Genome-wide identification and expression analysis of TCP transcription factors in Chrysanthemum indicum reveals their critical role in the response to various abiotic stresses

Abstract

Chrysanthemum indicum is an important medicinal plant that has a particularly strong effect on bacteria and viruses and has antioxidant, anti-inflammatory, and immunomodulatory properties. The genes of the TCP family, a group of plant-specific transcription factors (TFs), have been found to play a crucial role in the regulation of plant growth and development as well as resistance to abiotic stress. Nevertheless, no systematic analysis of the TCP family genes in C. indicum has been performed so far. In the present study, a total of 26 non-redundant CiTCP genes were identified in the genome of C. indicum. The TCP genes were categorized into three subgroups on the basis of the phylogenetic analysis: 7, 9, and 10 genes belonged to the CIN subgroup, CYC/TB1 subgroup, and PCF subgroup, respectively. All CiTCPs were unevenly distributed across the 9 chromosomes. TCP genes in the same subgroup showed similar gene structures and conserved motifs. Gene duplication analysis revealed that segmental duplications had a significant effect on the expansion of CiTCP genes. The analysis of cis-elements revealed that CiTCP genes may be involved in the regulation of plant development, hormone response and response to abiotic stress. Expression profile analysis of the transcriptome data indicated that CiTCP genes exhibited similar or distinct expressions within different tissues and under different abiotic stresses. According to the results of quantitative RT-PCR (qRT-PCR), the expression of 15 selected genes responded strongly to various abiotic stress factors. The results of our studies could provide comprehensive insights into the TCP family genes of C. indicum for further functional investigations.

Introduction

The Teosinte Branched1/Cycloidea/Proliferating Cell Factors (TCPs) are a group of plant-specific genes that encode TFs (transcription factors) with a TCP domain [1], and were named after the first four identified members: TEOSINTE BRANCHED1 (TB1) from Zea mays, CYCLOIDEA (CYC) from Antirrhinum majus, PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR 1 and 2 (PCF1 and PCF2) from Oryza sativa [2,3,4]. These members were characterized by the TCP domain, a highly conserved 59-amino acid basic helix-loop-helix (bHLH) structure at the N-terminus that is involved in DNA binding, protein–protein interaction and facilitating nuclear localization [5]. According to their conserved domain, TCP proteins can be divided into two classes: Class I (represented by PCF proteins) and Class II (represented by CYC and TB1) [6]. The most obvious difference between the two subfamilies was that class I members lack 4 amino acids in the basic region of the TCP domain. TCP members belonging to Class II can be further divided into two subclades: CIN and CYC/TB1 [1]. Most of the members of Class II contain a conserved R domain, which may be responsible for facilitating protein–protein interaction [7].

Previous studies showed that Class I genes have generally been associated with cell division and proliferation, whereas Class II genes have mainly been involved in plant stress resistance and lateral organ development [8, 9]. Class I-members have been shown to be involved in the regulation of seed germination [10], flowering [11], stem elongation [12], stamen filament elongation [13] and various hormone signaling pathways in Arabidopsis thaliana. TCP14 and TCP15 have been found to mediate GA-dependent activation of the cell cycle during germination and to regulate cell proliferation. In tomato, SlTCP12, SlTCP15 and SlTCP18 were preferentially expressed in the tomato fruit, suggesting a role during fruit development or ripening [14]. In Class II, the famous member TB1, has been found to participate in the determination of maize axillary meristem fate [3]. AtTCP18 and AtTCP12, were involved in suppressing bud outgrowth [15]. AtTCP1, a homologous gene of CYC, has been demonstrated to affect plant growth/development by regulating the expression levels of DWARF4, a brassinosteroid (BR) biosynthesis gene [16]. SlTCP9 and SlTCP7 have similar functions in the initiation and growth of axillary buds [17].

Although much of the research on TCP transcription factors has focused on plant growth and development and morphogenesis, in recent years there has been increasing evidence that TCP transcription factors also play an important role in plant responses to environmental stresses. TCP transcription factors regulate abiotic stress responses mainly by modulating hormone signaling, ROS scavenging, and stress-responsive gene expression. For instance, under high salt conditions, Arabidopsis transferred with PeTCP10 improved the antioxidant capacity of transgenic Arabidopsis plants by promoting catalase (CAT) activity and enhanced their tolerance to H₂O₂ compared with wild-type A. thaliana [18]. The heterologous expression of the rice gene OsTCP19 in A. thaliana has been shown to decrease water loss and the levels of reactive oxygen species in the plants. Additionally, this genetic modification enhances the accumulation of lipid droplets, thereby improving the stress tolerance of transgenic plants during both the seedling and mature stages of development [19]. Overexpression of ZmTCP42 in A. thaliana has been demonstrated to alter seed germination hypersensitivity to ABA and enhances its drought tolerance [20]. The GmTCP4 transcription factor plays an important role in signaling pathways such as ABA and ET, and the overexpression of GmTCP4 can increase the drought tolerance in soybean [21]. Above all, the TCP transcription factor family can actively participate in the plant response to abiotic stresses.

With the rapid development of genome sequencing technology, genome-wide identification and analysis of gene families have become common. To date, the TCP gene family has been identified in many different plant species. In eudicots, 24 TCP genes have been identified in Arabidopsis [22], 30 in tomato [14], 19 in strawberry [19], 34 in Pyrus bretschneideri [23], 36 in Populus trichocarpa [24], 31 in potato [25]. Among monocots, a total of 28, 66, 42, and 16 TCPs have been identified in Oryza sativa, Triticum aestivum, Panicum virgatum, and Phyllostachys edulis, respectively [26,27,28,29]. However, little is known about the TCP gene family in C. indicum.

C. indicum is an important germplasm resource for perennial herbs of the Asteraceae family. The main chemical components of the secondary metabolites are flavonoids, terpenoids and phenolic compounds, which have anti-inflammatory, antioxidant and immunoregulatory properties, so, the medicinal value of C. indicum is relatively high. Besides, Chrysanthemum is a traditionally famous flower in China, with rich colors and various shapes. It is widely used in landscaping and has high cultural value, ornamental value and economic value. C. indicum grows normally in the wild and is often affected by abiotic factors such as drought and low temperature during its growth, which greatly affect yield and quality. However, current research on C. indicum focuses on cultivation, breeding, chemical formation, genetic characteristics and medicinal value, and little is known about resistance to abiotic stress. Therefore, the study of the molecular regulatory mechanism of resistance to abiotic stress is of great importance for the breeding of new chrysanthemum cultivars. Unfortunately, systematic investigations of the TCP gene family in C. indicum have not been reported so far. Recently, the entire genome of C. indicum was sequenced [30], laying the foundation for identifying TCP genes in C. indicum. In the present study, 26 CiTCPs were identified. A systematic analysis and prediction on the structures and functions of TCPs was performed, including their physicochemical properties, phylogenetic relationships, gene structure, conserved motifs, chromosome locations, gene duplications, promoter cis-acting elements, and expression profiles of CiTCPs in different tissues and under different abiotic stresses. The results of this study provide a theoretical basis for further research into the potential functions and regulatory mechanisms of CiTCP genes in response to abiotic stress.

Materials and methods

Identification of TCP transcription factors in Chrysanthemum indicum

The whole genome data and annotation files were downloaded from the public website (https://figshare.com/projects/Chrysanthemum_indicum_genome_diploid/197683) [30]. To identify all potential TCP members in C.indicum, the TCP domain HMM profile (accession number PF03634) was downloaded from the InterPro database (https://www.ebi.ac.uk/interpro/) and then used as a query to perform an HMMER search against the C.indicum genome database using HMMER 3.0 with the threshold expectation value set to 1e- 20 [31]. All AtTCP and OsTCP proteins were then used as queries to search the C.indicum protein database using the BLASTP program with the default parameters [32]. Each matching sequence was then submitted to SMART (http://smart.embl-heidelberg.de/), Pfamscan and NCBI conserved domain search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to find the TCP domain. The sequences containing no or incomplete TCP domains were manually eliminated. The physicochemical protein properties of the CiTCPs were analyzed using the online website ExPASy (https://web.expasy.org/protparam). Prediction of the subcellular localization was performed using Softberry (http://www.softberry.com/).

Sequence alignment and phylogenetic analysis of CiTCP genes

Multiple protein sequences of CiTCPs, A.thaliana, O.sativa and P. trichocarpa were aligned using ClustalX 2.0 with default parameters. An unrooted phylogenetic tree based on the full-length protein sequence alignments was constructed with MEGA X software using the maximum likelihood (ML) method with the optimal replacement and 1000 bootstrap replications. The constructed phylogenetic tree was optimized using the online tool Evolview (http://www.evolgenius.info/evolview/#/). The TCP domain of all CiTCPs was visualized using the website Web logo online (http://weblogo.berkeley.edu/logo.cgi).

Chromosomal distribution and collinearity analysis

The genomic coordinates of the CiTCPs were extracted from the genome annotation files. The gene densities of the entire chromosomes were determined and visualized by MapGene2 Chrom (http://mg2c.iask.in/mg2c_v2.1/). The members of the different subgroups on the chromosomes are marked with different colors. Tandem duplicated genes were identified by their physical position on the individual chromosomes. Two or more genes located on the same chromosome were spaced 200 kb apart and showed more than 70% identity when analyzed with BLASTP, which can be defined as tandem duplication events. Segmental duplication events were identified using Multiple Collinearity Scan toolkit (MCScanX) (https://github.com/wyp1125/MCScanX) with default parameters [33]. The Circos program was used to draw collinearity maps to represent duplicated gene pairs [34]. The Ka/Ks calculator 2.0 was used to calculate the ratio between the non-synonymous rate (Ka) and the synonymous substitution rate (Ks) of duplicated genes [35].

Analysis of gene structures, conserved motifs and cis-element analysis of CiTCP genes

The corresponding CDS and DNA sequences of the candidate TCP genes in C. indicum were also retrieved from the genome database. The intron distributions, positions and phases of the CiTCP genes were extracted. Candidate CiTCP protein sequences were analyzed using MEME version 5.5.5 software (https://meme-suite.org/meme/) with the following parameters: maximum motif number was 10; minimum motif width was 6; maximum motif width was 50; and the distribution of motif occurrences was zero or one per sequence. Evolview was used to integrate and visualize images of the phylogenetic tree, gene structures and conserved motifs. The regions upstream of 2000 bp were used to search for regulatory elements.

Gene Ontology (GO) annotation and Protein–Protein Interaction (PPI) analysis

Gene Ontology (GO) analysis was carried out for the CiTCP genes from the EggNOG database (evolutionary genealogy of genes: Non-supervised Orthologous Groups (http://eggnogdb.embl.de/#/app/home). All genes of C. indicum served as a reference set. A Gene Ontology (GO) enrichment analysis was performed. TBtools was used to obtain the GO enrichment terms with corrected p-values (≤ 0.05) [36]. Statistical analyzes and mapping were performed via the Biozeron Cloud Platform (http://www.cloud.biomicroclass.com/CloudPlatform). The TCP protein sequences were uploaded to the STRING database (https://string-db.org/) for node comparison, and the relationships between major proteins were predicted based on Arabidopsis protein interactions.

Analysis of gene expression patterns of CiTCPs using RNA-Seq databases

RNA-Seq data were used to analyze the expression profiles of CiTCP genes in different tissues (roots, buds, tongue flowers, leaves, tubular flowers and stems) and under different abiotic stresses (cold, heat, UVB and Cd²⁺). Expression data of fragments-per-kilobase-per-million (FPKM) were retrieved from genome-wide RNA-Seq databases (unpublished). Heat maps were drawn based to gene expression values (FPKM) using the TBtools software.

Plant material and treatments

The wild type of C. indicum used in this study was obtained from the Chrysanthemum Research Center in Cold Land, Northeast Forestry University, Har bin, China (126°63′E, 45°72′N). The seeds that were full and uniform in shape were selected. The samples were then exposed to two layers of filter paper moistened with distilled water in round petri dishes at 25 °C for 3 days. The seedlings were transplanted into square plastic pots with vermiculite in a greenhouse for growth (16/8 h light/dark photoperiod, 60%–70% relative humidity). Four-week-old C. indicum seedlings were subjected to different treatments. For cold stress, C. indicum seedlings were transferred to an artificial climate chamber at 4 °C. For drought stress, the plants were irrigated with 10% polyethylene glycol (PEG) 6000. For salt stress, 100 mM NaCl was used to simulate salt stress. Leaves were sampled at 0, 0.5, 2, 6, 12 and 24 h, and the samples treated for 0 h were used as controls. Leaf samples from each treatment were collected three times. All samples were immediately stored at − 80 °C prior to RNA extraction.

RNA isolation and quantitative real-time PCR (qRT-PCR) for CiTCP genes

Total RNA was isolated from leaves using the E,Z,N,A,TotalRNAKitI-R6827, according to the manufacturer’s protocol. The Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2 was used to perform the reverse transcription experiment. The quantitative real-time PCR reaction was performed to analyze the relative transcript levels of selected genes with the Light-Cycle 96 instrument using the SYBR Green Premix Pro Taq HS qPCR Kit III (Low Rox Plus). The reaction was performed as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C, for 5 s, and 55 °C for 30 s, 72 °C for 30 s. Each reaction was performed in three biological replicates, and relative expression was calculated using the 2 − ^∆∆Ct method. The results were analyzed as mean ± SE. The primers used in this study were designed with Primer5.0 and are listed in Additional file 8.

Results

Identification of TCP genes in C. indicum

A total of 26 CiTCP genes were identified in the genome of C. indicum by HMMER and local BLASTP searches. These genes were designated as CiTCP1 to CiTCP26 based on their chromosomal distribution. Details of the physical and chemical properties of the CiTCP genes are given in Table 1. The length of the CiTCP proteins varied between 154 (CiTCP 11) and 440 (CiTCP 7) amino acid residues. The theoretical isoelectric point (PI) values ranged from 5.55 to 9.99. CiTCP11 showed the lowest molecular weight value (17.01 kDa), while the highest molecular weight (47.50 kDa) was observed for CiTCP7. The predicted subcellular localization revealed that most CiTCP proteins are localized in nuclears (14), while the rest are localized in the extracellular spaces (12).

Table 1 The detail information of 26 CITCP genes identified in present study

Phylogenetic analysis and classification of TCP members in C. indicum

To investigate the evolutionary and phylogenetic relationships among the C. indicum TCPs, the full-length amino acid sequences of the TCP members from C. indicum (26) and the model species A. thaliana (22), O. sativa (18) and P. trichocarpa (35) were used to construct a phylogenetic tree (Fig. 1). In accordance with the classification of TCPs from other known species, 26 CiTCPs can be divided into two subfamilies: Class I and Class II, Class I (PCF) contains 10 members, and 16 members are assigned to Class II which in turn can be divided into two subgroups: CIN (7 members) and CYC/TB1 (9 members). To confirm the reliability of our phylogenetic tree generated by the maximum likelihood (ML) method, we use the neighbor-joining (N-J) method. The result showed that the classification of CiTCP genes using the two methods was consistent.

Fig. 1

Phylogenetic analysis of TCP transcription factors from Chrysanthemum indicum, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa. ClustalW was applied for the alignment of protein sequences. Maximum-likelihood method with 1000 bootstrap replicates was utilized to construct the phylogenetic tree in MEGA X software. Different colors represent different sub-classes in the TCP gene family. The TCPs from Chrysanthemum indicum, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa are marked with blue star, green square, red circle and yellow triangle, respectively

To further explore the evolutionary relationships of the CiTCP genes, we also performed conserved domain sequence alignment analysis with all TCP protein sequences in C. indicum. The results (Additional file 1 and Fig. 2) revealed that all the CiTCP genes contained a conserved basic region at the N-terminus and a helix-loop-helix motif at the C-terminus. Consistent with the TCP domain structure in other plant species, the TCP members in Class I have four fewer amino acid residues than Class II members in the basic region. These results indicate that the TCP gene family, together with other plant species, has a high level of conservation. In addition to the TCP domain, only a few members in Class II have an R domain with 18 amino acid residues. As shown in Additional file 2, eight Class II TCP proteins contained the R domain at the C-terminus of the TCP domain, seven of which were CiTCPs (CiTCP3, CiTCP5, CiTCP6, CiTCP13, CiTCP21, CiTCP22 and CiTCP23) from the CYC/TB1 subgroup, CiTCP24 from the CIN subgroup. At the same time, we found that the R domain was more conserved in the CYC/TB1 subgroup compared to the CIN subgroup.

Fig. 2

Multiple sequence alignment of CITCPs. a Alignment of the TCP domain in all identified TCP TFs; b The sequence logo was constructed by Weblogo, the height of each letter representing the corresponding amino acid the frequency of occurrence

Analysis of chromosomal localization and duplication events of TCP gene in C. indicum

Our initial step was to map all identified CiTCPs on the chromosomes (Fig. 3) to investigate the molecular mechanisms behind C. indicum's expansion. During the analysis, it was found that 8 out of 9 chromosomes had 26 identified TCP genes distributed unevenly, apart from chr05. With the highest proportion of TCP genes, Chr08 was the most prominent TCP and contained 8 members (30.7%) of the total chromosomes. Chr07 harbored the second largest number of TCP genes, with a total of five. There were a small number of TCP genes present on chr01, chr03, and chr09, with each having three members. Two members were present in chr02, while chr04 and chr06 each had a single member.

Fig. 3

Chromosomal locations of Chrysanthemum indicum TCP genes. The chromosomal position of each CITCP was mapped according to the genome of C. indicum. The chromosome number is marked at the top and the ratio on the left represents the chromosome length with the unit for the scale is mega bases (Mb)

A syntenic analysis of the CiTCP family genes was performed to gain a more in-depth understanding of their expansion and evolution. The overarching collinearity analysis revealed that five pairs of segmental duplications were identified (Fig. 4), whereas no tandem duplication events were found (Fig. 3), indicating that segmental duplication was the primary driving factor for the expansion of the TCP gene family in C. indicum. Collinearity maps of the evolutionary relationships among 26 CiTCPs between C. indicum and Arabidopsis, G. max, P. trichocarpa and Vitis vinifera. According to the syntenic maps (Fig. 5), the TCP genes in C. indicum had the most homologous gene pairs in G. max (39 orthologous gene pairs), followed by V. vinifera (24 orthologous gene pairs), A. thaliana and P. trichocarpa (7 orthologous gene pairs), and fewer homologous gene pairs in O. sativa presented 1 orthologous gene pair (Additional file 3). The evolutionary selection pressure on the CiTCP family was investigated by calculating the Ka/Ks ratios. The results showed that the Ka/Ks ratio of the duplicated gene pairs was less than 1.0, indicating that the gene pairs were subjected to strong purifying selection pressure after duplication events (Table 2).

Fig. 4

Schematic representation of the inter-chromosomal relationships of TCP genes in C. indicum. Gray lines in the background indicate the synteny blocks within the whole C. indicum, and the redlines denote the segmental duplication TCP gene pairs

Fig. 5

Synteny relationships of TCPs in Chrysanthemum indicum and five representative species. Synteny relationships of TCPs between Chrysanthemum indicum with (a) Arabidopsis thaliana, (b) Oryza sativa, (c) Glycine max, (d) Vitis vinifera, (e) Populus trichocarpa

Table 2 The duplicated genes in CiTCPs

Analysis of the gene structure and conserved motifs of the CiTCP gene family

To enhance understanding of the evolutionary connections between the various members of the CiTCP gene family, a map was created that depicts the phylogenetic tree, exon–intron structure, and conserved motif. The organization of 26 CiTCP members is highly conserved and simple, as demonstrated in Fig. 6. The remaining members had only one exon, while three out of 26 CiTCP genes had two coding regions (CDS). CiTCP3 and CiTCP19 were particularly notable for having two introns, whereas the remaining genes had simple structures that could consist of either one or no introns. All members had one or more UTRs in untranslated regions. The distribution patterns of exons and introns in CiTCP genes within the same subgroup mirrored those of their counterparts in terms of length and number, which substantiated the classification of subgroups and evolutionary relationships.

Fig. 6

Phylogenetic relationships, gene structure analysis, conserved motifs of CITCPs. Details of clusters in the phylogenetic tree are shown in different colors. The left panel represents the motif composition of the CITCPs. The right panel shows the intron–exon structure of the CITCPs. Different motifs are indicated by different colors. Introns are represented by black lines, exons by green boxes and UTR by blue boxes

Analysis of possible conserved motif compositions in the TCP gene family was performed to gain insight into the diversity and functional properties of the TCP genes in C. indicum. The MEME program was utilized to identify and annotate the sequences of these motifs, and Additional file 4 contains detailed information about them. As shown in Fig. 6, the highly conserved TCP domain (motif 1) was detected in all CiTCP proteins. The conserved R domain (motif 3) was present in 10 members of Class II (Additional file 4). Also, the N-terminal TCP domain of motif 4 was detected in all class II members. It was found that certain subgroups contain multiple distinctive motifs, including motif 2, motif 6, motif 7, and motif 8, in PCF subgroup proteins according to the results. Although the motif organization of the TCPs differed among the different subgroups, it was consistent with the phylogenetic tree, indicating that functional similarity and motif compositions may have contributed to functional divergence in the evolutionary process.

Analysis of Cis-acting elements in the promoters of CiTCPs

Differences in the transcript expression and biological function of genes can be revealed by revealing the crucial regions in gene promoters that initiate transcription at transcription factor-binding sites. 26 CiTCPs were subjected to cis-element analysis in order to obtain more valuable information. The results (Additional file 5 and Fig. 7) showed that 36 cis-acting elements, which included light responsive, hormone responsive, development related, and environmental stress-related elements, were identified and categorized into four categories. Light responsive elements, which are frequently found in TCP genes, accounted for the highest proportion. The Box 4, G-box, and GT1-motif elements stand out in terms of prominence. The second most distributed element was found to be the hormonal response elements, following the light responsive elements. Except for CiTCP3, CiTCP16, and CiTCP23, all CiTCPs had one or more ABREs. The promoters of 20 CiTCPs contain the CGTCA and TGACG patterns that are associated with the MeJA response. Numerous elements that respond to auxin (AuxRR-core and TGA-elements), ethylene (EREs), salicylic acid (TCA-elements), and gibberellin (P-boxes) are present in certain CiTCPs. Moreover, six types of elements related to plant growth and development were present in 21 members in small numbers, such as the CAT-box in 11 members, and the CCGTCC-box, circadian, GCN4_motif, O2-site and RY-elements in 5, 5, 6, 6 and 4 CiTCP genes. Additionally, we discovered seven types of cis-acting elements that cause environmental stress, and ARE elements were the most plentiful (68). The second highest number of W-box elements (46) was found in 17 CiTCP genes after that. The presence of MBS elements in half of the members suggests that drought stress may regulate these genes. CiTCP5, CiTCP14, and CiTCP24 contain DRE, which is a component of dehydration, low-temperature, and salt stresses. 13 CiTCP members had LTRs that were responsible for the low-temperature responsiveness with a number of 18. In 12 genes, there were TC-rich repeats and WUN-motifs that were observed.

Fig. 7

Analysis of cis-elements in the promoter of the CITCP genes. The phylogenetic tree of 26 CITCPs was present on the left. The middle panel showed the heatmap of cis-elements in the promoter of the CITCP genes. The number of cis-elements for each CITCP gene in four categories

GO enrichment analysis and PPI prediction

Using gene ontology (GO) to categorize CiTCPs into functional groups, it was determined that they were only assigned to one category: biological processes (15 terms) (Fig. 8 and Additional file 6). In the biological process, the 22 genes were found to be associated with RNA biosynthetic process, RNA metabolic process, cellular biosynthetic process, macromolecule biosynthetic process, nucleobase-containing compound metabolic process, primary metabolic process, nitrogen compound metabolic process, biosynthetic process, macromolecule metabolic process, cellular metabolic process, metabolic process, biological process. It can be inferred from these annotations that the CiTCP genes are involved in biological processes.

Fig. 8

Functional analysis of CiTCPs. a GO enrichment analysis of CiTCP genes; vertical axis indicates GO terms; horizontal axis indicates Rich factor. the larger the Rich factor, the stronger the enrichment. The size of the dots indicates the number of genes in the GO terms; b Protein–protein interaction analysis among CiTCPs by the STRING database. The results were based on an Arabidopsis association model

A protein–protein interaction (PPI) network was constructed based on STRING download data to predict the function of the CiTCP family genes. As shown in Fig. 8, members in the same subgroup have the same homologous genes, such as, CiTCP3/5/6/12/13/21/22 belong to the CYC/TB1 subgroup have high homology with AtTCP12, CiTCP7/24 belong to the CIN subgroup have high homology with AtTCP14. It should be noted that SAP11, zinc finger AN1 and C2H2 domain-containing stress-associated protein 11, may be responsible for the environmental stress response. According to the map, SAP11 can interact with TCP12/18/4/15/17/5/6/24/20/3/13/14/2/SPEAR3, while CiTCP3/5/6/12/13/21/22 shared homology with AtTCP12, CiTCP18 shared homology with AtTCP18, CiTCP10/26 shared homology with AtTCP4, CiTCP17 shared homology with AtTCP15, CiTCP14/15/16 shared homology with AtTCP5, CiTCP19/11 shared homology with AtTCP20, CiTCP20 shared homology with AtTCP13, CiTCP2/4 shared homology with AtTCP14, CiTCP7/24 shared homology with AtTCP2, interacts with SAP 11. According to the map, SAP11 can interact with TCP12/18/4/15/17/5/6/24/20/3/13/14/2/SPEAR3, while CiTCP3/5/6/12/13/21/22 shared homology with AtTCP12, CiTCP18 shared homology with AtTCP18, CiTCP10/26 shared homology with AtTCP4, CiTCP17 shared homology with AtTCP15, CiTCP14/15/16 shared homology with AtTCP5, CiTCP19/11 shared homology with AtTCP20, CiTCP20 shared homology with AtTCP13, CiTCP2/4 shared homology with AtTCP14, CiTCP7/24 shared homology with AtTCP2, interacts with SAP 11. The above results indicate that CiTCP proteins tend to form protein complexes to exercise their functions, and it is hypothesized that the possible diversity of CiTCP gene functions is related to the diversity of interacting proteins. Although the interaction network of TCP proteins in C. indicum needs to be further verified, our results provide an important theoretical basis for exploring the molecular mechanism of TCP genes in C. indicum.

Tissue specific expression patterns of CiTCPs

Tissue-specific expression patterns of all CiTCP genes were analyzed using RNA-Seq data to gain a better understanding of their potential roles in C. indicum development. The CiTCP genes were found to have obvious tissue specificity in the results, as shown in Additional file 7 and Fig. 9. The expression of all CiTCP genes was detected in least one tissue, in which 12, 11, 10, 11, 13 and 13 genes presented high transcript abundance (FPKM > 5) in the bud, leaf, tubular flowers, tongue flowers, root, stem tissues, respectively. CiTCP genes, such as CiTCP1, CiTCP7, CiTCP8, CiTCP10, CiTCP17, CiTCP25, and CiTCP26, have identical expression profiles in different organs and tissues, as demonstrated by Fig. 9. These genes, which are ubiquitously high expressed in all tissues, include CiTCP1, CiTCP7, CiTCP8, CiTCP10, CiTCP17, CiTCP25, and CiTCP26. Alternatively, other CiTCPs exhibited tissue-specific transcript accumulation patterns, which could suggest the functional diversification of CiTCP genes during growth and development. For example, CiTCP3, CiTCP6, CiTCP9, CiTCP12, CiTCP13, CiTCP16, CiTCP18 were expressed at a very low level in all tested tissues. Additionally, certain CiTCP genes displayed a high level of expression in particular tissues (FPKM > 20). For instance, CiTCP15 and CiTCP17 displayed high expression level in buds than other tissues, CiTCP1 and CiTCP8 presented high transcript abundance than other tissues, demonstrating that key roles of these genes in tissue development. It should be noted that some genes that were duplicated exhibited similar expression patterns. For example, CiTCP3 and CiTCP21 showed relatively high expression level in stem, while very low expression in other tissues. The expression levels of CiTCP4 and CiTCP17 were more elevated in bud, tongue flowers, and root compared to other tissues.

Fig. 9

Expression profiles of CITCPs in different tissues under normal conditions (A), and in response to cold (4 ℃), heat (42 ℃), Cd (200 μmol) and UVB (B) from RNA-seq data. Color scale at the right of the heatmap shows the expression level, red indicates high transcript abundance while green indicates low abundance

Response of CiTCP genes to different abiotic treatments using RNA-Seq data

Cadmium (Cd²⁺), cold, hot and UV-B are common stresses that affect growth and reduce the production of C. indicum. To understand the potential functions of the CiTCPs in response to these different stresses, we extracted RNA-Seq data of the four stresses (cold, 4℃, 6 h, hot, 42℃, 6 h, UV-B, 24 h, Cd²⁺, 200 μmol, 24 h, listed in Additional file 7). According to the results, the CTTCP genes responded to cold and UV-B stress to a greater extent than to hot and Cd²⁺ treatments (Additional file 7 and Fig. 9). Among them, CiTCP4, CiTCP15 and CiTCP25 significantly induced against the cold treatment, while CiTCP1 and CiTCP26 were down-regulated. Under UV-B stress, most genes were significantly down-regulated, such as CiTCP15, CiTCP24, CiTCP3, CiTCP8 and so on, CiTCP4, CiTCP11, CiTCP17, CiTCP19 and CiTCP25 were up-regulated. In response to hot stress, eight genes showed an increased expression patterns and 14 genes were more or less reduced, the remaining members could not be detected in hot response. We deducted these genes as pseudogenes or may be expressed only at specific developmental stages or under special conditions. For the Cd²⁺ treatment, CiTCP4, CiTCP7, CiTCP17, CiTCP26 were significantly down-regulated, CiTCP1, CiTCP5, CiTCP8, CiTCP12, CiTCP20 were up-regulated. Most members of the CiTCP family showed diverse expression patterns under different abiotic stresses. Interestingly, we found that CiTCP25 was up-regulated under all treatments, suggesting that it might be a candidate gene for mitigating abiotic stresses.

Validation of the expression profile of selecting CiTCP genes by qRT-PCR under abiotic treatments

To delve deeper into the potential impact of different stress treatments on the expression of these CiTCP genes, a subset of 15 members from various subgroups were subjected to qRT-PCR (Fig. 10). The qRT-PCR results showed that most of the tested CiTCP genes were induced potentially under different stresses and the expression levels of these genes were correlated with the RNA-Seq data. Under cold stress, the tested TCP genes showed different expression patterns. For example, CiTCP4 and CiTCP13 were significantly up-regulated with 12-fold and twofold increase, while CiTCP1, CiTCP2, CiTCP8, CiTCP10 and CiTCP20 were down-regulated in response to low temperature treatment at 0.5 h, indicating these genes were regulated for a rapid response to cold stress. Compared with the untreated leaves, CiTCP2/9/12/13/16/22 were dramatically up-regulated and CiTCP1/4/8/10/14/21 were down-regulated after 2 h cold treatment, suggesting these genes may be cold acclimation genes. After 6 h treatment, which generated 4 genes (CiTCP1/9/12/13) were significantly up-regulated and 4 genes (CiTCP8/14/21/22) were down-regulated, suggesting that these genes may interact with other proteins to initiate a cascade of downstream signal pathway. CiTCP1/4/9/14/16 were up-regulated and CiTCP2/10 were down-regulated after 12 h cold treatment. CiTCP1/4/12/14/16/18 were up-regulated and CiTCP2/8/21/22 were down-regulated after 24 h treatment, indicating that these genes could be crucial for long-term cold acclimation.

Fig. 10

Expression pattern of C. indicum TCPs in response to cold stress determined by qRT-PCR. The Y-axis indicates the relative expression level and the X-axis represents different time points after stress treatment taken for expression analysis. The data presented are the average of three biological replicates, the bar represents the standard deviation

Under drought conditions (Fig. 11), nine members (CiTCP1/2/10/21/8/9/14/16/18) showed similar expression patterns, they each had a sharp increase and reached their peak value at early point, then had a down trend. For example, the expression of CiTCP1 and CiTCP8 increased at 0.5 h but as the treatment time increased, the expression level subsequently decreased. The expression of CiTCP16 and CiTCP21 reached the highest at 6 h and then decreased gradually. CiTCP2 and CiTCP22 reached the highest at 12 h. However, the expressions of CiTCP12 and CiTCP13 were significantly down-regulated at 2 h and 6 h, and then increased gradually. However, the expression level of CiTCP7 and CiTCP20 were not changed significantly.

Fig. 11

Expression pattern of C. indicum TCPs in response to drought stress determined by qRT-PCR. The Y-axis indicates the relative expression level and the X-axis represents different time points after stress treatment taken for expression analysis. The data presented are the average of three biological replicates, the bar represents the standard deviation

For the salt stress (Fig. 12), CiTCP1 was down-regulated at early points, then increased and peaked at 24 h. CiTCP16 and CiTCP10 were down-regulated at all time periods. CiTCP2/4/21/7/8/18/22 were all up-regulated and peaked at 6 h, with CiTCP4 peaking 12-fold up-regulated of salt stress. CiTCP12/13/9/14 were all up-regulated and peaked at 0.5 h, with tenfold up-regulated. CiTCP1 which was down-regulated and then up-regulated. CiTCP22 showed an up-regulated, down-regulated and then up-regulated expression pattern. NaCl had no significant effect on the expression levels of CiTCP20.

Fig. 12

Expression pattern of C. indicum TCPs in response to salt stress determined by qRT-PCR. The Y-axis indicates the relative expression level and the X-axis represents different time points after stress treatment taken for expression analysis. The data presented are the average of three biological replicates, the bar represents the standard deviation

Collectively, the results confirm that these CiTCP genes might be involved in response to stress, which is consistent with the result of cis-acting element analysis in the promoters.

Discussion

C. indicum is an important resource plant with high medicinal and ornamental value. TCP genes play important roles in plant developmental and physiological process, as well as various plant biotic and abiotic stress responses. In this study, we performed a comprehensive identification and analysis of TCP genes in C. indicum, with an aim of exploring the potential roles of this gene family in regulation of development and stress resistance in C. indicum.

Totally, we have reported a total number of 26 CiTCPs by complete genome screening of C. indicum using two search methods. There is a difference in the number of TCP gene family members between different species. A total of 26 TCP members were identified in the C. indicum genome, the size of gene number is closely related to Arabidopsis (24), tomato (30), potato (31), O. sativa (28), P. trichocarpa (36), but significantly differ with strawberry (19), P. edulis (16), T. aestivum (66), P. virgatum (42) [14, 19, 21, 23,24,25,26,27,28,29, 37, 38]. In general, there are two possible causes for the difference. Firstly, the size of the plant genome may affect the scale of the TCP gene family. As an illustration, C. indicum has a genome size of more than 3 Gb, and T. aestivum has a genome size of over 15 Gb. Despite having a genome that is over 1 Gb smaller than C. indicum, P. virgatum has a higher TCP gene count than C. indicum. The TCP family members in Arabidopsis and O. sativa are similar to those in C. indicum, despite having genomes of 135 Mb and 1.15 Gb. The duplication event is another factor contributing to the difference, and it could be the main reason for the expansion of the TCP gene family. C. indicum has encountered at most two WGD events. Likewise, Arabidopsis and P. trichocarpa have also encountered two and one WGD events, respectively [30, 39, 40].

Phylogenetic analysis (Fig. 1) and protein sequence alignment (Fig. 2) demonstrated that the 26 CiTCP members could be classified into two main classes (Class I and Class II) and three subgroups (PCF/CIN/CYC/TB1), which is similar with the classification of TCP genes in other species. We found that the homologous genes of 26 CiTCP genes were almost from either of Arabidopsis and P. trichocarpa (Fig. 1), indicating that CiTCP gene family has the evolutional conservation and closer homology relationship with closely related species. Furthermore, the distribution of TCP genes within each subgroup assumed that dicotyledonous plants occupy a larger proportion, suggesting that TCP genes underwent expansion from a common ancestor before angiosperm evolution and speciation. The TCP gene family featured by the conserved TCP domain, which forms a bHLH structure [7]. Be similar to the mechanism of the amphipathic helix (K region) in the MADS-BOX proteins, the bHLH domain may regulate protein–protein interaction in TCP genes. Also, most members in the CYC/TB1 clade possessed the R domain with unknown function might facilitate protein–protein interaction [6]. According to the phylogenetic analysis, CiTCPs shared similar motif compositions and gene structures were clustered together, further supporting the close evolutionary relationship among CiTCP genes.

Tandem, segmental, and whole-genome duplication are important sources of the functional diversity and evolution of gene families [37]. For detection of the expansion mechanism in CiTCP gene family, we first identified the tandem duplication and segmental duplication event. As shown in Fig. 3, most CiTCP genes distributed on chromosomes but clustered, there is no tandem duplication event was identified in these clusters with BLASTP method. In contrast, five segmental duplication events involving 26.9% (7/26) CiTCP genes were identified (Fig. 4 and Table 2). Also, all the CiTCP genes were segmentally duplicated within one subgroup to expand their members. Our results are largely similar with that described in orchard grass, also in A. thaliana and O. sativa, indicating that TCP duplication in plant genomes possibly has a common mechanism [26]. In P. trichocarpa TCP gene family, 13 pairs of duplicated genes were identified, accounting for about 72% of the P. trichocarpa TCP family, and they result from segment duplications rather than tandem duplications [24]. For the switchgrass, no tandem repeats occurred in the evolutionary process in switchgrass TCP genes, the large enrichment of switchgrass TCP genes was presumably due to the allotetraploid event [28]. The duplication mechanisms vary among different species, which suggests that different duplication events played a different role in the history of evolution. Comparative mapping (Fig. 5) established the orthologous and paralogous relationship among dicotyledonous and monocotyledonous species. Interestingly, the presence of 39, 24, 7 and 7 collinear pairs between C. indicum with G. max, V. vinifera, A. thaliana and P. trichocarpa, respectively, but only one pair in O. sativa, may suggest that these orthologous pairs may be involved in divergence of dicot and monocot plants. Different species have different collinear pairs with C. indicum, which suggests that these orthologous pairs were created during the divergence between dicot and monocot plants. The high conservation of TCP proteins across different species suggests that C. indicum shares significant functional similarity with other plants. This suggested that TCP genes could be closely related to different species, along with similar biological functions. In terms of every subgroup, the PCF subgroup contained the most segmental duplication events with three pairs, but the expansion of gene number in this subgroup was small, almost comparable to the other species in the phylogenetic tree (Fig. 1 and Table 2). In contrast, the CYC\TB1 subgroup only possessed one pair duplication event, but the CiTCP genes in this subgroup were larger than other species. In the remaining subgroup, the number of TCP members in C. indicum was less than or equal to that in Arabidopsis, O. sativa and P. trichocarpa. Indeed, the Ka/Ks ratio results showed that the duplicated CiTCP genes were under strong purifying selection.

Expression pattern analysis could provide more insights into the potential roles of the TCP gene family. According to the qRT-PCR results (Fig. 10), the 15 selected genes from different groups showed different expression patterns under different stress treatments. For example, the expression of CiTCP10 was enhanced by drought but was declined under salt and cold stress. Also, the expression of CiTCP8 was down-regulated under cold stress, while it was up-regulated by salt and drought. These results suggest that CiTCP10 and CiTCP8 might have different mechanisms to maintain protection against various abiotic signals. We also found that some CiTCP genes were responsive to specific stresses. For instance, CiTCP13 was significantly induced by drought and cold, but not by salt. CiTCP18 was significantly induced by salt and cold, but not in drought. Interestingly, most members in the same subgroup showed different expression patterns. For instance, CiTCP10/14/16, belong to the CIN subgroup, CiTCP10 and CiTCP16 were significantly down-regulated by salt stress, while CiTCP14 were strongly induced. CiTCP2/4/8/9, belong to the PCF subgroup, CiTCP2, CiTCP4, and CiTCP9 were up-regulated by cold stress, and in contrast, CiTCP8 were down-regulated. Moreover, we examined the expression patterns of duplicated genes in different tissues. The results indicate that TCP genes that are clustered in segmental duplication pairs generally have similar expression patterns. For example, the segmental duplication pairs, CiTCP3 and CiTCP21 were abundantly expressed in stem, CiTCP15 and CiTCP17 were characterized by high expression in leaf and tongue flowers. The analysis of functional genes in C. indicum revealed that these genes play various roles in different stress conditions.

TCP proteins orchestrate a lot of plant developmental processes and respond to environmental stimuli. According to the prediction results of cis-regulatory elements in CiTCPs promoter, we found that TCPs contained many elements involving in abiotic stress response, such as, CiTCP5, CiTCP14 and CiTCP24 possessed DRE element involved in dehydration, low-temperature, salt stresses, more than half of CiTCP genes contained LTR element, suggesting that TCP genes play a significant role in stress response. For example, CiTCP25 contained LTR, MBS and TC-rich repeats elements in promoter, the expression of CiTCP25 were induced more than threefold after cold treatment. Also seen in CiTCP9, one TC-rich repeats element exhibited in promoter region, the expression changed more than 3 times compared to the 0 h. In G. max, most GmTCPs appeared to respond to heat, cold, and defense stress, which was similar with the results in C. indicum [38]. Stress-responsive cis-acting elements were present in all 26 CiTCP genes, indicating their potential roles in response to abiotic stresses (Additional file 5 and Fig. 7). It has been proven by many studies that TCP genes are involved in responding to various plant abiotic stresses. For instance, overexpression of ZmTCP42 in Arabidopsis led to a hypersensitivity to ABA in seed germination and enhanced drought tolerance. Here, qRT-PCR was used to analyze the expression of CiTCPs under multiple stress treatment (Fig. 10). The results showed that CiTCP2/9/14/16/21/22 were significantly induced against salt, cold and drought conditions, and all of them contained DRE, LTR, MBS and TC-rich repeats elements in their promoters, indicating that these two genes may integrate different stress signals. The functional information of CiTCPs can be predicted according to their identified orthologous in Arabidopsis and Rice. According to the PPI prediction (Fig. 8), CiTCP2/10/14/16/22 may interact with SAP11, and participatein many abiotic stress responses. Also, CiTCP10/14/16 belong to the CIN subgroup, CiTCP10 shared homology with AtTCP4. Previous studies showed that TCP4 may play crucial roles in plant resistance to abiotic. In addition, TCP4 targeted by miR319 s is involved in abiotic stress response such as high salt and drought have been demonstrated [39, 40]. This suggests the potential involvement of CiTCP10 in multiple abiotic stress responses and warrants further exploration of its specific functions in these conditions. CiTCP8, had homology with OsTCP19, belonged to the PCF subgroup, and was highly induced under salt and drought stress. Overexpression of OsTCP19 in Arabidopsis could improve the drought tolerance and the overexpression of GmTCP4 can increased the drought tolerance in soybean [18]. So, we suspect that CiTCP8 may also take part in the drought response in C. indicum. Verification of the initial findings requires further study of the results. Analyzing the biological functions of these genes will provide a significant theoretical basis for improving C. indicum quality.

Conclusions

In this study, 26 TCP genes were identified through comparative genomics analyses of sugarcane. Systematic informatics analyses were performed, including phylogenetic, conserved motif and intron/exon analyses. Duplicated analysis revealed that segmental supplication event contributed to the expansion of the gene family, and purifying selection was the main force driving evolution. Moreover, the expression levels of CiTCPs were analyzed in response to various treatments (UVB, Cd²⁺, hot, cold, drought, NaCl) based on available RNA-seq data and qPCR validation, indicating that CiTCPs played important roles in C. indicum stress tolerance. Furthermore, our findings provide the foundation for further functional characterization and identification of the regulatory mechanism of TCP genes in plant stress responses. Additionally, candidate CiTCPs can be employed for germplasm improvement using molecular breeding techniques and genome editing tools to enhance production.