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Article

Genome-Wide Identification and Characterization Analysis of WUSCHEL-Related Homeobox Family in Melon (Cucumis melo L.)

by
Lingli Tang
1,2,
Yuhua He
1,2,
Bin Liu
3,
Yongyang Xu
1,2,* and
Guangwei Zhao
1,2,*
1
Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, China
2
National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya 572000, China
3
Hami-melon Research Center, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(15), 12326; https://doi.org/10.3390/ijms241512326
Submission received: 25 June 2023 / Revised: 27 July 2023 / Accepted: 28 July 2023 / Published: 1 August 2023
(This article belongs to the Special Issue Molecular Research in Fruit Crop)
Browse Figures
Versions Notes

Abstract

:
WUSCHEL-related homeobox (WOX) proteins are very important in controlling plant development and stress responses. However, the WOX family members and their role in response to abiotic stresses are largely unknown in melon (Cucumis melo L.). In this study, 11 WOX (CmWOX) transcript factors with conserved WUS and homeobox motif were identified and characterized, and subdivided into modern clade, ancient clade and intermediate clade based on bioinformatic and phylogenetic analysis. Evolutionary analysis revealed that the CmWOX family showed protein variations in Arabidopsis, tomato, cucumber, melon and rice. Alignment of protein sequences uncovered that all CmWOXs had the typical homeodomain, which consisted of conserved amino acids. Cis-element analysis showed that CmWOX genes may response to abiotic stress. RNA-seq and qRT-PCR results further revealed that the expression of partially CmWOX genes are associated with cold and drought. CmWOX13a and CmWOX13b were constitutively expressed under abiotic stresses, CmWOX4 may play a role in abiotic processes during plant development. Taken together, this study offers new perspectives on the CmWOX family’s interaction and provides the framework for research on the molecular functions of CmWOX genes.

1. Introduction

WUSCHEL-related homeobox (WOX) protein family is one of the subclades of the plants-specific homeodomain (HD) superfamily. The family members are distinguished through a conserved HD, which is typically formed of 60–66 amino acids with a specific structure. The HD domain, which is highly conserved among WOX protein families and essential to their functions, can bind particular DNA sequence to control the expression of downstream genes [1,2,3].
Plant evolution has led to a progressive expansion of the WOX family, for example, Physcomitrella patens possess three WOX, whereas unicellular green algae only have one [4]. Recent studies have identified 15 in Arabidopsis [5], 45 in tomato [6], 17 in A. chinensis genomes [7], and 11 in sorghum and 21 in Zea mays. The evolution and biological functions of WOX have been well-studied in Arabidopsis. WOX members were classified into the modern, intermediate, and ancient clade [8]. AtWOX proteins that belong to the same clade have highly conserved biological properties, whereas WOX members that belong to separate clades have significantly various properties.
Past research has presented that WOX family members are crucial for stem cell conservation and organ formation [9,10,11,12]. The WOX members which belong to intermediate clade were mainly regulated zygote and early embryo development. AtWOX8 is required for normal development of the proembryo, and AtWOX9 plays a key role in controlling the proliferation of meristem cells and furthering embryo growth [13,14]. PaWOX8/9 is the homolog of AtWOX8 and AtWOX9 and may play a conserved role as a regulator of the embryonic foundation [15,16]. CsWOX1 regulates leaf vein development and leaf size via the CsSPL-mediated pathway and/or modulate auxin polar transport and signaling, and may also affect the early reproductive process in cucumber [17,18].
Members of the modern clade contain a specific WUS box (TLXLFPXX), which is also an important component of the WUS gene regulating homeostasis for apical meristem and organ initiation [19,20,21,22]. Except for the cambium stem cell regulator WOX4, members of the WUS clade can partially or fully replace the WUS function in maintenance of shoot apical meristem and floral meristem. Ancient WOX13 and WOX9 are incapable of maintaining stem cells [23,24]. Otherwise, AtWOX11 and AtWOX12, both functionally redundant, have been reported to stimulate the expression of LBD16 and LBD29 and promote the initiation of adventitious root and root hair development [25,26,27]. AtWOX13 belonged to the ancient clade and promoted carpel maturation by inhibiting the activities of JAG/FIL [28]. In addition to controlling the growth of other organs and roots, AtWOX14 also cooperated with AtWOX4 to control the differentiation of vascular meristems [29]. Additionally, it had been shown that AtWOX14 played a stimulating role in the accumulation of biologically-active GA by increasing the production of GA3ox and preventing the breakdown of GA2ox, which in turn promoted the differentiation and lignification of vascular in inflorescence stem cells. Cis-elements analysis in the promoter regions found that the faculty of WOX family controlled by plant hormone signaling pathways might be conserved [30,31]. Plants with abnormal stamen and another dysplasia and dwarfed growth were caused by the overexpression of WOX1. PRESSED FLOWER (PRS)/WOX3 and WOX1 also took part in the establishment of leaf outgrowth and boundary development downstream of adaxial/abaxial polarity and controlling cell reproduction. [3,32,33,34,35].
WOX family also performs crucial roles in response to various environmental challenges in addition to plant growth and development. For example, eight WOX members (OsWUS, OsWOX3, OsWOX4, OsWOX5, OsWOX9B, OsWOX11, OsWOX12A, and OsWOX12B) respond to drought, two genes (OsWOX3 and OsWOX5) respond to salt, and four genes (OsWOX5, OsWOX9B, OsWOX12A, and OsWOX12B) respond to cold stress in rice [36]. Studies have reported that the direct homologous WOX13 in rice responds to various abiotic stresses such as drought, cold and salt [37]. AtWOX6 responds to freezing tolerance through studying the hos9-1 mutant in Arabidopsis [2]. MdWOX13-1 can directly bind to the promoter of the MdMnSOD to increase the callus weight and enhance ROS scavenging under drought stress [38]. Phvul-WOX-1, Phvul-WOX-9 and Phvul-WOX-16 genes respond to salt stress detected by qRT-PCR [39]. In cucumber, heat and cold shocks reduced the expression of CsWOX1a, CsWOX3 and CsWOX4,and the three WOX genes CsWOX1b, CsWOX3, and CsWOX5, respectively, suggesting that which might be involved in the regulation of cucumber stress tolerance [40]. Similarly, a previous study revealed that most of the WOX genes can respond to various stresses in cotton [41].
Melon is an important economic horticulture crop worldwide. Based on the ovary pubescence, melon can be classified into two subspecies, C. melo subsp. melo (hereafter as melo) and C. melo subsp. agrestis (hereafter as agrestis). The quality and yield of melon were affected by growth environment and development processes. Although the roles of WOX members in regulating many developmental aspects have been exhibited in many crops, there is no characteristic research on WOX members in melon. The updated high-quality genomic sequence of melon provides an opportunity to identify and characterize CmWOX family. In this study, we identified 11 CmWOX genes by using the bioinformatics methods based on the melon genome, their phylogeny and conserved domains, investigated their gene expression under cold and drought stress condition, which provides new insights into the evolution and biological functions of CmWOX genes in melon.

2. Results

2.1. Phylogenetic Analysis of the CmWOX Family

To explore the evolutionary relationships of WUSCHEL-related homeobox (WOX) family, we performed the phylogenetic analysis of 61 WOX genes from Arabidopsis, rice, tomato, cucumber and melon by homologous alignment and MEGA 7.0. Based on the phylogenetic tree, a total of 61 WOX proteins from 5 plants were divided into 3 branches, including the modern clade, the ancient clade, and the intergenic clade. By analyzing paralogous and orthologous relationships of WOX family, we found that most CmWOXs showed closer relationships to CsWOXs, which might be due to the close evolutionary relationship between cucumber and melon. Putative CmWOX family were also identified and classified into the modern clade, intermediate clade, and ancient clade (Figure 1).

2.2. Identification, Description, and Classification of CmWOX Family

To identify WOX family members in melon, a genome-wide analysis was carried out using BLASTp. A total of 11 CmWOX genes were identified, consistent with the previous study in identifying CsWOX family [42], and then we named the CmWOX genes after the CsWOX family according to the phylogenetic and evolutionary relationship. The phylogenetic tree included the WOX family of melon (Cm), cucumber (Cs), tomato (Sl), Arabidopsis (At) and rice (Os). The coding sequences of these genes range from 519 to 1140 bp (Base Pair) in the melon (DHL92) v4 Genome. The amino acid (AA) number ranges from 172 to 385, and the molecular weights between 14.66 and 43.64 kDa and isoelectric point (pI) values from 5.23 to 10.312, the number of amino acids of the whole family are from 172 to 385 (Table 1). Although CmWOXs belong to a family, the basic information varies greatly.

2.3. The Chromosomal Localization of CmWOX Genes

The chromosomal location of CmWOX genes was analyzed through the MapGene2Chrom online website [43], and the result exhibited that CmWOX genes were irregularly distributed on eight chromosomes (Figure 2; Supplementary Table S2). There was one CmWOX gene on chromosome 2 (CmWOX1b), 4 (CmWOX11), 6 (CmWOX13a), 7 (CmWOX13b), 11 (CmWOX3), and two CmWOX genes on chromosome 3 (CmWOX5 and CmWOX4), 8 (CmWOX9 and CmWUS), and 12 (CmWOX1a, CmWOX2), and no CmWOX genes were discovered on other four chromosomes of melon. Gene duplication events were investigated among the CmWOX genes, no segment duplication pairs were identified in this family.

2.4. Gene Structure Analysis of CmWOX Genes

Gene structure is important for determining the relationship between genomic evolution and functional differentiation of multi-genic family members. MEGA 7.0 was used to build the phylogenetic tree of 11 CmWOX proteins. CmWOX1a, CmWOX1b, CmWOX3, CmWOX4, and CmWOX5 were divided into the modern clade; CmWOX9 and CmWOX11 were divided into the intermediate clade; and CmWOX13a and CmWOX13b were divided into ancient clade. In order to investigate the structural diversity of CmWOX genes, the exon-intron map was constructed according to the genome and coding sequences of CmWOX genes (Figure 3a). The number of introns in CmWOXs varies from one to two. Combined with the results of phylogenetic trees, the modern/WUS group with 1-2 intron insertions can be further divided into three sub-clades. CmWOX5, CmWOX3, CmWOX1a, and CmWOX1b contained 1 intron. The other two members, CmWOX4 and CmWOX2, had two introns. Both intermediate (CmWOX9 and CmWOX11) and ancient group (CmWOX13a and CmWOX13b) contained two introns. Seven members of the CmWOX genes contain 3 exons, and 4 CmWOX genes possess 2 exons (Figure 3b). There was a big difference among CmWOX genes in numbers and lengths of exon and intron, which suggested that CmWOX family may play a specific or redundant role in evolution.

2.5. The Conserved Motifs Analysis of CmWOX Proteins

The conserved motifs of WOX members played a key role in regulating plant cell specificity, and the size of flower meristem and worked as a transcript repressor. CmWOX proteins were examined to find the motifs through PlantCARE, MEME suite and TBtools [44,45]. A total number of 10 conserved motifs were prefigured in CmWOX proteins, and the amino acid number were ranged from 15 to 50. The results revealed that groups classified by phylogenetic analysis contained similar motif compositions, but there were some differences between the different subgroups. Motifs 1 and 2 were existing in all the CmWOX proteins, and the motif 2 was the conserved homeodomain (Figure 4a). Motif 7 (WUS motif) only existed in modern clade, and was located in CmWUS, CmWOX1a, CmWOX2, CmWOX3, CmWOX4, and CmWOX5, whereas motifs 3 and 8 only existed in ancient CmWOX members. Motif 5 only existed in intergenic members. Motif 6 presented in CmWOX3, CmWOX4, CmWOX5, CmWOX1a, and CmWOX1b. The motif 4 presented in CmWOX13a, CmWOX2, and CmWOX5. Motif 9 presented in CmWOX1a and CmWOX1b, and the motif 10 presented in CmWOX9 and CmWOX1b. The homeobox seq-logo of the CmWOX was analyzed using the SeqLogo program (TBtools (v1.120)). R, W, P, Q, G, I, L, W and F residues were highly conserved in CmWOX proteins (Figure 4b). To some degree, these specific and conserved motifs of CmWOX proteins may lead to different functions or functional redundancy in plant growth and development, which need to be further explored.

2.6. The Cis-Elements Analysis in Promoter Regions of CmWOX Genes

Cis-elements in the promoter region of the WOX genes were involved in affecting plant growth and development through regulating expression of target gene. It is meaningful to analyze the cis-acting elements of the CmWOX family in their promoter regions. The analysis results revealed that many cis-motifs might be taken part in response to biotic and abiotic stresses, such as low temperature, cold, and photo stresses (Figure 5; Supplementary Table S3). Abiotic stress-responsive cis-elements were found in the majority regions of the CmWOX promoter. However, drought and low-temperature responsive elements were only detected in the promoter region of CmWOX13a, CmWOX3, and CmWOX2, respectively. Additionally, we discovered light-sensitive elements in nearly all the promoter regions of the CmWOX genes, suggesting that light signaling may take part in controlling the expression of CmWOX. Six out of the eleven CmWOX promoters had MeJA-responsive elements. Abscisic acid-responsive elements were identified in seven CmWOX promoters, whereas auxin-responsive elements were only found in the CmWOX13a promoter. These results indicated that CmWOX members may response to stresses and regulate the plant growth and development crossed with plant hormone.

2.7. Transcriptome Data Analysis under Cold and Drought Treatments

Research has reported that CmWOX genes are involved in abiotic stresses in horticultural crops, such as tomato and cucumber. To investigate the roles of the CmWOXs under cold and drought condition, we analyzed the gene expression of this family in the leaves of melon seedlings. After analysing of the cis-element in the promoter regions of CmWOXs, leaves in the seedlings stage were subjected to RNA-seq gene expression analysis under cold and PEG6000 treatments. Differential genes expression levels were calculated by FPKM, the expression data of all sequenced samples were then subjected to principal component analysis (PCA) and DEGs analysis (Figure 6; Supplementary Table S4). The PCA of the expressed biographies of the 12 libraries (6 samples with 2 replicates each, Figure 6a,b) revealed that samples collected at different hours can be obviously different. PCA1 explained 96.9% and 67.4% of the variance for cold and PEG6000, respectively. Thus, the results were verified to be highly dependable and acceptable for further analyses. Under the cold treatment, there were 4626 DEGs between ‘962-0 h’ and ‘674-0 h’, of which 2352 DEGs were up-regulated and 2274 DEGs were down-regulated. A total of 5850 DEGs between ‘962-6 h’ and ‘674-6 h’, of which 3031 DEGs were up-regulated and 2819 were down-regulated. A total of 6306 DEGs between ‘962-12 h’ and ‘674-12 h’, of which 3248 DEGs were up-regulated and 3058 were down-regulated, which are higher than that at 0 h and 6 h (Figure 6c). Moreover, under the PEG6000 treatment, there were 5430 DEGs between ‘962-0 h’ and ‘674-0 h’, of which 2718 DEGs were up-regulated and 2703 were down-regulated. The number of DEGs between ‘962-6 h’ and ‘674-6 h’ is similar to the sample at 0 h. While 4624 DEGs between ‘962-12 h’ and ‘674-12 h’, of which 2205 DEGs were up-regulated and 2419 were down-regulated, which are lower than that at 0 h and 6 h (Figure 6d). The number of DEGs under cold and PEG6000 treatment differed considerably between in melo and agrestis. Furthermore, studying the functions of the differential expressed genes among CmWOXs will help us to understand the molecular mechanism of melon response to stresses.

2.8. The Expression Pattern of CmWOX Gene in Melon Leaves under Cold and PEG6000 Treatments

Transcriptome data analysis showed that the expression level of CmWOX1a, CmWOX1b, CmWOX4, CmWOX5, CmWOX13a, and CmWOX13b changed with the treating time. While no obvious changes in transcript levels were observed in the CmWUS, CmWOX2, CmWOX3, CmWOX9, and CmWOX11 under cold and 20% PEG6000 condition. CmWOX13a and CmWOX13b were constitutively expressed in both ‘674’ and ‘962’. The log2 (fold change) of CmWOX13a and CmWOX13b were higher than other genes (Figure 7a,b; Supplementary Tables S4 and S5). The above results suggested that some CmWOX members were involved in abiotic stress response during melon growth and development.

2.9. The Expression Level of CmWOX Genes under Cold and PEG6000 Treatments

To confirm the expression level of CmWOX genes acquired by RNA-seq data, qRT-PCR assay was conducted. According to the log2 (value) analysis, four genes, including one orthologous pair, were supposed to be highly expressed under cold stress. The expression pattern of the orthologous genes (CmWOX13a and CmWOX13b) were detected by qRT-PCR in leaves of ‘674’ and ‘962’. The results showed a similar tendency with the RNA-seq data (Figure 8a,b). The expression of CmWUS was undetected at 0 h, 6 h, and 12 h under cold and drought treatments in both ‘674’ and ‘962’. These results revealed that CmWUS did not response to abiotic stresses, and may play a conserved role in controlling the proliferation of plant shoot and root stem cells. The expression of CmWOX4 was decreased both under cold and drought treatments in ‘962’. However, the expression levels of CmWOX13a and CmWOX13b were high in both ‘674’ and ‘962’ under cold and drought stresses, suggesting that which might be the key players and potential candidate genes in response progress to stresses in melon (Figure 8c,d).

3. Discussion

Transcript initiation and gene expression regulation are significantly influenced by transcription factors. The WOX gene family encodes transcription factors unique to plants that are important for maintaining shoot and root stem cell homeostasis, developing tissues, and growing organs [11,22]. The WOX gene family has been identified in many plants, and so far, detailed information on this family in melon has not been uncovered. The updating and optimized genome of melon provides a chance for us to systematically analyze the CmWOX family. In this study, we identified and characterized 11 CmWOXs through genome-wide analysis. The WOX family members in melon are less than that in Arabidopsis, tomato, rice, and maize, the same number as in cucumber and sorghum [12,42]. Phylogenetic analysis showed that CmWOX proteins had a closer relationship with that in cucumber (Figure 1; Supplementary Table S1), which is consistent with the discovery that melon and cucumber are most homologous in Cucubitaceae [46].
Although the CmWOX family has a conservative homeodomain and WUS domain based on the conserved motif analysis, each member also has their distinctive motifs. The genomic structure was highly varied even within the same family, according to homology analyses (Figure 4). Those findings implied that the CmWOX gene family may have both functional redundancy and specific regulatory effects. Moreover, various studies found that the WOX genes respond to plant hormones and abiotic stresses, suggesting that they have important functions in regulating plant growth and development [25,35,37,47]. According to the results analyzed by MEME and TBtools, we discovered that the promoter regions of the CmWOX1a, CmWOX1b, CmWOX13a, and CmWOX13b contain 2-3 cis-elements responded to auxin, gibberellin, and jasmonic acid. Furthermore, we discovered cis-elements that are responsive to auxin in the promoter regions of the CmWOX2 and CmWOX4, responsive to jasmonic acid in that of the CmWOX9 and CmWOX11, and responsive to GA in the case of the CmWUS, CmWOX4, CmWOX5, and CmWOX9 (Figure 5). According to the aforementioned results, CmWOX genes may participate in the process of plant hormone signal transduction and be crucial in controlling the initiation and development of plant organs.
A previous study revealed that the two subspecies of melon, melo and agrestis, were domesticated independently [48]. The independent domestications might greatly influence gene duplication, differentiation, expression, and function finally resulting in various characteristics between melo and agrestis. According to transcriptome databases, the expression of genes varies in ‘674’ and ‘962’. The different expressed gene number (DEGs) in melo and agrestis under the two treatments was different. The majority of them were DNA-binding proteins that participate in abiotic conditions, stimuli and biological development, and played a role in regulating the transcriptional levels of downstream genes (Figure 6). Many studies have reported that (CBF/DREB1)- and ABA -related genes were involved in cold and drought stresses [49,50]. Therefore, we detected that the CmPYR, CmPP2C-1, and CmPP2C-2 are ABA- related genes, and CmDREB2A-like, CmERF039-like, and CmDREB3-like are CmDREB family members in melon according to the homologous proteins alignment. The transcript level of the CmPYR, CmPP2C-1 and CmPP2C-2 were up-regulated under PEG6000 treatment (Figure S1a). CmERF039-like respond to cold, which was up-regulated in ‘674’ and down-regulated in ‘962’ under cold stress, while both of CmDREB2A-like and CmDREB3-like were up-regulated in ‘674’ and ‘962’ under cold treatment in the transcriptome data (Figure S1b, Supplementary Table S8). There were different genes responding to abiotic stress compared with cucumber. Several CmWOX genes were expressed in leaves, except for CmWUS, CmWOX2, CmWOX3, CmWOX9, or CmWOX11. Unlike the CsWOX1a and CsWOX5 response to drought treatment, there was no difference on transcript level in melon with the same treatment. Although the evolution relationship between melon and cucumber are close, the functions of WOX family members in two species are shown differently. The expression levels in leaves under cold stress were higher than those of other family members for the two CmWOX13a and CmWOX13b members, indicating that they had a regulatory influence on melon stress response (Figure 7), which is consistent with the previous study in cucumber [40,42,51]. These findings demonstrated the divergence of CmWOX genes as well as the pattern of expression under cold in two melon subspecies. However, it is necessary to study the biological function of the CmWOX genes in the future.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Melon seeds of ‘674’ (C. melo subsp. melo, derived from Japan) and ‘962’ (C. melo subsp. agrestis, derived from China) were provided by obtained from the National Mid-term Genebank, soaked at 55 °C for 3 h first and then transferred to the dark 28 °C incubator for germinating. Seedlings grew in a growth chamber under long-light condition (16 h light/8 h dark cycle with 100 μM photons m−2 s−1 at 28 °C for 2 weeks). Two-week-old seedlings were treated by 20% (w/v) PEG6000 and 8 °C in the chamber. Leaves were collected after time intervals at 0, 6, and 12 h, and non-treated seedlings as control. Leaf samples were frozen immediately in liquid nitrogen and subsequently stored at ultra-low temperature (−80 °C) freezer for RNA extraction and subsequent for the expression level analysis.

4.2. Identification, Characteristics, and Phylogenetic Analysis of CmWOX Genes

The identification and analysis of melon WOX genes were performed using genome sequence data of melon (Cucumis melo L., cv. DHL92 v4 Genome) deposited at the Cucurbit Genomics Data website (http://cucurbitgenomics.org/v2/organism/23; accessed on 24 March 2023) [52]. CmWOX homologous protein sequences of Arabidopsis, tomato and rice were downloaded from the genome database and used as query sequences to search for putative counterparts in melon by using BlastP. All CmWOX genes were named according to their homologous relationship with that in cucumber. The multiple sequence alignment for the full-length amino acid sequences of AtWOX, CsWOX, OsWOX, and SlWOX family members were chosen [53]. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with settings of pairwise deletion, Poisson correction, and 1000 bootstraps analysis for CmWOX proteins. The total 11 amino acid sequences of CmWOX family were used to be analyzed. All positions containing gaps and missing data were eliminated. There were a total of 86 positions in the final dataset. Evolutionary analyses were conducted through MEGA7 (Version 7.0), and the distributions of CmWOXs on chromosomal were rendered using MapGene2 Chrome (http://mg2c.iask.in/mg2c_v2.1/; accessed on 24 March 2023) [43].

4.3. Gene Structure and Motif Analysis

The genomic and CDS sequences of CmWOX genes were from the melon genome database, and the gene structures were analyzed and visualized according to the genome sequence by TBtools. The conserved motifs of CmWOX amino acids were identified with MEME 5.5 (https://meme-suite.org/meme/doc/meme.html; accessed on 24 March 2023). Totally, 10 conserved motifs were identified for the melon WOXs and designated as motif 1 to motif 10.

4.4. Putative Promoter Region Analysis of CmWOX Genes

To identify cis-elements in promoter regions, the 2.0 kb sequences upstream of the translational start site (TSS) of melon WOX genes were downloaded from the Cucurbit Genomics Data website. Conserved motifs were analyzed with the online tool PlantCARE (http://bioinformatics.psb.ugent.Be/webtools/plantcare/html/; accessed on 24 March 2023), visualized these elements through TBtools [44,54].

4.5. RNA Extracting and RNA-seq Data Analysis

Three-week-old melon seedlings of ‘674’ and ‘962’ were grown in a growth chamber and 8 °C for cold treatment for 12 h at normal light/dark condition. Leaves were collected at 0, 6 and 12 h after treatment. Total RNA was extracted from the treated leaves using an FT-plant RNA Isolation kit (NS-26, NONAZYME). The sequencing library was constructed using an Ultra RNA sample preparation kit and then sequenced using a Novaseq-S4-150PE according to the standard method (Berry Genomics, Fuzhou, China). Total reads were mapped to the DHL92 genome (V4.0). Differentially expressed genes were identified using Cuffdiff with default criteria (log2 (fold change) ≥ 1) and adjusted false discovery rate (p value < 0.05). Two independent biological replicates were used for the RNA-sequencing analysis.

4.6. Gene Expression and Real-Time PCR Analysis

The expression patterns of CmWOX genes were graphically represented in a heat map by cluster analysis using TBtools software (V1.120). Primers were designed for qRT-PCR verification of the selected CmWOX genes through NCBI, and CmACTIN gene was used as an internal control. The primers for qRT-PCR were synthesized by Sangon Biotech (Zhengzhou, China); the details are shown in Supplemental Table S7. Reverse transcription PCR (RT-PCR) was carried out with an All-in-one mix kit (Bioman, Shanghai, China). QRT-PCR detections for the target genes were performed with the SYBR Green FAST Mixture qPCR kit (GenStar, Shenzhen City, China) through the LightCycler480 real-time PCR detection System (Roche Diagnostics, Indianapolis, IN, USA), following by the PCR program of 95 °C for 30 s, 40 cycles of 95 °C for 15 s and 60 °C for 15 s, and finally 72 °C for 30 s. Relative expression levels of genes were measured through the 2−ΔΔCT method [55,56,57].

4.7. Statistical Analysis

A minimum of three biological replicates were used per experiment. Results are provided as means ± SD using GraphPad Prism 8.

5. Conclusions

In this study, a genome-wide analysis of CmWOX genes was conducted in melon. A total of 11 CmWOX genes were identified and described, and were divided into three subclades according to phylogenetic and structural analysis. Protein sequence alignment showed that CmWOX family had a typical homeodomain and conserved motifs. There are many cis-elements in the promoter region of CmWOXs which were respond to cold, drought and plant hormone. CmWOX genes displayed different expression patterns under cold and drought stresses in melo and agrestis. CmWOX13a and CmWOX13b were expressed at a higher level in comparison with other CmWOX genes. RNA-seq and qRT-PCR results imply that CmWOX4 may play a role in abiotic processes during plant development. Taken together, our work provides insights into the homology and characteristics of the CmWOX genes, which helps further research on their biological functions in tolerance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241512326/s1.

Author Contributions

L.T. and G.Z. Planned and designed the research. L.T. analyzed the bioinformatics of the CmWOX family and performed RNA extraction experiments, analyzed the transcriptome data, prepared the figures and wrote the original draft. Y.H. performed the material collection. Y.X., G.Z. and L.T.: Acquired Fundings; G.Z. and B.L. edited and approved the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2022-ZFRI), Agriculture Research System of China (CARS-25-2023-G6), Key Research and Development Project of Hainan Province (ZDYF2021XDNY164), Central Public-interest Scientific Institution Basal Research Fund (1610192023306), and Henan Province Science and Technology Research Projects (232102110185).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The BioProject accession number of the RNA-seq raw data of ‘674’ and ‘962’ under cold and PEG6000 treatments is PRJNA952866.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Van der Graaff, E.; Laux, T.; Rensing, S.A. The WUS homeobox-containing (WOX) protein family. Genome Biol. 2009, 10, 248. [Google Scholar] [CrossRef]
  2. Lian, G.; Ding, Z.; Wang, Q.; Zhang, D.; Xu, J. Origins and evolution of WUSCHEL-related homeobox protein family in plant kingdom. Sci. World J. 2014, 2014, 534140. [Google Scholar] [CrossRef] [Green Version]
  3. Nakata, M.; Matsumoto, N.; Tsugeki, R.; Rikirsch, E.; Laux, T.; Okada, K. Roles of the middle domain-specific WUSCHEL-RELATED HOMEOBOX genes in early development of leaves in Arabidopsis. Plant Cell 2012, 24, 519–535. [Google Scholar] [CrossRef] [Green Version]
  4. Bleckmann, A.; Weidtkamp-Peters, S.; Seidel, C.A.; Simon, R. Stem cell signaling in Arabidopsis requires CRN to localize CLV2 to the plasma membrane. Plant Physiol. 2010, 152, 166–176. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Alvarez, J.M.; Bueno, N.; Canas, R.A.; Avila, C.; Canovas, F.M.; Ordas, R.J. Analysis of the WUSCHEL-RELATED HOMEOBOX gene family in Pinus pinaster: New insights into the gene family evolution. Plant Physiol. Biochem. 2018, 123, 304–318. [Google Scholar] [CrossRef]
  6. Li, X.; Hamyat, M.; Liu, C.; Ahmad, S.; Gao, X.; Guo, C.; Wang, Y.; Guo, Y. Identification and Characterization of the WOX Family Genes in Five Solanaceae Species reveal their conserved roles in peptide signaling. Genes 2018, 9, 260. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Feng, C.; Zou, S.; Gao, P.; Wang, Z. In silico identification, characterization expression profile of WUSCHEL-Related Homeobox (WOX) gene family in two species of kiwifruit. Peer J. 2021, 9, e12348. [Google Scholar] [CrossRef] [PubMed]
  8. Vandenbussche, M.; Horstman, A.; Zethof, J.; Koes, R.; Rijpkema, A.S.; Gerats, T. Differential recruitment of WOX transcription factors for lateral development and organ fusion in Petunia and Arabidopsis. Plant Cell 2009, 21, 2269–2283. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Borchers, A.; Pieler, T. Programming pluripotent precursor cells derived from Xenopus embryos to generate specific tissues and organs. Genes 2010, 1, 413–426. [Google Scholar] [CrossRef] [Green Version]
  10. Kadri, A.; Grenier De March, G.; Guerineau, F.; Cosson, V.; Ratet, P. WUSCHEL Overexpression Promotes Callogenesis and Somatic Embryogenesis in Medicago truncatula Gaertn. Plants 2021, 10, 715. [Google Scholar] [CrossRef]
  11. Tvorogova, V.E.; Krasnoperova, E.Y.; Potsenkovskaia, E.A.; Kudriashov, A.A.; Dodueva, I.E.; Lutova, L.A. What Does the WOX Say? Review of Regulators, Targets, Partners. Mol. Biol. 2021, 55, 311–337. [Google Scholar] [CrossRef]
  12. Zhang, X.; Zong, J.; Liu, J.; Yin, J.; Zhang, D. Genome-wide analysis of WOX gene family in rice, sorghum, maize, Arabidopsis and poplar. J. Integr. Plant Biol. 2010, 52, 1016–1026. [Google Scholar] [CrossRef]
  13. Breuninger, H.; Rikirsch, E.; Hermann, M.; Ueda, M.; Laux, T. Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo. Dev. Cell 2008, 14, 867–876. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Haecker, A.; Gross-Hardt, R.; Geiges, B.; Sarkar, A.; Breuninger, H.; Herrmann, M.; Laux, T. Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana. Development 2004, 131, 657–668. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Kyo, M.; Maida, K.; Nishioka, Y.; Matsui, K. Coexpression of WUSCHEL related homeobox (WOX) 2 with WOX8 or WOX9 promotes regeneration from leaf segments and free cells in Nicotiana tabacum L. Plant Biotechnol. 2018, 35, 23–30. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Zhu, T.; Moschou, P.N.; Alvarez, J.M.; Sohlberg, J.J.; von Arnold, S. WUSCHEL-RELATED HOMEOBOX 8/9 is important for proper embryo patterning in the gymnosperm Norway spruce. J. Exp. Bot. 2014, 65, 6543–6552. [Google Scholar] [CrossRef] [Green Version]
  17. Niu, H.; Liu, X.; Tong, C.; Wang, H.; Li, S.; Lu, L.; Pan, Y.; Zhang, X.; Weng, Y.; Li, Z. The WUSCHEL-related homeobox1 gene of cucumber regulates reproductive organ development. J. Exp. Bot. 2018, 69, 5373–5387. [Google Scholar] [CrossRef] [Green Version]
  18. Wang, H.; Niu, H.; Li, C.; Shen, G.; Liu, X.; Weng, Y.; Wu, T.; Li, Z. WUSCHEL-related homeobox1 (WOX1) regulates vein patterning and leaf size in Cucumis sativus. Hortic. Res. 2020, 7, 182. [Google Scholar] [CrossRef]
  19. Laux, T.; Mayer, K.F.; Berger, J.; Jürgens, G. The WUSCHEL gene is required for shoot and floral meristem integrity in Arabidopsis. Development 1996, 122, 87–96. [Google Scholar] [CrossRef] [PubMed]
  20. Nardmann, J.; Werr, W. The shoot stem cell niche in angiosperms: Expression patterns of WUS orthologues in rice and maize imply major modifications in the course of mono- and dicot evolution. Mol. Biol. Evol. 2006, 23, 2492–2504. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  21. Bao, Y.; Dharmawardhana, P.; Arias, R.; Allen, M.B.; Ma, C.; Strauss, S.H. WUS and STM-based reporter genes for studying meristem development in poplar. Plant Cell Rep. 2009, 28, 947–962. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Zhang, C.; Wang, J.; Wang, X.; Li, C.; Ye, Z.; Zhang, J. UF, a WOX gene, regulates a novel phenotype of un-fused flower in tomato. Plant Sci. 2020, 297, 110523. [Google Scholar] [CrossRef] [PubMed]
  23. Dolzblasz, A.; Nardmann, J.; Clerici, E.; Causier, B.; van der Graaff, E.; Chen, J.; Davies, B.; Werr, W.; Laux, T. Stem Cell Regulation by Arabidopsis WOX Genes. Mol. Plant 2016, 9, 1028–1039. [Google Scholar] [CrossRef] [PubMed]
  24. Jha, P.; Ochatt, S.J.; Kumar, V. WUSCHEL: A master regulator in plant growth signaling. Plant Cell Rep. 2020, 39, 431–444. [Google Scholar] [CrossRef]
  25. Cheng, S.; Zhou, D.X.; Zhao, Y. WUSCHEL-related homeobox gene WOX11 increases rice drought resistance by controlling root hair formation and root system development. Plant Signal. Behav. 2016, 11, e1130198. [Google Scholar] [CrossRef] [Green Version]
  26. Liu, D.; Sun, W.; Yuan, Y.; Zhang, N.; Hayward, A.; Liu, Y.; Wang, Y. Phylogenetic analyses provide the first insights into the evolution of OVATE family proteins in land plants. Ann. Bot. 2014, 113, 1219–1233. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Liu, J.; Hu, X.; Qin, P.; Prasad, K.; Hu, Y.; Xu, L. The WOX11-LBD16 Pathway Promotes Pluripotency Acquisition in Callus Cells During De Novo Shoot Regeneration in Tissue Culture. Plant Cell Physiol. 2018, 59, 734–743. [Google Scholar] [CrossRef] [Green Version]
  28. Romera-Branchat, M.; Ripoll, J.J.; Yanofsky, M.F.; Pelaz, S. The WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana fruit. Plant J. 2013, 73, 37–49. [Google Scholar] [CrossRef]
  29. Deveaux, Y.; Toffano-Nioche, C.; Claisse, G.; Thareau, V.; Morin, H.; Laufs, P.; Moreau, H.; Kreis, M.; Lecharny, A. Genes of the most conserved WOX clade in plants affect root and flower development in Arabidopsis. BMC Evol. Biol. 2008, 8, 291. [Google Scholar] [CrossRef] [Green Version]
  30. Denis, E.; Kbiri, N.; Mary, V.; Claisse, G.; Conde, E.S.N.; Kreis, M.; Deveaux, Y. WOX14 promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis. Plant J. 2017, 90, 560–572. [Google Scholar] [CrossRef] [Green Version]
  31. Wu, C.C.; Li, F.W.; Kramer, E.M. Large-scale phylogenomic analysis suggests three ancient superclades of the WUSCHEL-RELATED HOMEOBOX transcription factor family in plants. PLoS ONE 2019, 14, e0223521. [Google Scholar] [CrossRef] [PubMed]
  32. Conklin, P.A.; Johnston, R.; Conlon, B.R.; Shimizu, R.; Scanlon, M.J. Plant homeodomain proteins provide a mechanism for how leaves grow wide. Development 2020, 147, dev193623. [Google Scholar] [CrossRef]
  33. Costanzo, E.; Trehin, C.; Vandenbussche, M. The role of WOX genes in flower development. Ann. Bot. 2014, 114, 1545–1553. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Honda, E.; Yew, C.L.; Yoshikawa, T.; Sato, Y.; Hibara, K.I.; Itoh, J.I. LEAF LATERAL SYMMETRY1, a Member of the WUSCHEL-RELATED HOMEOBOX3 Gene Family, Regulates Lateral Organ Development Differentially from Other Paralogs, NARROW LEAF2 and NARROW LEAF3 in Rice. Plant Cell Physiol. 2018, 59, 376–391. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Nakata, M.T.; Tameshige, T.; Takahara, M.; Mitsuda, N.; Okada, K. The functional balance between the WUSCHEL-RELATED HOMEOBOX1 gene and the phytohormone auxin is a key factor for cell proliferation in Arabidopsis seedlings. Plant Biotechnol. 2018, 35, 141–154. [Google Scholar] [CrossRef] [Green Version]
  36. Cheng, S.; Huang, Y.; Zhu, N.; Zhao, Y. The rice WUSCHEL-related homeobox genes are involved in reproductive organ development, hormone signaling and abiotic stress response. Gene 2014, 549, 266–274. [Google Scholar] [CrossRef]
  37. Minh-Thu, P.T.; Kim, J.S.; Chae, S.; Jun, K.M.; Lee, G.S.; Kim, D.E.; Cheong, J.J.; Song, S.I.; Nahm, B.H.; Kim, Y.K. A WUSCHEL Homeobox Transcription Factor, OsWOX13, Enhances Drought Tolerance and Triggers Early Flowering in Rice. Mol. Cells 2018, 41, 781–798. [Google Scholar] [CrossRef] [PubMed]
  38. Lv, J.; Feng, Y.; Jiang, L.; Zhang, G.; Wu, T.; Zhang, X.; Xu, X.; Wang, Y.; Han, Z. Genome-wide identification of WOX family members in nine Rosaceae species and a functional analysis of MdWOX13-1 in drought resistance. Plant Sci. 2023, 328, 111564. [Google Scholar] [CrossRef]
  39. Akbulut, S.E.; Okay, A.; Aksoy, T.; Aras, E.S.; Buyuk, I. The genome-wide characterization of WOX gene family in Phaseolus vulgaris L. during salt stress. Physiol. Mol. Biol. Plants 2022, 28, 1297–1309. [Google Scholar] [CrossRef]
  40. Han, N.; Tang, R.; Chen, X.; Xu, Z.; Ren, Z.; Wang, L. Genome-wide identification and characterization of WOX genes in Cucumis sativus. Genome 2021, 64, 761–776. [Google Scholar] [CrossRef]
  41. Yang, Z.; Gong, Q.; Qin, W.; Yang, Z.; Cheng, Y.; Lu, L.; Ge, X.; Zhang, C.; Wu, Z.; Li, F. Genome-wide analysis of WOX genes in upland cotton and their expression pattern under different stresses. BMC Plant Biol. 2017, 17, 113. [Google Scholar] [CrossRef]
  42. Gu, R.; Song, X.; Liu, X.; Yan, L.; Zhou, Z.; Zhang, X. Genome-wide analysis of CsWOX transcription factor gene family in cucumber (Cucumis sativus L.). Sci. Rep. 2020, 10, 6216. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Chao, J.-T.; Kong, Y.-Z.; Wang, Q.; Sun, Y.-H.; Gong, D.-P.; Lv, J.; Liu, G.-S. MapGene2Chrom, a tool to draw gene physical map based on Perl and SVG languages. Hereditas 2015, 37, 91–97. [Google Scholar] [CrossRef] [PubMed]
  44. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  45. Elkan, B.A. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1994, 2, 28–36. [Google Scholar]
  46. Ma, L.; Wang, Q.; Zheng, Y.; Guo, J.; Yuan, S.; Fu, A.; Bai, C.; Zhao, X.; Zheng, S.; Wen, C.; et al. Cucurbitaceae genome evolution, gene function, and molecular breeding. Hortic. Res. 2022, 9, uhab057. [Google Scholar] [CrossRef]
  47. Li, H.; Li, C.; Wang, Y.; Qin, X.; Meng, L.; Sun, X. Genome-Wide Analysis of the WOX Transcription Factor Genes in Dendrobium catenatum Lindl. Genes 2022, 13, 1481. [Google Scholar] [CrossRef]
  48. Zhao, G.; Lian, Q.; Zhang, Z.; Fu, Q.; He, Y.; Ma, S.; Ruggieri, V.; Monforte, A.J.; Wang, P.; Julca, I.; et al. A comprehensive genome variation map of melon identifies multiple domestication events and loci influencing agronomic traits. Nat. Genet. 2019, 51, 1607–1615. [Google Scholar] [CrossRef]
  49. Bhaskara, G.B.; Nguyen, T.T.; Verslues, P.E. Unique drought resistance functions of the highly ABA-induced clade A protein phosphatase 2Cs. Plant Physiol. 2012, 160, 379–395. [Google Scholar] [CrossRef] [Green Version]
  50. Zhang, X.; Fu, X.; Liu, F.; Wang, Y.; Bi, H.; Ai, X. Hydrogen Sulfide Improves the Cold Stress Resistance through the CsARF5-CsDREB3 Module in Cucumber. Int. J. Mol. Sci. 2021, 22, 13229. [Google Scholar] [CrossRef]
  51. Wang, P.; Guo, Y.; Chen, X.; Zheng, Y.; Sun, Y.; Yang, J.; Ye, N. Genome-wide identification of WOX genes and their expression patterns under different hormone and abiotic stress treatments in tea plant (Camellia sinensis). Trees 2019, 33, 1129–1142. [Google Scholar] [CrossRef]
  52. Yu, J.; Wu, S.; Sun, H.; Wang, X.; Tang, X.; Guo, S.; Zhang, Z.; Huang, S.; Xu, Y.; Weng, Y.; et al. CuGenDBv2: An updated database for cucurbit genomics. Nucleic Acids Res. 2023, 51, D1457–D1464. [Google Scholar] [CrossRef]
  53. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, A database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef]
  55. Green, M.R.; Sambrook, J. Analysis and Normalization of Real-Time Polymerase Chain Reaction (PCR) Experimental Data. Cold Spring Harb. Protoc. 2018, 2018, 769–777. [Google Scholar] [CrossRef] [PubMed]
  56. Huggett, J.; Dheda, K.; Bustin, S.; Zumla, A. Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005, 6, 279–284. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  57. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
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Figure 1. Phylogenetic relationships of the WUSCHEL-related homeobox (WOX). Phylogenetic relationships of the WOX family from Cucumber (Cs), tomato (Sl), Arabidopsis (At), melon (Cm), and rice (Os). The phylogenetic tree was built by MEGA 7.0. The WOX from 5 plants can be divided into 3 groups, each branch is indicated in a specific color.
Figure 1. Phylogenetic relationships of the WUSCHEL-related homeobox (WOX). Phylogenetic relationships of the WOX family from Cucumber (Cs), tomato (Sl), Arabidopsis (At), melon (Cm), and rice (Os). The phylogenetic tree was built by MEGA 7.0. The WOX from 5 plants can be divided into 3 groups, each branch is indicated in a specific color.
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Figure 2. Distribution of CmWOX genes on the chromosome in melon. Total 11 CmWOX genes are located in assembled chromosome. The number of chromosome was noted on the left side of each chromosome. The bar scale on the left is in Mega base (Mb).
Figure 2. Distribution of CmWOX genes on the chromosome in melon. Total 11 CmWOX genes are located in assembled chromosome. The number of chromosome was noted on the left side of each chromosome. The bar scale on the left is in Mega base (Mb).
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Figure 3. Phylogenetic analysis and gene structure of CmWOXs. (a) MEGA 7.0 was used to build the phylogenetic tree of 11 CmWOX proteins and divided them into the modern clade, the intermediate clade, and the ancient clade, each clade was indicated in red, blue and green line, respectively. (b) Exon-intron structures of CmWOX genes were drawn by the Gene Structure Display Server, Green boxes indicated exons and black lines indicated introns.
Figure 3. Phylogenetic analysis and gene structure of CmWOXs. (a) MEGA 7.0 was used to build the phylogenetic tree of 11 CmWOX proteins and divided them into the modern clade, the intermediate clade, and the ancient clade, each clade was indicated in red, blue and green line, respectively. (b) Exon-intron structures of CmWOX genes were drawn by the Gene Structure Display Server, Green boxes indicated exons and black lines indicated introns.
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Figure 4. The conserved motifs and homeodomain of CmWOX family. (a) The conserved motif 1 to motif 10 of CmWOX proteins were highlighted with different colored boxes. Motif 1 and motif 7 were homeodomain and WUS motif, respectively. (b) The seq-logo of conserved domains was analyzed using the SeqLogo program of TBtools. Different colors indicated highly conserved residues, which are R, W, P, Q, G, I, L, W, and F.
Figure 4. The conserved motifs and homeodomain of CmWOX family. (a) The conserved motif 1 to motif 10 of CmWOX proteins were highlighted with different colored boxes. Motif 1 and motif 7 were homeodomain and WUS motif, respectively. (b) The seq-logo of conserved domains was analyzed using the SeqLogo program of TBtools. Different colors indicated highly conserved residues, which are R, W, P, Q, G, I, L, W, and F.
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Figure 5. Predicted cis-elements in promoter regions of CmWOX genes. A promoter region of about 2-kb upstream of CmWOX genes are showed potential cis-elements, particularly those associated with stress responses (such as light induction, low temperature, and anaerobic induction) and plant hormones (such as auxin and gibberellin). Different cis-elements are stated with different colors.
Figure 5. Predicted cis-elements in promoter regions of CmWOX genes. A promoter region of about 2-kb upstream of CmWOX genes are showed potential cis-elements, particularly those associated with stress responses (such as light induction, low temperature, and anaerobic induction) and plant hormones (such as auxin and gibberellin). Different cis-elements are stated with different colors.
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Figure 6. Principle component analysis (PCA) and statistics on the number of DEGs between ‘674’ and ‘962’. (a,b) Principle component analysis of samples in ‘674’ and ‘962’under cold and PEG6000 treatment, respectively. (c,d) Statistics on the number of DEGs in ‘674’ and ‘962’ under cold and PEG6000 treatment. The number of differential expressed genes is counted at three different time points.
Figure 6. Principle component analysis (PCA) and statistics on the number of DEGs between ‘674’ and ‘962’. (a,b) Principle component analysis of samples in ‘674’ and ‘962’under cold and PEG6000 treatment, respectively. (c,d) Statistics on the number of DEGs in ‘674’ and ‘962’ under cold and PEG6000 treatment. The number of differential expressed genes is counted at three different time points.
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Figure 7. The heat map of CmWOX genes under cold and drought stresses. The heat map of the expression of typical CmWOX genes in melon. (a) The transcriptomes heat map of ‘674’ and ‘962’ CmWOX family under low-temperature treatment at 0 h, 6 h, and 12 h. (b) The transcriptomes heat map of ‘674’ and ‘962’ CmWOX family under PEG6000 treatment at 0 h, 6 h, and 12 h. Color scale represents fold changes (log2 fold change). Blue and green colors indicate low expression level, red and yellow color indicate high expression level, respectively. The data were processed from 2 replicates of transcriptomic data at different treatment times.
Figure 7. The heat map of CmWOX genes under cold and drought stresses. The heat map of the expression of typical CmWOX genes in melon. (a) The transcriptomes heat map of ‘674’ and ‘962’ CmWOX family under low-temperature treatment at 0 h, 6 h, and 12 h. (b) The transcriptomes heat map of ‘674’ and ‘962’ CmWOX family under PEG6000 treatment at 0 h, 6 h, and 12 h. Color scale represents fold changes (log2 fold change). Blue and green colors indicate low expression level, red and yellow color indicate high expression level, respectively. The data were processed from 2 replicates of transcriptomic data at different treatment times.
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Figure 8. The expression level of CmWOX genes under cold and drought stresses. (a) QRT-PCR verified the expression of CmWUS, CmWOX13a, and CmWOX13b at 0 h and 6 h under cold treatment in ‘674’. (b) QRT-PCR verified the expression of CmWUS, CmWOX4, and CmWOX13b at 0 h and 12 h under cold treatment in ‘962’. (c) QRT-PCR verified the expression of CmWOX1a, CmWOX4, CmWOX5, and CmWOX13b at 6 h and 12 h under PEG6000 treatment in ‘674’. (d) QRT-PCR verified the expression of CmWOX1a, CmWOX4, CmWOX13a, and CmWOX13b at 6 h and 12 h under PEG6000 treatment in ‘962’. CmACTIN was used as an internal control, and three or four biological replicates were calculated, error bars indicate the standard error of the mean. The different colors of symbols circle, square, and arrow denote different genes.
Figure 8. The expression level of CmWOX genes under cold and drought stresses. (a) QRT-PCR verified the expression of CmWUS, CmWOX13a, and CmWOX13b at 0 h and 6 h under cold treatment in ‘674’. (b) QRT-PCR verified the expression of CmWUS, CmWOX4, and CmWOX13b at 0 h and 12 h under cold treatment in ‘962’. (c) QRT-PCR verified the expression of CmWOX1a, CmWOX4, CmWOX5, and CmWOX13b at 6 h and 12 h under PEG6000 treatment in ‘674’. (d) QRT-PCR verified the expression of CmWOX1a, CmWOX4, CmWOX13a, and CmWOX13b at 6 h and 12 h under PEG6000 treatment in ‘962’. CmACTIN was used as an internal control, and three or four biological replicates were calculated, error bars indicate the standard error of the mean. The different colors of symbols circle, square, and arrow denote different genes.
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Table 1. Identification and characteristics of CmWOX family genes.
Table 1. Identification and characteristics of CmWOX family genes.
NameCladeCDS (bp)AAMW (kDa)PI
CmWUSModern Clade57619521.1810.228
CmWOX1aModern Clade114638543.135.699
CmWOX1bModern Clade51917220.0310.312
CmWOX2Modern Clade75024928.219.211
CmWOX3Modern Clade57919622.126.424
CmWOX4Modern Clade69023326.028.56
CmWOX5Modern Clade59119622.218.637
CmWOX9Intermediate Clade114038341.577.688
CmWOX11Intermediate Clade79227128.095.665
CmWOX13aAncient Clade85829332.516.204
CmWOX13bAncient Clade81027730.635.23
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Tang, L.; He, Y.; Liu, B.; Xu, Y.; Zhao, G. Genome-Wide Identification and Characterization Analysis of WUSCHEL-Related Homeobox Family in Melon (Cucumis melo L.). Int. J. Mol. Sci. 2023, 24, 12326. https://doi.org/10.3390/ijms241512326

AMA Style

Tang L, He Y, Liu B, Xu Y, Zhao G. Genome-Wide Identification and Characterization Analysis of WUSCHEL-Related Homeobox Family in Melon (Cucumis melo L.). International Journal of Molecular Sciences. 2023; 24(15):12326. https://doi.org/10.3390/ijms241512326

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Tang, Lingli, Yuhua He, Bin Liu, Yongyang Xu, and Guangwei Zhao. 2023. "Genome-Wide Identification and Characterization Analysis of WUSCHEL-Related Homeobox Family in Melon (Cucumis melo L.)" International Journal of Molecular Sciences 24, no. 15: 12326. https://doi.org/10.3390/ijms241512326

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