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Article

Genome-Wide Identification and Characterization of TCP Genes in Eight Prunus Species and Their Expression Patterns Under Cold Stress in P. tenella var. tenella

by
Qiang Zhang
1,
Cheng Qian
2,
Lulu Li
2,
Wei Li
2,
Yanhua Li
2,* and
Han Zhao
3,*
1
Institute of Marine Science and technology, Shandong University, Qingdao 266215, China
2
College of Landscape Architecture and Forestry, Qingdao Agricultural University, Qingdao 266109, China
3
Research Institute of Non-Timber Forestry, Chinese Academy of Forestry, Zhengzhou 450014, China
*
Authors to whom correspondence should be addressed.
Genes 2024, 15(11), 1443; https://doi.org/10.3390/genes15111443
Submission received: 13 September 2024 / Revised: 29 October 2024 / Accepted: 4 November 2024 / Published: 8 November 2024
(This article belongs to the Section Plant Genetics and Genomics)
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Abstract

:
Background/Objectives: Teosinte branched1/Cycloidea/Proliferating cell nuclear antigen factors (TCPs) are plant-specific transcription factors involved in leaf development, flowering, branching, hormone signaling, and stress responses. Prunus a key temperate fruit tree with ornamental spring blooms, still lacks comprehensive TCP gene studies across many species. Methods: We identified 154 TCP genes in eight Prunus species: 19 in Prunus tenella var. tenella, 19 in P. amygdalus, 17 in P. armeniaca ‘Rojo Pasion’, 19 in P. mira, 20 in P. jamasakura var. jamasakura, 19 in P. fruticosa, 19 in P. mume var. tortuosa, and 22 in P. × yedoensis ‘Somei-yoshino’. These genes were classified into PCF, CIN, and CYC/TB1 groups. We examined segmental duplication, conserved motifs, and cis-acting elements. Expression patterns of 12 TCPs in P. tenella var. tenella were tested under low-temperature stress (25 °C, 5 °C, −5 °C, and −10 °C), and PtTCP9’s subcellular localization was determined. Results: TCP genes within the same groups showed similar motifs and cis-acting elements. Cold stress analysis identified multiple low-temperature-responsive elements in gene promoters. Four genes (PtTCP2, PtTCP6, PtTCP14, and PtTCP16) increased expression under cold stress, while six genes (PtTCP1, PtTCP5, PtTCP8, PtTCP9, PtTCP17, and PtTCP19) decreased. PtTCP9 was localized to the nucleus. Conclusions: This was the first genome-wide study of the TCP gene family in these eight Prunus species, providing valuable insights into the characteristics and functions of TCP genes within this important genus.

1. Introduction

Transcription factors play a crucial role in plant growth, development, and response to environmental stress. TCP is a plant-specific transcription factor family named after its first characterized members: Teosinte branched1 from maize (Zea mays), Cycloidea from Antirrhinum, and PCF from rice (Oryza sativa) [1,2,3]. Sequence alignment analysis has revealed that members of the TCP gene family contain a unique domain known as the TCP domain, which features a nonclassical basic helix–loop–helix structure. This domain is mainly related to DNA binding, protein interaction, and protein nuclear localization [1,2,3,4]. The nonclassical bHLH (basic Helix–Loop–Hleix) motifs are located at the N-end of the TCP domain [1,2,3,5]. The TCPs from eight Prunus species were classified into Class I and Class II subfamilies. Class I is also known as the PCF subfamily, and Class II is further divided into CIN and CYC/TB1 groups. The R domain is found only in members of the CYC and TB1 subfamilies and plays a role in protein interactions [4].
The TCP gene family plays key roles in plant growth, development, and response to stresses, influencing processes such as seed germination, bud growth, flower organ development, leaf morphogenesis, apical dominance, axillary meristem development [6,7,8,9,10,11], and hormonal signal transduction [12,13]. TCP members can be regulated by endogenous signals such as plant hormones [14]. The functions of TCP proteins in the cold resistance of plants have been identified [15,16]. In O. sativa, the overexpression of OsTCP14 and OsTCP21 increases the sensitivity of plants to low temperatures, and silencing these two TCP genes through RNAi technology enhances low-temperature tolerance in O. sativa [17]. In addition, TCP members can respond to exogenous factors such as abiotic stress [18].
The Prunus genus comprises more than 200 species. Many Prunus plants are important fruit and nut crops as well as ornamental plants. Although various classification systems exist for Prunus species, the most widely accepted is Rehder’s classification, which divides this genus into five subgenera: Prunus, Amygdalus, Padus, Cerasus, and Laurocerasus [19]. P. mume and P. armeniaca, which belong to the Prunus subgenus, are fruit crops and ornamental trees in East Asia. P. jamasakura var. jamasakura, P. × yedoensis, and P. fruticosa belong to the Cerasus subgenus and are important ornamental woody plant that bloom in spring. P. fruticosa is a wild species in the temperate regions of Europe and Asia [20]. Studies have shown that P. fruticosa is one of the parents of cherry (P. cerasus) [21]. P. amygdalus, P. tenella var. tenella, and P. mira belong to the Amygdalus subgenus. P. amygdalus is an important nut crop and ornamental tree. P. tenella var. tenella is a related species of almond and is also known as the wild almond [22]. It is an excellent raw material for breeding new resistant varieties of almond and drought- and cold-resistant dwarf rootstock types of stone fruit trees [23,24,25]. TCP family members have been identified in many plants, excluding the aforementioned species.
This study aims to identify TCP family genes of eight species from three subgenera of Prunus. The phylogenetic analysis, conserved motifs, sequence alignment, chromosome localization, collinearity analysis, and cis-acting element analysis of TCP genes were conducted. Additionally, the study sought to investigate the expression pattern of PtTCPs under cold stress using quantitative real-time polymerase chain reaction (qRT-PCR) and to verify the subcellular localization of the PtTCP9 transcription factor. The overall goal was to preliminarily reveal the evolutionary correlation of the TCP family in Prunus and the gene expression changes in response to cold stress in P. tenella var. tenella.

2. Materials and Methods

2.1. Taxon Sampling

The genomes of P. amygdalus (GCA_902201215.1), P. armeniaca ‘Rojo Pasion’ (GCA_903112645.1), P. fruticosa (GCA_018703695.1), P. jamasakura var. jamasakura (GCA_020521455.1), P. mira (GCA_020226265.1), P. mume var. tortuosa (GCA_029339155.1), and P. × yedoensis ‘Somei-yoshino’ (GCA_005406145.1) were downloaded from the NCBI website. The whole genome sequence data of the P. tenella var. tenella genome have been deposited in the Genome Warehouse at the National Genomics Data Center (accession number: GWHCBGA00000000). P. tenella var. tenella seeds were collected from Toli County, Xinjiang, China (46°08′51.33″ N; 83°33′56.33″ E; Elevation 823 m), and the characteristics of P. tenella var. tenella are shown in Figure S1. Then the seeds were treated with a solution containing gibberellin (200 mM) for 24 h to release dormancy, after which they were planted in plastic pots (15 × 12 × 14 cm) with consistent conditions (22 °C, 16 h light/8 h dark). For the convenience of taxonomic sampling and discussion, we have used and applied the names of species and infraspecific taxa according to POWO (https://powo.science.kew.org/, accessed on 28 October 2024), which includes the latest classifications and information on global plant species.

2.2. Identification of TCP Genes

The TCP Hidden Markov model (PF03634) downloaded from the Pfam website (http://pfam.xfam.org/, accessed on 2 July 2024) was run on the whole-genome protein sequence of eight Prunus species (P. tenella var. tenella, P. amygdalus, P. armeniaca ‘Rojo Pasion’, P. fruticosa, P. jamasakura var. jamasakura, P. mira, P. mume var. tortuosa, P. × yedoensis ‘Somei-yoshino’). The obtained sequence was used to build the TCP Hidden Markov model, and a search was performed again with this model to obtain members of the TCP family. The Conserved Domains Tool and InterProScan v102.0 (https://www.ebi.ac.uk/interpro/, accessed on 2 July 2024) online tools were used to confirm the conserved domains in protein sequences of TCPs, and the proteins that did not contain TCP domains were removed.

2.3. Chromosomal Location and Structure Analysis of TCPs

The distribution of TCPs on chromosomes was plotted using the gff3 file from genomes and TBtools-II v2.085 software [26]. An online analysis of conserved motifs of different TCP proteins was conducted using MEME v5.5.6 (https://meme-suite.org/meme/, accessed on 29 August 2024) [27], with a motif search number of 10. The obtained motifs were visualized and analyzed using TBtools software.

2.4. Prediction of cis-Regulatory Elements and Transcription Start Sites in the Promoter of PtTCPs

TBtools software was used to extract the 2000 bp upstream sequence of ATG as the promoter subsequence [26]. At the same time, the PlantCARE v1.0 Database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 August 2024) was used to predict the type and number of cis-regulatory elements in each member’s promoter. The data were screened and plotted in Excel 2016.

2.5. Construction of Phylogenetic Tree

We downloaded the genomes of Arabidopsis thaliana and O. sativa from NCBI, extracted TCP protein sequences as outgroups, constructed an amino acid matrix containing TCP protein sequences from eight Prunus species (Supplementary Data S1), and conducted phylogenetic analysis using MEGA 6.0. The tree was optimized using the neighbor-joining method, 1000 bootstrap tests, and the online tool iTOL v6.0 (https://itol.embl.de/, accessed on 22 July 2024).

2.6. Cold-Stress Treatment and qPCR Analyses

To further understand the expression patterns of the TCP genes in P. tenella var. tenella in response to cold stress, based on previous research results, nine genes were selected: PtTCP1, PtTCP2, PtTCP3, PtTCP4, PtTCP5, PtTCP6, PtTCP8, PtTCP9, and PtTCP12. Two-month-old P. tenella var. tenella seedlings were transferred to an incubator, and the temperature was changed at 3 °C/h until the final target temperatures (25, 5, 0, −5, and −10 °C) were reached. All samples were immediately frozen in liquid nitrogen and stored at −80 °C for further experiments.
The total RNA was extracted from all samples using the Easy Fast reagent (Tiangen, Beijing, China). Subsequently, SuperMix (Vazyme, Beijing, China) was used to reverse transcribe RNA into cDNA. The primers were identified based on the protein sequence of the desired gene (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome, accessed on 30 August 2024) (Table S1). Then, qPCR experiments were conducted on the selected genes. Three biological replicates were employed, with the PtPP2A gene as the reference gene. The 2−ΔΔCt method was used to calculate the relative expression level of the PtTCP genes.

2.7. Subcellular Localization of PtTCP9 Protein

The full-length coding sequences (CDS) of the PtTCP9 gene were cloned using the complementary DNA (cDNA) of P. tenella var. tenella leaf as the template, and the PtTCP9 gene was connected to the vector 1300-GFP by the double enzyme digestion method. The primers used in the study are shown in Table S1. The overexpressed vector was transformed into DH5α Escherichia coli in receptor state, and the positive clones were identified by colony PCR and confirmed by sequencing. The correctly sequenced plasmid was then transformed into the Agrobacterium GV3101 in receptor state. Positive colonies were selected and expanded in Luria–Bertani (LB) liquid medium until the OD600 reached approximately 0.6. The infection solution (MS + 10 mM MES + 0.15 mM AS + 10 mM MgCl2) was resuspended to an OD600 of about 0.8 and incubated at room temperature for 2 h. The lower epidermis of Nicotiana. benthamiana leaves was injected with a syringe, incubated at 25 °C for 24 h, cultured in the presence of light for 24 h, and observed and photographed under a laser scanning confocal microscope (Agilent, Santa Clara, CA, USA).

3. Results

3.1. Identification and Chromosomal Location of TCP Genes

A total of 19, 19, 17, 19, 20, 19, 19, and 22 TCP genes were identified in the P. tenella var. tenella, P. amygdalus, P. armeniaca ‘Rojo Pasion’, P. fruticosa, P. jamasakura var. jamasakura, P. mira, P. mume var. tortuosa, and P. × yedoensis ‘Somei-yoshino’, respectively, using HMM maps (PF03634) (Table 1). These candidate proteins were then confirmed after further validation using the conserved domain database (CDD) and Pfam database. The TCP genes in P. tenella var. tenella were annotated as PtTCP1 to PtTCP19 based on their genome distribution and relative linear orders among the respective chromosomes (Figure 1). The proportion of TCPs ranged from 17 to 22, the highest in P. × yedoensis ‘Somei-yoshino’, followed by P. jamasakura var. jamasakura and P. fruticosa, while P. armeniaca ‘Rojo Pasion’ had the least (Table 1).
Due to variable shear, 154 genes encode 157 TCP proteins (Table 1). The length of TCP proteins varied from 145 (Pfe03g1497) to 966 (PmuVar_Chr4_1948) amino acid residues (Table S2). The molecular weight (MW) ranged from 15.0 to 104.9 kDa. The protein isoelectric point (pI) ranged from 4.63 (PtTCP12) to 11.22 (PtTCP15), with a mean of 7.23. The calculation range of the hydrophilicity index (GRAVY) values for all TCPs was between −1.015 and 0.128, with only positive values for PmuVar_Chr4_1948. This indicates that most TCPs were essentially hydrophilic. The range of the instability index was between 34.0 and 76.7. The value of the aliphatic index ranged from 51.4 to 90.9. Among those TCP proteins, 89.2% proteins were located in the nucleus, 15 TCP were on the chloroplast, and only PmuVar_Chr4_1948 and PmuVar_Chr5_3217 were found in the Plasma membrane and Cytoplasm, respectively.
Because the P. × yedoensis ‘Somei-yoshino’ genome is not assembled to the chromosomal level, chromosomal localization of their TCP genes is not possible. In seven Prunus species, TCP genes were unevenly distributed on 6–7 chromosomes. Except for P. mume var. tortuosa, which has no TCP gene on Chr01, the other six species have the maximum number of TCPs on Chr01. Except for P. tenella var. tenella and P. mume var. tortuosa, the other species have no TCP gene on Chr08.

3.2. Phylogenetic Analysis of TCP Proteins

A phylogenetic tree was constructed using TCP genes in eight Prunus species to examine the phylogenetic relationships of TCP genes in the Prunus species. Based on the phylogenetic tree, the TCP family was divided into PCF, CIN, and CYC/TB1 groups (Figure 2). Also, a phylogenetic tree was constructed by combining TCPs from P. tenella var. tenella, and O. sativa and A. thaliana also support the above classification (Figure S2). Among 157 TCPs, 82, 51, and 24 TCP transcription factors were divided into the PCF, CIN, and CYC/TB1 subgroups, respectively. Among all Prunus species, the PCF subfamily has the highest number of members (43.8–52.6%), while the CYC/TB1 subfamilies have the lowest numbers (12.5–20.0%).

3.3. Collinearity Analysis of TCP Genes

The collinearity analysis showed that most of the TCPs are segmentally duplicated genes (76.3%), followed by dispersed genes (23.7%) (Table S2). Only two tandem genes were found in P. amygdalus. Those results indicate that segmental duplication was the main driving force behind the amplification of TCPs in the Prunus species.
The Ka (non-synonymous substitution)-to-Ks (synonymous substitution) ratio for each pair of paralogous genes was calculated for a closer look at the rate of TCP gene evolution in eight Prunus species. In this study, the Ks values of P. × yedoensis ‘Somei-yoshino’ gene pairs were mainly distributed between 0.009 and 1.798, whereas those of other Prunus species were between 1.0 and 3.0 (Figure 3A). The Ka values of most P. × yedoensis ‘Somei-yoshino’ gene pairs were less than 0.03, except PQQ12916.1_PQP99671.1 (0.272) and PQQ03731.1_PQP94471.1 (0.235). The Ka values of other plum plants were mostly between 0.2 and 0.6. In addition, the Ka/Ks value of other gene pairs was less than 1, except PQM42156.1_PQM36679.1 (1.436), indicating that PQM42156.1 and PQM36679.1 genes may be subjected to positive selection (Figure 3B).
Subsequently, a phylogenetic tree of eight Prunus species was constructed, and the genome collinearity of TCPs within the Prunus genus was determined based on the evolutionary relationships among the various species. The findings indicated significant collinearity across different Prunus species (Figure 4). The number of orthologous pairs in the Cerasus subgenus (P. jamasakura var. jamasakura vs. P. × yedoensis ‘Somei-yoshino’ and P. × yedoensis ‘Somei-yoshino’ vs. P. fruticosa) was 31 each. Further, 29 orthologous pairs were found between P. mume var. tourtosa and P. armeniaca ‘Rojo Pasion’ (Prunus subgenera). For the Amygdalus subgenus, 40 and 41 orthologous pairs were found in P. amygdalus vs. P. mira and P. mira vs. P. tenella var. tenella, respectively.

3.4. Conserved Motif and Domain Analysis of TCPs

All TCPs displayed the TCP domain, characterized by a basic helix–loop–helix structure (Figure S3). A total of 20 conserved motifs of 154 TCPs were analyzed to understand their structural characteristics (Figure 5). The motifs owned or shared by most members of the gene family may be an integral part of this gene family and have important functions or structures. Although the conserved motifs of TCPs differed in composition, all TCP proteins contained motif 1 and motif 23. In addition, different conserved motifs were present in different subfamilies. For example, motif 11 existed only in some members of the PCF subfamily, while motif 19 was exclusive to certain members of the CYC/TB1 subfamilies. Most members of the PCF subfamily contained motif 3.

3.5. Prediction of cis-Elements in the Promoter of TCPs

PlantCARE was used to predict cis-acting elements in the promoter region of TCP genes (2000 bp upstream of the transcription start site) to explore the transcriptional regulation of TCPs (Figure 6). The findings indicate that 4225 cis-acting elements were identified. The number of cis-acting elements of the TCP promoters in eight Prunus species was variable (Figure 6). Among these, P. amygdalus contained the largest number of cis-acting elements (581), followed by P. × yedoensis ‘Somei-yoshino’ (542). These cis-acting elements primarily included the following: (1) light response-related elements, with an average of 11.5 elements per gene; (2) biological and abiotic stress response-related elements, such as drought inducibility (0.9 elements per gene), low-temperature responsiveness (0.5 elements per gene), defense and stress responsiveness (0.5 elements per gene), wound-responsive element (0.1 elements per gene), and anaerobic induction (2.7 elements per gene); (3) hormone response-related elements, such as abscisic acid (2.4 elements per gene), Methyl Jasmonate (MeJA) (2.5 elements per gene), auxin (0.9 elements per gene), gibberellin (1.0 elements per gene), salicylic acid (1.0 elements per gene); (4) development- and-tissue-specificity-related elements, including meristem expression (0.6 elements per gene), zein metabolism regulation (0.7 elements per gene), endosperm expression (0.3 elements per gene), circadian control (0.3 elements per gene), differentiation of the palisade mesophyll cells (0.1 elements per gene), seed specificity (0.1 elements per gene), and flavonoid biosynthesis (0.1 elements per gene) (Figure 6).

3.6. Expression of PtTCP Under Low Cold Stress

The expression profiles of PtTCPs were assessed after exposure to different temperatures (25 °C, 5 °C,−5 °C, and −10 °C) to investigate their expression under cold stress. The differential expression patterns of 12 PtTCPs under cold stress were detected (Figure 7). The expression levels of PtTCP2, PtTCP14, and PtTCP16 initially increased and then decreased. The expression of PtTCP1, PtTCP8, and PtTCP17 was inhibited, but the expression of PtTCP6 increased at –10 ℃. Compared with the control, the expression levels of PtTCP3 and PtTCP4 remained unchanged.

3.7. Subcellular Localization of PtTCP9 Proteins

The constructed pCambia1300-PtTCP9-GFP vector was transiently transfected into N. benthamiana leaves to verify the subcellular position of PtTCP9. The results of confocal laser microscopy reveal that the green fluorescence signal displayed in the lower epidermal cells of N. benthamiana leaves transformed with the empty pCAMBIA1300-GFP vector was detected throughout the whole cell (Figure 8). In contrast, the green fluorescence signal in pCAMBIA1300-PtTCP9-GFP transgenic leaves was detected exclusively in the nucleus. These results indicate that PtTCP9 is a transcription factor localized in the nucleus, consistent with previous findings.

4. Discussion

TCP transcription factors play an essential role in the growth and development of plants as well as in responses to abiotic stresses. The genome-wide analysis of TCP has been performed in a large number of species: 27 in rice [28], 23 in Andrographis paniculat [29], and 23 in Robinia pseudoacacia [30], respectively. In Rosaceae, the number of TCP family members in different subfamilies varied: 52 in Malus domestica, 34 in Pyrus bretschneideri, 18 in Rosa chinensis, 19 in Fragaria vesca, 19 in P. mume, 17 in Rubus occidentalis, 20 in P. persica, and 14 in P. avium [31,32]. M. domestica and P. bretschneideri underwent whole-genome duplication (WGD) 3–4 billion years ago, which led to an increase in their chromosome numbers and may explain the higher number of TCP genes in these species [33,34]. The number of TCP genes in Prunus was similar (17–22), probably because Prunus plants did not experience WGD events.
Based on the phylogenetic analysis, the TCP proteins were classified into the PCF, CIN, and CYC/TB1 groups. Among these, the PCF subfamily contained the maximum number of genes (52.2%), and the CYC/TB1 group contained the least (15.3%). These proportions were different from the observations in O. sativa [27], A. thaliana [35], and Alfalfa [36], but were similar to those in Melilotus albus [37].
The results of motif analysis indicate that the types and quantities of motifs among the three classes significantly differed. Each class had its own unique motif, as well as a common motif among the three classes. Motif 1 was present in all TCPs. This further confirmed that the differences in motifs between different groups may be the main reason for their functional differences.
Previous studies have reported that most TCP proteins in other plants are located in the nucleus [28,29,30,35,36]. In this study, subcellular localization prediction results reveal that most TCPs were located in the nucleus. The experiment showed that the PtTCP9 protein was located in the nucleus. It is speculated that this family of genes mainly plays a regulatory role as TFs in the nucleus.
The differences among TCP proteins may be related to the involvement of different TCP genes in various processes such as organ development, signal transduction, and stress response [38,39,40,41,42]. Analysis of the subsequence of the TCPs promoter revealed that the promoter of TCPs contains several light-, low temperature-, and hormonal-responsive elements, such as abscisic acid and jasmonic acid, similar to the distribution type of the action elements of the promoter in the TCP gene family of P. persica [43].
The TCP genes have been reported to regulate abiotic stress in many plants. In Sorghum bicolor, the expression level of SbTCP7 was upregulated under drought stress [44]. HrTCP20 can improve the drought resistance of sea buckthorn by mediating JA signaling pathway in Hippophae rhamnoides [45]. In Rosa Chinensis, the expression of RcTCP2 gradually increased under salt treatment, whereas the expression of RcTCP6, 8, 12, and 13 decreased [32]. Overexpression of the Phyllostachys edulis PeTCP10 gene enhanced the salt tolerance of transgenic plants in the vegetative growth stage and increased the salt sensitivity in the germination and seedling stage [46]. Moreover, the TCP gene has been reported to be involved in regulating cold tolerance. OsPCF6 and OsTCP21 were regulated by miR319 and can reduce the frost cold resistance in O. sativa [42]. The gene expression analysis showed that the expression levels of PtTCP6, PtTCP614, and PtTCP16 increased, whereas those of PtTCP5, PtTCP8, PtTCP17, and PtTCP19 decreased under cold treatment. These results indicate that the functions of PtTCPs were varied, and several PtTCPs may participate in the response of P. tenella var. tenella to cold stress.

5. Conclusions

This study analyzed the TCP gene family in Prunus in terms of physical and chemical properties, subcellular location, phylogenetic relationship, gene structure, chromosomal location, cis-regulatory element, and response to cold stress under gene overexpression. The expression of four genes (PtTCP2, PtTCP6, PtTCP14, and PtTCP16) increased under cold stress. Future studies should focus on analyzing the biological functions of these genes in P. tenella var. tenella in response to cold stress and exploring their potential as excellent genetic resources for developing cold-resistant varieties of P. tenella var. tenella.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes15111443/s1, Table S1. The primer sequences of genes used in qRT-PCR analysis; Table S2. Characteristics of the members of the TCP gene family of eight Prunus species; Table S3. Ka/Ks analysis of the TCP homologous gene pairs from eight Prunus species. ‘WGD’ or ‘segmental’ means that the gene might arise from Whole Genome Duplication or Segmental Duplication. ‘Dispersed’ means that the gene might arise from transposition; Figure S1. The characteristics of Prunus tenella var. tenella. (A) Leaf and flower; (B) fruit; (C) nutlet; Figure S2. Phylogenetic analysis of the TCP genes from Arabidopsis thaliana, Prunus tenella var. tenella, and Oryza sativa; Figure S3. Multiple sequence alignment of the TCP domain. Multiple sequence alignment was conducted with DNAMAN.

Author Contributions

All authors contributed to the study’s conception and design. Material preparation and data collection were performed by H.Z., Y.L. and Q.Z. Funding was provided by L.L. and H.Z. Software analysis was performed by W.L. and C.Q. The first draft of the manuscript was written by Q.Z. and all authors commented on earlier versions of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Young Scientists Fund of the National Natural Science Foundation of China (32201611) and by the Scientific and Technological Innovation Cooperation Project (BHYLJT-KT-24-0102).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

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

Data Availability Statement

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

Acknowledgments

The authors extend gratitude to all members of the research team for their help and inspiration, and to the reviewers for their language assistance during the preparation of this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Genes 15 01443 g001
Figure 1. Chromosomal location of the TCPs in eight Prunus species. (A) P. tenella var. tenella; (B) P. amygdalus; (C) P. fruticosa; (D) P. mira; (E) P. jamasakura var. jamasakura; (F) P. mume var. tortuosa; (G) P. armeniaca ‘Rojo Pasion’. The scale (Mb) represents the length of the chromosome. Chr represents chromosomes, and the colors on the chromosomes represent gene density, with red representing high gene density and blue representing low gene density.
Figure 1. Chromosomal location of the TCPs in eight Prunus species. (A) P. tenella var. tenella; (B) P. amygdalus; (C) P. fruticosa; (D) P. mira; (E) P. jamasakura var. jamasakura; (F) P. mume var. tortuosa; (G) P. armeniaca ‘Rojo Pasion’. The scale (Mb) represents the length of the chromosome. Chr represents chromosomes, and the colors on the chromosomes represent gene density, with red representing high gene density and blue representing low gene density.
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Figure 2. Phylogenetic analysis of the TCP genes in eight Prunus species. (A) Phylogenetic analysis of the TCP proteins; (B) the number of TCP proteins identified in the three groups.
Figure 2. Phylogenetic analysis of the TCP genes in eight Prunus species. (A) Phylogenetic analysis of the TCP proteins; (B) the number of TCP proteins identified in the three groups.
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Figure 3. The Ka, Ks, and Ka/Ks values of TCP gene pairs in eight Prunus species. (A) The distribution of Ka and Ks values among TCPs; (B) the Ka/Ks values of TCP gene pairs.
Figure 3. The Ka, Ks, and Ka/Ks values of TCP gene pairs in eight Prunus species. (A) The distribution of Ka and Ks values among TCPs; (B) the Ka/Ks values of TCP gene pairs.
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Figure 4. Phylogenetic tree and collinearity analysis of TCP genes in eight Prunus species. The triangle indicates the location of the gene, the chr represents the chromosome, and the blue line represents the TCP homologous gene.
Figure 4. Phylogenetic tree and collinearity analysis of TCP genes in eight Prunus species. The triangle indicates the location of the gene, the chr represents the chromosome, and the blue line represents the TCP homologous gene.
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Figure 5. Analysis of conserved motifs in TCP proteins from eight Prunus species. Colored boxes represented different conserved motifs with different sequences and sizes. The overall height of each stack represents the degree of conservation at this position, whereas the height of the individual letters within each stack indicates the relative frequency of the corresponding amino acids.
Figure 5. Analysis of conserved motifs in TCP proteins from eight Prunus species. Colored boxes represented different conserved motifs with different sequences and sizes. The overall height of each stack represents the degree of conservation at this position, whereas the height of the individual letters within each stack indicates the relative frequency of the corresponding amino acids.
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Figure 6. Distribution of cis-acting elements in the promoters of TCPs in eight Prunus species. Different colored squares show different cis-acting elements in the promoter. Pt: Prunus tenella var. tenella; Pam: Prunus amygdalus; Par: Prunus armeniaca ‘Rojo Pasion’; Pf: Prunus fruticose; Pja: Prunus jamasakura var. jamasakura; Pmi: Prunus mira; Pmvar: Prunus mume var. tortuosa; Pyn: Prunus × yedoensis ‘Somei-yoshino’.
Figure 6. Distribution of cis-acting elements in the promoters of TCPs in eight Prunus species. Different colored squares show different cis-acting elements in the promoter. Pt: Prunus tenella var. tenella; Pam: Prunus amygdalus; Par: Prunus armeniaca ‘Rojo Pasion’; Pf: Prunus fruticose; Pja: Prunus jamasakura var. jamasakura; Pmi: Prunus mira; Pmvar: Prunus mume var. tortuosa; Pyn: Prunus × yedoensis ‘Somei-yoshino’.
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Figure 7. The expression patterns of 12 PtTCPs at different temperatures (25, 5, −5, and −25 °C) as revealed by qRT-PCR. The mean values were from three independent biological replicates. The data were statistically analyzed using Student’s t-test (** p < 0.01). (a–l) The relative expression levels of PtTCP1, PtTCP2, PtTCP3, PtTCP4, PtTCP5, PtTCP6, PtTCP8, PtTCP9, PtTCP14, PtTCP16, PtTCP17, and PtTCP19 at different temperatures.
Figure 7. The expression patterns of 12 PtTCPs at different temperatures (25, 5, −5, and −25 °C) as revealed by qRT-PCR. The mean values were from three independent biological replicates. The data were statistically analyzed using Student’s t-test (** p < 0.01). (a–l) The relative expression levels of PtTCP1, PtTCP2, PtTCP3, PtTCP4, PtTCP5, PtTCP6, PtTCP8, PtTCP9, PtTCP14, PtTCP16, PtTCP17, and PtTCP19 at different temperatures.
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Figure 8. Subcellular localization of PtTCP9 proteins. Green fluorescent, green fluorescent signals; Bright field, bright field signals; Chloroplast, Chloroplast fluorescence signals; Combination, different fluorescent superimposed signals. Red fluorescence indicates chloroplasts, and grey images are bright field.
Figure 8. Subcellular localization of PtTCP9 proteins. Green fluorescent, green fluorescent signals; Bright field, bright field signals; Chloroplast, Chloroplast fluorescence signals; Combination, different fluorescent superimposed signals. Red fluorescence indicates chloroplasts, and grey images are bright field.
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Table 1. Number of TCPs in eight Prunus species.
Table 1. Number of TCPs in eight Prunus species.
SubgenusSpecies NameChromosome NumberGenome Size (Mb)Genome Protein NumberNumber of TCP GenesNumber of TCP ProteinsProportion of TCP Proteins (%)
AmygdalusP. tenella var. tenella8220.332,08819190.059
P. amygdalus8220.727,98419220.071
P. mira8242.828,51919190.067
CerasusP. jamasakura var. jamasakura8375.326,98620200.074
P. fruticosa8249.228,58719190.070
P. × yedoensis ‘Somei-yoshino’8299.541,29422220.053
PrunusP. armeniaca ‘Rojo Pasion’8251.340,06717190.045
P. mume var. tortuosa8237.829,70619190.067
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Zhang, Q.; Qian, C.; Li, L.; Li, W.; Li, Y.; Zhao, H. Genome-Wide Identification and Characterization of TCP Genes in Eight Prunus Species and Their Expression Patterns Under Cold Stress in P. tenella var. tenella. Genes 2024, 15, 1443. https://doi.org/10.3390/genes15111443

AMA Style

Zhang Q, Qian C, Li L, Li W, Li Y, Zhao H. Genome-Wide Identification and Characterization of TCP Genes in Eight Prunus Species and Their Expression Patterns Under Cold Stress in P. tenella var. tenella. Genes. 2024; 15(11):1443. https://doi.org/10.3390/genes15111443

Chicago/Turabian Style

Zhang, Qiang, Cheng Qian, Lulu Li, Wei Li, Yanhua Li, and Han Zhao. 2024. "Genome-Wide Identification and Characterization of TCP Genes in Eight Prunus Species and Their Expression Patterns Under Cold Stress in P. tenella var. tenella" Genes 15, no. 11: 1443. https://doi.org/10.3390/genes15111443

APA Style

Zhang, Q., Qian, C., Li, L., Li, W., Li, Y., & Zhao, H. (2024). Genome-Wide Identification and Characterization of TCP Genes in Eight Prunus Species and Their Expression Patterns Under Cold Stress in P. tenella var. tenella. Genes, 15(11), 1443. https://doi.org/10.3390/genes15111443

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