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Article

Genome-Wide Identification and Analysis of TCP Gene Family among Three Dendrobium Species

by
Yaoting Li
1,2,†,
Lingli Li
3,†,
Jiapeng Yang
3,
Zhitao Niu
3,
Wei Liu
3,
Yi Lin
1,
Qingyun Xue
3,* and
Xiaoyu Ding
3,*
1
School of Life Sciences, Anhui Agricultural University, Hefei 230036, China
2
School of Biological and Pharmaceutical Engineering, West Anhui University, Lu’an 237012, China
3
College of Life Sciences, Nanjing Normal University, Nanjing 210023, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2023, 12(18), 3201; https://doi.org/10.3390/plants12183201
Submission received: 25 July 2023 / Revised: 28 August 2023 / Accepted: 1 September 2023 / Published: 7 September 2023
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Abstract

:
Dendrobium orchids, which are among the most well-known species of orchids, are appreciated for their aesthetic appeal across the globe. Furthermore, due to their strict living conditions, they have accumulated high levels of active ingredients, resulting not only in their medicinal value but also in their strong ability to respond to harsh environments. The TCP gene family plays an important role in plant growth and development, and signal transduction. However, these genes have not been systematically investigated in Dendrobium species. In this study, we detected a total of 24, 23, and 14 candidate TCP members in the genome sequences of D. officinale, D. nobile, and D. chrysotoxum, respectively. These genes were classified into three clades on the basis of a phylogenetic analysis. The TCP gene numbers among Dendrobium species were still highly variable due to the independent loss of genes in the CIN clade. However, only three gene duplication events were detected, with only one tandem duplication event (DcTCP9/DcTCP10) in D. chrysotoxum and two pairs of paralogous DoTCP gene duplication events (DoTCP1/DoTCP23 and DoTCP16/DoTCP24) in D. officinale. A total of 25 cis-acting elements of TCPs related to hormone/stress and light responses were detected. Among them, the proportions of hormone response, light response, and stress response elements in D. officinale (100/421, 127/421, and 171/421) were similar to those in D. nobile (83/352, 87/352, and 161/352). Using qRT-PCR to determine their expression patterns under MeJA treatment, four DoTCPs (DoTCP2, DoTCP4, DoTCP6, and DoTCP14) were significantly upregulated under MeJA treatment, which indicates that TCP genes may play important roles in responding to stress. Under ABA treatment, seven DoTCPs (DoTCP3, DoTCP7, DoTCP9, DoTCP11, DoTCP14, DoTCP15, and DoTCP21) were significantly upregulated, indicating that TCP genes may also play an important role in hormone response. Therefore, these results can provide useful information for studying the evolution and function of TCP genes in Dendrobium species.

1. Introduction

Transcription factors (TFs) specifically bind to the promoter region of eukaryotic genes, and they play important roles in regulating the transcriptional initiation of specific sequences, which are fundamental to both plant development and responses to external environment stimulations [1,2]. TCP transcription factors, named after the four founding members, TEOSINTE BRANCHED1 (TB1) from Zea mays L., CYCLOIDEA (CYC) from Antirrhinum majus L., and PROLIFERATING CELL NUCLEAR ANTIGEN FACTORS 1 and 2 (PCF1 and PCF2) from Oryza sativa L. [3,4,5], feature the TCP domain, a highly conserved 59-residue-long basic helix–loop–helix (bHLH) structure at the N-terminus, which is associated with DNA binding, protein–protein interaction, and protein nuclear localization [6]. TCP proteins are a small family of plant-specific transcription factors and play important roles in multiple processes of plant growth and development by regulating cell growth and proliferation [7,8,9]. For example, in Arabidopsis thaliana, AtTCP4 regulates jasmonic acid (JA) biosynthesis via the interaction with the JGB to further mediate pollen germination and gametophyte development. TCP14 and TCP15, both members of TCP class I, were shown to function in cell proliferation and in several plant hormone signaling pathways.
According to the sequences of TCP conserved domains and phylogenetic relationships, members of the TCP gene family can be divided into two subfamilies: clade I and clade II. Clade II is also known as the PCF subfamily, while clade I TCP members are further divided into the CINCINNATA (CIN) and CYC/TB1 subfamilies [8,10]. The most obvious difference between these two subfamilies is that the basic region of the TCP domain of the clade I subfamily has four amino acids more than that of the clade II subfamily. In addition, several members of clade I have another conserved region outside the bHLH domains, named R domain, which is an arginine-rich motif containing 18–20 residues [8,11]. For instance, AtTCP1 and OsTB1 were identified due to the R domain in Arabidopsis thaliana and Oryza sativa, respectively.
The TCP gene family can participate in different processes of plant development, such as seed germination [12]; cell proliferation [13,14]; and leaf [15,16], flower [17], axillary bud [18], lateral branching [19], and pollen development [20]. For example, Tatematsu et al., using microarray and reporter gene analyses, indicated that AtTCP14 regulates the activation of embryonic growth potential in Arabidopsis thaliana seeds. Takeda et al., by constructing transgenic Arabidopsis thaliana plants and performing reporter gene analysis, revealed that TCP16 plays a crucial role in early processes in pollen development. In addition, the TCP gene family plays an important role in the response to various abiotic stresses, such as salt stress [21,22], drought stress [1,23], low temperature, and short photoperiod [24]. The TCP gene family also influences developmental and abiotic stress signaling via hormone pathways [25,26].
Dendrobium, one of the largest genera in Orchidaceae, includes approximately 1800 species, mainly distributed in tropical Asia, Australasia, and Australia [27]. There are about 120 species in China, and many of them are of great horticultural and medicinal value [27,28]. For example, both Dendrobium officinale Kimura et Migo and Dendrobium nobile Lindl. are famous traditional Chinese medicines (TCMs) due to their rich content of active ingredients, such as polysaccharides, flavonoids, and alkaloids [7,29], resulting in their excellent medicinal merits [27]. Dendrobium chrysotoxum Lindl. is popular for its high medicinal value and aesthetic appeal, while it can also be used in traditional Chinese food. With the rapid development of sequencing technology, the chromosomal-level genomes of these three species (D. officinale, D. nobile, and D. chrysotoxum) have been assembled with high quality [27,30,31], making it possible for researchers to explore the evolution of the Dendrobium TCP gene family.
In this study, TCP genes were identified in D. officinale, D. nobile, and D. chrysotoxum, and their chromosomal positions, structure features, conserved motifs, and cis-acting elements were analyzed. Phylogenetic analysis, collinearity analysis of the TCP genes, and prediction of their protein–protein interaction (PPI) networks were conducted. Lastly, on the basis of the D. officinale genome, tissue-specific expression analysis and transcriptome analysis were performed on the coding genes under MeJA treatment.

2. Results

2.1. Identification and Chromosomal Location of the TCP Genes in D. officinale, D. nobile, and D. chrysotoxum

To identify TCP family genes in D. officinale, D. nobile, and D. chrysotoxum, we used the BLAST method and hidden Markov model (HMM) to determine TCPs from their genome sequences, using 23 of the 24 known TCP protein sequences from Arabidopsis as references (Table S1). A total of 24, 23, and 14 candidate TCP members were identified in D. officinale, D. nobile, and D. chrysotoxum, named DoTCP1 to DoTCP24, DnTCP1 to DnTCP23, and DcTCP1 to DcTCP14, respectively (Table S2). The TCP genes differed in their amino acid sequence length, isoelectric point (pI), and molecular weight (MW) among the three Dendrobium species (Table S2). For example, the amino acid sequence length ranged from 179 (DoTCP12) to 584 (DoTCP3) in D. officinale, while it ranged from 211 (DnTCP13) to 786 (DnTCP15) in D. nobile and from 200 (DcTCP8) to 629 (DcTCP1) in D. chrysotoxum. The isoelectric point (pI) ranged from 5.82 (DoTCP10) to 11.28 (DoTCP7) in D. officinale, while it ranged from 6.16 (DnTCP23) to 10.39 (DnTCP2) in D. nobile and from 6.16 (DcTCP6) to 10.39 (DcTCP1) in D. chrysotoxum. The protein molecular weight (MW) ranged from 20.02 kDa (DoTCP12) to 64.87 kDa (DoTCP3) in D. officinale, while it ranged from 22.32 kDa (DnTCP23) to 63.93 kDa (DnTCP2) in D. nobile and from 21.19 kDa (DcTCP8) to 70.26 kDa (DcTCP1) in D. chrysotoxum (Table S2).
The density and distribution of TCPs on chromosomes are shown in Figure 1. All 23 candidate DnTCPs in D. nobile were anchored on the chromosomes. On the other hand, 22 of the 24 were located on chromosomes of D. officinale, with DoTCP23 and DoTCP24 being instead located in unmapped regions (Figure 1A). Consistently, most DcTCPs were located on chromosomes of D. chrysotoxum, with only DcTCP14 being located in an unidentified region (Figure 1B,C). In D. officinale, chromosome 6 contained the most TCP genes, followed by chromosomes 2, 11, 14, and 15, which contained two TCP genes, and chromosomes 1, 5, 7, 8, 9, 16, 17, and 19, with only one TCP gene (Table S3; Figure 1A). In D. nobile, chromosomes 9, 13, and 17, contained three TCP genes, followed by chromosomes 5, 12, 14, and 18, which contained two TCP genes, and chromosomes 1, 2, 4, 6, 10, and 19, with only one TCP gene (Table S3; Figure 1B). In D. chrysotoxum, chromosome 14 contained the most TCP genes, followed by chromosomes 5 and 12, which contained two TCP genes, and chromosomes 4, 6, 10, 13, 16, and 18, with only one TCP gene (Table S3; Figure 1C).

2.2. Phylogenetic Relationship Analysis of TCP Gene Family

To explore the relationships among Dendrobium TCP genes, we conducted a phylogenetic analysis on 84 amino acid sequences, including 24 genes from D. officinale, 23 genes from D. nobile, 14 genes from D. chrysotoxum, and 23 genes from A. thaliana (Figure 2). The results inferred from the maximum likelihood (ML) evolutionary tree showed that the TCP genes were classified into three main classical subfamilies (class Ⅰ, class Ⅱ, and class Ⅲ). Among them, 28 genes (9 DoTCPs, 10 DnTCPs, and 9 DcTCPs) belonged to class I (PCF) (Figure 2). Class II and class Ⅲ represented CYC/TB1 (4 DoTCPs, 1 DnTCP, and 2 DcTCPs) and CIN (11 DoTCPs, 12 DnTCPs, and 3 DcTCPs), respectively (Figure 2).

2.3. Gene Structure Analysis and Conserved Motifs of TCPs

To better understand the TCP family genes in the three species of Dendrobium, we constructed three new evolutionary trees using the amino acid sequences of DoTCP, DnTCP, and DcTCP, respectively. Then, we analyzed the gene structure and conserved motifs of TCPs. Each of these three evolutionary trees of TCPs was divided into three branches (Figure 3). According to the CDS/UTR structure of each gene from the genome annotation files, we found that the gene structures of TCP genes were conserved (Figure 3). For example, the CDS of most TCP genes only had one exon, with only three genes containing one or two introns in each Dendrobium species (DnTCP6 and DnTCP22 with two introns and DnTCP12 with one intron in D. nobile; DcTCP1 with two introns and DcTCP 6 and DcTCP12 with one intron in D. chrysotoxum) (Figure 3).
According to the MEME online program, 15 conserved motifs were identified for each Dendrobium species, while their motif content and distribution were diversified (Figure 3). For example, in D. officinale, Motif 1, Motif 2, and Motif 3 were widespread on almost all DoTCP genes (Figure 3A), while Motif 9 and Motif 12 were specific to class Ⅰ (Figure 3A). Motif 5 was specific to class Ⅲ (Figure 3A). In D. nobile, Motif 1 was widespread on almost all DnTCP genes (Figure 3B), while Motif 5 and Motif 9 were specific to class Ⅲ (Figure 3B). In D. chrysotoxum, Motif 1 was widespread on almost all DcTCP genes (Figure 3C), while Motif 5 and Motif 9 were specific to class Ⅰ (Figure 3C).

2.4. Gene Collinearity Analysis

To further explore the expansion and evolution of TCP gene families, we analyzed duplication events of TCP genes. However, the evolution of the TCP gene family was conserved, with only one tandem duplication event (DcTCP9/DcTCP10) being found in D. chrysotoxum (Figure 4B) and two pairs of paralogous DoTCP gene duplication events (DoTCP1/DoTCP23 and DoTCP16/DoTCP24) being identified in D. officinale (Figure 4A).
Collinearity analyses of the TCPs between D. officinale and the other plant species were used to explore evolutionary relationships. Notably, only three pairs of collinear gene pairs were found in the genomes of D. officinale and A. thaliana (Figure 5E). Collinear gene pairs of the TCPs between D. officinale and A. thaliana only existed on Chr 1 and Chr 5 of A. thaliana (Figure 5E). There were 11 pairs of collinear gene pairs of TCPs between D. officinale and O. sativa, which only existed on Chr 1, Chr 2, Chr 3, and Chr 12 (Figure 5D). There were 14 collinear gene pairs of TCPs between D. officinale and V. planifolia, and 15 pairs between D. officinale and D. chrysotoxum (Figure 5B,C). D. nobile was more closely related to D. officinale, with 26 pairs of collinear gene pairs (Figure 5A). The results showed that the TCP genes of D. officinale and D. nobile were highly conserved across their evolution history.

2.5. cis-Acting Element Analysis on Promotors of TCPs

To further explore the potential function of TCPs, we classified and identified cis-acting elements in the promoter regions. cis-Acting elements are closely related to gene expression patterns and functions. After removing the nonfunctional items, a total of 61 genes representing the remaining cis-acting elements of TCPs were divided into four categories, which were related to hormone response (ABRE, TCA-element, ERE, GARE-motif, P-box, TATC-box, TGACG-motif, TGA-element, etc.), light response (G-box, GATA-motif, 3-AF1-binding site, i-box, TCT-motif, AE-box, GT1-motif, etc.), stress response (Box 4, TC-rich repeats, MBS, Myc, Myb, S box, W box, etc.), and others (LTR, CAT-box, WUN-motif, etc.) (Table S4; Figure 6).
In D. chrysotoxum, most cis-acting elements were related to light response (79/159), followed by hormone response (57/159) and stress response (10/159) (Figure 6C). The proportions of hormone response, light response, and stress response in D. officinale (100/421, 127/421, and 171/421) were similar to those in D. nobile (83/352, 87/352, and 161/352) (Figure 6A,B). Among the cis-acting elements of two Dendrobium species (D. officinale and D. nobile), the cis-acting elements related to stress response were the most abundant, followed by light response and hormone response (Figure 6A,B).

2.6. Expression of DoTCPs in Different Tissues and Expression Analysis under MeJA and ABA Treatments

To further study the function of TCPs, we analyzed the specific expression of TCPs in different tissues. Among the 24 TCP genes of D. officinale, 8 DoTCPs were obviously expressed (DoTCP6, DoTCP9, DoTCP11, DoTCP13, DoTCP14, DoTCP15, DoTCP19, and DoTCP21) (Figure 7A). According to the expression of these genes in different tissues, most genes were highly expressed in stems, flowers, and leaves but hardly expressed in roots (Figure 7A).
Since cis-acting element analysis showed that most TCPs in D. officinale corresponded to the stress response category, we used qRT-PCR to determine their expression patterns under MeJA treatment. Among them, eight DoTCPs (DoTCP1, DoTCP3, DoTCP11, DoTCP13, DoTCP18, DoTCP19, DoTCP21, and DoTCP23) were significantly downregulated, and four DoTCPs (DoTCP2, DoTCP4, DoTCP6, and DoTCP14) were significantly upregulated under MeJA treatment; the other genes showed no significant changes (Figure 7B).
In DoTCPs, there were more cis-acting elements of hormone response (ABRE); thus, we used qRT-PCR to determine their expression patterns under ABA treatment. Among them, six DoTCPs (DoTCP1, DoTCP5, DoTCP8, DoTCP17, DoTCP20, and DoTCP22) were significantly downregulated, and seven DoTCPs (DoTCP3, DoTCP7, DoTCP9, DoTCP11, DoTCP14, DoTCP15, and DoTCP21) were significantly upregulated; the other genes showed no significant changes (Figure 7C).

2.7. Protein–Protein Interaction Network of TCP Proteins in D. officinale

To further understand the function of genes, we analyzed the PPI network and detected the interaction between TCP proteins and related proteins using the STRING website.
A total of 72 proteins and 622 connections were identified, with TCP8 interacting with other proteins at least 38 times. Among these proteins, we found that (i) three genes (DoTCP5, DoTCP6, and DoTCP13) were related to TCP20, (ii) three proteins (DoTCP8, DoTCP17, and DoTCP21) were related to TCP2, and (iii) two proteins (DoTCP1 and DoTCP24) were related to TCP3 (Figure 8). These results indicated that TCP proteins of D. officinale have severe vital roles in multiple functions (Table S5; Figure 8).

3. Discussion

3.1. The Loss of CIN Clade Genes Resulted in a Diversified TCP Gene Family among Dendrobium Species

TCP genes are well documented to regulate various physiological processes in plants, from hormone pathways [32,33], growth, and development [16,18,19], to the regulation of the circadian clock and defense [5,34]. For example, in A. thaliana, AtTCP23 regulates plant development and flowering time [35], and AtTCP14 regulates embryonic growth during seed germination [12]. With more and more plant genomes being published, numerous TCP gene families have been identified from many plants, with important roles in various physiological processes, especially related to their critical function in regulating plant growth and development. However, there remains a lack of information on the evolution and genome duplication of TCP genes in Dendrobium species.
In this study, a total of 24, 23, and 14 TCP members were identified in D. officinale, D. nobile, and D. chrysotoxum, respectively (Table S2; Figure 1). Inferred from a previous study, the TCP genes were highly conserved in terms of their gene number and gene sequence. For example, the gene number was highly conserved. The number of TCP genes ranges from 6 TCP genes in Physcomitrella patens [36] to 74 in Gossypium raimondii [37]. However, in angiosperms, there are about 20 TCP members. The evolution of the TCP gene sequence was conserved. Consistent with previous studies, the TCP genes of Dendrobium species also belonged to two clades according to our phylogenetic analysis (Figure 2). Gene duplication is considered important in the variation of gene families, which are mainly classified as tandem duplication and segmental duplication according to the duplication patterns. Although only three gene duplication events happened among Dendrobium species, the TCP gene numbers among Dendrobium species were still highly variable, ranging from 14 TCP genes in D. chrysotoxum to 24 TCP genes in D. officinale (Table S2; Figure 1). Inferred from our evolutionary tree of TCP genes, a total of 14 genes remained in D. chrysotoxum with only three genes in the CIN clade, indicating an independent loss of CIN genes in D. chrysotoxum.
Increasing studies have shown that CIN genes play important roles in the regulation of growth and development in plants, especially in lateral organ development. For example, in A. thaliana, TCP transcription factors negatively regulate the expression of specific genes to control stem side-organ morphology [38]. In tomato, miR319 regulates LANCEOLATE, which affects the development of composite leaves [39]. Compared with D. officinale and D. nobile, D. chrysotoxum has distinct characters in the habitat environment and photosynthesis pathway. For example, D. officinale and D. nobile are widely distributed in the southeastern part of China, while D. chrysotoxum is only distributed in Yunnan province in China [40]. Compared with the diversified habitat of D. officinale and D. nobile, which have a varied environment, D. chrysotoxum species are mainly distributed in warm regions. There are different photosynthesis pathways among the three species, whereby D. officinale and D. nobile are facultative CAM plants, while D. chrysotoxum is a C3 plant [41]. Considering the diversified habitats, photosynthesis pathways, and the critical functions of CIN genes, we suggest that the loss of CIN genes in D. chrysotoxum led to a compact TCP gene family of D. chrysotoxum, resulting a diversified TCP gene family among Dendrobium species.

3.2. The Harsh Habitats and Different Photosynthesis Pathways Have Resulted in the Diversity of Biological Functions of the TCP Gene Family among Dendrobium Species

Previous studies have shown that TCP proteins play a critical role in molecular mechanisms such as plant organ development, hormone signaling regulation, and defense against abiotic stress [42,43]. However, different genes in the TCP gene family have different functions, with the same gene in different species sometimes having different functions. For instance, the cis-acting element analysis showed that some TCP genes respond to hormones, light, or stress. Both DcTCP12 and DoTCP24 were subclaves of CYC; DcTCP12 responded to stress, whereas DoTCP24 did not (Figure 2 and Figure 6).
In this study, on the basis of our comparative analysis, we believe that the harsh habitats and different photosynthesis pathways resulted in the diversity of biological functions of the TCP gene family among Dendrobium species.
Firstly, a total of 25 cis-acting elements of TCPs related to hormone/stress and light responses were detected. Among them, 18 genes were related to hormone response, and 22 genes were related to light response, while 36 genes were related to stress response (Figure 6).
Secondly, the diverse gene expression patterns of TCP genes indicated that TCP genes were mainly a function of the harsh habitats and different photosynthesis pathways. For example, the gene expression under MeJA treatment revealed four upregulated genes related to defense against abiotic stress (Figure 7). Only DoTCP6 and DoTCP14 showed high gene expression levels in stems, leaves, and flowers (Figure 7). Dendrobium species grow under strict conditions, such as epiphytic environments on cliffs or tree trunks, or distributed at high altitudes, above 1200 m, leading to the accumulation of high content of active ingredients, i.e., polysaccharides, flavonoids, and alkaloids, in their stems. The high content of polysaccharides, flavonoids, and alkaloids also improves the stress response to harsh habitats, resulting in a higher gene expression of TCP genes than other organisms (Figure 7A). A total of five genes related to light stress were expressed in leaves (Figure 7A). Dendrobium species are diversified in their photosynthesis pathways; for instance, D. chrysotoxum, Dendrobium Chrysanthum Lindl., Dendrobium Lindleyi Stendel, Dendrobium Longicornu Lindl., and Dendrobium thyrsiflorum Rchb. f. are C3 plants; Dendrobium terminale Par. et Rchb. f., Dendrobium acinaciforme Roxb., and Dendrobium primulinum Lindl. are crassulacean acid metabolism (CAM) plants; D. nobile, D. officinale, and Dendrobium hercoglossum Rchb. f. are recognized as C3-CAM plants [41]. The higher expression of DoTCP genes may improve the response of D. officinale to light stress by changing the photosynthesis pathway from C3 to CAM upon exposure to light stress. Additionally, there were seven genes (DoTCP6, DoTCP9, DoTCP11, DoTCP13, DoTCP14, DoTCP15, and DoTCP21) expressed in the flowers of DoTCPs (Figure 7A), potentially related to hormone response. Seven DoTCPs (DoTCP3, DoTCP7, DoTCP9, DoTCP11, DoTCP14, DoTCP15, and DoTCP21) were significantly upregulated under ABA treatment (Figure 7C), suggesting that these genes play an important role in hormone response.
Thirdly, the protein–protein interaction network analysis showed a close relationship among the DoTCP proteins, indicating that TCP genes play important roles in response to PCF1. For example, the genes with the most connections, such as PCF1 and PCF2, were related to plant growth and development. Therefore, we speculate that TCP genes play a more important role in plant growth and development.

4. Materials and Methods

4.1. Identification of TCP Genes in D. officinale, D. nobile, and D. chrysotoxum Genome

The hidden Markov model (HMM) map of TCP (PF03634) was downloaded from the protein family database (Pfam) (http://pfam-legacy.xfam.org/ (accessed on 22 August 2022)), and candidate proteins with an E-value of 1 × 10−5 were identified using HMMER v3.2.1. The TCP protein sequences of A. thaliana were downloaded, and BLASTP was used to query the proteins of D. officinale, D. nobile, and D. chrysotoxum. The TCP protein sequences identified with the two methods were integrated. In order to exclude false-positive sequences, the Pfam, NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 22 August 2022)), and SMART (http://smart.embl-heidelberg.de/ (accessed on 22 August 2022)) databases that submitted the identified TCP protein sequences were selected for further analysis of the TCP gene family. The protein molecular weight (kDa), aliphatic index (AI), and theoretical isoelectric point (pI) were estimated using ExPASy software [44]. Lastly, the chromosome position was analyzed using TBtools v1.6 [45].

4.2. Phylogenetic Relationship

Multiple sequence alignments of TCP full-length amino acid sequences of D. officinale, D. nobile, D. chrysotoxum, and A. thaliana were compared using MAFFT v7.487 software [46], and the maximum likelihood evolutionary trees were constructed using RAxML v1.3 software with the CAT + GTR model and 1000 bootstrap replicates [47].

4.3. Motifs and Domains

Firstly, MEME (https://meme-suite.org/meme/ (accessed on 22 August 2022)) was used to determine conserved motifs, with the number of motifs set to 20 and all other parameters set to their default values. Then, the exon–intron structure of each sequence was displayed online using GSDS software (http://gsds.gao-lab.org/index.php (accessed on 22 August 2022)). Lastly, the characteristic motifs and domains of the TCP gene family were aligned using TBtools v1.6 [45].

4.4. Gene Duplication and Syntenic Analysis

DNA sequences of the TCP gene family of D. officinale, D. nobile, and D. chrysotoxum were compared using BLASTN with an e-value of 1 × 10−20. Gene duplication was identified using BLASTN results and analyzed using MCScanX, and the results were visualized using Circos. MCScanX (cscore ≥ 0.7) was employed to detect and display the syndesmosis between D. officinale and other plant genomes.

4.5. Promoter Analysis

The region 1500 bp upstream of the TCP gene translation initiation site was extracted as the presumed promoter and submitted to the PlantCare database online to analyze the assumed cis-acting elements (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 22 July 2023). The characteristic cis-acting elements of the TCP gene family were aligned using TBtools v1.6 [45].

4.6. Expression of TCPs in Different Tissues

To analyze the expression pattern of the TCP genes in D. officinale, we searched the NCBI SRA database for RNA-sequencing data from root (SSR2014227 and SSR2014230), stem (SSR1917040, SSR1917041, SSR1917042, and SSR1917043), leaf (SSR2012297 and SSR2014325), and flower (SSR2012396 and SSR2014476) as four different tissues. The FPKM value of DoTCPs was calculated using StringTie v2.2.0 (http://string-db.org/, accessed on 22 July 2023) to estimate the abundance of transcripts, and the heat map was built using TBtools v1.6 to visualize the expression [45].

4.7. Quantitative Real-Time PCR

Firstly, total RNA was extracted using the EASY spin Plant RNA Kit (Aidlab, Beijing, China). Then, HiScript® III-RT SuperMix for qPCR (Vazyme, Beijing, China) was used to synthesize the first-strand cDNAs. Snapgene software was used to design primers (Table S6), and the ABI-7500 Connect Real-Time PCR Detection System was used to perform qRT-PCR. With 1 μL of template in a reaction volume of 20 μL, cDNAs were diluted to 200 ng, and three technical repetitions were performed. The PCR amplification procedure was as follows: 95 °C for 30 s, 95 °C for 10 s, 60 °C for 30 s, and 60 °C for 15 s. GAPDH was used as the internal reference gene. Primer sequences are presented in Table S6.

4.8. Protein–Protein Interaction Network

The expression data were calculated using the 2−∆∆CT method [48]. The STRING database was used to align the DoTCP protein sequences and predict the relationships. The regulatory networks were visualized using Cytoscape v3.7.2 software [49].

5. Conclusions

This study explored the evolution of TCP genes in D. officinale, D. nobile, and D. chrysotoxum by analyzing the conserved motifs, gene structure, gene duplication, and collinearity. Gene motif and structure analysis showed that genes in the same clade had relatively conserved gene structures and similar protein motifs. The differentially expressed genes screened under MeJA and ABA treatment using qRT-PCR may play an important role in stress response and hormone response. On the basis of our comparative analysis, we suggest that the harsh habitats and different photosynthesis pathways resulted in the diversity of biological functions of the TCP gene family among Dendrobium species. This study lays a foundation for the genetic evolution and functional analysis of TCP genes in Dendrobium.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants12183201/s1, Figure S1: Sequence logs of the DoTCP protein motifs. The height of each amino acid represents the relative frequency of the amino acid at that position, Figure S2: Sequence logs of the DnTCP protein motifs. The height of each amino acid represents the relative frequency of the amino acid at that position, Figure S3: Sequence logs of the DcTCP protein motifs. The height of each amino acid represents the relative frequency of the amino acid at that position, Table S1: The sequence information on 23 reference A. thaliana proteins used in the BLASTP search, Table S2: The characteristics of TCP members identified in D. officinale (Do), D. nobile (Dn), and D. chrysotoxum (Dc), Table S3: The chromosome characteristics of D. officinale, D. nobile, and D. chrysotoxum, Table S4: Information on cis-acting elements in TCP genes of D. officinale, D. nobile, and D. chrysotoxum, Table S5: PPI networks of TCP proteins in D. officinale, Table S6: Primer sequences of RT-qPCR.

Author Contributions

Conceptualization, X.D. and Q.X.; formal analysis, Y.L. (Yaoting Li) and L.L.; investigation, Y.L. (Yaoting Li), L.L. and J.Y.; methodology, Y.L. (Yaoting Li), L.L., J.Y., Y.L. (Yi Lin) and W.L.; writing—original draft, Y.L. (Yaoting Li), L.L. and Z.N.; writing—review and editing, Y.L. (Yaoting Li), L.L. and Z.N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant Nos. 32070353 and 31900268), the Forestry Science and Technology Innovation and Promotion Project of Jiangsu Province (LYKJ[2021]12), and the Agricultural Science and Technology Independent Innovation Fund Project of Jiangsu Province (CX(22)3147).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

All data generated or analyzed during this study are included in the article and Supplementary Materials.

Acknowledgments

The authors thank the School of Life Sciences, Anhui Agricultural University, and the College of Life Sciences, Nanjing Normal University, for supporting this work. The authors are also grateful to the Jiangsu Provincial Engineering Research Center for Technical Industrialization for Dendrobium for technical support.

Conflicts of Interest

The authors declare no conflict of interest.

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Plants 12 03201 g001
Figure 1. Chromosomal distributions of the identified TCP genes in D. officinale (A), D. nobile (B), and D. chrysotoxum (C). Green, blue, and yellow-green colors represent D. officinale, D. nobile, and D. chrysotoxum, respectively. The red letters represent the names of TCP genes.
Figure 1. Chromosomal distributions of the identified TCP genes in D. officinale (A), D. nobile (B), and D. chrysotoxum (C). Green, blue, and yellow-green colors represent D. officinale, D. nobile, and D. chrysotoxum, respectively. The red letters represent the names of TCP genes.
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Figure 2. Phylogenetic relationships of TCPs in D. officinale, D. nobile, D. chrysotoxum, and A. thaliana. The maximum likelihood evolutionary tree was constructed with RAxML with 1000 bootstraps replicates. Light-blue, dark-blue, green, and red colors represent TCP protein sequences from D. officinale (Do), D. nobile (Dn), D. chrysotoxum (Dc), and A. thaliana (At), respectively. Different subfamilies are shaded with different colors.
Figure 2. Phylogenetic relationships of TCPs in D. officinale, D. nobile, D. chrysotoxum, and A. thaliana. The maximum likelihood evolutionary tree was constructed with RAxML with 1000 bootstraps replicates. Light-blue, dark-blue, green, and red colors represent TCP protein sequences from D. officinale (Do), D. nobile (Dn), D. chrysotoxum (Dc), and A. thaliana (At), respectively. Different subfamilies are shaded with different colors.
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Figure 3. Phylogenetic relationships, conserved motifs, and exon–intron structures of TCP genes in D. officinale (A), D. nobile (B), and D. chrysotoxum (C). Different colors represent the 15 different motifs. Different branches are shaded with different colors. The black lines indicate introns.
Figure 3. Phylogenetic relationships, conserved motifs, and exon–intron structures of TCP genes in D. officinale (A), D. nobile (B), and D. chrysotoxum (C). Different colors represent the 15 different motifs. Different branches are shaded with different colors. The black lines indicate introns.
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Figure 4. Schematic representations of the gene duplications of TCP genes from D. officinale (A) and D. chrysotoxum (B). Blue and green lines highlight the syntenic gene pairs in D. officinale (Do) and D. chrysotoxum (Dc), respectively.
Figure 4. Schematic representations of the gene duplications of TCP genes from D. officinale (A) and D. chrysotoxum (B). Blue and green lines highlight the syntenic gene pairs in D. officinale (Do) and D. chrysotoxum (Dc), respectively.
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Figure 5. Collinearity analysis of TCP genes between D. officinale and five other plants, including D. nobile (A), D. chrysotoxum (B), V. planifolia (C), O. sativa (D), and A. thaliana (E). Gray lines indicate the collinear blocks. Syntenic genes of the TCP gene family are exhibited with red lines. The numbers in the figure represent the number of the corresponding chromosome.
Figure 5. Collinearity analysis of TCP genes between D. officinale and five other plants, including D. nobile (A), D. chrysotoxum (B), V. planifolia (C), O. sativa (D), and A. thaliana (E). Gray lines indicate the collinear blocks. Syntenic genes of the TCP gene family are exhibited with red lines. The numbers in the figure represent the number of the corresponding chromosome.
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Figure 6. Information on cis-acting elements in TCP genes of D. officinale (A), D. nobile (B), and D. chrysotoxum (C). The different colors in the histogram indicate the number of cis-elements in each category.
Figure 6. Information on cis-acting elements in TCP genes of D. officinale (A), D. nobile (B), and D. chrysotoxum (C). The different colors in the histogram indicate the number of cis-elements in each category.
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Figure 7. Expression analysis of DoTCPs in different tissues under MeJA treatment. (A) Expression profiles of TCP genes of D. officinale in different tissues including root, stem, leaf, and flower. Z-score-transformed FPKM values. (B) Relative expression levels of DoTCPs under MeJA treatments. (C) Relative expression levels of DoTCPs under ABA treatments.
Figure 7. Expression analysis of DoTCPs in different tissues under MeJA treatment. (A) Expression profiles of TCP genes of D. officinale in different tissues including root, stem, leaf, and flower. Z-score-transformed FPKM values. (B) Relative expression levels of DoTCPs under MeJA treatments. (C) Relative expression levels of DoTCPs under ABA treatments.
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Figure 8. Protein–protein interaction (PPI) networks of TCP proteins in D. officinale. The gradient circle size and color indicates the degree of importance. The important of yellow to a greater extent than blue.
Figure 8. Protein–protein interaction (PPI) networks of TCP proteins in D. officinale. The gradient circle size and color indicates the degree of importance. The important of yellow to a greater extent than blue.
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MDPI and ACS Style

Li, Y.; Li, L.; Yang, J.; Niu, Z.; Liu, W.; Lin, Y.; Xue, Q.; Ding, X. Genome-Wide Identification and Analysis of TCP Gene Family among Three Dendrobium Species. Plants 2023, 12, 3201. https://doi.org/10.3390/plants12183201

AMA Style

Li Y, Li L, Yang J, Niu Z, Liu W, Lin Y, Xue Q, Ding X. Genome-Wide Identification and Analysis of TCP Gene Family among Three Dendrobium Species. Plants. 2023; 12(18):3201. https://doi.org/10.3390/plants12183201

Chicago/Turabian Style

Li, Yaoting, Lingli Li, Jiapeng Yang, Zhitao Niu, Wei Liu, Yi Lin, Qingyun Xue, and Xiaoyu Ding. 2023. "Genome-Wide Identification and Analysis of TCP Gene Family among Three Dendrobium Species" Plants 12, no. 18: 3201. https://doi.org/10.3390/plants12183201

APA Style

Li, Y., Li, L., Yang, J., Niu, Z., Liu, W., Lin, Y., Xue, Q., & Ding, X. (2023). Genome-Wide Identification and Analysis of TCP Gene Family among Three Dendrobium Species. Plants, 12(18), 3201. https://doi.org/10.3390/plants12183201

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