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Article

Identification and Expression Analysis of TCP Transcription Factors Under Abiotic Stress in Phoebe bournei

by
Wenzhuo Lv
1,†,
Hao Yang
2,†,
Qiumian Zheng
2,
Wenhai Liao
2,
Li Chen
2,
Yiran Lian
3,
Qinmin Lin
3,
Shuhao Huo
4,
Obaid Ur Rehman
4,
Wei Liu
5,
Kehui Zheng
6,
Yanzi Zhang
7,* and
Shijiang Cao
2,*
1
College of Jun Cao Science and Ecology (College of Carbon Neutrality), Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
4
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
5
Shandong Analysis and Test Center, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250014, China
6
College of Computer and Information Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
7
Metabolomics Center, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2024, 13(21), 3095; https://doi.org/10.3390/plants13213095
Submission received: 13 October 2024 / Revised: 29 October 2024 / Accepted: 31 October 2024 / Published: 3 November 2024
(This article belongs to the Special Issue Molecular Biology and Bioinformatics of Forest Trees)
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Versions Notes

Abstract

:
The TCP gene family encodes plant transcription factors crucial for regulating growth and development. While TCP genes have been identified in various species, they have not been studied in Phoebe bournei (Hemsl.). This study identified 29 TCP genes in the P. bournei genome, categorizing them into Class I (PCF) and Class II (CYC/TB1 and CIN). We conducted analyses on the PbTCP gene at both the protein level (physicochemical properties) and the gene sequence level (subcellular localization, chromosomal distribution, phylogenetic relationships, conserved motifs, and gene structure). Most P. bournei TCP genes are localized in the nucleus, except PbTCP9 in the mitochondria and PbTCP8 in both the chloroplast and nucleus. Chromosomal mapping showed 29 TCP genes unevenly distributed across 10 chromosomes, except chromosome 8 and 9. We also analyzed the promoter cis-regulatory elements, which are mainly involved in plant growth and development and hormone responses. Notably, most PbTCP transcription factors respond highly to light. Further analysis revealed three subfamily genes expressed in five P. bournei tissues: leaves, root bark, root xylem, stem xylem, and stem bark, with predominant PCF genes. Using qRT-PCR, we examined six representative genes—PbTCP16, PbTCP23, PbTCP7, PbTCP29, PbTCP14, and PbTCP15—under stress conditions such as high temperature, drought, light exposure, and dark. PbTCP14 and PbTCP15 showed significantly higher expression under heat, drought, light and dark stress. We hypothesize that TCP transcription factors play a key role in growth under varying light conditions, possibly mediated by auxin hormones. This work provides insights into the TCP gene family’s functional characteristics and stress resistance regulation in P. bournei.

1. Introduction

Plants, being sessile organisms, are exposed to various degrees of abiotic stresses during their growth and development. These stresses arise when the external environmental conditions become unsuitable and deviate from the optimal range required for plant growth. Consequently, plants transduce environmental signals into internal signals to regulate physiological processes through numerous regulatory proteins, enabling continual adaptation to changing conditions [1,2]. Transcription factors (TFs), as essential proteins, play a crucial role in plant stress responses by interacting with cis-elements to regulate gene expression within specific DNA sequences of target gene promoters [3,4].
The TCP gene family, first identified in 1990, derives its name from the initials of three members: Teosinte Branched1 (TB1) in maize (Zea mays), Cycloidea (CYC) in snapdragon (Antirrhinum majus), and Proliferating Cell Factors 1 and 2 (PCF1 and PCF2) in rice (Oryza sativa) [5,6,7,8]. The TCP domain comprises 59 amino acids and features an atypical basic helix–loop–helix (bHLH) motif at the N-terminus, crucial for mediating protein–protein interactions through the coiled helix structure [9]. The protein crystal structure of rice TCP OsPCF6 reveals that the TCP domain forms a dimer, maintaining a stable conformation [10].
Based on differences within their domains, TCPs are categorized into two main classes: Class I (PCF subfamily) and Class II (CIN and CYC/TB1 clades). Class I domains lack four amino acids, whereas the Class II CYC/TB1 clade features an arginine-rich R domain [11,12,13]. Research indicates that these clades exhibit distinct DNA recognition mechanisms. Class I TCPs bind to GGNCCC or CCNNCC sequences, while Class II TCPs specifically bind to the GGNCCC sequence [14,15]. Functionally, Class I transcription factors are involved in leaf and flower organ development, seed germination, and cell proliferation, thereby promoting plant growth. Conversely, Class II transcription factors are associated with hormone regulation, defense responses, and branching regulation, inhibiting plant growth [16,17,18].
TCP transcription factors play significant roles in seed germination, leaf senescence, defense responses, circadian rhythm regulation, and cell proliferation by modulating plant hormones [9,19,20,21,22]. Auxin, a vital hormone, influences organ size, differentiation, and morphology, serving as a key player in plant growth, development, and stress responses [23]. YUC enzymes, which are rate-limiting enzymes in the tryptophan-dependent auxin biosynthesis pathway, are regulated by the PIF transcription factor, which promotes shade response gene expression under low-light conditions. TCP17, TCP5, and TCP13 enhance auxin signaling by upregulating PIF and YUCs, thereby boosting auxin biosynthesis [24]. Additionally, TCP14 and TCP15 are known to promote proper plant elongation by regulating a set of auxin-induced genes associated with cell expansion (Figure 1) [25]. Therefore, TCP transcription factors are crucial in the regulation of auxin biosynthesis.
The TCP gene family has been identified in various plants [26], including Arabidopsis thaliana with 24 TCP transcription factors [22], tomato (Solanum lycopersicum) with 30 TCP genes [27], Dendrobium streptolum with 25 transcription factors [11], and oats (Avina sativa) with 49 TCP genes [28]. In ginseng (Panax ginseng), the PgTCP26-02 gene is involved in ginsenoside synthesis and regulates secondary metabolism [29]. The CaTCP16 gene mediates bud phylogeny in pepper (Capsicum annuum) [3], and NGAs regulate auxin-related genes in apical pistils through their interaction with TCP4 [30]. In tomato, SlTCP12, SlTCP15, and SlTCP18 influence fruit ripening [1], while PavTCP1, PavTCP2, and PavTCP3 affect total anthocyanins, soluble sugars, and soluble solids in sweet cherries (Prunus avium) [31]. Leaf development in larch (Larix decidua) is regulated by the interaction of class I and class II LcTCP genes [32]. In birch (Betula pendula), BpTCP20 enhances salt tolerance by regulating stomatal closure and reducing reactive oxygen species (ROS) accumulation [33]. Additionally, PeTCP expression in passionflower is induced by hormone treatment and various stresses such as cold, heat, and salt [34]. The diverse biological functions of TCP genes warrant further study due to their extensive roles.
Forests are the key to the global carbon balance and are a key solution to climate change [35]. Compared to plantations, natural forests have more sustained and stronger carbon sequestration capabilities [36]. Phoebe bournei (Hemsl.), one of the most valuable native tree species in southern China, is widely used in shipbuilding, woodworking, and other industries due to its excellent properties, providing significant ecological and economic benefits [37,38]. However, overexploitation and its inherently slow growth have led to a decline in P. bournei habitat adaptability [39], and global warming is exacerbating abiotic stresses such as high temperatures and drought, which adversely affect its physiological activities [40]. Studies have shown that different light stress conditions impact the leaf area, chlorophyll a, and chlorophyll b in P. bournei [41,42]. Increases in temperature and light intensity can enhance the emission of biogenic volatile organic compounds (BVOCs) from P. bournei, prolonging the atmospheric residence time of greenhouse gases like CH4 and creating a positive feedback loop [43]. Drought stress inhibits the synthesis of Chla and Chlb in P. bournei, reducing photosynthesis, and under prolonged drought conditions, stem weight decreases significantly, and seedlings undergo extensive transcriptomic changes [44]. Therefore, understanding the mechanisms underlying P. bournei response to abiotic stress is crucial.
Despite extensive research demonstrating that TCP transcription factors play a key role in plant growth, development, and stress response, no systematic analysis has been conducted in P. bournei. In this study, we used bioinformatics and expression analysis to investigate TCP transcription factors in P. bournei. We analyzed the physicochemical properties, gene structure, chromosomal distribution, exon–intron organization, conserved motifs, and promoter regions of the TCP gene family. Additionally, we employed qRT-PCR to assess their expression patterns under high temperature, drought, and light stress conditions. The findings will contribute to understanding the functional characteristics and potential roles of the TCP gene family, providing high-quality candidate genes for genetic engineering in P. bournei at the molecular level.

2. Results

2.1. Identification of the PbTCP Protein Characterization

We identified 29 PbTCP proteins from the P. bournei genome database, naming them PbTCP1 to PbTCP29 (Table 1). Analysis revealed that the amino acid (aa) lengths of the TCP gene family members ranged between 177 and 679 amino acids (aa) and had an average length of 362 aa. The relative molecular weights of these proteins varied between 17.80 kDa (PbTCP10) and 72.15 kDa (PbTCP9), averaging 39.15 kDa, indicating significant variability in the size of TCP transcription factor proteins. The isoelectric points of these proteins range from 5.68 to 10.15, with nine of the 29 TCP proteins being slightly acidic, while the remaining 20 are basic. Except for PbTCP9, all identified proteins are considered unstable (instability index > 40), with a range from 37.11 to 69.1. The aliphatic index of TCP proteins ranges from 51.61 to 92.99, with an average value of 68.38, suggesting that these proteins are thermostable. Additionally, all TCP proteins are hydrophilic (GRAVY < 0), indicating strong hydrophilicity. Subcellular localization predictions show that most PbTCP family members are in the nucleus (Table 1), except PbTCP9, which is found in the mitochondria, and PbTCP8, which is located in both the chloroplast and the nucleus. This suggests that the primary regulatory function of these proteins occurs in the nucleus. Chromosomal localization indicates that the 29 TCP genes are unevenly distributed across 10 chromosomes, with no TCP genes found on chromosomes 8 and 9 (Figure 2). Notably, chromosome 3 contains the highest number of genes, with nine (PbTCP8 to PbTCP16), which are closely arranged in this region, occupying approximately 43 Mb. Chromosomes 6, 7, and 12 each contain a single gene, with some located near the chromosome ends.

2.2. Evolutionary Analysis of PbTCP Gene Family

To investigate the evolution of the PbTCP gene family, we constructed a phylogenetic tree using protein sequences from P. bournei and A. thaliana (Figure 2). Based on the multiple sequence alignment of 29 PbTCP and 24 AtTCP proteins, all TCP proteins were classified into three branches: PCF, CIN, and CYC/TB1, belonging to the two subfamilies, Class I TCP and Class II TCP. Among these, the PCF branch contains 15 PbTCP proteins, representing 52% of the total PbTCP proteins, making it the largest among the four subfamilies. The CIN subfamily is the smallest, with only six proteins. Although the CYC/TB1 subfamily has a modest number of proteins (eight), PbTCP proteins constitute a significant proportion (73%). In comparison, the PCF subfamily in A. thaliana has 13 AtTCP genes, the CIN subfamily has eight, and the CYC/TB1 subfamily has three, indicating that the number of TCP genes in each subfamily varies between species (Figure 3). The study also reveals that the evolution rate of PbTCP genes is higher than that of AtTCP genes, resulting in a greater diversity of gene types. This accelerated evolution in P. bournei may be an adaptive response to environmental changes, enhancing the species’ survival rate.

2.3. PbTCP Protein Sequence Analysis

We further analyzed the sequence characteristics of the 29 PbTCP proteins by comparing their conserved domain sequences (Figure 4). The results reveal that PbTCP proteins contain four conserved motifs: basic, helix I, loop, and helix II. The degree of stacking suggests that these motifs are highly conserved within the same subfamily. Compared to Class II, Class I is missing four amino acids in the basic region but exhibits higher overall conservation, allowing for effective differentiation between subfamilies. The basic region is the most conserved, followed by the helix regions, while the loop region shows the greatest diversity. Specific amino acids, such as glycine (G) in the basic region, leucine (L) in the helix region, and tryptophan (W), are completely conserved across all sequences. These residues likely play critical roles in protein function by participating in different structural domains. Additionally, the PbTCP14 protein exhibits significant amino acid deletions in both the loop and helix II regions, which may contribute to functional differences between Class I and Class II proteins, potentially affecting their regulatory roles and the expression of target genes.

2.4. Protein Structure Analysis of PbTCP Transcription Factors

We conducted a secondary structure analysis of all PbTCP proteins, using color coding to represent different levels of confidence. As shown in Figure 5, the PCF branch exhibited a higher confidence level compared to CIN and CYC/TB1 branches. The distinct structures of different proteins further confirm the diverse functional expression of the TCP gene family. The tertiary structure was further predicted, and a total of 10 conserved motifs were identified, which were named motif1–motif10. Comparative analysis revealed that these motifs were conserved across different subfamilies. All PbTCP proteins contain motif 1, indicating that they were highly conserved. The members from the PCF subfamily all contained motif5, and the motifs within the family were highly similar. The members of the CIN subfamily consistently contain motif 10 (Figure 6), which is located at the N-terminus, while motif 9 was only present in the PCF and CIN subfamilies and located in the C-terminus (Figure 6). The special location may be related to a specific function, but the specific role remains unclear.
In addition, motifs 3 and 7 were only found in the CYC/TB1 subfamily, specifically in PbTCP12, PbTCP13, PbTCP14 and PbTCP15, and motifs 4 and 6 were found in the CYC/TB1 subfamily, indicating the diversity and uniqueness of the CYC/TB1 subfamily (Figure 6). In the comparison of the exon and intron structure of the PbTCP gene family, it was found that all 29 genes contained exons of different lengths, and 52% of PbTCP did not have introns.

2.5. Collinearity Analysis of TCP Gene Family in P. bournei

Intraspecific collinearity analysis of the PbTCP gene was performed. Twenty-nine PbTCP genes were mapped to 10 chromosomes except chromosomes 8 and 9 (Figure 7). A total of 15 pairs of fragments of repeat events were found, involving 20 PbTCP genes. The TCP gene on chromosome 3 had the most genes (9) and was involved in most of the repeat events (five pairs, among which PbTCP9 involves two pairs with PbTCP23 and PbTCP28 respectively). Most of the remaining duplication events occurred on chromosomes 1 and 5. Among them, nine pairs of collinearity appeared in the PCF genome, four pairs appeared in the CIN genome, and only two pairs appeared in the CYC/TB1 genome. It is worth noting that many PbTCP genes have a collinearity relationship with more than one pair of genes, suggesting that TCP genes are highly conserved during evolution in P. bournei. In addition, no tandem duplication events were analyzed. The results of collinearity analysis revealed the functional diversity of TCP transcription factors in P. bournei.

2.6. Interspecific Collinearity Analysis

To further explore the evolutionary mechanisms, an inter-species collinearity analysis of TCP gene pairs was conducted between the genomes of P. bournei and A. thaliana, O. sativa, and Populus trichocarpa (black cottonwood) (Figure 8A). The results indicate that the strength of association with PbTCP genes, from highest to lowest, is as follows: AtTCP, PtTCP, and OsTCP. Most collinear relationships are found within the PCF subfamily, followed by the CIN subfamily. Consistent with previous analyses, the CYC/TB1 branch shows lower similarity across species. Furthermore, 17 PbTCP genes were found to be homologous with those in A. thaliana, 11 with O. sativa, and 16 with P. trichocarpa. Six genes are shared among all three species. Notably, two unique homologous genes (Figure 8) were absent in A. thaliana and O. sativa, which may be related to the evolutionary history of plant genomes (Figure 8), as well as factors such as gene family expansion and recombination.

2.7. Analysis of Cis-Acting Elements

To clarify the role of cis-acting elements in the PbTCP promoter regions in light response and environmental stress response processes, we identified and aligned the cis-acting elements present in the PbTCP promoters. A total of 723 cis-acting components belonging to 19 functional groups/types were identified in the TCP gene promoter serial (Figure 9). There were eight types of growth and development response elements, six stress response elements and five hormone response elements. Among them, the number of plant growth and development elements was the largest, with a total of 398, accounting for 55%; followed by hormone-responsive elements, accounting for 29%, while stress-responsive elements accounted for only 16% (Figure 9A). It is worth noting that cis-elements related to light response function were widely distributed in PbTCP gene promoters, indicating that TCP genes play an important role in the light response process of plants. Also, elements related to hypoxia-specific induction, circadian rhythm, and differentiation of palisade mesenchymal cells were abundant in 29 PbTCP. In addition, abscisic acid response elements and methyl jasmonic acid elements were also widely distributed in the TCP genes of P. bournei, suggesting that the TCP genes may be involved in the regulation of the signaling processes of these two plant hormones, plant stress, and plant growth and development (Figure 9B).

2.8. Gene Expression Heat Map of TCP Gene in Different Tissues

Transcriptome data analysis from five different tissues—leaves, root bark, root xylem, stem xylem, and stem bark—reveals significant tissue-specific expression of PbTCP genes in P. bournei. The results show that PCF genes are expressed at much higher levels than CYC/TB1 genes. Among the PCF genes (Figure 10), PbTCP16, PbTCP26, and PbTCP23 exhibit high expression levels in both roots and stems, indicating strong tissue specificity and suggesting that these three TCP genes may play crucial roles in the growth and development of P. bournei. In contrast, CYC/TB1 genes generally show low expression across roots, stems, and leaves. Within the CIN genes, PbTCP2, PbTCP20, and PbTCP7 have zero expression in roots and very low expression in stems. Notably, PbTCP18 is highly expressed in leaves and root xylem, suggesting its importance in these tissues. Among the CYC/TB1 genes, PbTCP5 also exhibits higher expression in the stem xylem, which may be closely related to the growth of the stem xylem. The remaining TCP genes have very low expression levels across roots, stems, and leaves (Figure 10).

2.9. Expression of the PbTCP Gene Under Abiotic Stress

Six genes were selected from each of the three subfamilies for analysis under four stress conditions: high temperature, drought, light stress, and shading (Figure 11). PbTCP23 and PbTCP16 are members of the PCF subfamily; PbTCP7 and PbTCP29 belong to the CIN subfamily; and PbTCP15 and PbTCP14 are part of the CYC/TB1 subfamily. The results indicated that all stress conditions significantly impacted PbTCP gene expression, with distinct responses observed across the PCF, CIN, and CYC/TB1 subfamilies.
In the PCF subfamily, PbTCP14 showed a marked increase under drought stress (simulated by immersion in a 10% PEG6000 solution to mimic short-term drought conditions), with expression levels rising approximately 300-fold at 8 h post-treatment compared to controls. Similarly, PbTCP7 displayed an approximately 100-fold increase at this point, indicating a robust upregulation response to drought stress in this subfamily. Further studies extending treatment duration and measuring relative water content could provide deeper insights into the drought response of these woody plants. In the CIN subfamily, PbTCP23 and PbTCP16 showed varying expression suppression under shading stress, suggesting that CIN members may be particularly sensitive to light reduction. For PbTCP7, PbTCP29, PbTCP14, and PbTCP15, upregulation was observed approximately 24 h after exposure to high temperature and light stress, with CYC/TB1 subfamily genes (PbTCP14 and PbTCP15) exhibiting a delayed yet strong response. This pattern suggests that the CYC/TB1 subfamily may be especially responsive to prolonged stress exposure. Overall, these findings indicate that PCF subfamily genes typically respond rapidly to drought, CIN subfamily genes are more sensitive to shading, and CYC/TB1 subfamily genes exhibit delayed but substantial expression changes under high temperature and light stress.

3. Discussion

Plants encounter various abiotic stresses during growth, sparking interest in how they regulate their signaling pathways under such conditions. TCP, a plant-specific transcription factor gene family, plays a crucial role in development, influencing traits such as floral symmetry, leaf morphology, plant branching, and hormone signaling [26]. While TCP genes have been extensively studied in various plants, including A. thaliana [22], Chrysanthemum morifolium [13], Dendrobium officinale [45], Catharanthus roseus [46], and Citrus sinensis [47], they have not yet been identified and characterized in P. bournei.
In this study, we identified 29 TCP genes from P. bournei, classified into two subfamilies: Class I (PCF) and Class II (CIN and CYC/TB1), consistent with findings in other species like Helianthus annuus [12], Zingiber officinale [48], Dendrobium officinale [49], Petunia hybrida [49], and Melastoma candidum [50]. This conservation suggests that TCP genes in P. bournei are likely to perform similar functions to those in other species. Subcellular localization analysis revealed that all P. bournei TCP family members, except PbTCP8 and PbTCP9, primarily function in the nucleus. PbTCP8 is expressed in both chloroplasts and the nucleus, like SsTCP13 in sugarcane and SbTCP15 in sorghum [51,52]. Notably, PbTCP8 shows high expression in root epidermis, root xylem, stem epidermis, and stem xylem, suggesting a potential role in chloroplast regulation. PbTCP9, the only stable protein among the TCP genes, is predominantly expressed in mitochondria and demonstrates the highest heat stability, indicating its ability to maintain function under abiotic stress and support mitochondrial stability. However, the regulatory mechanism of PbTCP9 requires further investigation.
Structural similarities across TCP genes are evolutionarily conserved and functionally significant. The presence of conserved motifs and gene structures supports this conclusion. For instance, motif 1 is widespread among PbTCP genes, while PbTCP1 (PCF), PbTCP2 (CIN), and PbTCP3 (CYC/TB1) share similar motifs. PbTCP18 (CIN) and PbTCP19 (CYC/TB1) have similar exon lengths, indicating that genes from different branches may have similar functions, preventing phenotypic changes due to single gene mutations under abiotic stress. Additionally, each subfamily possesses unique characteristics, such as motifs 3, 4, 6, 7, and 8 being exclusive to the CYC/TB1 branch, and motif 10 being present in all CIN members, highlighting the complementary roles these motifs may play in protein function.
In the analysis of conserved domains, we identified that PbTCP transcription factors possess a typical bHLH domain, with high conservation within subfamilies. A notable finding is the significant loss of amino acids in the loop and Helix 2 regions of the PbTCP14 protein. Compared to PbTCP14, PbTCP15 contains additional motifs 8 and 3, suggesting that the loss of these motifs in PbTCP14 may influence plant growth, development, or stress responses. The second conserved region in CYC/TB1, the R domain, is known to be rich in polar residues such as arginine, lysine, and glutamic acid. The type and sequence of amino acids in this domain significantly affect its hydrophilicity [53], which is consistent with the high hydrophilicity observed in other P. bournei gene families [54,55,56,57]. This specificity may be related to the adaptation of P. bournei to its moist environment.
Gene duplication events play a crucial role in providing genetic material and promoting gene evolution. Our synteny analysis of the 29 PbTCP genes revealed that they all exhibit syntenic relationships with multiple genes. Among the 15 segmental duplication events, nine occurred in the PCF subfamily, four in the CIN subfamily, and only two in the CYC/TB1 subfamily. Previous studies have identified 12 segmental duplication events in ginger, and the number of PbTCP segmental duplications far exceeds that in Orchard grass [58], Dendrobium officinale [45], and Capsicum annuum [3], suggesting a potential evolutionary advantage in P. bournei that may enhance its survival and reproductive success. Additionally, interspecies synteny analysis between P. bournei and A. thaliana, Z. mays, and P. trichocarpa revealed lower synteny and sequence similarity with rice, potentially reflecting differences in evolutionary history, gene family expansion, and recombination.
To further explore the response of PbTCP genes to stress, we analyzed their expression profiles and cis-acting elements. PbTCP genes are expressed in roots, stems, and leaves, with the highest expression levels in the PCF subfamily. In contrast, most genes in the other two subfamilies are reduced, like the expression pattern observed in Helianthus annuus [12]. However, unlike H. annuus, where only a few PCF genes are highly expressed, P. bournei shows significantly higher expression in the PCF subfamily. In Capsicum annuum, the CIN subfamily exhibits the highest expression [3], indicating species-specific differences in TCP transcription factor expression. PbTCP16 and PbTCP23 show prominent expression in roots, stems, and leaves, with PbTCP23 notably responsive to MeJA, which promotes defensive protein production in plants to concentrate resources against environmental stress [59], suggesting their significant roles in abiotic stress response. Additionally, PbTCP18, a member of the CIN subfamily, is upregulated in leaves and roots, like the expression patterns of AtTCP2 and AtTCP3, which are associated with active cell division in floral meristems [53]. Given the strong phylogenetic relationship between PbTCP18 and AtTCP2/AtTCP3, it is plausible that PbTCP18 promotes meristematic tissue differentiation in leaves and roots.
We conducted abiotic stress experiments on PbTCP16, PbTCP23, PbTCP7, PbTCP29, PbTCP14, and PbTCP15, representing the three subfamilies. The results revealed differential expression of these genes under high temperature, drought, light stress, and shading conditions. Genes involved in stress response predominantly belonged to the CYC/TB1 subfamily, differing from other species like Betula platyphylla and Phyllostachys edulis [60,61], where the PCF subfamily showed more active expression under stress. Notably, PbTCP16 exhibited downregulation across all stress conditions, possibly due to its low confidence in secondary structure prediction, making it less favorable for stress response. PbTCP14 and PbTCP15, on the other hand, demonstrated strong expression under various stresses, particularly in response to light and shading, where they showed significantly higher expression than other representative genes, indicating greater resistance to light and dark conditions. Previous studies have shown that TCP transcription factors activate PIFs under shade conditions, further promoting auxin synthesis and plant growth (Figure 12) [24,25]. The cis-element analysis also revealed that all PbTCP transcription factors are highly expressed in response to light stress. In A. thaliana, TCP2 positively regulates key factors in the light signaling pathway, such as HY5 and HYH, altering plant morphology under light conditions [62]. Similar conclusions have been drawn in other studies [51], highlighting the critical role of TCP transcription factors in adapting to changes in environmental light conditions.

4. Materials and Methods

4.1. Identification of PbTCP Genes in P. bournei

The genome assembly file for P. bournei was downloaded from the NCBI Conserved Domain Database (NCBI-CDD) [63,64]. To search for TCP gene candidates within the P. bournei genome, we utilized a specific Hidden Markov Model (HMM) of the TCP transcription factor (Pfam number: PF03634) [64]. Subsequently, we utilized TB tools to compare the protein sequences of A. thaliana and P. bournei with default parameters. A total of 30 genes were identified, while 29 belong to the TCP transcription factor family, and were renamed according to their position on the chromosomes. Additionally, we used Batch CD-Search (default parameters) from the website (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 6 August 2024))) to confirm the conserved domain of PbTCP [65]. The physicochemical characteristics of all PbTCP proteins, encompassing amino acid number (size), molecular weight (MW), theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY), were assessed through the utilization of the ExPASy online tool (https://www.expasy.org/ (accessed on 6 August 2024)). Subsequently, the website Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/ (accessed on 6 August 2024)) was employed to forecast the subcellular localization of the PbTCP proteins.

4.2. Phylogenetic Analysis

Sequence alignment was conducted on 29 amino acid sequences of the PbTCP protein using the MUSCLE function, with default parameters, in MEGA 7.0.21 [66]. The multiple sequence alignment results were then swiftly trimmed using Quick Run TrimAL in TBtools (V1.120) [67]. Subsequently, a dependable intraspecific phylogenetic tree was generated using the trimmed sequences. Furthermore, a phylogenetic tree of A. thaliana and P. bournei was constructed using the neighbor-joining approach (NJ) with 1000 bootstrap replications in MEGA 7.0.21. The interspecific phylogenetic tree was improved with the Evolview website (https://www.evolgenius.info/evolview/#/treeview (accessed on 6 August 2024)). Additionally, Jalview 2.11.2.7 was used to enhance the sequence alignment results [68].

4.3. Protein Motifs Analysis and Gene Structures, Conserved Domain

Using the Multiple Em for Motif Elicitation (MEME) suite (http://meme-suite.org/tools/meme (accessed on 6 August 2024)) [69], conservative motifs of the protein sequences were exhibited with two parameters: the maximum motif number was 10 and the motif site occurrences were distributed at zero or one per sequence. Distribution data for exons and introns was obtained from the genome GFF files of P. bournei. The conserved domain of PbTCP proteins was uploaded and verified by the NCBI-CDD database. Finally, the intron–exon structure, conserved domain, and 10 motifs of the PbTCP proteins were visualized using TBtools (V1.120) [67].

4.4. Chromosomal Location, Gene Duplication, and Collinearity Relationship

The location of the PbTCP genes was determined using the GFF annotation files of the P. bournei genome [63]. Utilizing MG2C v2.1 (http://mg2c.iask.in/mg2c_v2.1/ (accessed on 7 August 2024)), we generated a pattern of chromosome location. Gene duplication models of the PbTCP family gene were identified and analyzed using Tbtools. To obtain the genome files of four other plant species, namely Arabidopsis, rice, and poplar, we retrieved them from the NCBI. The collinearity relationships between PbTCP and the three mentioned species were analyzed and visualized using the “Advanced Circos” tool in Tbtools.

4.5. Cis-Elements in the Promoter and Expression Analysis of PbTCP Genes

The 2000 bp upstream sequence of PbTCPs was extracted and served as the promoter sequence used to identify cis-elements and prediction using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 7 August 2024)) [70]. The positions and numbers of the identified cis-elements were visualized by Tbtools. RNA-seq data of different tissues in P. bournei were downloaded from the NCBI database using BioProject accession number PRJNA628065 [65].

4.6. Plant Materials and Abiotic Stresses Treatment

Seedlings were carefully selected from one-year-old P. bournei specimens. Before planting the seedlings, the soil was prepared by mixing peat moss, humus soil, sandy soil, and perlite in the ratio of 5:2:2:1, and the organic matter content was between 2.57% and 6.07%. The annual average temperature in the growth area was between 16 °C and 20 °C, the annual precipitation was between 900 mm and 2100 mm, and the annual relative humidity was about 77%. In our experiment, samples were taken from the drought and heat treatment groups at 0, 4, 8, 12, and 24 h, from the light treatment group at 0, 24, 48 and 72 h, and from the dark treatment group at 0, 12, 24, 48 and 72 h. The seedlings collected at 0 h were used as the control group. Each treatment has three biological replicates, meaning each treatment includes three individuals (at 0 h). A sample taken from one seedling (3–5 mature leaves) constitutes one biological replicate. Each group was subjected to the appropriate experimental conditions. During the stress treatment process, the parameters set in the artificial climate chamber were as follows: the light period was 12 h/d, LED lamps were used for lighting, the photosynthetically active radiation was set at 1200 μmol·mol−1·s−1, and the temperature was 25 °C. The experimental plan was designed to simulate drought stress, in which the treatment groups were transplanted into beakers containing 10% PEG 6000, which was a means of simulating drought conditions. For temperature treatment, individuals were incubated at 40 °C. For light stress, the control group was sampled under natural conditions at 0, 24, 48, and 72 h, while the treatment group was continuously exposed to light and sampled at 0, 24, 48, and 72 h. For dark treatment, except for the control group, samples were collected at 12, 24, 48, and 72 h in darkness. After the treatment was completed, the leaves were immediately collected and stored at −80 °C in liquid nitrogen for subsequent RNA extraction.

4.7. RNA Extraction and qRT-PCR Analysis

RNA was extracted from both control and stress-treated leaf tissue samples using a HiPure Plant RNA Mini Kit (Magen), followed by cDNA synthesis with a PrimeScript RT reagent Kit (Perfect Real Time) (TaKaRa). qRT-PCR was then conducted to determine gene (PCF: PbTCP16 and PbTCP23, CIN: PbTCP7 and PbTCP29, CYC/TB1: PbTCP14 and PbTCP15) expression profiles in response to stress. The qRT-PCR experiment utilized specific primers which were designed through the Primer 3 website (http://bioinfo.ut.ee/primer3-0.4.0/ (accessed on 7 August 2024)). PbEF1α was selected as the reference gene (GenBank number, KX682032.1) [43]. The raw Cq values were assessed using the 2−ΔΔCT method and then compared to the reference gene [71]. All experiments were conducted with three biological replicates (Taking one sample (three to five mature leaves) from each of three seedlings) and three technical replicates. The relative levels of gene expression were analyzed using one-way ANOVA, with multiple comparisons with the control group at a significance threshold of 5%. The expression graphs were generated via GraphPad Prism 8.3.0 software.

5. Conclusions

This study identified 29 PbTCP genes in P. bournei and analyzed their properties, relationships, structures, functions, and expression patterns. The TCP gene family was classified into two classes: Class I (PCF) and Class II (CIN and CYC/TB1). The PCF subgroup was the most conserved, while the CYC/TB1 subgroup exhibited the greatest evolutionary divergence. PCF genes showed notable expression in roots, stems, and leaves, suggesting a potential involvement in growth and development. Further analysis revealed that PbTCP14 and PbTCP15 had high expression in both light and dark environments, suggesting a potential role in light stress resistance. However, the mechanisms by which TCP transcription factors regulate growth and development in P. bournei require further investigation. This study systematically analyzed the expression patterns of TCP genes in different tissues and their response to abiotic stress, providing valuable insights into their role in regulating plant growth and development. These findings lay the foundation for future research on stress resistance and functional genomics in this species.

Author Contributions

Writing-original draft preparation, W.L. (Wenhai Liao) and H.Y.; Conceptualization, Q.Z.; Software, W.L. (Wei Liu) and L.C.; Conceptualization and Methodology, Y.L.; Writing—review & editing. Q.L.; Writing—review & editing, S.H., O.U.R. and W.L. (Wenzhuo Lv); Editing, K.Z.; Formal analysis, Y.Z.; Writing—Reviewing and funding acquisition, S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fujian Agriculture and Forestry University Forestry peak discipline construction project (71201800739 to S.C.).

Data Availability Statement

Data are contained within the article.

Acknowledgments

We are grateful to the reviewers for their helpful comments on the original manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Plants 13 03095 g001
Figure 1. Conceptual framework of regulation of auxin signaling by TCP transcription factors. Note: The role of TCP transcription factors in regulating auxin biosynthesis and plant responses to abiotic stress. TCP5, TCP13, and TCP17 enhance auxin synthesis by upregulating PIF, which in turn increases the expression of YUC enzymes, key players in the auxin biosynthesis pathway. Elevated auxin levels contribute to plant growth and stress adaptation. TCP14 and TCP15 specifically promote plant elongation by regulating auxin-induced genes associated with cell expansion. Together, these TCP factors support plant resilience under various abiotic stresses.
Figure 1. Conceptual framework of regulation of auxin signaling by TCP transcription factors. Note: The role of TCP transcription factors in regulating auxin biosynthesis and plant responses to abiotic stress. TCP5, TCP13, and TCP17 enhance auxin synthesis by upregulating PIF, which in turn increases the expression of YUC enzymes, key players in the auxin biosynthesis pathway. Elevated auxin levels contribute to plant growth and stress adaptation. TCP14 and TCP15 specifically promote plant elongation by regulating auxin-induced genes associated with cell expansion. Together, these TCP factors support plant resilience under various abiotic stresses.
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Figure 2. Chromosomal localization analysis of the TCP gene family of Phoebe bournei (Hemsl.). Distribution of PbTCP genes in the P. bournei chromosome. (A) Each chromosome figure shows the chromosome number at the top. The scale on the left can be used to assess chromosome length and gene position. (B) The number of TCP genes on the chromosome.
Figure 2. Chromosomal localization analysis of the TCP gene family of Phoebe bournei (Hemsl.). Distribution of PbTCP genes in the P. bournei chromosome. (A) Each chromosome figure shows the chromosome number at the top. The scale on the left can be used to assess chromosome length and gene position. (B) The number of TCP genes on the chromosome.
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Figure 3. Phylogenetic analysis of TCP protein. (A) Genome and protein sequences P. bournei. (B) The percentage of 3 subfamilies of PbTCP genes. (C) Phylogenetic tree of PbTCP and AtTCP proteins. The arcs of different colors indicate a subfamily of the TCP family. One thousand times with MEGA11 and Bootstrap respectively. The tree was constructed by 29 PbTCPs identified in P. bournei and 25 AtTCPs identified in A. thaliana.
Figure 3. Phylogenetic analysis of TCP protein. (A) Genome and protein sequences P. bournei. (B) The percentage of 3 subfamilies of PbTCP genes. (C) Phylogenetic tree of PbTCP and AtTCP proteins. The arcs of different colors indicate a subfamily of the TCP family. One thousand times with MEGA11 and Bootstrap respectively. The tree was constructed by 29 PbTCPs identified in P. bournei and 25 AtTCPs identified in A. thaliana.
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Figure 4. Multiple sequence alignment of TCP domains. Note: TCP domain serial alignment of P. bournei TCP family members. At the bottom, the highly conserved amino acid position is indicated by the length of the rectangle; The serial indicator is displayed at the bottom.
Figure 4. Multiple sequence alignment of TCP domains. Note: TCP domain serial alignment of P. bournei TCP family members. At the bottom, the highly conserved amino acid position is indicated by the length of the rectangle; The serial indicator is displayed at the bottom.
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Figure 5. PbTCP protein structure analysis. Note: The categories of three branches are marked on the left, and the confidence level of the protein’s secondary structure is indicated by different colors, and the four levels of confidence are shown in the lower right corner.
Figure 5. PbTCP protein structure analysis. Note: The categories of three branches are marked on the left, and the confidence level of the protein’s secondary structure is indicated by different colors, and the four levels of confidence are shown in the lower right corner.
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Figure 6. PbTCP conserved domain and motif analysis. (A) Phylogenetic tree of PbTCPS. (B) The motif of PbTCPS. Patterns 1–10 are displayed in rectangles of different colors. Protein length can be estimated using the scale at the bottom. (C) PbTCP protein with conserved domains. (D) Gene structure of the PbTCPS gene. Yellow boxes indicate exons (CDS), black lines indicate introns, and blue boxes indicate 5′ and 3′ untranslated regions. (E) The sequence logo of Motif1.The colored letters indicate the specific sequence of motif1.
Figure 6. PbTCP conserved domain and motif analysis. (A) Phylogenetic tree of PbTCPS. (B) The motif of PbTCPS. Patterns 1–10 are displayed in rectangles of different colors. Protein length can be estimated using the scale at the bottom. (C) PbTCP protein with conserved domains. (D) Gene structure of the PbTCPS gene. Yellow boxes indicate exons (CDS), black lines indicate introns, and blue boxes indicate 5′ and 3′ untranslated regions. (E) The sequence logo of Motif1.The colored letters indicate the specific sequence of motif1.
Plants 13 03095 g006
Plants 13 03095 g007
Figure 7. Genomic location, replication events, and homology of the PbTCP gene. Note: Synteny analysis of the PbTCP family in P. bournei. The gray line represents all isotope blocks in the P. bournei genome, while the red line represents the gene pairs of the duplicate PbTCP. The chromosome number is displayed in a rectangular box for each chromosome.
Figure 7. Genomic location, replication events, and homology of the PbTCP gene. Note: Synteny analysis of the PbTCP family in P. bournei. The gray line represents all isotope blocks in the P. bournei genome, while the red line represents the gene pairs of the duplicate PbTCP. The chromosome number is displayed in a rectangular box for each chromosome.
Plants 13 03095 g007
Plants 13 03095 g008
Figure 8. Orthologous analysis of TCP genes in A. thaliana, O. sativa, P. trichocarpa and P. bournei. (A) Genome homology analysis of A. thaliana and P. trichocarpus. The grey line represents the genome pairs between homologous blocks, and the blue line highlights the TCPS gene pairs synthesized in the three species. (B) Number of genome pairs of three clades of different species. (C) Number of shared genes of three genome pairs of three species.
Figure 8. Orthologous analysis of TCP genes in A. thaliana, O. sativa, P. trichocarpa and P. bournei. (A) Genome homology analysis of A. thaliana and P. trichocarpus. The grey line represents the genome pairs between homologous blocks, and the blue line highlights the TCPS gene pairs synthesized in the three species. (B) Number of genome pairs of three clades of different species. (C) Number of shared genes of three genome pairs of three species.
Plants 13 03095 g008
Plants 13 03095 g009
Figure 9. Analysis of the cis-acting element of the gene for the promoter. (A) Cis-component predictions of 29 PbTCP gene promoter serial (−2000 bp) were analyzed using PlantCARE technology. Here are the 19 categories of cis-elements. (B) Number of 19 cis-components for the 29 PbTCP genes.
Figure 9. Analysis of the cis-acting element of the gene for the promoter. (A) Cis-component predictions of 29 PbTCP gene promoter serial (−2000 bp) were analyzed using PlantCARE technology. Here are the 19 categories of cis-elements. (B) Number of 19 cis-components for the 29 PbTCP genes.
Plants 13 03095 g009
Plants 13 03095 g010
Figure 10. Expression spectrum of PbTCP. Note: Different colors are used to indicate the level of expression, and there is an expression value on the right. At the bottom, there are three sub-categories with gene names.
Figure 10. Expression spectrum of PbTCP. Note: Different colors are used to indicate the level of expression, and there is an expression value on the right. At the bottom, there are three sub-categories with gene names.
Plants 13 03095 g010
Plants 13 03095 g011
Figure 11. The expression of PbTCPs under high temperature, drought, light stress and dark stress was detected by qRT-PCR. (A) Relative gene expression levels at high temperature (40 °C) and control (25 °C). (B) Relative gene expression levels at the same point (4, 8, 12, and 24 h) were treated with 10% PEG nutrient solution in a simulated arid environment. The control group is treated in distilled water. (C) Relative gene expression levels under light stress. (D) Relative gene expression levels under dark stress. (* p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001).
Figure 11. The expression of PbTCPs under high temperature, drought, light stress and dark stress was detected by qRT-PCR. (A) Relative gene expression levels at high temperature (40 °C) and control (25 °C). (B) Relative gene expression levels at the same point (4, 8, 12, and 24 h) were treated with 10% PEG nutrient solution in a simulated arid environment. The control group is treated in distilled water. (C) Relative gene expression levels under light stress. (D) Relative gene expression levels under dark stress. (* p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001).
Plants 13 03095 g011
Plants 13 03095 g012
Figure 12. The different responses of PIFs and HY5 under Pfr and Pr form. Note: Light promotes the degradation of PIF and the expression of HY5 in the nucleus through photosensitive pigments (Pfr); Under dark conditions, the Pr form promotes the transcription and accumulation of PIFs and the degradation of HY5, which together regulate the normal physiological state of plants.
Figure 12. The different responses of PIFs and HY5 under Pfr and Pr form. Note: Light promotes the degradation of PIF and the expression of HY5 in the nucleus through photosensitive pigments (Pfr); Under dark conditions, the Pr form promotes the transcription and accumulation of PIFs and the degradation of HY5, which together regulate the normal physiological state of plants.
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Table 1. Analysis of amino acid sequence characteristics of TCP gene family of Phoebe bournei (Hemsl.).
Table 1. Analysis of amino acid sequence characteristics of TCP gene family of Phoebe bournei (Hemsl.).
Gene AccessionProtein NameAA/aaMW/kDapIIIAIGRAVYSubcellular Localization
OF00845-RAPbTCP144346.016.6759.4266.5−0.491Nucleus
OF19791-RAPbTCP230133.728.2764.2271.3−0.642Nucleus
OF22744-RAPbTCP325928.659.8861.3358.84−0.777Nucleus
OF11465-RAPbTCP429531.836.7251.4663.53−0.693Nucleus
OF11860-RAPbTCP532236.069.2755.3951.61−0.774Nucleus
OF04159-RAPbTCP658363.365.7354.9673.95−0.54Nucleus
OF04032-RAPbTCP741045.388.6351.7669.46−0.74Nucleus
OF25895-RAPbTCP836439.118.7961.5967.86−0.563Chloroplast, Nucleus
OF25557-RAPbTCP967972.159.4937.1192.99−0.092Mitochondrion
OF23858-RAPbTCP1017717.8010.156966.89−0.117Nucleus
OF23646-RAPbTCP1127730.787.6344.6774.69−0.307Nucleus
OF23645-RAPbTCP1237742.239.5143.1271.17−0.62Nucleus
OF23643-RAPbTCP1343548.758.9442.5878.23−0.575Nucleus
OF23640-RAPbTCP1423125.559.6545.2458.74−0.943Nucleus
OF23639-RAPbTCP1543848.419.5748.3769.06−0.654Nucleus
OF24987-RAPbTCP1625025.866.1148.7371.8−0.399Nucleus
OF21041-RAPbTCP1744045.757.3163.0662.25−0.453Nucleus
OF01693-RAPbTCP1843847.806.5847.4362.95−0.743Nucleus
OF02672-RAPbTCP1933137.119.1342.3161.66−0.872Nucleus
OF11303-RAPbTCP2040644.719.4851.8970.86−0.596Nucleus
OF02247-RAPbTCP2136338.358.1158.3965.21−0.572Nucleus
OF05103-RAPbTCP2246451.055.9557.6757.87−0.809Nucleus
OF18329-RAPbTCP2323825.288.8951.8576.34−0.321Nucleus
OF26947-RAPbTCP2430932.915.6853.6173.88−0.469Nucleus
OF00332-RAPbTCP2529030.599.3966.1272.07−0.364Nucleus
OF29850-RAPbTCP2634436.009.5568.4272.12−0.426Nucleus
OF21638-RAPbTCP2737840.606.6456.6265.61−0.536Nucleus
OF17792-RAPbTCP2823424.439.9856.5178.08−0.279Nucleus
OF09077-RAPbTCP2941445.106.5769.157.39−0.746Nucleus
Note: AA: number of amino acids; MW: molecular weight; pI: theoretical isoelectric point; II: instability index; AI: aliphatic index; GRAVY: grand average of hydropathicity.
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MDPI and ACS Style

Lv, W.; Yang, H.; Zheng, Q.; Liao, W.; Chen, L.; Lian, Y.; Lin, Q.; Huo, S.; Rehman, O.U.; Liu, W.; et al. Identification and Expression Analysis of TCP Transcription Factors Under Abiotic Stress in Phoebe bournei. Plants 2024, 13, 3095. https://doi.org/10.3390/plants13213095

AMA Style

Lv W, Yang H, Zheng Q, Liao W, Chen L, Lian Y, Lin Q, Huo S, Rehman OU, Liu W, et al. Identification and Expression Analysis of TCP Transcription Factors Under Abiotic Stress in Phoebe bournei. Plants. 2024; 13(21):3095. https://doi.org/10.3390/plants13213095

Chicago/Turabian Style

Lv, Wenzhuo, Hao Yang, Qiumian Zheng, Wenhai Liao, Li Chen, Yiran Lian, Qinmin Lin, Shuhao Huo, Obaid Ur Rehman, Wei Liu, and et al. 2024. "Identification and Expression Analysis of TCP Transcription Factors Under Abiotic Stress in Phoebe bournei" Plants 13, no. 21: 3095. https://doi.org/10.3390/plants13213095

APA Style

Lv, W., Yang, H., Zheng, Q., Liao, W., Chen, L., Lian, Y., Lin, Q., Huo, S., Rehman, O. U., Liu, W., Zheng, K., Zhang, Y., & Cao, S. (2024). Identification and Expression Analysis of TCP Transcription Factors Under Abiotic Stress in Phoebe bournei. Plants, 13(21), 3095. https://doi.org/10.3390/plants13213095

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