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Article

Genome-Wide Identification of the WUSCHEL-Related Homeobox (WOX) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii

by
Fei Wang
,
Xiuming Chen
,
Mengya Cheng
,
Chengcheng Zhou
,
Ruiyue Zheng
,
Xiaopei Wu
,
Yanru Duan
,
Sagheer Ahmad
,
Zhongjian Liu
,
Jinliao Chen
* and
Donghui Peng
*
Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization, College of Landscape Architecture and Art, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2024, 10(6), 645; https://doi.org/10.3390/horticulturae10060645
Submission received: 18 May 2024 / Revised: 11 June 2024 / Accepted: 14 June 2024 / Published: 16 June 2024
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
Browse Figures
Versions Notes

Abstract

:
Numerous members of the WOX gene family play pivotal roles during the processes of growth and development in many plants, as has been demonstrated. Cymbidium goeringii, Cymbidium ensifolium, and Cymbidium sinense are ornamental plants with a fascinating floral morphology that are economically important in China. However, there is limited knowledge about the members of the WOX gene family and their functions in these three Cymbidium species. Hence, the WOX genes in three Cymbidium species were identified on the ground of the genomes data of C. goeringii, C. ensifolium, and C. sinense in this study. These identified WOX genes were further studied for their physicochemical properties, evolutionary relationship, gene structure, protein structure, and cis-acting elements of promoters, as well as the expression pattern of the WOX genes in different tissues of C. goeringii. The findings revealed that eight WOX genes in C. goeringii, twelve WOX genes in C. ensifolium, and nine WOX genes were identified. These WOX genes were further subdivided into WUS, ancient, and intermediate clades. The length of the coding region ranged from 149 to 335 aa, and it was predicted that all WOX genes would be located on the cell nucleus. The promoter cis-acting elements primarily comprised stress response, phytohormone response, plant growth and development, and transcription factor elements. Furthermore, both the transcriptomic data and RT-qPCR analysis showed that most WOX genes may be involved in multiple developmental stages of C. goeringii. To sum up, these results may serve as a theoretical foundation for further study of the function analysis of WOX genes in orchids.

1. Introduction

WUSCHEL-related homeobox (WOX) transcription factors are pivotal in the regulation of plant tissue and organ growth and development. These TFs are specifically expressed in plants and are instrumental in determining the fate of cells [1]. So, WOX genes have been the subject of extensive research in various plant species. In 1996, the first WOX gene of plants was found from Arabidopsis thaliana, namely WUSCHEL (WUS) [2]. To date, 15 WOX genes have been identified in A. thaliana, which are crucial for numerous developmental processes, especially during the stages of embryogenesis, organogenesis, and florogenesis [2,3,4,5,6]. In the case of Oryza sativa, a total of 13 WOX genes have been discovered [7]. Previous study showed that OsWOX13 affects flower development and drought resistance [8]. Additionally, OsWOX11 has been shown to regulate the growth and development of adventitious roots [9], and OsWOX3 is known to impact the development of shoot apical meristem and leaf [10].
The orchids is considered one of the most significant flowering plant groups in plant kingdom, and most species have been exploited commercially [11]. The recent genome-wide sequencing of selected orchids has enabled the identification of several gene families with potential economic values. In Dendrobium catenatum, the expression levels of DcWOX4 and DcWOX5 were mainly expressed in the roots, suggesting these two genes may be involved in root development [12]. Ramkumar et al. [13] discovered that there are 14 and 10 WOX genes in Phalaenopsis equestris and D. catenatum, respectively. Of those, two paralogous genes (PeWOX9A and PeWOX9B) exhibit flower-specific expression. Apart from that, the ortholog DcWOX9 in D. catenatum also shows similar preferential expression for its flowers, reaching maximum values especially in the flower bud and gynostemium. These discoveries have the potential to aid in comprehending how the WOX genes play a crucial part in orchids, thereby enabling the functional characterization and selection of WOX genes for the directive breeding of orchid plants with excellent traits for commercial utilization.
Numerous studies have established the essential role of WOX genes in the growth and development in plants. However, the evolutionary history and functional roles of the WOX gene family within the orchids remain largely unexplored. There is a notable absence of research that delves into the adaptive evolution and functional diversification of WOX genes in Cymbidium species. Hence, this study utilized bioinformatics approaches to identify WOX genes from the publicly available whole-genome sequences of three Cymbidium species: C. goeringii, C. ensifolium, and C. sinense. Following the identification, a comprehensive analysis was conducted on these WOX genes, which included examining their sequence structures, regulatory cis-acting elements, phylogenetic relationships, chromosomal distribution, and syntenic relationships. Finally, the expression patterns of WOX genes across various tissues in C. goeringii were determined by real-time fluorescence quantitative PCR. The objective was to lay the groundwork for future investigations into the biological roles of WOX gene family members in orchids.

2. Materials and Methods

2.1. Identification of the WOX Genes

The protein sequences for the WOX genes of C. goeringii, C. ensifolium, and C. sinense were derived from their respective whole-genome sequencing projects as reported in previous research [14,15,16]. Blast screening was performed to search the WOXs protein database of three Cymbidium species based on the sequences of WOX genes in A. thaliana using TBtools software (V2.084). The WOX sequences for A. thaliana were retrieved from the Plant Transcription Factor Database, which is accessible at http://planttfdb.gao-lab.org/ (accessed on 10 August 2023). Subsequently, the filtered results were further analyzed using the Conserved Domain Database (CDD) search tool available on the NCBI website at https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 10 August 2023). Through these processes, the members of the WOX gene family within the three Cymbidium species were successfully identified.

2.2. Phylogenetic Analysis and Physicochemical Properties

Except for the protein sequences for the WOX genes of C. goeringii, C. ensifolium, and C. sinense, corresponding WOX protein sequences for A. thaliana, and P. equestris were sourced from the Plant Transcription Factor Database (PlantTFDB), accessible at http://planttfdb.gao-lab.org/ (accessed on 10 August 2023). Subsequently, multiple sequence alignment for these WOX proteins was performed utilizing the MEGA 7.0 software [17]. The phylogenetic analysis was conducted employing neighbor-joining (NJ), applying the Jones–Taylor–Thornton (JTT) model for sequence substitution and carrying out 1000 bootstraps to assess the robustness of the phylogenetic tree [18,19]. The final phylogenetic tree was then visually enhanced using the Interactive Tree of Life (iTOL) web tool, available at https://itol.embl.de/ (accessed on 16 August 2023). Furthermore, the physicochemical characteristics of the WOX genes were evaluated with the aid of the ExPASy ProtParam website, found at https://web.expasy.org/protparam/ (accessed on 20 August 2023). The prediction of subcellular localization for these WOX genes was accomplished using the Plant-mPLoc tool, which can be accessed at http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ (accessed on 20 August 2023).

2.3. Conserved Motif, Gene Structure Analysis, and Cis-Acting Element Prediction

The ClustalW function of Mega 7.0 was used to generate alignment of the CgWOX, CeWOX, and CsWOX protein sequences, and the residues were then colored using Jalview [20]. The conserved motifs of the WOX genes in three Cymbidium species were performed and visualized through the MEME online tool [21]. Additionally, the gene structure of these WOX genes was examined using Tbtools software [22]. This software was also employed to isolate the upstream sequences extending 2000 base pairs from the WOX genes. Subsequently, the potential cis-acting regulatory elements within the WOX gene family of the three Cymbidium species were scrutinized with the aid of the PlantCARE database [23]. Finally, the results were visualized using Microsoft Excel 2010.

2.4. Chromosome Location and Collinearity Analysis

The chromosomal localization and visualization of WOX genes of three Cymbidium species were performed using Tbtools software, and their interspecies collinear relationships were mapped within the WOX gene family [22].

2.5. RNA Extraction and RT-qPCR Analysis

The samples of C. goeringii were collected from the individuals grown at Fujian Agriculture and Forestry University, including the root, rhizome, leaf, and flower bud at the flower bud stage. The total RNA from the C. goeringii samples was extracted using the FastPure® Plant Total RNA Isolation Kit (polysaccharides and polyphenolics-rich) (Vazyme, Nanjing, China). Then, the Nanodrop 2000 spectrophotometer (Thermo Scientific, Shanghai, China) was used to determine the concentration of the total RNA of the C. goeringii samples, and agarose gel electrophoresis method was used to assess the integrity of the total RNA. The HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China) was employed to reverse transcribe RNA into cDNA. The primers were designed using Oligo 7.0 software and the actin gene was used as a reference [24]; detailed information is provided in Table S1. The qRT-PCR experiment was conducted using the Taq Pro Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). Finally, the relative expression levels were assessed using the 2−∆∆Ct method [25]. One-way ANOVA was used to evaluate the comparisons among different tissues using IBM SPSS Statistics 22.0 software.

3. Results

3.1. Phylogeny Analysis of the WOX Genes

The phylogenetic tree was derived from the protein sequence of WOXs of five species, including A. thaliana (15), P. equestris (13), C. goeringii (8), C. ensifolium (12), and C. sinense (9). As shown in Figure 1, the 57 WOX genes from these five selected species are categorized into three main clades, referred to as the WUS clade, ancient clade, and intermediate clade. These results reveal an uneven distribution of the WOX proteins from five species across the three clades. The WUS clade possessed the highest number of 29 WOX genes, the intermediate clade contained 15, and the ancient clade had the fewest number with 13 WOX genes. Specifically, the WUS clade included the following numbers of WOX genes for each species: A. thaliana (8), P. equestris (6), C. goeringii (3), C. ensifolium (7), and C. sinensis (5). In contrast, the ancient clade comprised 3 WOX genes from A. thaliana, 4 WOX genes from P. equestris, and each species had 2 WOX genes for three Cymbidium species. Additional detailed information regarding the WOX proteins of these five species are available in Text S1.

3.2. Identification and Characterization of the WOX Genes in Three Cymbidium Species

A total of eight WOX genes in C. goeringii, twelve WOX genes in C. ensifolium, and nine WOX genes in C. sinense were found, and were named CgWOX1-CgWOX8, CeWOX1-CeWOX12, and CsWOX1-CsWOX9 according to their chromosome location information, respectively (Figure S1). As shown in Table 1, the analysis of the characteristics of these 29 WOX genes reveals that they contain 102–335 amino acids. Their theoretical PI varied from 4.89 to 11.63, whereas the instability index (II) ranged from 40.74 to 79.61. Additionally, the molecular weight (Mw) varied from 12,008.36 to 36,197.62, while the aliphatic index (AI) ranged from 50.57 to 86.89. The values of WOX genes’ GRAVY ranged from −1.154 to −0.194. The protein hydropathy coefficients were all negative, indicating that all the WOX proteins were hydrophilic. In addition, predicted subcellular localization results illustrate that all WOXs of three Cymbidium species are predicted to be located in the nucleus, suggesting that the nucleus may be the location where the WOX genes perform their functions.

3.3. Sequence Alignment, Gene Structure, and Conserved Motif Analysis of WOX Genes

Multiple sequence alignments were employed to generate sequence logos for the WOX domains found in C. goeringii, C. ensifolium, and C. sinense. As illustrated in Figure 2A, all 29 WOX genes are found to possess a conserved homeodomain, which includes several invariant amino acids. Specifically, the homeodomain featured conserved residues such as glutamic acid (E) and glutamine (Q) in helix 1; glycine (G) in the loop region; proline (P), isoleucine (I), and a second leucine (L) in helix 2; another glycine (G) in the turn region; and asparagine (N), valine (V), tyrosine (Y), tryptophan (W), phenylalanine (F), glutamine (Q), asparagine (N), alanine (A), and arginine (R) in helix 3 (Figure 2). These findings are congruent with the conserved amino acid residues previously reported in the literature [7]. Furthermore, the current study identified additional conserved residues, including arginine (R), tryptophan (W), and proline (P) before helix 1. These additional conserved residues were observed among the CgWOX, CeWOX, and CsWOX proteins, suggesting a potential for novel functional insights into the WOX genes of these Cymbidium species. These findings also indicate that the homo-domains of WOX proteins of C. goeringii, C. ensifolium, and C. sinense are highly conserved.
The WOX genes exhibiting similar gene structures and sharing identical conserved motifs tend to group together within the same clade on the phylogenetic tree (Figure 3A). The results of conserved motif predictions reveal the presence of 10 distinct conserved motifs among the 29 WOX genes examined. Notably, the genes CeWOX12, CgWOX7, and CsWOX9 were found to harbor the highest number of these conserved motifs, totaling eight each (Figure 3B and Table S2). The clustering of these genes with similar structure and motif characteristics suggested that there might be a potential for conserved functional roles or regulatory mechanisms in three Cymbidium species. Also, all WOX proteins in three Cymbidium species contain motifs 1 and 2, indicating that motif 1 and motif 2 were conserved in these WOX genes and could be regarded as the HD domains of CgWOXs, CeWOXs, and CsWOXs. Three WOX genes (CsWOX6, CeWOX2, and CeWOX8) represented the most conserved protein in three Cymbidium species, which only had motif 1 and motif 2. Except for the highly conserved motif 1 and motif 2, CeWOX9 and CeWOX10 uniquely contain only motif 10 or motif 4. In addition, motif 5 is only present in CgWOX1, CgWOX7, CeWOX1, CeWOX4, CeWOX12, CsWOX3, and CsWOX9, while motif 6 was only present in CeWOX3, CsWOX4, and CgWOX2. The gene structure analysis showed that all 28 WOX genes possessed introns ranging from 1 to 4 (Figure S1). Among them, CsWOX7 had the longest intron, followed by CgWOX5. Only CeWOX12, CgWOX8, and CeWOX11 were found to have one intron both in the N-terminal and C-terminal regions. CgWOX1, CeWOX1, CeWOX5, CgWOX8, CeWOX11, CsWOX8, and CeWOX10 contained three exons, and only CeWOX12 and CsWOX7 contained four exons.

3.4. Chromosomal Localization and Collinearity Analysis of WOX Genes

The gene distribution maps were drawn to evaluate the chromosome distribution of WOX genes in three Cymbidium species (Figure S2). And the findings suggest that 29 WOX genes are found on five, seven, and five chromosomes in C. ensifolium, C. goeringii, and C. sinense, respectively. In C. goeringii, CgWOX3, CgWOX4, and CgWOX5 were located on the same chromosome (Chr15), while most WOX genes, C. ensifolium, and C. sinense were found to distribute on Chr12.
In addition, collinear correlation analyses of WOX genes in three Cymbidium species were also performed. As shown in Figure 4, there are eight, twelve, and nine WOX genes in C. goeringii, C. ensifolium, and C. sinense, respectively. Except for C. sinense, the WOX genes displayed corresponding one-to-one relationships in two other Cymbidium species. A pair of segmental duplicated genes was found in the genome of C. goeringii, which were CgWOX1 on Chr02 and CgWOX4 on Chr15 (Figure 4A and Figure S2), while a total of two pairs of segments duplicated genes was identified in the genome of C. ensifolium, which were CeWOX1 on Chr01 and CeWOX12 on Chr9, and CeWOX6 on Chr12 and CeWOX9 on Chr17 (Figure 4B and Figure S2). However, the C. sinense genome did not contain any segmental duplicated genes (Figure 4C).

3.5. Cis-Acting Element Analysis of Three Cymbidium Species

To further explore the regulatory mechanisms of the WOX genes in three Cymbidium species, the 2000 bp promoter regions of these genes were extracted. The objective was to discern potential cis-regulatory elements, which were identified using the PlantCARE database. This analysis led to the discovery of 185, 257, and 194 promoter elements within the genomes of C. goeringii (as illustrated in Figure 5A), C. ensifolium (as illustrated in Figure 5B), and C. sinense (as illustrated in Figure 5C), respectively. Across the three Cymbidium species, four main functional categories of cis-regulatory elements were identified: those involved in the stress response, phytohormone response, plant growth and development, and transcription factor (Figure S3). The stress-responsive elements included those associated with light response, anaerobic induction, low temperature, and defense and stress responsiveness, all of which can significantly impact plant growth and development. Additionally, elements linked to plant growth and development were identified, such as those regulating meristem expression, zein metabolism, cell cycle, and endosperm development. These findings suggest that the WOX genes in C. goeringii, C. ensifolium, and C. sinense may play roles in meristem growth, stress responses, and phytohormone signaling pathways, thereby contributing to their growth and adaptability to environmental stresses. Moreover, the most frequently occurring regulatory elements were cis-acting regulatory elements related to light response in C. goeringii (78%), C. ensifolium (84%), and C. sinense (83%), respectively, followed by MeJA responsiveness (43%, 57%, and 36%, respectively) and MYB binding site (87%, 86%, and 92%, respectively) (Figure S3). It indicated that light may be important for controlling the functions of WOX during the growth and development of these three Cymbidium species and even other orchids.

3.6. Expression Patterns and qRT-PCR Analysis

According to the FPKM values, the expression levels of eight WOXs of C. goeringii in different tissues suggested that CgWOX2, CgWOX4, and CgWOX8 express in all tissues (Figure 6, Table S4). CgWOX2 and CgWOX8 had higher expressions in the rhizome than in the other six tissues, while CgWOX4 was highly expressed in gynostemium. CgWOX1 showed a high expression level in the rhizome. Moreover, CgWOX3, CgWOX5, and CgWOX6, all belonging to the WUS clade, exhibited similar expression patterns in different organs. CgWOX7 was found to express only in the rhizome, petal, and gynostemium. These results suggest that the WOX genes may be involved in different processes during the growth and development of C. goeringii.
CgWOX2, CgWOX3, and CgWOX8 were chosen for qRT-PCR analysis in order to investigate the expression patterns of the WOXs in different tissues in C. goeringii. The results show that the three genes are expressed in multiple tissues (Figure 7). The constitutive expression of CgWOX2, CgWOX3, and CgWOX8 was observed on the root, rhizome, leaf, and flower bud, which indicated that they may be involved in diverse development stages of C. goeringii. CgWOX2 and CgWOX3 showed significant expression in the leaf, followed by in the root, rhizome, and flower bud. Moreover, CgWOX8 had the highest expression level in the leaf, root, and flower bud, but possessed relatively less expression in the rhizome.

4. Discussion

Orchids possess significant economic and ornamental significance. The ornamental value of orchids is significantly influenced by the growth and development of their organs, including the development of floral organs [26,27,28]. However, it takes several years from seed to flowering and they must go through five stages: seed germination, rhizome growth, leaf–bud differentiation, plant growth, and flowering. Such a long growth cycle has severely limited the breeding of novel varieties [29]. Previous studies have demonstrated that WOX transcription factors are closely associated with plant development, including but not limited to embryogenesis, callus formation and maintenance, stem cell maintenance, and flower and root development [8,30,31,32,33]. Moreover, new findings have also shown that altering the WOX genes can change genetic requirements for plant regeneration and transformation, which could lead to a more efficient transgenic pathway for ornamental plants [34,35]. So, further investigations of the WOX gene family in plants may have the potential to enhance ornamental traits in orchids. However, there are few studies of the WOX gene family in orchids, especially the role of the WOX genes in Cymbidium species.
A comprehensive analysis of the WOX genes of C. goeringii, C. ensifolium, and C. sinense within their genomes were performed in this study. A total of 29 WOXs was identified from three Cymbidium species, including eight CgWOXs, twelve CeWOXs, and nine CsWOXs (Table 1). A previous study showed that plant WOX proteins can be subdivided into three clades, referred to as the WUS, intermediate, and ancient clades [3]. Similarly, CgWOXs, CeWOXs, and CsWOXs were also categorized into three distinct clades following the classification of WOX genes in Arabidopsis, namely WUS, ancient, and intermediate clades (Figure 1). And it was worth noting that the WUS clade had the most members of all three clades. There were 29 WOXs members in this clade, including three CgWOXs from C. goeringii, seven CeWOXs from C. ensifolium, five CsWOXs from C. sinense, eight AtWOXs from A. thaliana, and six PeWOXs from P. equestris. This means that the WUS clade appears to be highly conserved across diverse plant species like C. goeringii, C. ensifolium, and C. sinense, supporting previous studies [3,36].
Furthermore, we also identified CgWOX1 and CgWOX4, CeWOX1 and CeWOX5 as members of the intermediate clade, while CeWOX6 and CeWOX9 belong to the WUS clade. These results are suggestive of duplication events, and these genes might enhance the adaptability of C. goeringii and C. ensifolium to environmental changes through gene replication [4,31]. Moreover, ten conserved motifs in the WOXs of three Cymbidium species were found (Figure 3, Table S3). The arrangement of these motifs and the exon–intron structures were in accordance with the evolutionary relationship, suggesting that the functional integrity of WOX genes might have been effectively conserved throughout their evolutionary history. The CgWOX genes all contain elements related to light response, accounting for 78% in elements related to stress response, followed by MeJA responsiveness elements of phytohormone response (Figure S3). Consequently, it was plausible to infer that CgWOX genes may play an important role in the regulation of growth and developmental processes in C. goeringii, particularly in response to stress conditions.
The expression levels of specific genes across various tissues can provide insights into their potential functions within those tissues. In our study, we focused on eight candidate CgWOX genes and analyzed their expression profiles in the root, rhizome, leaf, and flower bud of C. goeringii, as depicted in Figure 6. The findings reveal that CgWOX8 exhibits high levels of expression in numerous tissues; it is closely related to AtWOX13 in the evolutionary analysis. Prior studies have demonstrated that AtWOX13 is prominently expressed in the primary and lateral roots of A. thaliana, as well as in the inflorescence and flower buds, suggesting its involvement in lateral root development and the formation of floral organs in plants [31]. This parallel in expression patterns and evolutionary proximity between CgWOX8 and AtWOX13 implied that CgWOX8 may share similar roles in the growth and developmental processes of C. goeringii. This deserves further study. The qPCR results also confirm that CgWOX8 displays the highest levels of expression in the root and flower buds (Figure 7). In addition, the WUS gene is essential for the formation and maintenance of the shoot apical meristem, and the process of ovaries and anther [9,37,38]. It is worth noting that WUS acts mainly as a repressor in the maintenance of stem cell populations in shoot and floral meristems, but its function changes from that of a repressor to that of an activator in the case of the regulation of the expression of the AGAMOUS protein [38]. CgWOX3, CgWOX5, and CgWOX6 were homologous to WUS, but they had little expression or did not show up in different parts. It is speculated that the differences in expression among genes could be due to distinct sampling stages [39].

5. Conclusions

The WOX gene family in the plant kingdom is relatively conserved in both sequence and function, playing a pivotal role in regulating a myriad of developmental processes in plants. In the present study, an initial discovery was made, revealing the presence of 29 WOX genes within the genomes of three distinct Cymbidium species. These WOX genes were categorized into three clades based on their evolutionary relationships. Then, a comprehensive analysis was undertaken, including identifying the conserved motifs and gene structure, examining the cis-acting elements associated with these WOX genes. Additionally, the chromosomal distribution of the WOX genes of three Cymbidium species was assessed, along with an analysis of their collinearity within their genomes. Finally, the expression and RT-qPCR data were combined to speculate that CgWOX8 might play a regulatory role in the root growth and formation of flower organs in C. goeringii. Overall, this study has the potential to aid in comprehending the mechanisms of the WOX gene family in orchids, thereby facilitating the functional characterization and selection of WOXs for the development of a new variety with desired traits for commercial utilization.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10060645/s1, Text S1: WOX protein sequences of A. thaliana, and P. equestris, C. goeringii, C. ensifolium, and C. sinense; Table S1: The primers for qRT-PCRs; Table S2: WOX genes used for the phylogenetic tree in A. thaliana, P. equestris, C. goeringii, C. ensifolium, and C. sinense; Table S3: The Seqlogo of motifs; Table S4: The FPKM values of the CgWOX genes in different tissues; Figure S1: Analysis of WOX gene structure in three Cymbidium species. (A) Phylogenetic tree of WOX genes in C. goeringii, C. ensifolium, and C. sinense. (B) Gene structure WOX genes in C. goeringii, C. ensifolium, and C. sinense. The coding sequences (CDSs) and untranslated regions (UTRs) are denoted by distinct colored boxes, whereas introns are indicated by lines; Figure S2: Distribution of CgWOXs, CeWOXs, and CsWOXs on chromosomes; Figure S3: The percentage of each cis-acting regulatory element of WOX promoters in three Cymbidium species: (A) C. goeringii, (B) C. ensifolium, and (C) C. sinense.

Author Contributions

Conceptualization and writing—original draft preparation, F.W., J.C. and D.P.; form analysis and visualization, C.Z., R.Z. and X.W.; Resources and data curation, Y.D., X.C. and Z.L.; writing—review and editing, F.W., S.A. and M.C.; Supervision, J.C. and D.P. 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 (32071815).

Data Availability Statement

All the data can be found in the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic tree of the WOX genes of five species. Proteins sequences from A. thaliana, P. equestris, C. goeringii, C. ensifolium, and C. sinense are marked with solid triangles, solid squares, red solid circle, dark-green solid circle, and dark blue solid circle, respectively. The phylogenetic tree was constructed using MEGA 7.0 software and was divided into three sub-clades based on the classification of WOXs from A. thaliana. The three sub-clades are shown in different colors. Yellow is used for the ancient clade, pink for the intermediate clade, and light purple for the WUS clade. The WOX genes ID of the five species are supplemented in Table S2.
Figure 1. Phylogenetic tree of the WOX genes of five species. Proteins sequences from A. thaliana, P. equestris, C. goeringii, C. ensifolium, and C. sinense are marked with solid triangles, solid squares, red solid circle, dark-green solid circle, and dark blue solid circle, respectively. The phylogenetic tree was constructed using MEGA 7.0 software and was divided into three sub-clades based on the classification of WOXs from A. thaliana. The three sub-clades are shown in different colors. Yellow is used for the ancient clade, pink for the intermediate clade, and light purple for the WUS clade. The WOX genes ID of the five species are supplemented in Table S2.
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Figure 2. The amino acid multiple-sequence alignment results of CgWOX, CeWOX, and CsWOX proteins. (A) The conserved domain of WOXs; (B) the Seqlogo of WOXs.
Figure 2. The amino acid multiple-sequence alignment results of CgWOX, CeWOX, and CsWOX proteins. (A) The conserved domain of WOXs; (B) the Seqlogo of WOXs.
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Figure 3. Analysis of WOX gene structure and conserved motifs in three Cymbidium species. (A) Phylogenetic tree. (B) Conserved motifs. The conserved motifs of the WOX genes in three Cymbidium species are represented by boxes with different colors. The sequence of 10 conserved motifs are listed in Table S3.
Figure 3. Analysis of WOX gene structure and conserved motifs in three Cymbidium species. (A) Phylogenetic tree. (B) Conserved motifs. The conserved motifs of the WOX genes in three Cymbidium species are represented by boxes with different colors. The sequence of 10 conserved motifs are listed in Table S3.
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Figure 4. Collinear correlation analysis in three Cymbidium species. (A) Synteny analysis of WOX genes in C. goeringii. (B) Synteny analysis of WOX genes in C. ensifolium. (C) Synteny analysis of WOX genes in C. sinense. The red lines represent the pairs of segmental duplicated genes.
Figure 4. Collinear correlation analysis in three Cymbidium species. (A) Synteny analysis of WOX genes in C. goeringii. (B) Synteny analysis of WOX genes in C. ensifolium. (C) Synteny analysis of WOX genes in C. sinense. The red lines represent the pairs of segmental duplicated genes.
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Figure 5. The regulatory elements in the promoter region of three Cymbidium species. (A) The cis-acting elements of C. goeringii. (B) The cis-acting elements of C. ensifolium. (C) The cis-acting elements of C. sinense.
Figure 5. The regulatory elements in the promoter region of three Cymbidium species. (A) The cis-acting elements of C. goeringii. (B) The cis-acting elements of C. ensifolium. (C) The cis-acting elements of C. sinense.
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Figure 6. The expression pattern of CgWOXs in C. goeringii. The heat map of CgWOXs in different parts of C. goeringii. The color scale on the right side of the heatmap represents the relative expression level of CgWOXs, and the expression was increased with the color gradient from white to orange. The FPKM values of CgWOXs in C. goeringii are listed in Table S4.
Figure 6. The expression pattern of CgWOXs in C. goeringii. The heat map of CgWOXs in different parts of C. goeringii. The color scale on the right side of the heatmap represents the relative expression level of CgWOXs, and the expression was increased with the color gradient from white to orange. The FPKM values of CgWOXs in C. goeringii are listed in Table S4.
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Figure 7. Analysis of gene expression of three CgWOXs in C. goeringii through real-time quantitative fluorescence. The significance levels: **: p < 0.01, and ***: p < 0.001.
Figure 7. Analysis of gene expression of three CgWOXs in C. goeringii through real-time quantitative fluorescence. The significance levels: **: p < 0.01, and ***: p < 0.001.
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Table 1. Physicochemical property analysis of the WOX genes in three Cymbidium species.
Table 1. Physicochemical property analysis of the WOX genes in three Cymbidium species.
Gene NameGene IDNumber of
Amino
Acids
(AA)		Theoretical PIMolecular
Weight
(Mw)		Instability
Index
(II)		Aliphatic
Index
(AI)		Grand Average of Hydropathicity
(GRAVY)		CladeSubcellular
Localization
CgWOX1GL107722675.1728,478.8564.8273.52−0.194IntermediateNucleus
CgWOX2GL124802466.4628,129.6866.8065.45−0.725AncientNucleus
CgWOX3GL010221858.521,245.1956.3764.81−0.685WUSNucleus
CgWOX4GL175092706.1629,728.1768.6277.59−0.366IntermediateNucleus
CgWOX5GL121042655.6930,696.4272.5159.66−0.908WUSNucleus
CgWOX6GL179501939.0421,862.4669.9350.57−0.798WUSNucleus
CgWOX7GL206833347.7636,147.5652.4478.02−0.295IntermediateNucleus
CgWOX8GL212891995.7822,446.374.0269.20−0.716AncientNucleus
CeWOX1JL0104692595.1327,488.566.4470.15−0.283IntermediateNucleus
CeWOX2JL0083691649.7319,245.0948.1586.89−0.416WUSNucleus
CeWOX3JL0024732466.2628,201.7467.9365.45−0.737AncientNucleus
CeWOX4JL0004372498.2928,049.8771.7570.88−0.576WUSNucleus
CeWOX5JL0171012495.9527,247.2868.0570.84−0.418IntermediateNucleus
CeWOX6JL0141412126.0524,084.0479.6157.08−0.753WUSNucleus
CeWOX7JL01181614310.1316,583.2860.0272.94−0.691WUSNucleus
CeWOX8JL0118151497.0617,178.4245.6562.15−0.692WUSNucleus
CeWOX9JL01764710810.2812,931.875.1056.85−0.780WUSNucleus
CeWOX10JL0009931984.8922,612.7568.7854.80−0.956WUSNucleus
CeWOX11JL0004832555.2628,775.2469.5265.45−0.709AncientNucleus
CeWOX12JL0154703357.7636,218.6352.3178.09−0.289IntermediateNucleus
CsWOX1cymsin_Mol0092451029.8812,008.3662.3960.29−1.154WUSNucleus
CsWOX2cymsin_Mol0211681779.1420,024.3360.6751.30−0.801WUSNucleus
CsWOX3cymsin_Mol0075122498.7627,992.970.2869.32−0.573WUSNucleus
CsWOX4cymsin_Mol0179482466.0928,160.7168.7164.27−0.735AncientNucleus
CsWOX5cymsin_Mol0196432126.2324,098.1179.3957.08−0.767WUSNucleus
CsWOX6cymsin_Mol00673512811.6314,521.3677.4872.42−0.766IntermediateNucleus
CsWOX7cymsin_Mol0127951949.621,959.2740.7476.86−0.375WUSNucleus
CsWOX8cymsin_Mol0121742285.925,686.9766.9568.46−0.606AncientNucleus
CsWOX9cymsin_Mol0054063347.7736,197.6251.7078.02−0.302IntermediateNucleus
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Wang, F.; Chen, X.; Cheng, M.; Zhou, C.; Zheng, R.; Wu, X.; Duan, Y.; Ahmad, S.; Liu, Z.; Chen, J.; et al. Genome-Wide Identification of the WUSCHEL-Related Homeobox (WOX) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii. Horticulturae 2024, 10, 645. https://doi.org/10.3390/horticulturae10060645

AMA Style

Wang F, Chen X, Cheng M, Zhou C, Zheng R, Wu X, Duan Y, Ahmad S, Liu Z, Chen J, et al. Genome-Wide Identification of the WUSCHEL-Related Homeobox (WOX) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii. Horticulturae. 2024; 10(6):645. https://doi.org/10.3390/horticulturae10060645

Chicago/Turabian Style

Wang, Fei, Xiuming Chen, Mengya Cheng, Chengcheng Zhou, Ruiyue Zheng, Xiaopei Wu, Yanru Duan, Sagheer Ahmad, Zhongjian Liu, Jinliao Chen, and et al. 2024. "Genome-Wide Identification of the WUSCHEL-Related Homeobox (WOX) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii" Horticulturae 10, no. 6: 645. https://doi.org/10.3390/horticulturae10060645

APA Style

Wang, F., Chen, X., Cheng, M., Zhou, C., Zheng, R., Wu, X., Duan, Y., Ahmad, S., Liu, Z., Chen, J., & Peng, D. (2024). Genome-Wide Identification of the WUSCHEL-Related Homeobox (WOX) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii. Horticulturae, 10(6), 645. https://doi.org/10.3390/horticulturae10060645

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