Transcription factors (TF), also known as trans-acting factors, are a class of protein factors that can bind to specific DNA sequences (cis-acting elements) in the upstream promoter region of target genes through their DNA binding domain (DBD) and have essential roles in regulating plant growth and development (Li et al., 2019). The TCP protein, as a group of plant-specific transcription factors, evolved from algae and bryophytes and is named after the earliest identified members of the family: TB1 (TEOSINTE BRANCHED 1) in maize, CYCLOIDEA (CYC) in Cynoglossum, and Proliferating Cell Factor 1/2 (PCF1/2) in rice (Cubas et al., 1999). All members of the TCP protein contain a highly conserved basic helix-loop-helix (bHLH) structure consisting of 59 amino acid residues and can be divided into two subclasses based on the structural differences of TCP, including Class I (TCP-P/PCF) and Class II (TCP-C/CIN, TCP-C/CYC and TB1) (Viola et al., 2023). The TCP gene family is widespread in the plant kingdom, and its family members can directly or indirectly bind to regulatory target genes, which regulate multiple processes of plant growth and development (Zhou et al., 2022). In recent years, with the completion of whole genome sequencing of various species, the identification and analysis of TCP family genes have been carried out one after another on some species, and some related studies on the functions of the TCP gene family have been done (Cubas et al., 1999). ZmTCP42 in maize (Zea mays) regulates some ABA or drought stress-regulated genes and is an essential positive regulator of drought tolerance (Ding et al., 2019). MaPCF10 and MaPCF13 of banana (Musa acuminata) may be functionally important in fruit development and ripening (Wang et al., 2022). SmTCP40 of passion fruit (Solanum melongena) was stimulated by the jasmonic acid analog methyl jasmonate and is involved in its pigment signaling (Si et al., 2023). DchTCP2 and DchTCP13 in Dendrobium chrysotoxum significantly affect lateral organ development (Huang et al., 2023). Negative regulation of the growth of BpTCP8, BpTCP14, and BpTCP19 meristems of Broussonetia papyrifera (Zhao et al., 2020). CsTCP14, CsTCP16, CsTCP18, CsTCP19, and CsTCP20 were identified as TCP transcription factors associated with drought stress response in Citrus sinensis (Liu D. H. et al., 2022). OsTCP19, identified in Oryza sativa, responds to drought and salt stress and is an essential node in cell signaling that cross-links stress and developmental pathways (Mukhopadhyay and Tyagi, 2015). Thus, the TCP family of related genes is extensively involved in plant growth and development processes and is essential in response to abiotic stress regulation.

Oat (Avena sativa L.), as an annual grain-feeding crop, belongs to the grass family and is grown in about 42 countries around the world due to their excellent traits such as coolness, high nutritional value, high-stress resistance, and good palatability (Singh et al., 2013). Oat germplasm is mainly grown on marginal lands to avoid competition with grain crops for arable land. As a result, oat are often exposed to unfavorable growing conditions such as high salinity, drought, extreme temperatures, and low nitrogen stress. Nitrogen, as one of the essential nutrients in plants, is widely used in current agricultural production because its application significantly promotes plant growth and increases crop yields (Liang et al., 2021). Statistics show that the total amount of nitrogen fertilizers produced globally to increase crop yields exceeds 1 million tons annually. Although the production of N fertilizers consumes only 1-2% of global energy consumption, the utilization rate of N fertilizers is less than 50% (Dobermann and Cassman, 2005). As the global population continues to increase, overuse of nitrogen fertilizers has triggered severe environmental problems such as environmental pollution and global warming. Therefore, without increasing the use of nitrogen fertilizers, improving the nitrogen utilization rate of pasture grasses and cultivating pasture grass species with high nitrogen utilization rates or low nitrogen tolerance are of great significance for the sustainable development of the pasture industry.

The publication of the whole genome information of oat provides theoretical primary data for identifying family genes and gene function-related studies at the whole genome level. In recent years, functional genomics-related studies of oats have become a hot spot. Currently, in the available analysis of transcription factors in oat, the gene families of GRAS (Pan et al., 2023), WRKY (Liu K. et al., 2022), and PYL (Mi et al., 2023) have been characterized. Many genes have been unearthed in resistance to abiotic stresses, such as salinity, drought, cold, and high temperature. Although the TCP gene family members were widely used for abiotic stress response in other plants, the AsTCP gene family has not been characterized, and its function in response to low nitrogen stress remains unclear.

In this study, we identified and analyzed the AsTCP gene family members at the whole genome level and systematically analyzed the physical and chemical properties, conserved motif analysis, phylogeny, chromosomal localization, covariance, and cis-acting elements of the TCP gene family members. Meanwhile, this study analyzed the expression patterns of TCP gene family members in different tissues based on the genetic validation of qRT-PCR under low nitrogen stress, which provides a theoretical basis for further in-depth research on the molecular mechanism of abiotic stress resistance of AsTCP genes and the mining of excellent oat genes.

Recently, TCP gene family members have begun to be identified in different plant species with the publication of genome sequencing data from other plant species. In forages, the identification of TCP gene families in Medicago sativa (Zhang et al., 2023) and Dactylis glomerata (Wang C. et al., 2023) has also been completed. Oat is an annual crop in the grass family, with the advantages of stress resistance and high nutritional value. It is widely used in the grass and pasture industry (Fu and Yang, 2017). However, there are no reports on AsTCP gene family members at the genome-wide level. Therefore, the AsTCP genes were systematically analyzed using published oat genome-wide information and bioinformatics techniques in this study.

This study identified 49 AsTCP gene family members from the oat genome. Compared with plants such as Zea mays (46 genes), Triticum aestivum (66 genes), Hordeum vulgare (20 genes), and Cucumis sativus (27 genes), the number of AsTCP gene family members was on the high side which demonstrated that the number of TCP genes was closely related to the genome size and varied among species. Species with similar gene sequences to the AsTCP genes are mainly from grasses such as wheat and rice, suggesting that the AsTCP gene family members have similar evolutionary characteristics and affinities with the genes of these closely related species. Based on the tree-like branching structure of TCP proteins between Arabidopsis and oat, we further classified these AsTCP genes into three subfamilies: PCF, CIN, and CYC/TB1. In this study, we found that the AsTCP genes were unevenly distributed across 18 chromosomes in the oat genome ( Figure 3 ), and it is noteworthy that chromosome 5A contained the most AsTCP genes (9 genes), which are mainly from the PCF and CIN subfamilies. Many studies have been conducted to review the functions of plant TCP genes, revealing their diversity: PCF subfamily-related genes promote the proliferation of young internodal cells and activate seed embryo growth potential (Tatematsu et al., 2008). The CIN subfamily regulates plant leaf, root, and petal development (Palatnik et al., 2003). Therefore, some critical AsTCP genes may play a role in regulating plant growth and development.

Earlier findings showed that gene duplication transferred TCP gene family members from prokaryotes to land plants. Our study found some AsTCP genes (19 genes, about 39%) did not contain introns. This structural feature is similar to that found in Camellia sinensis (87%) (Shang et al., 2022), Cymbidium goeringii (57%) (Liu D. et al., 2022), and Dactylis glomerata (18%) (Wang C. et al., 2023). Most AsTCP genes do not have introns, meaning their transcription times are shorter than those containing introns. Evidence shows these intronless genes can produce more rapid protein (Ma et al., 2020). This study explains why the AsTCP genes can respond quickly and develop a response mechanism under abiotic stress. With the increase in gene sequencing data, there is growing evidence that the ancestors of all eukaryotes had genes containing introns but that most eukaryotes experienced intron loss as their species evolved. The high proportion of genes that have lost introns in these plants (e.g., Avena sativa, Camellia sinensis, and Cymbidium goeringii) suggests that an intron loss event may have occurred during their evolution. Most transcription factors perform functional regulation by modulating protein interactions and DNA binding activity. These regulations mainly depend on modifying structural domains and motifs of transcription factors (Liu et al., 1999). In this study, 10 conserved motifs were identified in oat, and most AsTCP proteins contained all the conserved motifs, and the types, numbers, and order of conserved motifs of proteins encoded by members of the same subfamily were relatively consistent. Notably, there are only 2 motifs (motif 1 and motif 2) in AsTCP42, suggesting that these motifs may have indispensable functions in the CIN subfamily. This study indicates that these 2 motifs may have essential functions for the CIN subfamily. When 2 or more genes are sequentially arranged within 200 kb of each other on a chromosome, we call this a tandem duplication event (Holub, 2001). Compared with other species, the results for AsTCP genes showed no significant correlation between chromosome length and the number of AsTCP genes.

To explore the evolutionary relationship between AsTCP gene family members and different plant species. We selected three monocotyledonous and one dicotyledonous plant species and analyzed the homology of their TCP genes. The results showed an excellent homology correlation between oat and wheat. Overall, monocotyledonous plants’ TCP gene family members showed better homology concordance than the identified AsTCP genes. Therefore, it is reasonable to speculate that the covariance between TCP genes correlates with the evolutionary divergence trends of the plant species in which they were found. The AsTCP genes originated from the same ancestor as the TCP gene in wheat. The results of phylogenetic clustering in angiosperms may be nuanced among species but are generally consistent. This study suggests a high degree of diversity and complexity of TCP genes in angiosperms (Zhao et al., 2018). Our phylogenetic tree is consistent with the phylogeny of TCP proteins from a wide range of Gramineae species (Chai et al., 2017). Oat and wheat are similar in the number of TCP proteins, but the genomic data for oat is much larger, indicating many repetitive DNA sequences in their genomes (Zhao et al., 2018). From the chromosome location map of oat ( Figure 4 ), we can see the tandem duplication of multiple AsTCP genes, which suggests a high rate of gene duplication in oat.

The promoter region of the AsTCP gene family members contains many action elements, which can be classified into three major categories according to their mechanisms of action: light-responsive, stress-responsive, and hormone-responsive action elements. The results of cis-acting element analysis showed that there were six types of light-responsive elements in the promoter regions of AsTCP gene family members. However, different subfamily members’ types and numbers of elements varied greatly. This result indicates that various subfamily members respond differently under other light conditions. The analyses in this study showed that the AsTCP gene family promoters contain a variety of adversity stress-acting elements and hormone response-acting elements. Hormones were also shown to act as a trans-acting factor capable of binding to hormone-responsive elements on promoters to regulate the transcriptional expression of target genes (Gunes et al., 2007). Hormones are not only directly involved in the growth and development of organisms but also act as signal transducers (e.g., ABA) in response to adversities such as low temperature, high temperature, and salt stress (Mundy et al., 1990). Therefore, it is reasonable to speculate that the expression of AsTCP gene family members may be regulated by hormones, which in turn are involved in scavenging excessive reactive oxygen species from their cells in vivo, increasing their resistance to abiotic stresses.

With the publication of plant whole genome sequencing results, TCP genes involved in abiotic stress response have been identified from a wide range of plants, including species such as Zingiber officinale (Jiang et al., 2023), Panicum virgatum (Zheng et al., 2019), Camellia sinensis (Shang et al., 2022) and Solanum melongena (Li et al., 2022). This study investigated the expression patterns of AsTCP genes in different tissues under low nitrogen stress conditions. By performing qRT-PCR experiments, we analyzed the expression patterns of AsTCP gene family members under low-nitrogen stress conditions. The experimental results showed that all AsTCP gene family subfamilies have members that can respond to low nitrogen stress. The AsTCP gene family plays an essential regulatory role in the response of plants to abiotic stresses (Parapunova et al., 2014). With the publication of plant whole genome sequencing results, TCP genes involved in abiotic stress response have been identified from a wide range of plants, including species such as Zingiber officinale (Jiang et al., 2023), Panicum virgatum (Zheng et al., 2019), Camellia sinensis (Shang et al., 2022) and Solanum melongena (Li et al., 2022). This study investigated the expression patterns of AsTCP genes in different tissues under low nitrogen stress conditions. The TaTCP9 identified were expressed throughout the development of young and immature spikes, and most TaTCP genes were expressed in multiple tissues and developmental stages. Expression in various tissues and developmental stages suggests that these TCP genes play an essential role in wheat development (Zhao et al., 2018), ZmTCP42 identified from maize is a critical TCP gene in maize and plays a crucial role in response to drought stress (Ding et al., 2019), so in-depth studies on the members of the AsTCP gene family members are necessary for mining their superior traits and implementing molecular breeding. Therefore, in-depth research on the members of the AsTCP gene family is of great significance for mining its particular characteristics and implementing molecular breeding. To tap the AsTCP genes responsive under low nitrogen stress, we conducted a preliminary screening and found some AsTCP genes with significantly up-regulated expression under NH₄NO₃ treatment. The results showed that the expression levels of AsTCP01, AsTCP03, AsTCP22, and AsTCP38 genes tended to increase during the gradual extension of low nitrogen stress. In leaf tissues, AsTCP01 and AsTCP03 showed higher expression levels after seven days of stress. On the other hand, AsTCP22 remained at relatively high expression levels after 14 days of stress. In root tissues, the expression patterns were similar except for AsTCP05 and AsTCP22, which showed the highest expression levels after 14 days of stress. The results of this study can further confirm that the AsTCP gene family has an essential role in response to low nitrogen stress. Therefore, a genome-wide survey of the AsTCP gene family will provide a necessary reference for the search for high-quality stress-resistant AsTCP genes, further deepen the understanding of the molecular mechanism of stress resistance in oat, and provide a theoretical basis for the development of excellent stress-resistant oat materials through genetic engineering technology.

In summary, in this study, members of the AsTCP gene family were identified at the genome-wide level using a bioinformatics approach, and their gene structures, conserved motifs, and evolutionary relationships were analyzed. Meanwhile, the potential regulatory mechanisms of the identified AsTCP genes in response to low nitrogen stress were revealed by determining the chromosomal distribution of the genes and their expression patterns in different tissues. In addition, the results of cis-acting element and gene expression analyses provide a basis for further investigation of AsTCP gene family members in abiotic stress response and other biological functions. However, the current study has only touched upon the preliminary characterization of AsTCP genes, and further functional validation studies are still needed to explore the roles of AsTCP genes in different biological processes in depth. The analysis of gene families relies on the quality of species genomes, and the quality of species genomic information affects the results of gene family analysis. As our research progresses, we will synthesize and analyze future updates of oat genomic information to sincerely resolve the theory of oat response to abiotic stress and lay the foundation for creating new highly resilient oat germplasm through genetic engineering technology.

In this study, we identified 49 AsTCP genes distributed unevenly on different chromosomes of the oat genome. Further categorizing these AsTCP genes, we classified them into three subfamilies: CIN, PCF, and CYC/TB1. We analyzed the gene structures and found that most AsTCP genes lacked introns, indicating their gene structures were very conserved. By analysis of conserved structural domains and motif patterns, we discovered that TCP gene family members of the same subfamily or branch have similar features, which may imply that they have identical gene functions. By qRT-PCR analysis, we confirmed that some AsTCP genes (AsTCP01, AsTCP03, AsTCP22, and AsTCP38) may play regulatory roles under low-nitrogen stress conditions, and these genes may become the focus of future research. In conclusion, this study comprehensively investigated the characterization of AsTCP genes. It provided a theoretical basis for further understanding the biological functions of AsTCP genes through the in-depth analysis of the screened particular AsTCP genes.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

JP: Data curation, Writing – original draft, Writing – review & editing. ZLJ: Methodology, Software, Writing – review & editing. XM: Writing – review & editing. LD: Data curation, Writing – review & editing. ZFJ: Project administration, Supervision, Writing – review & editing.