Genome-wide identification and stress-responsive expression profiling of the TCP gene family in Cenchrus fungigraminus under drought and cold stress

Abstract

Background

TCP transcription factors play a crucial role in various biological processes, including plant growth, development, and response to abiotic stress. However, few related studies investigated the characteristics of TCP genes of Cenchrus and how it plays a role in abiotic stress responses.

Results

Here, we identified 40 CfuTCPs unevenly distributed across 13 chromosomes of Cenchrus fungigraminus and classified them into three subfamilies based on the conserved domain and phylogenetic analysis. Sequence analysis revealed that all CfuTCPs contain basic Helix-Loop-Helix conserved regions, and the majority of the CfuTCP genes were intronless. Twelve genes in the CIN subclade had potential miR319 target sites. Cis-acting element analysis showed that most CfuTCP genes contained many light-, phytohormone-, and developmental stress-responsive elements in their promoter regions. Furthermore, CfuTCPs play an important role in biological activities such as transcription regulation, regulation of biosynthetic processes, and metabolic processes, and were involved in plant circadian rhythms and environmental adaptation pathways. Notably, RNA-seq data showed that most CfuTCPs were involved in stress response under drought and cold treatments. Through RNA-seq analysis and qPCR validation, CfuTCP27/31 were found to play a positive regulatory role in drought stress, while CfuTCP06 plays a negative potentially.

Conclusions

In total, 40 CfuTCP genes were identified in C. fungigraminus. Functional predictions suggested the roles of CfuTCPs in modulating multiple C. fungigraminus physiological processes and metabolic pathways. The induced expression of CfuTCPs under drought and cold stress indicated their involvement in abiotic stress response in C. fungigraminus. These findings lay the groundwork for further research on TCP genes in C. fungigraminus.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-11798-1.

Background

Cenchrus fungigraminus, a perennial herbaceous plant of the Poaceae family, is highly valued for its high biomass yield, rich crude protein and carbohydrate content, rapid growth rate, strong stress tolerance, wide adaptability, and lack of invasive risk. C. fungigraminus has been widely cultivated for its multifaceted benefits as a high-quality forage, a vital pasture grass, and an ecological restoration material for ecological restoration efforts. Commonly known by the name JUJUNCAO [1] (Pennisetum giganteum Zhan X. Lin, syn. Cenchrus fungigraminus Zhan X. Lin), this plant is primarily distributed in tropical, subtropical, and temperate regions, showcasing its adaptability across various climatic zones. It exhibits tolerance to drought, salt stress, and heavy metal pollution, making it a pioneer plant for ecological restoration. Given the limited drought tolerance of C. fungigraminus as evidenced by severe drought stress (25% field capacity) can affect the normal growth of C. fungigraminus [2], elucidating its stress response mechanisms becomes critical for developing improvement strategies. Such research is crucial for the sustainable development of animal husbandry in arid areas and for maintaining the earth’s ecological balance. Understanding and enhancing the plant’s ability to withstand drought can lead to more resilient agricultural practices, particularly in regions where water scarcity is a significant challenge.

Gene family research is indeed essential for deepening our understanding of the mechanisms behind plant growth and stress responses [3]. The Teosinte branched1/Cincinnata/Proliferating cell factor (TCP) family is a group of genes encoding plant-specific transcription factors that respond to hormones, nutrient elements, and biotic and abiotic stress stimuli, playing a crucial role in hormone-mediated plant cell proliferation and organ development [4]. TCPs can be divided into two major classes: Class I (PCF) members, which mainly promote cell division, and Class II (CIN and CYC/TB1) members, which primarily inhibit plant growth and development [5]. The TCP protein contains two conserved domains: the bHLH (basic Helix-Loop-Helix) domain and the R domain. The bHLH domain, consisting of 59 amino acids, is crucial for plant growth metabolism, signal transduction, and response to abiotic stress [6]. Only a small subset of the TCP proteins possess the R domain, which is predicted to adopt a coiled structure, potentially mediating protein-protein interactions and other domain functionalities [7].

Since the TCP gene family was first defined in 1999, members have been identified across various crops, including wheat, rice, maize, sorghum, and Arabidopsis thaliana [8–10]. Extensive research has demonstrated that TCP transcription factors play a pivotal role in plants’ response to abiotic stress by modulating cell osmotic pressure, altering cell permeability, reducing harmful substances, and adjusting hormone sensitivity to enhance plant reactions to stress stimuli [11]. For instance, in A. thaliana, the TCP genes AtTCP5/13/17 have been shown to interact with PIF4 (PHYTOCHROME INTERACTING FACTOR 4), which enhances plant activity and promote the development of thermal morphogenesis to adapt to high temperatures [12, 13]. Overexpression of the PeTCP10 gene from bamboo has been found to enhance salt tolerance in transgenic plants during their vegetative growth phase, and to increase salt sensitivity during germination and seedling stages. Similarly, in A. thaliana, the overexpression of the ZmTCP42 gene from Zea mays triggers a hypersensitive response in seed germination to abscisic acid, which in turn enhances drought tolerance [14]. In rice, overexpression of OsTCP19 has been demonstrated to reduce plant water loss and reactive oxygen species production, leading to increased lipid droplet accumulation and enhanced stress resistance in transgenic plants [15]. Additionally, repression of the expression of OsPCF5 and OsPCF8 in rice led to increased cold tolerance of rice seedlings after chilling acclimation [16]. Despite the well-established regulatory roles of the TCP genes in plant stress response networks, research on the TCP gene family within the genus Cenchrus is lacking. This knowledge gap highlights the necessity for an in-depth investigation into the evolutionary relationships and functional roles of the TCP family members in Cenchrus, with a particular focus on the economically and ecologically significant forage species C. fungigraminus as a representative case. In this study, we undertook a systematic identification and analysis of the TCP transcription factor family utilizing the genome sequence of C. fungigraminus. Our approach encompassed the prediction of physicochemical properties, gene structure, phylogenetic relationships, and miRNA target sites, followed by an in-depth analysis of their cis-regulatory elements. To gain insights into their biological roles, we conducted a functional enrichment analysis using KEGG/GO pathways. Moreover, we investigated the expression profiles of the TCP family members, designated as CfuTCPs (Cenchrus fungigraminus TCP genes) in C. fungigraminus under conditions of drought and cold stress. The collective findings from this study lay the groundwork for comprehending the functional roles of TCP family transcription factors and offer valuable insights for devising strategies in molecular breeding aimed at enhancing plants’ resilience to resistance.

Materials and methods

Identification and analysis of the TCP genes

The whole genome sequence and annotation files for C. fungigraminus and Setaria italica were obtained from the National Genomics Data Center (https://ngdc.cncb.ac.cn/, accessed on 18 March 2024). The TCP proteins sequences of A. thaliana were retrieved from TAIR2 database (https://www.arabidopsis.org, accessed on 18 March 2024) [17], and the corresponding sequences for Oryza sativa TCP proteins were acquired from the Rice Genome Annotation Project (http://rice.uga.edu/, accessed on 24 March 2024).

The BLAST table feature in TBtools software was utilized to compare and screen the genomic data of C. fungigraminus, enabling the identification of the TCP protein candidate sequences. The hidden Markov model (HMM) profile of the TCP domain (PF03634) was retrieved from the Pfam protein family database [18], which facilitated further identification of the TCP proteins using TBtools [18]. By integrating the outcomes of BLAST and Hmmsearch, all TCP genes were subjected to analysis with NCBI CDD (https://www.ncbi.nlm.nih.gov/cdd, accessed on 28 March 2024), and proteins harboring TCP conserved domains were classified as members of the TCP family. The ExPASy online tool [19] was employed to predict the physicochemical properties of the TCP genes, while the CELLO website(http://cello.life.nctu.edu.tw/) [20] was used to predict their subcellular localization.

Phylogenetic and miRNA − binding site recognition analysis of the TCPs

The TCP protein sequences from A. thaliana (24 TCPs), Oryza sativa (21 OSTCPs), Setaria italica (23 SITCPs), and C. fungigraminus (40 CfuTCPs) were imported into MEGA 7.0 software for analysis. Multiple sequence alignment was conducted using the MAFFT program, and the phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with a bootstrap value of 1000 replicates. Additionally, we utilized the online software Evolview v3.0 to refine the visualization of the phylogenetic tree (https://evolgenius.info//evolview-v2/, accessed on 1 May 2024) [21]. Potential TCP genes targeted by miRNA319 in C. fungigraminus were predicted by the online software psRNATarget (http://www.zhaolab.org/psRNATarget/) with default parameters [22].

Gene structure and motif analysis of CfuTCPs

We utilized the Conserved Domain Database (CDD) tool available in the NCBI online software suite (https://www.ncbi.nlm.nih.gov/, accessed on 15 May 2024) to analyze the conserved domain architecture of the TCP gene. Additionally, we employed the online software MEME (http://meme-suite.org/, accessed on 20 April 2024) to analyze and download the conserved motifs of the TCP gene, setting the predicted value threshold to 10 [23]. Finally, we integrated phylogenetic trees, conserved protein motifs, and gene structure maps using TBTools v1.120 [24].

Collinearity, Ka/Ks and chromosomal localization of CfuTCPs

We further employed the MCScanX tool to investigate the collinear relationships between C. fungigraminus, A. thaliana, and O. sativa, mapping the collinear gene blocks of the TCP within their respective genome sequences. Subsequently, we utilized TBTools v1.120 software to analyze the chromosome localization of the TCP gene in C. fungigraminus. We extracted location information from genome and gene annotation files, which allowed us to construct a comprehensive map of the TCP gene’s position on the chromosome. Non-synonymous (Ka) and synonymous (Ks) substitutions of each duplicated gene pairs were calculated using KaKs_calcu in TBtools (accessed on 25 October 2024).

Cis-elements analysis of CfuTCPs

To investigate the regulatory function of CfuTCP genes in plant growth and development, we used TBtools to extract the promoter sequence, encompassing 2000 base pairs upstream of the transcription initiation site for each CfuTCP gene. We then utilized the online tool PlantCARE to predict cis-acting elements within these promoter regions [25]. Finally, TBTools was employed to visualize the map of these cis-acting elements.

Protein interaction network and the GO/KEGG annotation of CfuTCPs

The interactions among CfuTCP proteins were predicted using the STRING database. Subsequently, the interaction network was visualized using Cytoscape v3.7.1 [26]. The CfuTCP genes were then subjected to Gene Ontology (GO) and KEGG pathway annotation through the eggNOG-mapper v2.1.1 website (http://eggnog-mapper.embl.de/, accessed on 20 June 2024) [27]. The protein files of the C. fungigraminus genome were submitted to the eggNOG 5.0 database to retrieve annotations, including GO terms and KEGG pathways. Following this, GO enrichment and KEGG pathway enrichment analyses were conducted using TBtools. Finally, the results were visualized using the Enrichment Bar Plot module within TBtools.

Expression patterns of CfuTCPs

Publicly available transcriptome data were selected for analysis to comprehensively understand the expression patterns of the TCP genes in different drought and chilling periods of C. fungigraminus and to reveal its potential roles in C. fungigraminus growth and abiotic stress. The expression levels of CfuTCP genes in response to drought treatments were analyzed using RNA-seq data (Accession number: SRR11800371-SRR11800388) obtained from the NCBI SRA database (http://www.ncbi.nlm.nih.gov/sra) [28]. The expression data of the TCP genes in the different drought periods of C. fungigraminus was extracted from Zhou et al., 2021 supplemental data. Additional transcriptome data of C. fungigraminus were obtained from EGDB [29], a comprehensive multi-omics database for energy grasses.

The expression profiles of chilling temperature were determined using RNA-seq data(Accession number: PRJNA544770) obtained from the NCBI SRA database. Extracted from the study data of Li et al. in 2020, which the first group served as the RT sample, while the other group was transferred and cultured at 4 °C as the CT sample. In the SRA toolkit v3.1.0, fasterq-dump was used to convert SRA files to FASTQ format. Trimmomatic v0.39 is used for filtering low-quality sequences. Hisat2 v2.1.0 was used to construct a genome index for C. fungigraminus and align the quality-control data. Samtools v1.19 converts SAM to BAM and sorts files, while StringTie v2.2.0 performs transcription assembly. In addition, the gene expression matrix was generated using the prepDE.py3 script with StringTie. The expression data was normalized to every million transcripts, log2-transformed, and visualized as a heatmap using TBtools.

Plant materials, RNA isolation, and qPCR analysis

In this study, C. fungigraminus plants were obtained from the Germplasm Resource Garden at Fujian Agriculture and Forestry University (Fuzhou, Fujian Province, China). Seedlings of uniform growth vigor and approximately 70 cm in height were selected and transplanted into plastic pots. The greenhouse temperature was maintained at approximately 25 °C. After one week of indoor cultivation, two groups were established: a control group and a drought stress group, each consisting of 3 pots with 3 plants per pot. The control group received 300 mL of water every 2 days, whereas the drought stress group was withheld from watering. Fresh leaves were harvested on the 1 st, 7th and 14th days of the drought period and the 1 st and 5th days following rewatering. The samples were then flash-frozen in liquid nitrogen and stored at − 80 °C for subsequent analysis.

RNA was isolated from the leaves of C. fungigraminus using the FastPure Plant Total RNA Isolation Kit, which was designed for polysaccharide and polyphenol-rich tissues (Vazyme Biotech Co, Ltd., Nanjing, China). Reverse transcription was performed using Hifair^® AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR (Yeasen Biotechnology Co., Ltd, Shanghai, China) to eliminate contaminated genomic DNA and generate cDNA. The Hieff UNICON^® Universal Blue qPCR SYBR Green Master Mix was utilized for qPCR. Primers targeting the CfuTCP genes were designed with Primer Premier 5 software, and their specificity was verified using primer blast on the NCBI website. The qPCR analysis was conducted using PerfectStart™ Green qPCR SuperMax (TransGen Biotech, Beijing, China). Finality, the actin gene from S. italica was selected as the reference gene [30]. Each sample included three biological replicates, the relative expression level of the target genes was calculated using the 2^−∆∆CT method, and GraphPad Prism 7.0 was used for data normalization.

Results

Identification and protein traits of CfuTCPs

A total of 40 members of the TCP gene family were identified in the genome of C. fungigraminus and were designated as CfuTCP1 to CfuTCP40. Their physicochemical characteristics are presented in Table 1. The proteins comprise 184 to 616 amino acids, with CfuTCP13 having the less (184) and CfuTCP29 having the most (616). The molecular weight (MW) ranged from 19264.73 kDa (CfuTCP13) to 63806.61 kDa (CfuTCP29), and the isoelectric point (pI) varied from 4.87 (CfuTCP37 and CfuTCP38) to 11.36 (CfuTCP24). All CfuTCP gene family members were predicted to be unstable proteins, with an instability index greater than 40. Additionally, prediction results indicate that CfuTCP7 was located in the mitochondria, while the other 39 CfuTCPs were nuclear-localized (Table 1).

Table 1.

Analysis of amino acid sequence characteristics of the CfuTCP genes family in C. fungigraminus

Gene name	Gene ID	AA	PI	Mw	PI	GRAVY	Subcellular localization
CfuTCP29	Pgi06A00044490	616	6.64	63806.61	49.86	−0.624	Nuclear
CfuTCP34	Pgi06B00064150	602	7.58	62403.02	51.67	−0.675	Nuclear
CfuTCP32	Pgi06B00014230	515	6.45	55416.88	52.43	−0.753	Nuclear
CfuTCP28	Pgi06A00013740	501	9.38	53496.41	64.00	−0.629	Nuclear
CfuTCP9	Pgi02A00005900	389	8.95	40096.12	42.48	−0.505	Nuclear
CfuTCP10	Pgi02A00005920	389	7.96	40165.16	41.06	−0.505	Nuclear
CfuTCP15	Pgi02B00005610	389	9.45	40003.03	40.78	−0.493	Nuclear
CfuTCP40	Pgi07B00043830	477	9.38	49748.02	50.24	−0.542	Nuclear
CfuTCP14	Pgi02B00005580	455	8.52	47473.77	43.15	−0.387	Nuclear
CfuTCP20	Pgi04A00002400	300	6.07	31198.94	52.03	−0.214	Nuclear
CfuTCP36	Pgi07A00040600	470	9.18	49224.35	56.05	−0.580	Nuclear
CfuTCP4	Pgi01B00002340	533	9.71	56899.47	73.37	−0.394	Nuclear
CfuTCP27	Pgi06A00013290	283	7.02	30101.5	43.10	−0.378	Nuclear
CfuTCP2	Pgi01A00039670	296	8.72	30815.52	45.64	−0.312	Nuclear
CfuTCP7	Pgi01B00048280	274	7.89	28346.82	40.82	−0.292	Mitochondrial
CfuTCP31	Pgi06B00013670	372	8.74	39388.25	50.47	−0.291	Nuclear
CfuTCP39	Pgi07B00026130	269	5.93	28424.34	50.05	−0.682	Nuclear
CfuTCP35	Pgi07A00022610	269	5.87	28786.69	49.32	−0.770	Nuclear
CfuTCP16	Pgi02B00011210	369	7.59	39513.07	52.85	−0.625	Nuclear
CfuTCP11	Pgi02A00011130	371	7.59	39722.4	52.14	−0.589	Nuclear
CfuTCP1	Pgi01A00022220	257	7.75	27085.25	56.87	−0.513	Nuclear
CfuTCP5	Pgi01B00026560	259	7.02	27336.45	54.74	−0.526	Nuclear
CfuTCP26	Pgi06A00003450	322	6.24	33965.49	63.69	−0.691	Nuclear
CfuTCP30	Pgi06B00003130	328	6.27	34518.03	63.80	−0.719	Nuclear
CfuTCP18	Pgi03A00005860	403	9.58	40637.75	65.40	−0.514	Nuclear
CfuTCP19	Pgi03B00005410	413	9.42	41499.57	62.61	−0.511	Nuclear
CfuTCP25	Pgi05B00020200	394	6.55	39892.91	59.26	−0.489	Nuclear
CfuTCP3	Pgi01A00055380	415	5.72	42070.78	71.78	−0.701	Nuclear
CfuTCP12	Pgi02A00034690	391	6.76	39861.97	61.38	−0.510	Nuclear
CfuTCP22	Pgi04A00038210	396	8.07	40762.84	58.65	−0.590	Nuclear
CfuTCP33	Pgi06B00044300	396	8.06	41010.21	59.76	−0.585	Nuclear
CfuTCP8	Pgi01B00063210	412	5.75	41500.22	70.23	−0.651	Nuclear
CfuTCP6	Pgi01B00034730	362	5.72	37106.29	68.53	−0.380	Nuclear
CfuTCP21	Pgi04A00024910	354	5.68	36304.28	71.01	−0.387	Nuclear
CfuTCP17	Pgi03A00000470	232	9.88	23288.22	43.79	−0.084	Nuclear
CfuTCP23	Pgi04B00017480	211	10.01	21634.46	62.80	−0.234	Nuclear
CfuTCP13	Pgi02A00035880	184	9.62	19264.73	66.95	−0.331	Nuclear
CfuTCP24	Pgi04B00027740	274	11.36	29096.27	56.97	−0.514	Nuclear
CfuTCP37	Pgi07B00013650	326	4.87	33342.12	60.20	−0.342	Nuclear
CfuTCP38	Pgi07B00018730	328	4.87	33510.32	61.07	−0.339	Nuclear

AA Number of amino acid; Mw Molecular weight; pI Theoretical isoelectric point; II Instability index; GRAVY Grand average of Hydropathicity

We also conducted a sequence alignment analysis of CfuTCP genes. The domain of all CfuTCP genes can be divided into four distinct parts (Fig. 1). The basic region was the most conserved, followed by two helix regions, while the loop region exhibited more variation than the other regions. We observed that base substitutions within each subfamily are highly conserved. For instance, all members of the CYC/TB1 subfamily displayed a consistent amino acid substitution pattern.

Fig. 1.

Multiple sequence alignment and protein sequence of the TCP domain. A Alignment of the TCP domain containing the Basic, Helix and Loop sequences for the predicted C. fungigraminus TCP proteins. B Sequence logo of motif 1, which encodes the TCP domain

The phylogenetic analysis and miRNA319 − binding site recognition of CfuTCPs

Phylogenetic analysis was conducted on TCP genes from four species: A. thaliana, S. italica, O. sativa and C. fungigraminus). A total of 108 genes were identified and classified into two major branches and three subfamilies as shown in Fig. 2: Class I includes PCF genes, while Class II comprises CIN genes and CYC/TB1 genes. The PCF subfamily was the most represented, with 18 members in C. fungigraminus, 13 in A. thaliana, 10 in O. sativa, and 10 in S. italica. The CIN subfamily comprised 16 members in C. fungigraminus, 10 in S. italica, 8 in O. sativa, and 8 in A. thaliana. The CYC/TB1 subfamily had the least members, with 6 in C. fungigraminus—twice the number found in the other three species, which each had 3 members. Notably, the CIN subfamily was twice as large in O. sativa and A. thaliana compared to the CYC/TB1 subfamily.

Fig. 2.

Phylogenetic tree based on the TCP protein sequences of A. thaliana, S. italica, O. sativa and C. fungigraminus. Phylogenetic analysis indicated that the TCP gene family was classified into the following two clades: Class I (PCF) and Class II (CIN and CYC/TB1)

Twelve CfuTCP genes (CfuTCP4/9/10/14/15/20/28/29/32/36/40) in the CIN clade contained putative binding sites for miRNA319 (Table S1). In other plants, putative binding sites for miRNA319 were also exclusively found in CIN-like genes [31]. Our findings suggest that the sequences of miR319-binding sites have been conserved throughout plant evolution.

Gene structure and motif analysis of CfuTCPs

The protein-conserved sequences and sequence logos of CfuTCP members were analyzed using MEME online analysis software, resulting in the identification of 10 conserved motifs, designated as motif 1 through motif 10 (Fig. 3). Notably, each CfuTCP protein contained motif 1. By comparing and analyzing the phylogenetic classification results of the TCP genes across four species, we observed that the protein motif types, numbers, and order were highly conservative among the same subfamily members. Closely related members shared similar motifs: Class I (PCF) primarily included motifs 1 and 2, while Class II (CIN and CYC/TB1) primarily included motifs 1 and 3. This conservation suggests that these motifs are not only highly conserved but also likely to play a significant role in the evolutionary history of the CfuTCP gene family.

Fig. 3.

Structural analysis of CfuTCPs. A The distribution of motifs in CfuTCPs. B The exon-intron structure of CfuTCPs

To elucidate the structural composition of the CfuTCPs, we compared the genomic DNA sequences to determine the exon and intron structure of these genes. Our analysis revealed that 26 of the CfuTCP genes (comprising 63%) lack introns. In contrast, 14 CfuTCPs contained one to three introns: specifically, CfuTCP31 and CfuTCP29 each contained 3 introns, while CfuTCP7/20/4 each contained 2 introns. Additionally, some CfuTCP genes included a non-coding region (UTR). As depicted in the Fig. 3, members within the same subfamily predominantly exhibited similar gene structures.

Chromosomal distribution, synteny and selection pressure analysis of the TCP genes

We further analyzed the chromosomal localization of CfuTCP genes and found that 40 CfuTCP genes were unevenly distributed across 13 chromosomes, with a higher frequency at the ends of the chromosomes. Interestingly, no CfuTCP genes were detected on chromosome 09 (Fig. 4A). Based on Holub ‘s definition [32], regions on chromosomes containing two or more genes within a 200 kb interval are considered to represent tandem duplication events. Within the CfuTCP genes, we identified 2 pairs of tandemly duplicated genes. Some genes were found to be closely adjacent on chromosomes, forming clusters on phylogenetic trees, which suggests they may share similar functions, such as CfuTCP9/10 and CfuTCP14/15.

Fig. 4.

Chromosome localization, gene duplication, and collinearity analysis of CfuTCP genes. A The distribution of the TCP genes on the chromosomes of C. fungigraminus. B The genomic locations and segmental duplication of the CfuTCP genes, the red lines indicating paralogous relationships among TCP genes. C The collinearity analysis of the TCP genes among O. sativa, C. fungigraminus and A. thaliana, the gray lines represent genome-wide collinearity between different species, and the red lines specifically show the collinear relationships among TCP genes

Furthermore, we identified 45 pairs of segmentally duplicated genes within the TCP gene family in C. fungigraminus (Fig. 4B). Among these, 43 pairs are paralogous genes. To gain insights into the evolutionary rate of 45 duplicated gene pairs, the nonsynonymous (Ka) over synonymous (Ks) ratio was calculated to evaluate the selection pressure during evolution(Table S2). The Ka/Ks of duplicated gene pairs were all less than 1, indicating that all duplicated gene pairs were under strong purification selection. This is consistent with the observation results of other plants such as soybean and sunflower [33, 34].

To further deduce the phylogenetic mechanisms of JUNCAO TCP family, we constructed a syntenic maps comparing C. fungigraminus with two representative species, a dicotyledon, A. thaliana, and a monocotyledon, O. sativa (Fig. 4C). The analysis revealed that A. thaliana and C. fungigraminus share 12 pairs of collinear gene pairs across 7 chromosomes, while C. fungigraminus and O. sativa have a higher degree of collinearity, with 21 pairs of collinear gene pairs distributed across 11 chromosomes(Table S3). The homology of the TCP gene between C. fungigraminus and O. sativa is greater than that between C. fungigraminus and A. thaliana. Collectively, these findings suggest that TCP genes may have a conserved evolutionary history within monocots, and that there is a high degree of evolutionarly conservation between the TCP genes of C. fungigraminus and O. sativa. The collinearity analysis between C. fungigraminus and these species was pivotal in elucidating the evolutionary patterns of the TCP genes.

Analysis of Cis-acting elements in CfuTCPs promoter

To explore the function and expression pattern of the CfuTCP genes, we extracted cis-regulatory elements (CAREs) within the 2000 bp promoter region upstream of the initiation codon of each TCP genes and analyzed these promoter sequence using the PlantCARE database (Fig. 5). A total of 1,177 CAREs were identified in C. fungigraminus (Fig. 5A). Functional annotation categorized these CAREs into three main types: light-responsive elements (431, 36.62%), plant development and stress physiology elements (321, 27.28%), and phytohormone-responsive elements (425, 36.11%).

Fig. 5.

Analysis of Cis-acting Regulatory Elements in CfuTCP genes. A The number of each cis-acting element within the promoter region of CfuTCP genes. Functional annotation classified these elements into three main categories: light responsive, development and stress physiology, and phytohormone responsive. B Cis-elements in the promoter region of CfuTCP gene family. These cis-acting elements are situated in the promoter region, specifically within 2000 bp upstream of the transcription start site of the CfuTCP genes

The identified cis-elements suggest that CfuTCP genes were involved in a range of responses, including those to phytohormones such as auxin, abscisic acid(ABA), gibberellin(GA), methyl jasmonate (MeJA), and salicylic acid. Additionally, these genes were associated with elements related to growth and development like light response, cell cycle regulation, endosperm expression, meristem expression and circadian control. Furthermore, the promoter regions of the CfuTCP genes contain one or more stress-related elements, indicating responses to anoxic, anaerobic, drought, and low-temperature stress. Notably, 21 of the CfuTCP genes contained drought-inducible elements.

Light responsiveness was the most prevalent function among the cis-elements in TCP genes, suggesting that light is crucial for regulating TCP function throughout plant growth and development. Following light responsiveness, elements responsive to MeJA and ABA were the second and third most common types, respectively, and were widely distributed among the C. fungigraminus TCP gene family (Table S4). These findings indicated that CfuTCPs play a significant role in the response to light, growth and development, and environmental stress in C. fungigraminus. Additionally, we identified components involved in meristem expression and meristem-specific activation, which aligns with the key function of the TCP genes in maintaining the dynamic balance of meristems. Elements related to cell cycle regulation, flavonoid biosynthetic gene regulation, and circadian control were less prevalent in our analysis. This analysis offers valuable insights for subsequent stress treatments in this study.

The GO/KEGG and protein-protein interaction analysis of CfuTCPs

We further annotated the CfuTCP genes in C. fungigraminus to elucidate their classification within the TCP gene family across Molecular Function, Cellular Component, and Biological Process categories (Fig. 6C and Table S5). Our results revealed enrichment of these genes across 78 GO terms, with significant enrichments in “RNA biosynthetic process” within the Biological Process category, “nucleus” within the Cellular Component category, and “transcription regulatory activity” within the Molecular Function category. These findings underscore the substantial role of the TCP gene family in plant growth and development, particularly in RNA biosynthesis and transcriptional regulation.

Fig. 6.

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation and prediction of interaction networks among CfuTCPs. A Analysis of interaction networks among CfuTCPs; B Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of CfuTCPs; C Gene Ontology (GO) enrichment analysis of CfuTCPs. The P-value is calculated with the hypergeometric test

Our analysis identified a total of 12 enriched KEGG metabolic pathways associated with CfuTCP genes, with significant enrichment observed in pathways such as “Circadian rhythm”,“Environmental adaptation”, and “Organismal Systems” (Fig. 6B and Table S6). These findings underscore the substantial involvement of the TCP gene family in the circadian rhythm and environmental adaptation of C. fungigraminus. Such insights are crucial for enhancing our comprehension of plant responses to environmental stresses.

Furthermore, the interaction relationships among the 40 CfuTCP proteins were found to be complex, with 15 members interacting with each other (Fig. 6A and Table S7). Notably, CfuTCP28/29/31 each interacted with 12 different members; CfuTCP4 and CfuTCP36 each interacted with 11 members, while CfuTCP8 interacted with 10. CfuTCP21/13/12 each interacted with nine members, and CfuTCP11/17/24/30 each interacted with eight members. CfuTCP38 interacted with seven members, and CfuTCP5 interacted with four. Moreover, the highest interaction score was observed between CfuTCP13 and CfuTCP17.

CfuTCP Genes exhibit diverse expression patterns in response to drought and cold stress

In investigating the potential roles of CfuTCP genes in abiotic stress response, their expression profiles were analyzed under drought and cold treatments using publicly available RNA-seq data (Fig. 7 and Table S8) [35]. The expression patterns of the TCP gene family members vary under drought and cold stress. Under drought stress, 12 CfuTCP genes (CfuTCP27/29/23/04/31/19/32/37/28/38/20/36) exhibited the highest expression level at either day 7 (D7) or day 14 (D14) of drought treatment. This finding suggests that these genes are associated with plant development and drought stress tolerance. Conversely, the expression levels of 10 CfuTCP genes (CfuTCP02/06/21/07/08/26/25/40/11/30) were downregulated, indicating their possible roles in negative regulation or stress-specific suppression.

Fig. 7.

Expression patterns of the TCP genes under drought and cold stress in C. fungigraminus. A Expression heatmap of CfuTCP genes under varying degrees of drought stress. CK is used as the control. D7, D14, R1, and R5 represent drought treatment for 7 day, 14 day, 1 rehydration and 5 rehydration, respectively. B Expression heatmap of CfuTCP genes under different cold time treatments. RT, CT 2 h, CT 8 h and CT 24 h represent cold temperature (4 °C) treatment for 0 h, 2 h, 8 h, and 24 h, respectively

C. fungigraminus having a chilling-sensitive nature, low-temperatures also elicited differential expression patterns among CfuTCP genes. The upregulation of 13 CfuTCP genes (CfuTCP40/03/27/02/17/08/36/18/01/19/28/32/23) at 2 h post-treatment indicates their role in early chilling stress responses. Conversely, the downregulation of 6 CfuTCP genes (CfuTCP29/34/38/37/25/12) indicated their potential negative regulatory roles within cold environments. Most genes showed a significant increase in expression levels between 8 and 24 h, indicated that the majority of the TCP genes play a critical role under cold stress conditions.

Additionally, the expression levels of CfuTCP13/24/39/35/5 did not significantly differ among the various stages under drought and cold stress (Fig. 7 and Table S9). The expression trend of CfuTCP1 showed slight changes under cold stress, but its expression levels were very low.

Quantitative Real-Time PCR (qPCR) analysis of CfuTCP genes in C. fungigraminus

To investigate the response of the CfuTCP genes to drought stress, this study conducted experiments on C. fungigraminus plants at 0, 7, and 14 days after the onset of drought treatment, as well as on the 1 st and 5th days after rewatering. Based on the analysis of cis-acting elements in the genes promoters, we selected eight genes from various branches (PCF, CIN, and CYC/TB1) that contain MYB binding sites involved in drought induction for qPCR validation. The research results were roughly consistent with the trend of changes in RNA-seq data (Fig. 8 and Table S10). In qPCR, CfuTCP11 was significantly downregulated under drought stress compared to the control. In contrast, the expression of the CfuTCP06 did not significantly change, showing only a transient decrease before returning to normal levels. Notably, there are only 5 genes (CfuTCP19/20/27/29/31) were significantly upregulated in qPCR and RNA-seq analysis under drought stress. Interestingly, CfuTCP19/27/31 was upregulated in qPCR detection on the 7th day of drought, but they showed a decrease on the 14th day.

Fig. 8.

RT − qPCR verification of eight CfuTCP genes under drought stress in C. fungigraminus. The Y-axis represents the relative expression values (2^-△△CT), and the X-axis represents the time points of drought treatment. The red line represents the expression levels of RNA-seq at different time periods. Statistically significant differences were conducted using the one-way ANOVA test and indicated with *(P < 0.05), **(P < 0.01), ***(P < 0.001), and ****(P < 0.001)

Discussion

As a traditional gramineous forage, C. fungigraminus and its varieties have made significant contributions to animal husbandry and environmental management. The TCP gene family is well recognized for its crucial role in various biological processes, including plant growth, development, and responses to abiotic stress, and has been identified across a variety of plant species, such as Arachis hypogaea [36], Musa acuminata [37], Medicago sativa [38], and Sorghum italica [39]. However, few studies have investigated the traits of TCP genes and their role in abiotic stress responses within the grass family. With the recent publication of the C. fungigraminus genome, we now have the opportunity to delve into the evolutionary dynamics of this gene family in detail. In this study, we identified TCP genes in C. fungigraminus for the first time and subsequently conducted a comprehensive analysis. Our objective is to provide valuable insights that will facilitate further exploration into the roles of CfuTCPs in regulating plant growth and adaptation to environmental stresses in C. fungigraminus.

Our results revealed a total of 40 members of the TCP gene family, classified into three subfamilies, within the genome of C. fungigraminus (Table 1; Fig. 2). In comparison with other Poaceae plants, the number of the TCP genes in C. fungigraminus is less than that in wheat (60), but similar to Avena sativa (49), maize (46) and switchgrass (42) [6, 40–42], and nearly twice as many as in O. sativa (22), Sorghum italica (22), Setaria viridis (22), and Coix Lacryma-jobi (16) species [30, 43, 44]. The observed differentiation among TCP gene family members between C. fungigraminus and other Poaceae plants is likely a consequence of the allopolyploid nature of C. fungigraminus, potentially resulting in rapid gene family expansion and duplication. In addition, the isoelectric point of the majority of these proteins (62.5%) is greater than 7, suggesting that most CfuTCP proteins are stable alkaline proteins, a trait common in most plants. Subcellular localization analysis revealed that, with the exception of CfuTCP7, which is localized to the mitochondria, all other CfuTCP proteins are nuclear-localized, hinting at their potential involvement in novel functions.

It is well established that genes structure, chromosomal localization and genome comparisons are integral to understanding genes function. In this study, multiple sequence alignment reveals similar nucleotide substitutions among protein sequences across the three subfamilies. Protein motif analysis shows that all TCP genes families possess the motif 1 domain, with motif 1 and 2 being shared in the PCF subfamily, and motif 1 and 3 in the CYC/TB1 and CIN subfamilies. Motif 2 was a distinguishing feature for identifying the PCF subfamily, aligning with the research findings in Solanum Murigatum and Gossypium barbadense. This suggests conservation within the same subfamily among CfuTCPs and the differences in protein motifs may contribute to functional divergence among subfamilies. Moreover, our results indicated that most CfuTCP genes in C. fungigraminus lack introns, while 14 CfuTCP genes contain 1 to 3 introns. This observation is consistent with Liu’s findings regarding the intron number of theTCP genes in Cymbidium goeringii. Their study suggests that genes with fewer introns may enhance more efficient and flexible gene expression, which can facilitate a rapid response to environmental changes. Our Ka/Ks analysis showed that all duplicated genes were subjected to purification selection, suggesting a tendency to maintain original function and highlighting the conservation of TCP transcription factors during species evolution. Additionally, genome comparisons between C. fungigraminus, A. thaliana and O. sativa revealed that C. fungigraminus shares 12 TCP gene pairs with A. thaliana and 21 with O. sativa. These findings indicated a closer collinear relationship between the chromosomes of C. fungigraminus and O. sativa than with A. thaliana, suggesting a shared evolutionary history and varying degrees of gene expansion [45].

Research on cis-acting regulatory elements within promoter regions that transcriptionally upregulate gene expression has significantly advanced our fundamental understanding of gene regulation and expanded the toolbox of available promoters [46]. In our study, we analyzed the cis-acting DNA elements of the CfuTCPs’ promoters (Fig. 6). A substantial proportion of light-responsive elements were identified in these promoter regions, suggesting that CfuTCP genes are regulated by light signals, with important implications for the coordination of plant growth and development (Table S4). This finding aligns with the fact that C. fungigraminus possess a typical C4 Kranz structure and exhibits high photosynthetic efficiency [47]. Furthermore, the CfuTCP genes also contained hormone regulatory elements, including those responsive to abscisic acid (ABA), methyl jasmonate (MeJA), and MYB transcription factors. The plant hormone ABA plays a crucial role in drought response by minimizing water loss through stomatal closure and initiating protective mechanisms to counteract water scarcity. In C. fungigraminus, with the exception of CfuTCP15 and CfuTCP25, all other genes contain ABA cis-acting elements, and 21 genes possess drought-induced response elements, indicating that most members of CfuTCP gene family are involved in drought stress response. This finding is consistent with research conducted on orchid, sorghum, and rice [26, 48, 49]. In summary, approximately 17% of cis-acting DNA elements in CfuTCP gene family are associated with abiotic stress responses in plants, particularly with defense and stress responsiveness, low-temperature responsiveness, abscisic acid responsiveness and drought-inducibility.

TCP genes are well documented to regulate various physiological processes in plants, including hormone pathways [4], growth, and development [50], as well as the regulation of the circadian clock and defense mechanisms [51]. In this study, GO analysis revealed that the molecular functions of CfuTCP genes were primarily associated with transcriptional regulation and the activity of DNA-binding transcription factors. Furthermore, these genes were implicated in biological processes, particularly the regulation of metabolic processes, which were essential for plant growth and physiological functions. KEGG analysis results highlighted the involvement of CfuTCP genes in key metabolic pathways, specifically the Circadian rhythm-plant, Environmental adaptation, and Organismal Systems pathways. These pathways are crucial for regulating biological rhythms, enabling plants to anticipate and respond to daily and seasonal changes. Additionally, this implies that modulating TCP activity could enhance metabolic efficiency (e.g., carbon allocation) or optimize photoperiod adaptation—critical traits for breeding high-yield cultivars in changing climates. For example, CRISPR-mediated editing of miR319-targeted CfuTCPs might improve leaf morphology and stress resilience [52].

Accumulated evidence suggests that TCP genes regulated by miR319 are widely involved in plant development and response to environmental stress [53]. By comparing the transcriptome of C. fungigraminus under cold stress and drought stress, it was found that miR319 targeted 12 CfuTCPs with different response expression patterns. The expression levels of CfuTCP9/40 and CfuTCP29/34 were both lower than normal conditions under drought and cold stress, respectively. The expression levels of CfuTCP15/10/28/32/4/20 under drought and cold stress were more than twofold increase than those under normal conditions. This can provide a molecular basis for further elucidating the regulatory network mediated by the miRNA-target module and revealing the regulatory mechanism of C. fungigraminus in environmental resistance. In molecular breeding, targeted mutagenesis of miR319 binding sites using gene-editing technologies (overexpression or CRISPR/Cas9) can attenuate its inhibitory effect on TCP genes, thereby enhancing stress tolerance. For instance, Overexpression of miR319a in Populus tomentosa leads to a significant increase in leaf epidermal hair density and improve insect defenses; Inhibition of endogenous miR319a leads to a reduction in epidermal hair and insect resistance [54].

TCP transcription factors play an important regulatory role in the growth and development of plants, not only related to plant growth, but also affecting organ development, environmental adaptability, and morphogenesis [55]. In cassava, over 20% of the TCP family members respond to drought and cold stress treatments [56]. In chrysanthemum, miR319 and its predicted target genes CnTCP2/4/14 can rapidly respond to cold treatment at the expression level. Furthermore, overexpression of CnTCP4 in Arabidopsis leads to excessive sensitivity of the plants to cold stress, indicating that CnTCP4 may play a negative regulatory role in the response of chrysanthemum to cold stress [57]. Among the 40 CfuTCP genes, 24 were upregulated under low-temperature stress, with 13 showing more than twofold upregulation. CfuTCP32/28/36/27/01/18/19/08/17/23 exhibited the highest expression levels after 2 h of low-temperature treatment, whereas CfuTCP09/10/15/40/20/16/11/03/22 showed the highest expression levels after 24 h of low-temperature treatment. This is similar to the findings from the study of the TCP gene family in Lonicera japonica [58], indicating that these CfuTCP transcription factors may play different roles at various stages of the cold stress response. Although TCP responses to low-temperatures have been observed in many plants, the mechanisms by which TCP responds to cold signals and regulates downstream gene expression remain largely unknown. Further research is needed to elucidate the functions of these genes under low-temperatures and their relationship with cold signaling pathways.

Our analysis of the expression heat maps for the 40 CfuTCPs revealed that the majority of these genes were down-regulated under drought stress but up-regulated during rehydration. This pattern suggested that most members of the TCP gene family may share a conserved function in the plant’s response to drought stress. However, the expression patterns of certain CfuTCPs, including CfuTCP27/29/23/04/31/20/36 were significantly up-regulated on the 7th day of drought stress; Similarly, CfuTCP19/32/37/38/28 exhibited high expression levels on the 14th day of drought. These findings suggested that these specific genes may play crucial roles in the plant’s response to drought conditions at different stages, underscoring the complexity of the TCP gene family’s involvement in stress adaptation. Additionally, Eight CfuTCP genes containing MYB cis-elements implicated in drought-inducibility were selected from different branches for qPCR validation. The results confirmed that CfuTCP19/20/27/29/31 genes exhibited a positive regulatory effect under drought stress, among them, the expression patterns of CfuTCP27/31 genes were consistent with the expression patterns observed in the heatmap; Conversely, the CfuTCP06 gene exhibits reverse regulation under drought stress (Fig. 7). Similar results have also been obtained in plants such as maize, Glycine max, Solanum tuberosum and Cicer arietinum [59, 60]. For example, five CaTCP genes (CaTCP3/13/15/20/21) containing MYB cis-elements in C. arietinum are strongly induced under drought conditions [61]. Our results highlight the potential of CfuTCP19/20/27/29/31 as key genetic targets for improving drought tolerance in crops through molecular breeding.

Interestingly, The expression levels of some genes showed a trend of initially rising and then declining under drought stress. The reason for this may be that the plant is the first to sense the stress experienced by the plant, inducing the expression of these genes to respond to the stress. As the severity of the stress increases, the upstream microRNA is activated, reducing the transcription levels of its target TCP. Similar results were also obtained in the study by Fan et al. [54]. The conservation of the TCP gene family’s function across different plant species, along with the specific expression patterns of CfuTCPs under stress conditions, underscores their importance in the adaptation to various environmental challenges.

Conclusions

TCP transcription factor proteins are known to play crucial roles in various aspects of plant growth and development. In this study, we identified 40 TCP genes in C. fungigraminus through phylogenetic analysis and classified them into two main branches and three subfamilies. Our comprehensive analysis, which included sequence alignment, motif analysis, gene structure, and chromosome localization revealed that CfuTCPs were conserved and exhibited four instances of gene tandem duplication events. Collinearity analysis suggested that gene duplication may be a primary factor influencing the expansion and evolution of the TCP gene family in C. fungigraminus. Cis-element and GO/KEGG analyses indicated that CfuTCPs are regulated by light and various hormones, participating in biochemical metabolic pathways such as circadian rhythm and environmental adaptation. The differential expression of CfuTCPs under drought and cold stress conditions indicated that these genes are involved in stress response, thereby contributing to the plant’s ability to withstand environmental challenges. qPCR and RNA-seq analysis indicated that CfuTCP27/31 may play a positive regulatory roles in response to drought stress, while CfuTCP06 plays a negative. In summary, this study provides insights into the structure and expression patterns of the TCP genes in C. fungigraminus, serving as a reference for further understanding the involvement of TCP genes in plant growth, development, and environmental adaptation [10].

Authors' contributions

S.K.: Conceptualization, Methodology, Investigation, Formal analysis, Writing-original draft, Writing- review & editing; X. H. and M. Z.: Methodology, Writing-review; L. L.: Data curation and Sample collection. Y. C. and Y. C.: Data curation, Writing-review; Y. H.: Conceptualization, Supervision, Writing-review & editing, and Funding acquisition; All authors reviewed the manuscript.

Funding

This study was supported by Guangdong Basic and Applied Basic Research Foundation (2023A1515012772, 2024A1515011721) and College Students’ Innovation and Entrepreneurship Training Program in Sun Yat-sen Univeristy.

Data availability

Genome files of Cenchrus fungigraminus and Setaria italica were obtained from National Genomics Data Center (https://ngdc.cncb.ac.cn/); TCP proteins of Arabidopsis thaliana and Oryza sativa were obtained from TAIR2 (https://www.arabidopsis.org) and the Rice Genome Annotation Project (http://rice.uga.edu/), respectively. RNA-seq data were obtained from the NCBI SRA database, accession number: PRJNA632455(https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA632455), and PRJNA544770(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA544770/).

Declarations

Ethics approval and consent to participate

Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, was carried out in accordance with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.