Naked oat Nei Avena 6 (NY6) and hulled oat Qing Yin 1 (QY1), provided by the Han Bing Oat Breeding Team at Inner Mongolia Agricultural University, were chosen as the primary experimental materials for this study. Uniformly shaped and sized oat seeds were surface-sterilized by immersing them in 2% NaClO for 5 min, rinsed with sterile water, and placed on filter paper soaked in sterile water. The seeds were then dark-incubated at 16°C in a temperature-controlled incubator until 5 cm shoots and primary roots developed. The seedlings were subsequently transferred to a 96-well hydroponic incubator containing Hoagland’s culture medium and grown for 20 days under day/night temperatures of 22/16°C and a photoperiod of 16/8 h. On the 21st day, seedlings were treated with 20% polyethylene glycol (PEG) 6,000 and 100 mM NaCl, while Hoagland’s solution served as the control. Samples from the treatment groups and control group were collected at 0, 2, 4, 8, 12, and 24 h. After sampling, the tissues were rapidly frozen in liquid nitrogen and stored at −80°C. Each sample included three independent biological replicates and technical replicates for qRT-PCR analysis.

Genomic data of three oat varieties “SFS”, “Sang” and “OT3098v2” used to identify the oat TCP gene family were obtained from the OatBioDB Biology database (http://waoOat.cn/). Among them, “Sang” and “OT3098v2” are skin oats and “SFS” is a naked oat. Genome files and genome annotation files for rice, maize, wheat, A. thaliana, Brachypodium distachyon, A. tauschii, were obtained from the Ensembl Plants database (http://plants.ensembl.org/index.html). The HMM file for the TCP structural domain (PF03634) was derived from the Pfam database (https://pfam.xfam.org), and generated by training and constructing a large number of sequences known to be in this gene family. Using the built-in algorithm of HMMER 3.0 software, the input gene sequences were compared with the HMM model to generate the Score value for match strength and the E-value for statistical significance. According to the screening criteria, genes with E-value below 0.01 were initially labelled as candidate genes (Finn et al., 2011). To improve the identification accuracy, all genes identified by HMMER 3.0 (including a few genes with E-value greater than 0.01) were submitted to the NCBI CDD database (https://www.ncbi.nlm.nih.gov/cdd/) for structural domain validation (Lu et al., 2020). Eventually, genes with TCP structural domains were confirmed as members of the TCP gene family. TCP genes from the “SFS” were designated AsTCP001 to AsTCP083, while genes from the “Sang” and “OT3098v2” retained their respective genomic gene IDs.

DNAman was used to compare selected TCP amino acid sequences and construct a phylogenetic tree for oat TCPs, along with wheat, rice, and A. thaliana using MEGA11, with 1,000 bootstrap replicates. The phylogenetic tree was further refined using the Evolview online tool (https://www.evolgenius.info/evolview/#/treeview) to enhance visualization and clarity (He et al., 2016).

Conserved motifs in the oat TCP gene family were predicted using the MEME suite (https://meme-suite.org/meme/), with the number of predicted motifs set to 10 (Bailey et al., 2009). TCP protein structural domains were identified via the NCBI Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/cdd/). Gene structure analysis was conducted using the online tool GSDS 2.0 (http://gsds.cbi.pku.edu.cn/). Visualization of motifs, domains, and gene structures was carried out using TBtools (v2.042) software (Chen et al., 2020). Subcellular localization was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/) and the Molecular Bioinformatics Center (MBC) website (http://cello.life.nctu.edu.tw/) (Horton et al., 2007). Oat TCP family amino acid sequences were analyzed for molecular weight and isoelectric point (pI) using the Expasy website (https://web.expasy.org/compute_pi/) (Artimo et al., 2012). Cis-acting elements within 2 kb upstream of the start codon of oat TCP genes were analyzed using the PlantCARE online tool (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot et al., 2002).

Chromosomal localization of oat TCP genes was performed using TBtools software (Chen et al., 2020). Nonsynonymous (Ka) and synonymous (Ks) substitution rates were calculated for oat TCP genes using TBtools, and the Ka/Ks ratio was computed to assess evolutionary pressures influencing gene trends (>1 indicates positive selection, = 1 neutral selection, <1 purifying selection) (Chen et al., 2020). Duplication events of AsTCP genes were analyzed using the Multicollinearity Scanning Toolkit (MCScanX) with default parameters (Wang et al., 2012). The Dual Synteny Plotter tool within TBtools was employed to visualize synteny relationships of oat TCP genes with those from rice, maize, wheat, two-spike phragmites, and knapweed genomes (Chen et al., 2020). STRING (https://cn.string-db.org/) was employed to predict interacting proteins using Arabidopsis as the reference species. Additionally, psRNATarget (https://www.zhaolab.org/psRNATarget/analysis) was used to predict the miRNAs targeting AsTCP proteins (Dai et al., 2018). All results were visualized using Cytoscape 3.10.0 software.

Transcriptome data related to silicon-mediated drought stress alleviation and salt stress in oats were sourced from the public NCBI (https://www.ncbi.nlm.nih.gov/sra/?term=Oat) (data number SRP237902, SRP093940) (Bray et al., 2016). FPKM values (log2 transformed) were used to analyze the expression of TCP family genes under the 2 treatments, and heatmaps were drawn using the Heatmap program of TBtools (Chen et al., 2020).

Total RNA extraction from plants was performed using the Transzol Up Plus kit from Beijing All Style Gold. The first strand of cDNA was synthesized via reverse transcription, following the instructions provided with the PrimeScript RT kit from Takara. Quantitative PCR primers for AsTCP021, AsTCP025, AsTCP033, AsTCP044, AsTCP053, and AsTCP058 were designed using Primer 5.0 software (Supplementary Table S1) and synthesized by Beijing Liuhe Huada Gene Science and Technology Co. Oat β-Actin was used as the internal reference gene (Zhang, 2023). The expression level of each AsTCP gene was quantified using the 2^-(ΔΔCt) method. The PCR reaction conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 20 s, and extension at 72°C for 20 s.

Using the genomic data of oat cultivars “SFS”, “Sang”, and “OT3098v2”, we identified 83, 65, and 30 members of the AsTCP gene family, respectively (Supplementary Tables S2, S3). In “SFS” and “Sang”, the amino acid sequences of 34 AsTCPs showed 100% identity, and those of 13 AsTCPs had over 90% identity. In “SFS” and “OT3098v2”, the amino acid sequences of 16 AsTCPs exhibited 100% identity, and those of 5 AsTCPs had over 90% identity. In “Sang” and “OT3098v2”, the amino acid sequences of 16 AsTCPs presented 100% identity, and those of 6 AsTCPs had over 90% identity. The “OT3098v2” contained three unique AsTCP genes (AVESA.00001b.r1.3Cg0000521.1, AVESA.00001b.r1.5Ag0002232.1, AVESA.00001b.r1.2Dg0002764.1). The “Sang” had four unique AsTCP genes (AVESA.00010b.r2.2DG0346510.1, AVESA.00010b.r2.2DG0402990.1, AVESA.00010b.r2.6DG1166000.1, and AVESA.00010b.r2.7AG1210110.1). The “SFS” had 22 unique AsTCP genes (AsTCP020 to AsTCP022, AsTCP033, AsTCP035, AsTCP040, AsTCP045, AsTCP056, AsTCP064, AsTCP065, AsTCP068, AsTCP070, AsTCP072, AsTCP075 to AsTCP083). Analysis of the amino acid sequence lengths of AsTCP genes in the “SFS” showed substantial variation, ranging from 71 to 609 amino acids (aa). The shortest protein, AsTCP081, comprised 71 aa, whereas the longest protein, AsTCP037, comprised 609 aa. The molecular weights of the 83 AsTCP proteins varied from 7954.97 to 64420.89 Da, and their isoelectric points (pI) ranged from 4.3 to 10.78 (Supplementary Table S4). Subcellular localization analysis indicated that 72 (86.75%) of the AsTCP proteins were localized in the nucleus. The remaining proteins were distributed as follows: five in the extracellular region, two at the plasma membrane, two in the cytoplasm, one in the chloroplast, and one in the mitochondrion (Supplementary Table S4).

Phylogenetic analysis of TCP proteins from oat, A. thaliana, rice, and wheat revealed that the 83 AsTCP proteins encoded by the “SFS” could be classified into three subfamilies: class I PCF, class II CYC/TB1, and CIN. Specifically, 40 proteins were categorized under the PCF subfamily, 13 under CYC/TB1, and 30 under CIN (Supplementary Figure S1A). In the “Sang”, 37 AsTCP proteins were classified as PCF, 10 as CYC/TB1, and 18 as CIN (Supplementary Figure S1B). Similarly, the “OT3098v2” showed 16 proteins in the PCF subfamily, 3 in CYC/TB1, and 11 in CIN (Supplementary Figure S1C). In the evolutionary relationship of TCP genes among oat, wheat, rice and Arabidopsis, the TCP genes in oat have a closer phylogenetic relationship with the TCP genes in wheat. A total of 14 AsTCP genes were found in the hulled and naked oat varieties “SFS”, “Sang” and “OT3098v2”, and the amino acid sequences encoded by them showed complete consistency (Supplementary Tables S2, S3). Within the oat genome, there are also different evolutionary relationships among members of the TCP gene family. Most oat TCP genes, such as AsTCP024/AsTCP023/AsTCP025, which are homologous genes distributed in subgroups A/C/D, are the closest in evolution, and these three genes are all distributed in the three oat genomes. A small number of oat TCP genes, such as AsTCP015/AsTCP017/AsTCP018 show a closer relationship to AsTCP021/AsTCP020/AsTCP022, AsTCP001/AsTCP003/AsTCP002 with AsTCP009/AsTCP008/AsTCP010, AsTCP043/AsTCP035 with AsTCP049/AsTCP048/AsTCP050, and AsTCP013/AsTCP012 with AsTCP014/AsTCP005. These types of partially homologous gene families have similar evolutionary relationships (Supplementary Figure S1; Supplementary Table S2).

Analysis of amino acid sequences revealed that 65, 59, and 27 AsTCP proteins in the “SFS”, “Sang”, and “OT3098v2”, respectively, possessed a complete helix-loop-helix (bHLH) structure, indicating a high conservation of the TCP structural domain in oats. Notably, the basic region within the bHLH of CYC/TB1 and CIN subfamilies contained a bidirectional nuclear localization signal (NLS), crucial for protein translocation to the nucleus. In contrast, the PCF subfamily exhibited a partial NLS in its basic region (Supplementary Figure S2A). These sequence differences likely contribute to the observed conservation pattern within the PCF subfamily, and suggest functional divergence between the subfamilies. Furthermore, variations were observed in the basic region of the bHLH domain. The CYC/TB1 and CIN subfamilies have four additional amino acids compared to the PCF subfamily. Specific AsTCP proteins, such as AsTCP69 and AsTCP71 in both “SFS” and “Sang”, exhibited amino acid deletions in the basic region (Supplementary Figures S2A, B). Additionally, 16, 3, and 3 AsTCP proteins in “SFS”, “Sang”, and “OT3098v2”, respectively, showed deletions spanning the entire bHLH domain (Supplementary Figure S3).

The phylogenetic tree results show that the 83 AsTCP genes are divided into three subfamilies: PCF, CIN, and CYC/TB1 (Figure 1A). Conservative base sequencing analysis identified 10 motifs present in the 83 AsTCP proteins (Supplementary Table S5). Several TCP genes exhibited deletions within the bHLH structural domain: AsTCP051, AsTCP067, AsTCP072, AsTCP077, AsTCP080, AsTCP078, and AsTCP081 lacked Motif 1 and Motif 2. While AsTCP070, AsTCP073, and AsTCP074 were missing Motif 1 (Figure 1B). Similarly, the AsTCP gene of “Sang” (AVESA.00010b.r2.5CG0872470.1, AVESA.00010b.r2.6CG1108510.1) and the AsTCP gene of “OT3098v2” (AVESA.001b.r1.2Dg0002764.1, AVESA.001b.r1.5Ag0002232.1,AVESA.00001b.r1.3Cg0000521.1) is also in a similar situation (Supplementary Figures S4, S5). Genes within the same subfamily exhibited similar motif compositions, indicating conserved motif types and distributions among closely related genes. Motif 4 is specific to the Class II (CYC/TB1 and CIN subfamilies), Motif 8 is specific to the CIN subfamily, and Motif 9 is specific to the PCF subfamily. In addition to the TCP structural domain, AsTCP061, AsTCP073, and AsTCP074 also contained two additional structural domains, flgK and HAD (Figure 1C). Gene structure analysis revealed that among the 18 AsTCP genes analyzed, intron numbers ranged from 1 to 4, with 78% of genes being intronless. Notably, the CIN subfamily exhibited the highest intron count among AsTCP genes (Figure 1D). Intronless AsTCP genes accounted for 65% and 40% of the total in the “Sang” and “OT3098v2”, respectively, significantly influencing the untranslated region (UTR) annotations of AsTCP genes (Supplementary Figures S4, S5). Additionally, differences in intron lengths within genes of the same subfamily contributed to significant variations in gene lengths.

A total of 4 types of elements, including 51 different cis-acting elements, were discovered in the promoter region of the AsTCP gene in “SFS” (Supplementary Table S6). 19 hormone response elements were widely distributed in the promoter regions of the AsTCPs (Figure 2), with the highest number of ABREs attributed to abscisic acid response elements, accounting for about 21% of the total number of hormone elements. In addition, the promoter regions harbored 14 types of stress-responsive elements, including those responsive to drought, endosperm-specific expression, low-temperature, cell cycle regulation, meristem expression, maize protein metabolism regulation, and defense stress responses (Figure 2). MYC and MYB elements, crucial for environmental adaptation, were particularly abundant, accounting for approximately 23.7% and 23.2% of total stress-responsive elements, respectively. 13 types of light-responsive elements were also identified, with G-box elements (Figure 2), which can bind MYC proteins, being the most prevalent at about 34.2% of all light-responsive elements. Furthermore, 5 types of physiological response elements were found, widely distributed across AsTCP gene promoters (Figure 2). The CCGTCC motif was the most abundant, comprising approximately 30.6% of all physiological response elements. The types of elements in the promoter regions of the AsTCP gene in “SFS”, “Sang” and “OT3098v2” are similar (Supplementary Figures S6– S8).

In the “SFS”, 21 AsTCP genes were localized on chromosome A, 29 on chromosome C, 31 on chromosome D, and 2 on Un (Figure 3). In the “Sang”, 22 AsTCP genes were localized on chromosome A, 16 on chromosome C, 23 on chromosome D, and 4 on Un (Supplementary Figure S9A). For the “OT3098v2”, 12 AsTCP genes were localized on chromosome A, 5 on chromosome C, 11 on chromosome D, and 2 on Un (Supplementary Figure S9B). Generally, the distribution of AsTCP genes across the A, C, and D subgenomes was relatively even, with the highest number of AsTCP genes found on subgenomes 4D and 5D, each containing 9 genes. Notably, only one gene was found on subgenome 6D, and none were present on 1C.

Gene duplication event analysis revealed 89, 73, and 20 homologous pairs of AsTCP gene family members in the “SFS”, “Sang”, and “OT3098v2”, respectively. Among these, two pairs (AsTCP058/AsTCP059 and AsTCP023/AsTCP025) were identified as tandem duplications across all three genomes, while the remaining 87, 71, and 18 pairs were segmental duplications (Figure 4; Supplementary Figure S10). Notably, 89 homologous gene pairs in the “SFS” exhibited Ka/Ks < 1, indicating that these genes were primarily subject to purifying selection, with no pairs evolving under strong positive selection (Ka/Ks > 1) post-duplication (Supplementary Table S7). These findings suggest that oat TCP genes underwent both segmental and tandem duplications, with segmental duplication being the predominant mode, and two pairs of tandem duplications remaining stable across the three oat genomes.

Comparative collinearity analysis between oat (“SFS”) and other species, including A. thaliana, rice, Brachypodium, Setaria, maize, and wheat, revealed the highest TCP gene homology with wheat, comprising 175 collinear pairs between 54 AsTCPs and 54 TaTCPs. Conversely, the lowest homology was observed with A. thaliana, where 14 AsTCPs exhibited collinearity with 4 AtTCPs, resulting in 15 collinear pairs (Figure 5; Supplementary Table S8). 42 AsTCP genes in the oat genome showed collinearity with TCP genes in all other species, excluding Arabidopsis. 9 out of the 12 AsTCP genes collinear with Arabidopsis were also part of these 42 genes (Supplementary Table S8). These duplicated AsTCP genes, exhibiting collinearity with other species, likely participated more frequently in gene duplication events, playing significant roles in the evolutionary trajectory of oats.

The predicted protein interaction relationships of AsTCP genes as well as miRNA realisation results were visualised by Cytoscape software. The results showed that 35 AsTCP proteins were predicted to have interaction relationships with other proteins, among which, 8 proteins, AsTCP005, AsTCP016, AsTCP019, AsTCP030, AsTCP037, AsTCP038, and AsTCP051, had a higher number of interacting proteins. Among those interacting proteins, SAUR65, SRFR1, APRR1, SAP11, and CYCB1-1 have higher chance to interact with these 8 AsTCP proteins as mentioned above (Figure 6A). When predicting the upstream regulator miRNAs of AsTCP genes, 197 upstream regulator miRNAs were found for 15 AsTCP genes, including AsTCP004, AsTCP006, AsTCP007, AsTCP026, and AsTCP027. When the expectation value was less than 2.5, all the upstream regulatory factors were miR319 (Figure 6B; Supplementary Table S9). Therefore, miR319 may play an important regulatory role in the AsTCP gene family.

Given the critical role of the TCP gene family in regulating plant stress resistance, the expression patterns of 83 AsTCP genes under silicon-mediated drought stress alleviation and salt stress treatments were analyzed (Supplementary Tables S10, S11). 83 AsTCP genes (excluding AsTCP068, AsTCP020, AsTCP076, AsTCP078, AsTCP077, AsTCP082, AsTCP075, AsTCP079) had significant responses to drought and salt stresses (Figure 7). Under drought treatment, AsTCP005, AsTCP010, AsTCP011, AsTCP021, AsTCP053, AsTCP056, AsTCP065, AsTCP069, and AsTCP072 were significantly downregulated, whereas in the control group, these genes were generally significantly upregulated. On the other hand, AsTCP007, AsTCP033, AsTCP044, AsTCP045, AsTCP071, AsTCP073, and AsTCP080 were significantly upregulated under drought treatment but significantly downregulated silicon-mediated drought stress alleviation. These genes generally exhibited low expression levels in the control group (Figure 7A). These results suggest that the aforementioned genes may play key roles in responding to drought stress. Under short-term salt stress, different oat varieties exhibited varying sensitivities to salt stress. There were significant differences in the timing and expression patterns of some AsTCP genes between the two cultivars, Huazao-2 and Hanyou-5. In addition, the majority of AsTCPs were significantly upregulated at 8 h and 12 h of salt stress (Figure 7B).

To investigate the expression levels of AsTCP genes in different oat varieties under drought stress (20% PEG6000) and salt stress (100 mM NaCl), six stress-responsive genes were selected from the oat TCP expression profile for qRT-PCR analysis. Among them, AsTCP021 and AsTCP033 were identified in the “SFS”, AsTCP044 and AsTCP053 were identified in both the “SFS” and “Sang”, while AsTCP025 and AsTCP058 were identified in the “SFS”, “Sang” and “OT3098v2”. In NY6, AsTCP044 and AsTCP058 were significantly upregulated at 4 h under NaCl treatment, whereas AsTCP021, AsTCP033, and AsTCP053 were significantly downregulated at 4 h (Figure 8A). In QY1, AsTCP021, AsTCP033, and AsTCP053 were significantly upregulated at 24 h under NaCl treatment, while AsTCP033, AsTCP044, and AsTCP058 were significantly downregulated at 0 h (Figure 8B). Comparison with transcriptome results from Huazao-2 and Hanyou-5 revealed that AsTCP025, AsTCP033, AsTCP044, and AsTCP058 displayed similar expression trends at 0 h and 2 h across the four varieties. Furthermore, AsTCP053 showed consistent expression trends in the four varieties, being downregulated at 0 h, 2 h, and 4 h, and upregulated at 8 h, 12 h, and 24 h, except for QY1, where it was upregulated at 2 h. In NY6, AsTCP021, AsTCP025, and AsTCP053 were significantly upregulated at 24 h under PEG treatment, while AsTCP021, AsTCP025, AsTCP033, and AsTCP053 were significantly downregulated at 4 h (Figure 8A). In QY1, AsTCP033, AsTCP044, and AsTCP053 were significantly upregulated at 24 h under PEG treatment, while AsTCP021 and AsTCP033 were significantly downregulated at 0 h (Figure 8B). The qRT-PCR results for AsTCP021 and AsTCP053 in NY6 under drought stress from 0 h to 12 h were largely consistent with transcriptome results, both showing a trend of downregulation.

In both NY6 and QY1, the six AsTCP genes exhibited significant responses to both salt and drought stress, with the most notable changes observed at 0 h, 4 h, and 24 h. At 0 h under drought and salt stress, AsTCP058 was significantly downregulated in both varieties. At 4 h, AsTCP044 was significantly upregulated in both varieties, while AsTCP053 was significantly downregulated. At 24 h, AsTCP025, AsTCP033, and AsTCP053 were significantly upregulated in both varieties. Additionally, in the QY1 variety, AsTCP033 and AsTCP053 showed the most significant upregulation at 24 h under both drought and salt stress. The expression patterns of different genes at different time points varied significantly between the two varieties, particularly at 24 h, where the notable upregulation of some genes in QY1 reflected a stronger response to stress. These findings indicate that varietal differences in stress sensitivity significantly influence the expression patterns of these genes.