2C gene family, a key regulator of plant stress responses, has been systematically identified and functionally characterized in multiple plant species, including A. thaliana, O. sativa, M. truncatula, B. rapa, C. sativus, Z. mays, and T. aestivum [12,17,19,22,23,24,25]. Despite this progress, the PP2C family in oat (Avena sativa L.), an economically important crop with remarkable stress resilience, has remained largely unexplored, leaving a critical gap in our understanding of its stress-adaptation mechanisms.

In this study, we present the first comprehensive genome-wide analysis of PP2Cs in oat (Avena sativa), identifying 194 AsaPP2C genes and classifying them into 13 phylogenetically distinct subfamilies. Through integrative bioinformatics, we characterized their gene structures, chromosomal distributions, and evolutionary relationships, uncovering both conserved features and lineage-specific expansions that reflect oat’s unique adaptation history. Expression profiling under multiple abiotic stresses (salt, ABA, drought, cold, and heat) revealed the dynamic, stress-specific regulation of select AsaPP2C genes, elucidating the specialized roles of these proteins in oat stress signaling pathways, significantly enhancing the comprehension of PP2C-mediated stress responses in cereal crops. These findings establish a critical genomic framework for subsequent functional investigations. Furthermore, this study identifies promising AsaPP2C gene candidates for targeted genetic modifications, providing novel strategies to enhance oat stress resilience and improve crop productivity under shifting environmental pressures.

2. Material and Methods

2.1. Plant Material and Treatments

Oat cultivar Longyan No.3 was used in this study. Seeds were planted in a 3:1 (w/w) mixture of soil and sand, germinated, and irrigated with a half-strength Hoagland solution once every 2 days [24]. The seedlings were grown at a night temperature of 18 °C and a day temperature of 22 °C, accompanied by a photoperiod of 16 h of light and 8 h of darkness, and a light intensity of 200–230 µmol m⁻²s⁻². After 4 weeks, heat and cold temperature stress treatments consisted of exposing the seedlings to 42 °C or 4 °C, respectively. Based on previous research by others, the seedlings were exposed in 200 mM NaCl [18] or 20% (w/v) [15] polyethylene glycol (PEG) 6000 for high salt or drought treatment, respectively. Additionally, other seedlings were sprayed with 100 µM ABA [24] until the leaves were completely moist. The leaf, leaf sheath, and root from each plant were collected at 6, 12, 24, 48, and 72 h after treatment and then frozen in liquid nitrogen before storage at −80 °C. The leaf, leaf sheath, and root from each plant of untreated seedlings were similarly collected as control material (0 h). Each treatment had three biological replicates.

2.2. Genome-Wide Identification of the PP2C Genes in Oat

To identify candidate PP2C genes in oat (Avena sativa) and Arabidopsis (Arabidopsis thaliana) (Tables S1 and S2), the Hidden Markov Model (HMM) profile of the PP2C protein domain (accession: PF00481) was obtained from the InterPro database (https://www.ebi.ac.uk/interpro/ (accessed on 4 November 2024)). Using HMMERv3.1 software, the entire genome-derived protein sequences of both species were screened with a stringent E-value threshold of 0.001 to ensure high-confidence matches.

To determine the accuracy of the sequences, the protein sequences of the screened candidate genes were submitted to the PFAM (http://pfam.xfam.org/ (accessed on 6 November 2024)) and SMART (http://smart.embl.de/ (accessed on 8 November 2024)) databases for the search of conserved protein structural domains, and the sequences with incomplete PP2C protein structural domains were eliminated to ensure that the screened protein sequences had PP2C structural domains.

2.3. Phylogenetic Construction, Conserved Motifs, and Exon–Intron Structure

A multiple sequence comparison of PP2C protein sequences was carried out using MAFFT (https://mafft.cbrc.jp/alignment/software/ (accessed on 18 November 2024)), followed by the removal of non-conserved amino acid sites in the compared protein sequences, deletion of sequences that were too short or too long, and retention of PP2C protein structural domains using Trimal (https://vicfero.github.io/trimal/ (accessed on 1 February 2025)). The maximum likelihood method was selected for the construction of the phylogenetic tree algorithm, the phylogenetic tree was constructed using iqtree (https://iqtree.github.io/ (accessed on 1 February 2025)), and the model selection was made using the JTT + R model. The branching credibility was checked based on 1000 times of bootstrap, the Newick Tree file was exported, and the phylogenetic tree was beautified by using the itol online tool (https://itol.embl.de/ (accessed on 1 February 2025)) [26,27].

The exon–intron structures of AsaPP2C genes were determined by comparing the coding sequences and the corresponding genomic sequences on the GSDS website (http://gsds2.cbi.pku.edu.cn (accessed on 3 February 2025)) [28].

MEME software (Version 4.11.4) was used to identify conserved motifs in AsaPP2C protein sequences according to the following parameters: -protein, -oc, -nostatus, -mod zoops, -nmotifs 15, -minw 6, -maxw 50 [29].

2.4. Protein Features, Chromosome Location, and Cis-Acting Element Analysis of AsaPP2C Genes

The compute pI/MW tool of the ExPASy server (http://web.expasy.org/compute (accessed on 30 February 2025)) was used to calculate the molecular weight (MW) and the theoretical isoelectric point (pI) of AsaPP2C proteins. According to the starting positions on chromosomes, MapInspect (https://mapinspect.software.informer.com/ (accessed on 8 April 2025)) was used to draw the chromosomal distribution images of AsaPP2C genes. The 1500 bp sequences upstream from the initiation codon (ATG) of all AsaPP2C genes were obtained from Phytozome v12.151.

2.5. Expression Pattern Analysis of AsaPP2Cs Obtained from Transcriptome Sequencing

To explore the AsaPP2C genes related to the drought response, the transcriptional abundance of AsaPP2C genes under drought stress was studied using transcriptome data (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1056521/ (accessed on 20 February 2025)). Fragments per kb per million reads (FPKM) were used as the standard for the expression pattern, and the omicshare tool was used to generate the associated heatmap figures (https://www.omicshare.com/tools/Home/Soft/heatmap, (accessed on 1 June 2025)).

2.6. RNA Extraction and Expression of AsaPP2Cs Under Different Stresses

Total RNA was isolated from all of the samples using the total RNA extraction kit (Tiangen, Sichuan, China). The quality and quantity of RNA was evaluated via agarose gel electrophoresis and with a Quawell micro volume spectrophotometer (Q5000, San Jose, CA, USA), respectively. Then, 1 µg of total RNA, after DNase I digestion, was reverse transcribed into cDNA using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Beijing, China).

The cDNA was amplified using a LightCycler 480 SYBR Green Master, with a Roche LightCycler 480 Real Time PCR system (Roche, Basel, Switzerland). The thermal cycling program was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 15 s. The melting curves were analyzed at 60–95 °C after 40 cycles. All qRT-PCRs were carried out for three technical replicates. The primer of the GAPDH gene was chosen as the internal reference gene to normalize cDNA concentrations [30]. The primers used in this study are listed in Supplementary Table S3. The relative expression levels of AsaPP2C genes was calculated by utilizing the 2^−ΔΔCq algorithm.

2.7. Statistical Analysis

Three independent sample replicates were used for qRT-PCR analysis at each time point. All data were compiled using Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA). One-way analysis of variance (ANOVA) was conducted in IBM SPSS Statistics 22.0 (IBM Corp., Armonk, NY, USA) to assess statistical significance. Graphs and visualizations were generated using OriginPro 2021 (OriginLab Corporation, Northampton, MA, USA).

3. Results

3.1. Identification of the PP2C Gene Family in Oat

To identify the AsaPP2C genes, the genome and annotation data of A. sativa were downloaded from the InterPro database (https://www.ebi.ac.uk/interpro/ (accessed on 4 November 2024)). Using the InterPro PP2C domain “PF00481”as the keyword, 194 putative PP2C genes were found. The physicochemical properties of AsaPP2C proteins are shown in Table S4. The sizes of the AsaPP2C proteins ranged from 130 to 1086 amino acids, since only 4 AsaPP2C proteins had a protein size of more than 1000 amino acids, of which Asa_chr6Ag00568, Asa_chr6Cg02400, and Asa_chr6Dg00303 had the longest amino acid sequences. In addition, the molecular weights of AsaPP2Cs ranged from 14.33 kDa (Asa_chr2Ag02741) to 120.95 kDa (Asa_chr4Cg02194), and the average molecular weight was 44.018 KDa. The predicted isoelectric point (pI) ranged from 4.21 (Asa_chr1Dg03162) to 11.5 (Asa_chr4Cg03743), with an average pI of 6 (Table S4).

3.2. Chromosomal Location of AsaPP2C Genes

Based on physical locations on A. sativa chromosomes, the 194 AsaPP2C genes were displayed using MapInspect (https://mapinspect.software.informer.com/ (accessed on 29 April 2025)). One hundred and ninety-four AsaPP2C genes are distributed across all 21 chromosomes, ranging from 1 to 15 per chromosome (Figure S1). The number of AsaPP2Cs located on each chromosome varies dramatically; chromosomes 1A and 4C contain the largest number of AsaPP2C family members with 15 genes, whereas the least number was detected on chromosome 22, containing only one AsaPP2C gene. Furthermore, one AsaPP2C (Asa_ChrUng00420) is located on an unassembled genomic scaffold and thus cannot be mapped to any particular chromosome according to what we currently know about this genome. These results showed that the AsaPP2C genes are unevenly distributed on different chromosomes and that each subfamily gene is also unevenly distributed.

3.3. Phylogenetic Analysis

To evaluate the evolutionary relationships of 194 PP2C proteins in A. sativa, a phylogenetic analysis was conducted (Figure 1), which revealed the evolutionary relationships between PP2C genes from A. sativa and A. thaliana, classifying them into 13 distinct subfamilies (A–L). Among them, the E subfamily had the most members, containing 28 genes and accounting for 14.3%; the J subfamily had the fewest members, containing only 1 gene. Notably, several PP2C subfamilies (A, H, D, G, and F2) contain more AsaPP2Cs than AthPP2Cs; for example, subfamily A contains 23 AsaPP2Cs but only 11 AthPP2Cs; this 2.1-fold expansion (p < 0.01) may enhance ABA signaling plasticity in oat, consistent with its drought adaptation (Figure 1). Subfamily H and G contain 20 AsaPP2Cs but only 4 AthPP2Cs; subfamily D contains 22 AsaPP2Cs but only 9 AthPP2Cs; subfamily F2 contains 19 AsaPP2Cs but merely 4 AthPP2Cs, the 4.75-fold expansion suggests purifying selection maintaining duplicates for grass-specific roles. In contrast, B subfamilies contain more AthPP2Cs than AsaPP2Cs. Additionally, subfamilies F1 and F2 exhibit oat-specific expansions, potentially linked to adaptive traits in this hexaploid crop. The presence of AsaPP2Cs in all subfamilies, including those with unknown functions (subfamily K), highlights the genetic diversification of this family in oats. This suggests that the PP2C gene family may have evolved from a common ancestor.

3.4. Gene Structure and Motif Analysis of AsaPP2C Genes

To further understand the relationship between gene structure and evolution among the AsaPP2C genes, the exon–intron distribution and conserved motifs were analyzed according to their full-length phylogenetic relationship. The number of introns in oats ranges from 0 to 19 (Figure S2A), of which Asa_chr4Cg02194 contains 19 introns and has the highest number of detected introns. Moreover, AsaPP2C gene family members have exon structures, with 6 members (Asa_chr5Dg02941, Asa_chr4Cg00365, Asa_chr6Ag02847, Asa_chr4Cg03743, Asa_chr5Ag00079, and Asa_chr5Ag02223) without intron structures, mainly concentrating in the A, B, and D subfamilies, and the I subfamily has more introns than the remaining subfamily, with a range of 8–13, suggesting that subfamily I may retain some of its evolutionary conservation. Although most members of the F1 and F2 subfamily contain introns, these introns are very short. Conversely, the H, L, and E subfamily contains the long introns. In terms of intron–exon conservation, it has the characteristics of subfamily distribution. For example, the number of similar intron–exon structures in subfamily A, C, E, G, F1, F2, and K is greater than that of subfamily L and H. The AsaPP2C gene family gene structure map (Figure S2B) shows that 10 motifs were identified in AsaPP2C genes, and motif 2 and 4 appeared in every subfamily. Within the same group, AsaPP2C members usually have similar motif compositions that ranged from 1-5 to 8-10. Except for C and D subfamilies, almost all proteins contain motifs 6 and 7 that define PP2C domains structures, respectively. However, some motifs belong to a special group, such as motif 2, 4, 1, and 8, which are only found in K subfamilies. Notably, there are similarities among subgroups such as the group subfamily H, L, E, B, and C, D and F1, and F2. These results indicate that proteins with the same or similar structures may be functionally or evolutionarily similar and demonstrate the reliability of the classification.

3.5. Cis-Element Analysis of the AsaPP2C Promoter in Oat

Abundant responsive regulatory elements were found in the promoter regions of AsaPP2Cs through PlantCARE analysis (Figure S3). The cis-elements screened were divided into four categories. The first type of element was the stress response, such as MYB, MYC, ARE, and MBS. The second type of element was the hormone response, such as ABRE (ABA response element) and the CGTCA-motif and TGACG-motif (MeJA-responsiveness response element), among others. The ABA-responsive (ABRE) elements were identified abundantly in the promoter regions of AsaPP2Cs, among which Asa_chr5Dg00515, Asa_chr2Cg01991, and Asa_chr3Ag01235 contained 16, 19, and 17 ABREs, which was the largest number, followed by Asa_chr6Cg03142 and Asa_chr6Dg00834; it was the most abundant element in the promoter region. The third type of element was the plant growth and development response, such as A-box and Box-4, among others. The fourth type of element was the light-responsive response, such as the G-box, among others. This suggests that most AsaPP2C genes may respond to various abiotic stresses and hormones.

3.6. Analysis of AsaPP2C Gene Expression Patterns Under Drought Stress

In order to gain insight into the potential function of AsaPP2C in the response to stress, we analyzed the expression patterns of AsaPP2C using transcriptomic data (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1056521/ (accessed on 20 February 2025)) obtained from Avena sativa subjected to these conditions (Figure 2; Table S5). AsaPP2C genes in subfamily L was not detected in any sample, whereas other subfamilies showed significant differences in expression patterns in response to the drought stress treatments. AsaPP2C genes exhibited differential gene expression patterns at different time points of PEG treatment. For example, Asa_chr3ag01998 was only upregulated at 24 h and 72 h of the drought stress treatment, while Asa_chr5Ag00079 and Asa_chr6Dg00217 showed a downward trend at 12 h after drought treatment. In B subfamilies, Asa_chr4Cg03270 was upregulated in the late stage (72 h) of drought treatment. However, in the E subfamily, Asa_chr6Cg02197 was significantly upregulated in the early stage (6 h) of drought treatment, followed by a decrease in expression levels in the later stages (72 h) of drought treatment. Additionally, in G and I subfamilies, Asa_chr6Ag01950 and Asa_chr7Dg02992 genes were significantly upregulated at all five time points of drought treatment. Therefore, these genes are subject to further validation and analysis.

3.7. Expression Profile Analysis of AsaPP2C Genes Under Abiotic Stresses and ABA Treatments in Leaves

A further analysis revealed that AsaPP2C gene expression levels varied under different abiotic stress treatments in leaf tissue, under cold treatment at 24 h; regarding Asa_chr6Dg00217, its expression level decreased by 1.5-fold (p < 0.01) compared to 0 h, while it was significantly increased under salt, ABA, drought, and heat treatments (Figure 3A). In the case of Asa_chr6Ag01950, it was significantly increased under salt, ABA, drought, cold, and heat treatments. Under ABA stress at 6 and 12 h, gene Asa_chr6Ag01950 expression increased by 22-fold and 24-fold (p < 0.01) compared to 0 h (Figure 3B). Asa_chr3Ag01998 was increased under salt, ABA, drought, cold, and heat stress, but it was decreased by 1.66-fold and 11.11-fold (p < 0.01) compared to 0 h under drought treatment at 6 h and 12 h, suggesting that Asa_chr3Ag01998 exhibited stress-specific suppression, and there might be negative feedback regulation (Figure 3C). Meanwhile, Asa_chr5Ag00079 remained almost unchanged after cold and heat treatment, while its expression was increased under other treatments (Figure 3D). Asa_chr4Cg03270 was notably increased by 132-fold, 118-fold, and 124-fold (p < 0.01) compared to 0 h under salt treatment, reaching its highest expression levels at 6 h, 12 h, and 24 h, respectively. Meanwhile, under drought stress at all time points, gene Asa_chr4Cg03270 expression increased by 62.2-, 85-, 80-, 75.6-, and 76.1-fold (p < 0.01) compared to 0 h (Figure 3E). Asa_chr6Cg02197 and Asa_chr7Dg02992 were increased under all treatments, with Asa_chr6Cg02197 reaching its highest expression level in response to ABA at 6 h and cold at 24 h, while Asa_chr7Dg02992 reached its highest expression level with drought at 24 h and heat at 12 h (Figure 3F,G).

3.8. Expression Profile Analysis of AsaPP2C Genes Under Abiotic Stresses and ABA Treatments in Leaf Sheath

To verify the function of AsaPP2C in the leaf sheath tissue, we analyzed the expression patterns of seven selected AsaPP2C genes (Asa_chr6Dg00217, Asa_chr6Ag01950, Asa_chr3Ag01998, Asa_chr5Ag00079, Asa_chr4Cg03270, Asa_chr6Cg02197, and Asa_chr7Dg02992) under salt, ABA, drought, cold, and heat stresses at different time points (Figure 4). Asa_chr6Dg00217 was increased by 23-fold (p < 0.01) at 24 h under salt treatments compared with 0 h; meanwhile, under ABA, drought, and heat treatments, gene Asa_chr6Dg00217 expression was increased (Figure 4A). The expression level of Asa_chr6Ag01950 significantly increased by 10- and 13.4-fold at 12 h under ABA and cold treatments, but it was decreased under salt and heat treatment at 12 and 72 h compared with 0 h (Figure 4B). Asa_chr6Dg01998 expression increased by 12- and 29.8-fold at 72 h under salt and drought treatments (Figure 4C). Regarding Asa_chr5Ag00079 and Asa_chr6Cg02197, their expression level was decreased under ABA, cold, and heat treatment at 12, 24, 48, and 72 h, while it was significantly increased under salt and drought treatments at 24, 48, and 72 h (Figure 4D,F). Asa_chr4Cg03270 expression increased by 13.8- and 24.5-fold at 6 and 12 h under ABA treatment, while it was decreased under salt and cold treatment at 6 and 12 h, and its expression level remain almost unchanged by heat treatment at 6, 12, 24, and 72 h compared with 0 h (Figure 4E). Under salt stress at 24, 48, and 72 h, gene Asa_chr7Dg02992 expression increased by 13-, 16.1-, and 17-fold (p < 0.01) compared to 0 h; meanwhile, its expression increased by 12-, 24.09-, and 5.21-fold (p < 0.01) compared to 0 h under drought at 12, 24, and 48 h (Figure 4G).

3.9. Expression Profile of AsaPP2C Genes Under Abiotic Stresses and ABA Treatments in Roots

In root tissues under salt stress, all examined genes exhibited increasing trends compared to 0 h, except for Asa_chr5Ag00079, which showed downregulation. The expression levels of Asa_chr6Dg00217, Asa_chr6Ag01950, Asa_chr3Ag01998, Asa_chr6Cg02197, and Asa_chr7Dg02992 were all upregulated under salt, ABA, drought, cold, and heat at all time points. Additionally, Asa_chr5Ag00079 was significantly upregulated under ABA, drought, and heat treatments, but it was downregulated under salt and cold treatments at 6, 12, 24, and 72 h. Meanwhile, Asa_chr4Cg03270 expression remained stable under cold and heat treatments between 6 and 24 h, while showing significant upregulation under other stress conditions at 6, 12, 24, and 48 h. In summary, most examined genes (Asa_chr6Dg00217, Asa_chr6Ag01950, Asa_chr3Ag01998, Asa_chr6Cg02197, Asa_chr7Dg02992) exhibited upregulation under salt stress at all time points, indicating a consistent positive regulatory role in salt adaptation. Asa_chr5Ag00079 showed downregulation under salt stress, suggesting a unique regulatory mechanism distinct from other PP2Cs. Meanwhile, Asa_chr6Dg00217, Asa_chr6Ag01950, Asa_chr3Ag01998, Asa_chr6Cg02197, and Asa_chr7Dg02992 were universally upregulated under all treatment stresses (salt, ABA, drought, cold, heat), highlighting their roles as central hubs in stress signaling networks (Figure 5).

4. Discussion

With the rapid advancements in plant genome sequencing, the PP2C gene family has been systematically characterized across diverse plant species, revealing its critical role as a key regulator of signal transduction, growth, and stress responses [31,32]. Comparative genomic analyses reveal significant variation in the PP2C family size across species: A. thaliana (80 members), O. sativa (90), M. truncatula (94), B. rapa (131), C. sativus (56), Z. mays (104), and T. aestivum (95) [12,17,19,22,23,24,25]. In this study, we identified 194 PP2C genes in A. sativa, the largest repertoire reported to date. This expansion likely reflects oat’s hexaploid genome (2n = 6x = 42) and the prevalence of genome-wide duplications, which have driven the diversification of stress-adaptive pathways in this crop [33]. The pronounced size of the AsaPP2C gene family underscores its evolutionary and functional significance, positioning oat as an exemplary model system for elucidating PP2C-mediated regulatory mechanisms in polyploid species.

Chromosomal mapping revealed that the AsaPP2C genes are unevenly distributed across the oat genome, with notable enrichment on Chr1A (15 genes), Chr1D (13), Chr4C (15), and Chr6C (12) (Figure S1), suggesting potential hotspots for gene duplication and functional diversification. Protein analysis further demonstrated that members within the same subfamily exhibit conserved molecular characteristics, including similar motif compositions, intron numbers, molecular weights (MWs), and isoelectric points (pIs), except for subfamily H, which displayed greater variability. Most subfamilies (e.g., A, B, D) showed a concentrated MW range (30–60 kDa) and broad pI distribution (5–11), while others exhibited distinct profiles: subfamilies F1/F2 had lower MWs (14.3–53.3 kDa), subfamily C displayed narrow MW (45.5–69.7 kDa) and pI (4.75–6.53) ranges, and subfamily L spanned a wide MW spectrum (37.4–120.7 kDa) (Table S4). These conserved subfamily-specific features imply shared functional roles among closely related members, likely driven by evolutionary selection for stress adaptation.

The PP2C gene family exhibits high evolutionary conservation from prokaryotes to eukaryotes, with the gene number and diversity increasing during speciation [34]. The phylogenetic analysis classified oat and Arabidopsis PP2Cs into 13 subfamilies (Figure 1), consistent with the patterns observed in B. rapa, M. truncatula, T. aestivum, and C. sativus [17,19,24,25]. While most subfamilies contained members from both species, oat and Arabidopsis genes generally clustered separately, suggesting lineage-specific diversification. The gene structure analysis revealed that AsaPP2Cs within the same subfamily share similar exon/intron architectures, with exceptions likely arising from evolutionary events like intron loss [35]. Notably, six intronless genes (Asa_chr5Dg02941, Asa_chr4Cg00365, Asa_chr6Ag02847, Asa_chr4Cg03743, Asa_chr5Ag00079, and Asa_chr5Ag02223) were identified, predominantly in subfamilies A, B, and D (Figure S2), potentially accelerating gene duplication and expansion [36]. The absence of introns in genes may accelerate evolution through gene duplication [37], which might explain the larger number of PP2C genes in oat compared to rice and Arabidopsis. In contrast, subfamily I exhibited higher intron numbers (8–13), possibly reflecting functional constraints. The conserved motif analysis identified 10 signature motifs, with motif 2 and 4 universally present across all AsaPP2Cs, likely constituting the catalytic core [38,39]. The subfamily-specific motif distributions provide additional evidence for intra-group functional conservation, establishing a conceptual framework for elucidating the underlying molecular mechanisms in future studies.

In plants, PP2C genes have been identified in numerous tissues [19,24,25]. Studies on cucumber [24], cotton [32], and maize [40] demonstrate that PP2Cs are differentially expressed in various organs, with some showing ubiquitous expression while others display tissue preferences. Similarly, in oat, we observed distinct spatiotemporal expression patterns, where Asa_chr6Dg00217, Asa_chr5Ag00079, and Asa_chr4Cg03270 showed leaf-predominant expression, while exhibiting moderate expression in the roots and stems (Figure 3, Figure 4 and Figure 5). These findings suggest that PP2Cs have evolved specialized roles in different tissues, potentially regulating critical processes like photosynthesis in leaves and root system development. The conserved yet diversified expression patterns across species highlight the evolutionary importance of PP2Cs in plant growth and adaptation.

Cis-acting elements play crucial roles in regulating plant development and stress responses [41]. Our analysis revealed that ABRE and MYC elements are most abundant in AsaPP2C promoter regions (Figure S3), consistent with their established roles in stress adaptation [42]. Notably, subfamilies A and B, which are plant-specific, showed distinct stress-response patterns: subfamily A members mediated ABA-dependent signaling, while subfamily B regulated MAPK activities [12]. Expression profiling demonstrated that subfamilies A, B, D, E, F2, and G were particularly responsive to abiotic stresses (Figure 4), mirroring findings in Arabidopsis and rice, where subfamily A PP2Cs are crucial for ABA signaling [42,43]. Specifically, Asa_chr3Ag01998 and Asa_chr4Cg03270 (subfamilies A/B) were strongly upregulated under salt and drought stresses, with subfamily A genes showing ABA-inducible expression, confirming their role in ABA-dependent pathways. These results align with observations in M. truncatula, where subfamily A PP2Cs were similarly stress-responsive [44], highlighting conserved regulatory mechanisms across species.

The PP2C gene family plays pivotal roles in phytohormone signaling, with particular importance in ABA-mediated stress responses across plant species [13,31]. Core ABA signaling components like ABI1, ABI2, AHG1, and PP2CA have been well-characterized [45,46], while HAI-type PP2Cs demonstrate unique drought resistance through both ABA-dependent and ABA-independent pathways [47]. For instance, alfalfa homologs MtPP2C8/37/67/73 show differential stress-induction patterns, with MtPP2C8 responding to both drought and cold [48]. PP2Cs exhibit tissue-specific regulation in ABA-mediated stress responses, reflecting their specialized functional adaptation. In roots, ABA rapidly induces PP2C expression (e.g., MtPP2C8 and HAI homologs) to maintain hydraulic conductivity and ion homeostasis under osmotic stress [47]. In contrast, leaf tissues show transient PP2C suppression (e.g., ABI1/ABI2) to sustain SnRK2 kinase activity, thereby promoting stomatal closure and reducing water loss [49]. Our study revealed distinct tissue-specific expression patterns among AsaPP2C genes under ABA treatment. Asa_chr6Ag01950 showed strong induction in leaves and leaf sheaths, suggesting its role in aerial stress adaptation through stomatal regulation and ABA signal amplification. Conversely, Asa_chr5Ag00079 and Asa_chr4Cg03270 were predominantly upregulated in roots, indicating their involvement in root-specific ABA responses, potentially modulating hydraulic conductivity, stress signaling, and soil exploration during drought (Figure 5). These results highlight the functional diversification of PP2Cs in mediating stress-responsive signaling pathways, thereby facilitating whole-plant adaptive strategies to environmental perturbations.

As vital regulators of plant growth and development, the PP2C gene family has been reported to respond to adverse environments in numerous species [50]. Under drought conditions, PP2Cs exhibit tissue-specific responses: ABI1 is transiently suppressed in guard cells to enable SnRK2-mediated stomatal closure, while HAI homologs are upregulated in roots to maintain growth under water deficits [24]. Similar stress-specific regulation occurs in other species: MtPP2C92 shows drought induction but cold repression [12], while OsPP2C53 mediates salt tolerance through SOS-independent ion homeostasis [43]. Temperature fluctuations further diversify responses: cold stress induces AtPP2C6 to maintain membrane stability, while heat represses TaPP2C62 to prevent signal over-attenuation [25]. Notably, individual PP2C isoforms show unique stress-response profiles: AP2C1 responds strongly to chilling, drought, and wounding, whereas AP2C2 shows minimal induction under identical conditions [50]. This functional divergence extends to cucumber, where specific CsPP2C members (CsPP2C3, 11, 17, 23, 45, 54, 55) respond significantly to multiple stresses including ABA, drought, salt, and cold [24]. In this study, seven of the AsaPP2C genes exhibited substantial transcriptional variations followed by drought, salt, cold, and heat stress treatments, suggesting their regulatory role in abiotic stress tolerance. However, whether or not each gene plays a critical role in abiotic stress tolerance still needs the functional characterization of an individual gene.

5. Conclusions

Based on the importance of the PP2C family in plants and the strong adaptability of A. sativa to stressed environments, the genome-wide identification and analysis of the PP2C gene family in A. sativa were performed for the first time in this study (Figure 6). The search for PP2C family members in the A. sativa genome led to the identification of 194 putative AsaPP2C family members. The AsaPP2C family was clustered into 13 subfamilies with AtPP2Cs, according to phylogenetics and their motif patterns. In addition, we found that the 194 AsaPP2C genes were unevenly distributed across the 22 chromosomes of A. sativa. Expression analysis with transcriptomic data and qRT-PCR showed that the candidate AsaPP2C family displays a wide range of sensitivities to abiotic stress and hormones. Several AsaPP2C were involved in the plant responses to both saline and ABA stresses to varying degrees, indicating that these AsaPP2Cs may have regulatory functions in tolerance to saline and ABA stress and deserve further investigation. Our results provide a foundation for further researching PP2C gene functions in A. sativa. Future researchers should establish a CRISPR-based genome editing technique to fast-track the development of stress-proof oat cultivars, merging genome-wide PP2C characterization with precision gene-editing blueprints for abiotic stress resilience.