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Article

The Tissue Expression Divergence of the WUSCHEL-Related Homeobox Gene Family in the Evolution of Nelumbo

by
Juanjuan Li
1 and
Yue Zhang
2,3,*
1
Hubei Province Research Center of Engineering Technology for Utilization of Botanical Functional Ingredients, Hubei Key Laboratory of Resource Utilization and Quality Control of Characteristic Crops, College of Life Science and Technology, Hubei Engineering University, Xiaogan 432000, China
2
Aquatic Plant Research Center, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
3
State Key Laboratory of Plant Diversity and Specialty Crops, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
*
Author to whom correspondence should be addressed.
Plants 2025, 14(13), 1909; https://doi.org/10.3390/plants14131909 (registering DOI)
Submission received: 9 May 2025 / Revised: 17 June 2025 / Accepted: 18 June 2025 / Published: 21 June 2025
(This article belongs to the Section Plant Genetics, Genomics and Biotechnology)
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Abstract

:
The yellow flower lotus (Nelumbo lutea) is the sister species of the sacred lotus (N. nucifera). The evolution of gene expression patterns across multiple tissues during the species divergence of these two lotuses remains unexplored. The WUSCHEL-related homeobox (WOX) family, a plant-specific transcription factor family, plays a crucial role in tissue development and stress responses. In this study, utilizing a chromosome-level reference genome and a transcriptome database covering multiple tissues, we identified and categorized 11 NlWOX genes into three subfamilies. We identified seven syntenic gene pairs between NnWOXs and NlWOXs that originated from whole-genome duplications. Through conserved motif analysis, we found subfamily-specific motifs in the protein sequences of NnWOXs and NlWOXs. Variations in the three-dimensional conformations of homologous WOX genes indicate function divergences between the two lotus species. The gene expression matrix of NlWOX across tissues reveals expression divergences within N. lutea and between the two lotus species. By employing a weight gene co-expression network analysis pipeline, we developed eight NlWOX co-expression networks that differed from the co-expression networks of their syntenic genes. Overall, our findings suggest that genomic variations in the WOX orthologs contribute to the distinct expression patterns and regulatory networks observed during the evolution of these two lotuses.

1. Introduction

Transcription factors (TFs) regulate gene expression by binding to cis-acting elements in the upstream promoter region, thereby playing vital roles in various plant growth and development pathways [1,2]. Comparative genomics typically examines changes in appearance or loss within different transcription factor families [3]. For instance, the evolution of transcription factor families involved in drought tolerance and phytohormone response between the early land plant Physcomitrella patens and Arabidopsis thaliana highlights their significance in the emergence of land plants [4]. However, the molecular functions of TFs are closely linked to specific cellular pathways and morphological features [5,6,7], necessitating further investigations at various levels [8]. Conserved regulatory relationships between TFs and their targeted genes exhibit similar compositions in gene co-expression networks across diverse species [9,10]. The evolution dynamics of TFs at the expression level have garnered considerable attention in research.
WUSCHEL-related homeobox (WOX) genes are part of a large family of plant-specific transcription factors containing a homeodomain (HD). The main identifying feature of the WOX gene is a conserved helix-loop-helix-turn-helix structure consisting of 60 amino acids [11]. Utilizing extensive transcriptomic and protein–protein interaction data from the model plant Arabidopsis, both forward and reverse genetic studies on 15 identified Arabidopsis WOX (AtWOX) proteins have highlighted their roles in organ formation and tissue development, including functions in the shoot apical meristem [12,13], ovule development [14], and floral transition [15]. Phylogenetic analyses have categorized AtWOX genes into three subfamilies: the ancient clade, the intermediate clade, and the WUS clade [16,17]. Recent investigations into the biological functions of WOX members have revealed their involvement in responses to abiotic stresses and transcriptional regulation [18,19,20]. For instance, in tomatoes, WOX gene expression variations were linked to responses to cold, salt, and drought stresses [21]. In rice, the overexpression of OsWOX11 enhanced tolerance to potassium scarcity and promoted root growth [22]. Additionally, the complex formed by OsWOX3B and OsSPL10 regulates the transcription of HL6, which is essential for trichome formation [23]. Genome-wide identifications of WOX genes have been conducted in various species [24,25], and distinct regulatory interactions of WOX orthologs exhibited similar responses, such as PagWOX11/12a-SAUR36 in poplar [26], OsWOX13-OsDREB1A/1F in rice [27], and MdWOX13-1-MdSOD in Rosaceae [19]. These findings suggest that the diverse interactions of WOX orthologs across species may represent different components in a molecular network governing the development of specific traits. With the widespread use of RNA-seq, gene co-expression network analysis is commonly used to cluster genes with similar expression patterns into modules [28]. Establishing connections between TFs and their co-expressed genes is effective in identifying the downstream-regulated genes of TFs, as genes within a module often participate in similar biological pathways [29]. Nevertheless, the evolution of the co-expression networks of WOX genes during plant species divergence remains largely unknown.
Following the last ice age, the Nelumbonaceae family currently comprises only two extant sister species globally: Nelumbo nucifera and N. lutea. A notable distinction in tissue morphology between these two species lies in their flower color, as N. nucifera exhibits flowers ranging from white to red, while N. lutea lotus flowers are yellow [30]. N. nucifera, commonly known as the sacred lotus, serves as a popular aquatic vegetable in East Asia, valued for its edible and medicinal properties. Consequently, extensive genomic studies on various lotus tissues have generated a substantial amount of genomic data [31,32], which is archived in the Nelumbo Genome Database [33]. Leveraging this resource, our prior research identified the complete set of NnWOX genes in N. nucifera and discerned their expression variations across diverse tissues [34]. Recently, a high-quality assembly of N. lutea and RNA-seq from multiple tissues at various developmental stages became available [35,36]. This development allows for a comparative analysis of gene co-expression networks in these closely related species, offering a novel perspective on the evolutionary trajectory of WOX genes. In this investigation, we characterized the WOX family members in N. lutea, examining their physicochemical attributes, genomic positions, and expression patterns across different tissues. Through a comparative assessment of the co-expression networks of WOX genes in N. nucifera and N. lutea, we observed relatively consistent evolutionary patterns, suggesting that orthologous WOX genes likely play analogous regulatory roles in both species.

2. Results

2.1. Identification of WOXs in N. lutea

Genome assembly and gene annotation of N. lutea were downloaded from the Nelumbo Genome Database (http://nelumbo.cngb.org/nelumbo/, accessed on 17 June 2025), and we further mapped the protein sequences to the orthologous public database to annotate their biological functions. To obtain the comprehensive WOX gene family members in N. lutea, the WOX protein sequences of the model plant A. thaliana and the basal angiosperm Amborella trichopoda were downloaded as the query sequences. We compared the sequence ortholog between the query WOX sequences and the protein of N. lutea using BlastP. According to the function annotation and orthologous mapped results, we filtered a candidate group of N. lutea WOX proteins (NlWOXs). We further eliminated candidate proteins without typical conserved domains and redundancies and identified 11 WOX gene family members in N. lutea. These include NL1g_04069, NL1g_04550, NL1g_06482, NL2g_10541, NL2g_11918, NL2g_11942, NL2g_12810, NL4g_22694, NL5g_27723, NL5g_28623, and NL6g_29420. Unlike the sequential nomenclature of WOXs in other species based on the location of chromosomes, we named the NlWOXs according to their orthologs in A. thaliana. As the nomenclature of NnWOXs is also based on their orthologs in A. thaliana, it is convenient to compare the orthologs between species. Finally, 11 NlWOX genes were obtained, including NlWOX1, NlWOX2, NlWOX3, NlWOX4, NlWOX5a, NlWOX5b, NlWOX9, NlWOX11, NlWOX13a, NlWOX13b, and NlWUS (Table 1). Compared to the number of WOX genes in N. nucifera, the WOX gene family is contracted.
The number of amino acids for the NlWOXs ranges from 124 to 362, with an average length of 244.72. Meanwhile, in the NlWOXs, the maximum molecular weight is 39945.94 kDa, and the minimum molecular weight is 14769.82 kDa (Table 1). The length of the longest WOX protein in N. nucifera and N. lutea is similar and is the ortholog of WOX9 in A. thaliana. The shortest WOX protein in N. lutea is NlWOX3, and in N. nucifera, it is NnWOX5, suggesting the sequence evolution of WOX members in Nelumbo. Similarly, we analyzed the chemical and physical characteristics of NlWOX proteins, including the theoretical PI values, instability index, aliphatic index, and grand average of hydropathicity (Table 1). Most NlWOX proteins (10/11) were predicted to have at least one N-glycosylation site (Figure S1). NlWOX1 has the most, with four N-glycosylation sites, followed by NlWOX9, which has three N-glycosylation sites. Interestingly, NlWOX13a was identified to have no N-glycosylation sites on the sequence, but its paralogous NlWOX13b was predicted to have one, suggesting the sequence evolution in NlWOX genes. In addition, we analyzed the hydrophobicity and hydrophilicity for each NlWOX protein (Figure S2).

2.2. Phylogenetic Tree of NlWOX Proteins

Previous studies suggest that lotus is one of the basal angiosperms and occupies an important position in phylogenetic evolution. To explore the evolutionary relationships of WOX genes in lotus and other plants, phylogenetic analysis was performed based on the sequences from N. lutea (11), N. nucifera (15), A. thaliana (15), and Amborella trichopoda (9) (Figure 1). In line with previous studies [37], the WOX genes across various species were categorized into three clades: ancient clade (AC), intermediate clade (IC), and WUS clade (WC) (Figure 1). Seven NlWOX genes were divided into WC clades, and the other two clades had two NlWOX genes, respectively. We found the AmtrWOX gene in each small branch, suggesting that these WOX genes originate from basal angiosperms. In comparison to the WOX genes in A. thaliana or A. trichopoda, NlWOXs show a closer evolutionary relationship to NnWOXs. This suggests that these two lotus species share a similar evolution in the WOX gene family. Notably, we found that NlWOX13a is closer to NnWOX13a rather than NlWOX13b, indicating a stronger sequence variation in paralogs of N. lutea than in orthologs of the two lotus species.

2.3. Gene Duplication and Synteny Analysis

To investigate the genetic distances of NlWOX genes, the chromosomal positions of 11 NlWOX genes are exhibited in Figure 2a. In terms of eight pseudochromosomes, the NlWOX genes are distributed in only five pseudochromosomes (i.e., chr1, chr2, chr4, chr5, and chr6). We found four NlWOX genes (NlWOX2, NlWOX3a, NlWOX5a, and NlWOX5b) located on chr2, indicating their close genetic distances. To further explore whether the physical bunching of NlWOX genes in chr2 was caused by whole-genome duplications, five different gene duplication types were identified using MCScanX. Only two duplicated gene pairs were found in the whole-genome duplication block of paralogs in N. lutea. The NlWOX5a/NlWOX5b duplicated gene pair was in the same chromosome (chr2), while the NlWOX13a/NlWOX13b duplicated genes were located in different chromosomes (Figure 2b). Our results indicate that whole-genome duplication events on chr2 may have contributed to the largest number of NlWOX genes. However, most NlWOX genes were not copied along with the whole-genome duplication or lost their duplications during evolution.
Orthologous genes change their chromosomal locations during the formation of species. To study the evolution of WOX genes on the genome during the divergence of two lotus species, an interspecies collinearity analysis of orthologous genes was carried out between N. nucifera and N. lutea. Based on the synteny regions, we identified a total of 13 collinearity gene pairs of WOX (Figure 2c). We found seven conserved syntenic gene pairs, including NlWOX13a-NnWOX13a, NlWOX1-NnWOX6b, NlWOX9-NnWOX9b, NlWOX4-NnWOX4a, NlWOX5a-NnWOX5b, NlWOX3-NnWOX3a, and NlWOX5b-NnWOX5a. Notably, three duplicated gene pairs, NnWOX3a/3b, NnWOX4a/4b, and NnWOX6a/6b, in N. nucifera were uniquely syntenic to NlWOX3a, NlWOX4, and NlWOX1, respectively. This indicates the direction of whole-genome duplication events in the N. nucifera genome, i.e., NnWOX3b was duplicated from NnWOX3a, NnWOX4b was duplicated from NnWOX4a, and NnWOX6a was from NnWOX6b. On the contrary, the duplicated genes of NlWOX3a, NlWOX4, and NlWOX1 might be lost after whole-genome duplication. In addition, no synteny genes were identified for NlWUS, NlWXO2, NlWOX11, and NlWOX13b, indicating their unique evolution of genome sequences.

2.4. Conserved Motifs and Protein Conformation of WOX Genes in Nelumbo

To explore the sequence evolution of syntenic and non-syntenic WOX genes between two lotus species, the conserved motifs were predicted in both NlWOX and NnWOX proteins. For all WOX proteins in the lotuses, Motif1 and Motif2 were identified but in different locations of WOX proteins (Figure 3a). In the WUS subfamily, except for NlWOX3, NlWOX2, and NnWOX2, NlWOX and NnWOX proteins contained Motif8 (Figure 3a). Motif10 was specifically identified in NlWUS and NnWUS (Figure 3a). In the AC subfamily, only NlWOX13b was found to lose Motif5, and all WOX proteins contained the subfamily-specific Motif7 (Figure 3a). Motif4 was identified to be IC subfamily-specific (Figure 3a). Notably, we found that syntenic WOX gene pairs show a high similarity of conserved motifs, suggesting that they might have the same functions in lotuses.
Based on the arrangement of amino acids in the polypeptide chain, the secondary structures of NnWOX and NlWOX proteins were predicted (Figure 3b). Random coils are the predominant secondary structure in the WOX protein sequences, accounting for 62.31% to 83.88% of all protein sequences (Figure 3b). Interestingly, alpha helices are more prevalent in the sequences of NlWOX3, NlWOX13a, and NnWOX13a (Figure 3b). Furthermore, a three-dimensional structure analysis of NnWOX and NlWOX proteins was performed (Figure 3c). The conserved helix-loop-helix-turn-helix structure was identified in the three-dimensional conformation of NnWOX and NlWOX proteins (Figure 3c). We found specific three-dimensional structures at the end of NlWOX9, NlWOX11, NlWOX13a, NnWOX9a/9b, NnWOX11, and NnWOX13a/13b, indicating that they may be involved in a specific protein complex. Most of the syntenic genes showed similar three-dimensional structures. However, NlWOX3 exhibited more loose protein conformation than its syntenic NnWOX3a, suggesting weaker noncovalent interactions.

2.5. Tissue Expression Pattern Divergence of WOX Proteins in Lotuses

Previous studies indicate that WOX genes are widely expressed in multiple tissues in the model plant Arabidopsis [38] and rice [39]. Meanwhile, our study found the expression divergence of NnWOX genes among different tissues [34]. To further compare the expression patterns of WOX genes in two lotuses, the RNA-seq datasets of different tissues in N. lutea were downloaded. A transcriptome analysis pipeline was carried out to construct the NlWOX gene expression matrix across 36 tissue samples (Figure 4a). Our results show the specific expression pattern of NlWOX1 in the carpel (Figure 4a), while the orthologous NnWOX6b was highly expressed in the carpel and cotyledon [34], suggesting the expression divergence of orthologous gene pairs between the two lotus species. NlWOX3 was specifically expressed in the apical meristem (Figure 4a), but the syntenic NnWOX3a had a wider tissue expression pattern. This highlights the potential role of NlWOX3 in the development of the apical meristem, which is associated with the asexual propagation of rhizomes in N. lutea. NlWOX4 is not only highly expressed in the apical meristem and carpel, like its orthologous gene NnWOX4, but also in the leaf, receptacle, and petal, indicating that the evolution of the tissue expression pattern might result in neofunctionalization between orthologous gene pairs. The NnWOX5a/5b were root-specifically expressed, but their orthologous NlWOX5b/5a were silent (FPKM < 1) in N. lutea tissues (Figure 4a). We speculate that NlWOX5a/5b lost its biological function or was downregulated by post-transcriptional regulatory mechanisms. We found that NlWOX13b was highly expressed in most tissue samples but is downregulated in the cotyledon at 12 and 15 days after pollination. Interestingly, the paralog NlWOX13b is only highly expressed in certain tissues, including the apical meristem, rhizome node, seed coat, radicle, internode, and petiole. This suggests that the complementary tissue expression patterns of these two duplicated genes might result from the biological function redundancy and subfunctionalization after duplication. Compared to the widely high expression levels of NnWOX13a across different tissues [34], the orthologous NlWOX13a was specifically expressed in certain tissues, indicating that the syntenic gene pairs underwent different functional divergences. We also found the conserved tissue expression patterns of NlWOX9 and NnWOX9b; therefore, these two orthologous genes from the AC subfamily continued the same biological function during evolution.
Since the transcription of the gene is activated by the trans-acting factors that bind the cis-regulatory elements, we further predicted the cis-regulatory elements in the promoter regions of NlWOX and NnWOX genes (Figure 4b). We found that cis-regulatory elements were different between the duplicated genes in both N. nucifera and N. lutea, suggesting that genetic variations in the promoter regions might result in the expression divergence of paralogous genes (Figure 4b). Similarly, both the type and number of cis-regulatory elements between the promoter regions of orthologous gene pairs were distinct (Figure 4b). Our results indicate that the tissue expression patterns of WOX genes are highly associated with the cis-regulatory elements that affect the transcriptional activity.

2.6. qRT-PCR Experiments of NlWOX Genes

To verify the tissue expression patterns of the NlWOX genes, we first collected five N. lutea tissue samples. According to the expression matrix of NlWOX genes, we selected six expressed genes in the collected tissues to perform the qRT-PCR experiments. Our results suggest that NlWOX1, NlWOX3, and NlWOX4 showed significantly (ANOVA test, p-value < 0.01) higher relative expression levels in the apical meristem than in other tissues (Figure 5a–c). NlWOX11 and NlWOX13b have significantly higher relative expression levels in the root (Figure 5d,f). The duplicated NlWOX13a showed higher relative expression levels in the leaf and petiole, which is different from its duplicates (Figure 5e,f). This indicates the expression divergences between duplicates. In addition, the qRT-PCR results were consistent with the RNA-seq sequencing, demonstrating the accuracy of our results.

2.7. Co-Expressed Relationships of NlWOX Genes

Transcription factors can regulate multiple downstream genes that play important roles in plant growth and development. Gene co-expression network analysis is an efficient method to identify gene clusters in the same biological pathway. Given the fact that the weight gene co-expression network analysis (WGCNA) pipeline is widely performed to construct the gene co-expression networks, we first filtered out the silent genes from the gene expression matrix in N. lutea. A total of 31,264 expressed genes were input into the WGCNA pipeline and aggregated into fourteen color modules (Figure S3). Based on the WGCNA results, these gene modules were significantly (p-value < 0.01) related to unique tissues (Figure S3). Only seven NlWOX genes were clustered into four color modules, including NlWOX1/3/4 (MEturquois), NlWOX9/11 (MEpink), NlWOX13a (MEgrey), and NlWOX13b (MEgrey60) (Figure 6a, Table S1). The MEturquois is significantly associated with the carpel, MEpink is significantly associated with the cotyledon, MEgrey is significantly associated with the rhizome node, and MEgrey60 is significantly associated with the root (Figure S3).
Our previous study constructed the co-expression networks for NnWOX genes [34], and we further compared the tissue bias of syntenic WOX gene pairs (Table S2). Due to the similar expression patterns of NnWOX3a/4a/6b, these three NnWOX genes from the WC subfamily were identified in one gene module that is significantly related to the cotyledon and apical meristem, whereas their syntenic NlWOX1/3/4 genes are clustered into the carpel-specific MEturquois, suggesting that the syntenic genes might be involved in different tissue developments by constructing distinct co-expression networks (Figure 6a). Similarly, NnWOX13a was in the gene module associated with the seed coat, but its syntenic NlWOX13a was significantly related to the rhizome node. However, the syntenic gene pair NnWOX9b-NlWOX9 is significant in the same tissue, the cotyledon, indicating its conserved expression pattern and co-expression relationship. Furthermore, a GO enrichment analysis of the co-expressed genes for the NlWOXs was performed (Figure 6b). The top three significantly enriched GO terms in the biological process are “DNA metabolic process”, “cell cycle”, and “cellular component organization”. Notably, the most enriched GO terms between the co-expressed genes for NnWOXs and NlWOXs were distinct, suggesting the WOX genes participated in different biological pathways between the two lotus species.

3. Discussion

Plant genome assemblies have facilitated the study of gene family evolution, encompassing expansion or shrinking across various species. The WOX gene family is a highly conserved and plant-specific transcription factor family that plays a pivotal role in governing tissue development and responses to abiotic stresses [40,41,42,43]. Recognizing the significance of the WOX family, our previous study carried out genome-wide analysis in N. nucifera, resulting in the identification of 15 NnWOX genes. Aimed at delving deeper into the evolution of WOX in Nelumbo, a total of 11 NlWOX genes were identified in N. lutea, the only sister species to N. nucifera. Bioinformatic assessments of the NlWOX genes, including phylogenetic trees, conserved motifs, the physicochemical properties of proteins, protein structures, cis-regulatory elements, and tissue expression patterns, provide a comprehensive framework for molecular experiments in N. lutea.
Being one of the basal angiosperms, the lotus species underwent a single ancient whole-genome duplication (WGD) event, as evidenced in the N. nucifera genome [31]. Changes in the lotus gene family composition, whether through gains or losses, are linked to their adaptive responses to environmental shifts. The comparatively lower number of WOX genes in N. lutea, in contrast to N. nucifera, hints at distinct evolutionary trajectories of genomes in these two lotus species. In this investigation, building upon the identified NnWOXs, we adopted nomenclature for lotus WOX genes based on homologous genes from the model plant A. thaliana, diverging from the chromosome position-based naming convention in other species [44]. Despite variations in the size of the WOX gene family, all genes consistently segregate into three subfamilies [45,46].
Within the AC subfamily, the WOX genes of both lotus species underwent a whole-genome duplication event. Notably, only the gene pair Nl/NnWOX13a remains collinear, suggesting that the genomic region near Nl/NnWOX13b underwent mutations after the genome duplication. This led to its loss of collinearity. Remarkably, the lotus lacks an orthologous gene to AtWOX14, which regulates the development and cell differentiation in the vascular bundle [47,48], aligning with the hollow vascular tissue in the lotus leaf petiole utilized for gas transport [49]. In the IC subfamily, a solitary duplication event occurred in N. nucifera (NnWOX9a/9b). While the WC subfamily stands as the largest within the WOX gene family, only one duplicated gene pair was identified in N. lutea, contrasting with three pairs in N. nucifera (Figure 1). In Boehmeria nivea, five duplication events expanded the WOX gene family [50]. We posit that differences in whole-genome duplication events and various duplication types contribute to WOX gene family expansion, with functional redundancy post-genome duplication potentially leading to gene losses during evolution. Notably, WOX5a/b exhibits collinearity between the two genomes, indicating they underwent a shared ancient duplication event. Compared to N. nucifera, N. lutea has fewer duplicated WOX genes, suggesting that N. lutea may have experienced WOX gene loss after whole-genome duplication. Therefore, further comparative genomics investigations on WGD in the two lotus species are essential to clarify the relationship between WGD events and the gain or loss of WOX gene family members.
Due to the hypothetical surface of a protein consisting of the regions defined by various three-dimensional structures detected by amino acid sequences [51], the protein sequences of homologous WOX genes would have distinct folded states and functional regions. Sequence motif results indicate that WOX genes in two lotus species have two conserved motifs (Motif1 and Motif2). The AC and IC subfamily members contained unique motifs, such as Motif7 in AC and Motif4 in IC. Subfamily-specific motifs were also identified in other plants [52], suggesting the conserved motifs in the same subfamily. We found that the percentage of protein secondary structures in paralogous and syntenic genes was also different (Figure 3b), revealing the genomic variations contributing to the changes in protein structures. Notably, we predicted the three-dimensional structures for both NlWOX and NnWOX genes, highlighting the same conformation of the conserved homeodomain. The three-dimensional conformations between duplicated genes of different duplication types are different. This result indicates that all duplicated genes undergo neofunctionalization during the evolutionary process. Otherwise, they would be lost due to functional redundancy. Differentiation in the three-dimensional conformations between the syntenic gene pairs (NlWOX1-NnWOX6b, NlWOX9-NnWOX9b, and NlWOX3-NnWOX3a) has been observed, especially the very loose conformation of NlWOX3. However, a high similarity in the three-dimensional conformations between the syntenic gene pairs (NlWOX4-NnWOX4a, NlWOX5a-NnWOX5b, NlWOX5b-NnWOX5a, and NlWOX13a-NnWOX13a) has also been found. This suggests that the two genomes of the lotus have undergone variation in specific regions while also retaining some conserved collinear regions.
Transcriptional factors contribute to certain cellular pathways or morphological features in the context of downstream targeted genes [53]. However, how TF expression patterns and regulatory networks evolved during the divergence of plant species is largely unclear. We compared the tissue expression patterns and co-expression networks of syntenic WOX gene pairs. Notably, we found the significant expression divergence of syntenic genes across different tissues at different developmental stages, indicating their distinct roles in regulating the phenotypes of lotuses (Figure 4a). Recently, studies suggested that WOX13 promotes cellular reprogramming and organ regeneration involved in various plant tissues [54,55]. NnWOX13a/13b exhibited consistently high expression levels in multiple tissues. However, NlWOX13a/13b has complementary high expression patterns, i.e., NlWOX13a is highly expressed in specific tissues where NlWOX13b is lowly expressed. Our results indicate that NnWOX13a/13b might have redundant functions, whereas NlWOX13a/NlWOX13b has completed the mutually exclusive assignment of ancestor functions or subfunctionalization. Furthermore, we compared the WGCNA co-expression network of syntenic WOX genes between N. nucifera and N. lutea. Syntenic genes were clustered into tissue-specific gene modules, indicating their different regulatory networks. We hypothesize that differences in the promoter regions of these collinear genes may contribute to their tissue specificity. The enriched biological functions of co-expressed genes for NnWOXs and NlWOXs diverged, suggesting that they participated in different pathways. Therefore, significant divergences of WOX genes in both tissue expression patterns and co-expression networks contributed to their distinct roles in regulating the growth and development of tissues, along with the evolution of the lotus species.

4. Materials and Methods

4.1. Identification of WOX Genes in Nelumbo lutea

The protein database of N. lutea was downloaded from the Nelumbo Genome Database [33,35] (http://nelumbo.cngb.org/nelumbo/, accessed on 17 June 2025). Biological function annotations of these proteins were predicted using EggNOG v5.0 [56]. To identify the WOX gene family members in N. lutea, we first filtered a candidate pool according to the annotation of the WUSCHEL-related homeobox. Meanwhile, a total of fifteen AtWOX genes in the model plant Arabidopsis thaliana and nine AmtrWOX genes in the basal angiosperm Amborella trichopoda were retrieved from the Plant Transcription Factor Database (PlantTFDB, https://planttfdb.gao-lab.org/, accessed on 17 June 2025) and further mapped to the protein sequences of N. lutea using BlastP. The mapped results were filtered with a p-value < 1 × 10−5 and a mapped ratio of targeted sequence length >0.8. Previous studies indicated that WOX orthologs contained a conserved HB domain, which specifically bound DNA sequences. Consequently, the candidate NlWOX genes were mapped to genome databases, including the NCBI Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, accessed on 17 June 2025), the SMART database (http://smart.embl-heidelberg.de/, accessed on 17 June 2025), and the Pfam database (https://pfam.xfam.org/, accessed on 17 June 2025). Only mapped results that were identified to have the HB domain region, i.e., cl00084 in CDD, SM000389 in SMART, and PF00046 in Pfam, were retained for subsequent analysis. Finally, the identified NlWOX genes were named according to their orthologs in Arabidopsis thaliana, and duplicates were named alphabetically.

4.2. Chromosome Location and Physicochemical Characteristics of NlWOX Genes

Genome sequences and gene locus information of the physical location of N. lutea were downloaded from the Nelumbo Genome Database [33,35] (http://nelumbo.cngb.org/nelumbo/, accessed on 17 June 2025). Zhang et al. annotated the genome of N. lutea with multiple transcripts in the gene loci; the longest transcript sequence from each gene was used to represent the protein-coding regions. The chromosome position of NlWOX genes was drafted using TBtools-II software [57]. To understand the physical and chemical characterizations of the NlWOX proteins, we used the Proparam tool of ExPASy (http://weB.expasy.org/protparam/, accessed on 17 June 2025). In the basal feature analysis, we examined several indicators, including the count of amino acids, molecular weights, theoretical PI values, instability index, aliphatic index, and the grand average of hydropathicity. Furthermore, NlWOX sequence features were analyzed using NetNGlyc (https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/, accessed on 17 June 2025) to predict the candidate N-glycosylation sites. Protein hydrophobicity and hydrophilicity were estimated using the ProtScale tools of ExPASy (https://web.expasy.org/protscale/, accessed on 17 June 2025). In addition, the sequence motifs of NlWOX genes were analyzed using the MEME tools (http://meme-suite.org/meme/tools/meme, accessed on 17 June 2025) with a maximum of ten motifs.

4.3. Phylogenetic Analysis of NlWOXs

WOX protein sequences from N. lutea (NlWOX), N. nucifera (NnWOX), A. thaliana (AtWOX), and A. trichopoda (AmtrWOX) were obtained. We first aligned these WOX protein sequences using the muscle. For the aligned sequences, a phylogenetic neighbor-joining tree was constructed using MEGA v7.0, with the parameters “pairwise deletion” and “1000 replicates in bootstrap” [58]. To improve the readability of the phylogenetic tree, iTOL online tools (https://itol.embl.de/, accessed on 17 June 2025) were used to add legends and colors.

4.4. Interspecies and Intraspecies Synteny Analyses

As N. lutea is the only sister species of N. nucifera, we further studied the interspecific synteny of orthologs. The genome sequences and location of genes of N. nucifera were also downloaded from the Nelumbo Genome Database. The protein sequences of N. nucifera and N. lutea were, respectively, extracted according to their gene annotation, and we aligned these proteins using BLASTP with a maximum of six mapped results. The synteny of orthologs between two lotuses was analyzed using MCSanX [59]. We filtered the synteny of WOX genes and displayed them with a dual synteny plot using TBtools software [57]. Then, MCSanX was also used to identify the synteny block regions on the N. lutea genome. According to the chromosomal distance between duplicated gene pairs, the duplicated types of genes were classified into five groups: singletons, WGD/segmental duplications, dispersed duplications, proximal duplications, and tandem duplications. Further, we identified the duplicated type of NlWOX genes and showed them in a circus plot using the RCircos package [60].

4.5. GO Enrichment Analysis

Based on the functional annotations of N. lutea, the GO enrichment analysis of syntenic genes of NlWOXs was carried out using TBtools [57]. The enriched GO terms, i.e., p-values < 0.01, are shown in a bar chart.

4.6. Tissue Expression Profiles of NlWOXs

To explore the expression divergences of NlWOX genes among multiple tissues, the RNA-seq database of 18 tissue samples in N. lutea was downloaded from the NCBI SRA dataset under accession number PRJNA705058. To remove the adapter and low-quality reads, the raw reads were filtered using Trimmomatic [61]. Clean reads were mapped to the N. lutea reference genome using Hisat2 [62]. Based on the gene loci annotation, the gene expression levels were estimated using the FPKM (fragments per kilobase of exon model per million mapped fragments) value for each tissue sample using StringTie [63]. The tissue expression patterns of NlWOXs were extracted from the gene expression profiles. The circlize v0.4.16 R package was used to generate the heatmap circle of NlWOX gene expression. The gene expression profile of 54 tissue RNA-seq samples in N. nucifrea was downloaded from the Nelumbo Genome Database.

4.7. Quantitative Real-Time PCR Experiments

The N. lutea plants were cultivated in the Wuhan Botanical Garden. The apical meristem, internode, leaf, petiole, and root tissues were collected. Total RNA was extracted from each tissue using an RNAprep Plant Kit, and high-quality RNA was reverse-transcribed into cDNA. Based on the expression pattern of NlWOX genes from RNA-seq, we filtered six expressed NlWOX genes (i.e., NlWOX1, NlWOX3, NlWOX4, NlWOX11, NlWOX13a, and NlWOX13b) in the collected tissues. The specific primers designed for these six genes are shown in Table S3. qRT-PCR experiments were conducted on the six genes under the following conditions: 95 °C for 30 s; 40 cycles of 95 °C for 5 s; 60 °C for 30 s; and 72 °C for 15 s; and 95 °C for 10 s. The relative expression levels were calculated using the 2−ΔΔCt method.

4.8. Comparative Co-Expression Gene Network Analysis

To study the evolution of co-expressed genes in collinear WOX genes in Nelumbo, the gene co-expression networks for N. nucifera and N. lutea were constructed. Briefly, genes that show low expression levels (an average of FPKM in the tissue expression profile of <0.1) were filtered out. Based on the pipeline of weighted gene co-expression network analysis (WGCNA) R-package, we built the WGCNA networks with a min-module of 500 genes using the tissue expression profile in N. nucifera and N. lutea. The correlations between gene modules and tissues were estimated; the modules that showed a significant (p-values < 0.001) correlation to one tissue were defined as tissue-specific modules. We defined the co-expressed genes as the top 5% of genes based on their weight proportions in the co-expression network. The NlWOX co-expression networks were extracted and visualized using Cytoscape v3.10 [64].

5. Conclusions

This study focuses on the evolution of the WOX family in Nelumbo. A total of 11 NlWOX genes were identified in N. lutea and were classified into three subfamilies. Phylogenetic analysis reveals the evolutionary relationships of lotus WOX members with basal angiosperms and model plants. Gene duplication and synteny analyses show that whole-genome duplications affected the distribution and evolution of these genes. Conserved motif and protein conformation analyses indicate variations between the two lotus species and among different subfamilies. In terms of tissue expression patterns, NlWOX genes exhibited significant divergence compared to their orthologs in N. nucifera, in line with their distinct cis-regulatory elements in promoters. Moreover, the co-expression networks of NlWOX genes were distinct from those of NnWOX genes, suggesting different regulatory roles in tissue development. Overall, our research provides valuable insights into the evolution and function of the WOX family in Nelumbo, highlighting the importance of genomic variations in gene expression and regulatory networks during the evolution of these two lotus species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14131909/s1, Figure S1: N-glycosylation analysis of NlWOX genes; Figure S2: The hydrophily and hydrophobicity of NlWOX proteins; Figure S3: A heatmap shows the correlations between WGCNA gene modules and tissues; Table S1: A summary of the coexpressed genes for NlWOXs; Table S2: A summary of the WGCNA gene module for syntenic WOX genes in Nelumbo; Table S3. A summary of the specific primers in this study.

Author Contributions

Formal analysis, methodology, writing—original draft preparation, J.L.; conceptualization, writing—review and editing, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Department of Education of Hubei Province Project (Q20202702) and the Outstanding Young and Middle-aged Science and Technology Innovation Team Project in the Colleges and Universities of Hubei Province (T2022030). This work was supported by Hubei Province Natural Science Foundation of China (no. JCZRYB202501075).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Plants 14 01909 g001
Figure 1. Phylogenetic tree of WOX protein in N. lutea, N. nucifera, A. thaliana, and A. trichopoda. Three subfamily clades were identified: ancient clade (AC, purple), intermediate clade (IC, green), and WUS clade (WC, blue).
Figure 1. Phylogenetic tree of WOX protein in N. lutea, N. nucifera, A. thaliana, and A. trichopoda. Three subfamily clades were identified: ancient clade (AC, purple), intermediate clade (IC, green), and WUS clade (WC, blue).
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Figure 2. The genome distribution and collinearity relationship of NlWOX genes. (a) The chromosome location of NlWOX genes. The left axis stands for the length of the chromosome. (b) Whole-genome duplications of genes in the N. lutea genome. The blue lines are the duplicated NlWOX genes in the collinearity block. (c) The interspecies collinearity gene pairs between N. lutea and N. nucifera. The WOX genes are highlighted as purple lines.
Figure 2. The genome distribution and collinearity relationship of NlWOX genes. (a) The chromosome location of NlWOX genes. The left axis stands for the length of the chromosome. (b) Whole-genome duplications of genes in the N. lutea genome. The blue lines are the duplicated NlWOX genes in the collinearity block. (c) The interspecies collinearity gene pairs between N. lutea and N. nucifera. The WOX genes are highlighted as purple lines.
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Figure 3. The conserved motif and protein sequence structures of WOX in Nelumbo. (a) The conserved motifs of NlWOX and NnWOX genes were identified and are listed according to the phylogenetic tree on the left. (b) The percentage of protein secondary structures in NlWOX and NnWOX genes. (c) The three-dimensional conformations of homologous WOX genes were predicted. The green highlights are the conserved homeodomains.
Figure 3. The conserved motif and protein sequence structures of WOX in Nelumbo. (a) The conserved motifs of NlWOX and NnWOX genes were identified and are listed according to the phylogenetic tree on the left. (b) The percentage of protein secondary structures in NlWOX and NnWOX genes. (c) The three-dimensional conformations of homologous WOX genes were predicted. The green highlights are the conserved homeodomains.
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Figure 4. The Tissue expression patterns of NlWOXs and cis-regulatory elements in WOX promoters. (a) A heatmap circle showing the expression level (log FPKM) of NlWOXs across tissue samples. (b) The distribution of cis-regulatory elements. Immature anther, IA; mature anther, MA; apical meristem, AM; pollinated carpel, PC; unpollinated carpel, UC; cotyledon-12d, C12d; cotyledon-15d, C15d; internode, I; leaf, L; immature receptacle, IR; mature receptacle, MR; rhizome node, RN; seed coat, SC; sepal, S.
Figure 4. The Tissue expression patterns of NlWOXs and cis-regulatory elements in WOX promoters. (a) A heatmap circle showing the expression level (log FPKM) of NlWOXs across tissue samples. (b) The distribution of cis-regulatory elements. Immature anther, IA; mature anther, MA; apical meristem, AM; pollinated carpel, PC; unpollinated carpel, UC; cotyledon-12d, C12d; cotyledon-15d, C15d; internode, I; leaf, L; immature receptacle, IR; mature receptacle, MR; rhizome node, RN; seed coat, SC; sepal, S.
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Figure 5. qRT-PCR and RNA-seq show the expression levels of NlWOX in five tissues. (a–f) Six expressed NlWOX genes were tested. The black bars show the qRT-PCR results, and the grey bars show the FPKM value. The error bars show the standard error of the mean. Significance was tested by an ANOVA test; ** means p-value < 0.01.
Figure 5. qRT-PCR and RNA-seq show the expression levels of NlWOX in five tissues. (a–f) Six expressed NlWOX genes were tested. The black bars show the qRT-PCR results, and the grey bars show the FPKM value. The error bars show the standard error of the mean. Significance was tested by an ANOVA test; ** means p-value < 0.01.
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Figure 6. The co-expression network analysis of NlWOXs. (a) The co-expression networks for each NlWOX gene. Blue squares are the NlWOX genes, and pink squares are their co-expressed genes. The color in the lines represents their weighted correlation. (b) The GO enrichment analysis of NlWOX co-expressed genes.
Figure 6. The co-expression network analysis of NlWOXs. (a) The co-expression networks for each NlWOX gene. Blue squares are the NlWOX genes, and pink squares are their co-expressed genes. The color in the lines represents their weighted correlation. (b) The GO enrichment analysis of NlWOX co-expressed genes.
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Table 1. Physicochemical properties of NlWOX proteins in Nelumbo lutea.
Table 1. Physicochemical properties of NlWOX proteins in Nelumbo lutea.
IDNameNumber of Amino AcidsMolecular WeightTheoretical PIInstability IndexAliphatic IndexGrand Average of Hydropathicity
NL1g_04069NlWOX936239,945.947.1553.1667.62−0.464
NL1g_04550NlWOX421624,502.739.4654.7763.61−0.946
NL1g_06482NlWUS27330,043.366.8363.7154.32−0.746
NL2g_10541NlWOX5a18220,712.296.9160.5767.47−0.795
NL2g_11918NlWOX225327,883.176.7150.0163.2−0.611
NL2g_11942NlWOX312414,769.8210.0181.8365.4−0.99
NL2g_12810NlWOX5b18521,006.718.742.9568.49−0.657
NL4g_22694NlWOX134539,317.676.4357.2553.45−0.904
NL5g_27723NlWOX13a26630,660.556.0858.3167.82−0.812
NL5g_28623NlWOX1128230,602.155.4275.0469.79−0.291
NL6g_29420NlWOX13b20422,930.456.0653.5768.38−0.793
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Li, J.; Zhang, Y. The Tissue Expression Divergence of the WUSCHEL-Related Homeobox Gene Family in the Evolution of Nelumbo. Plants 2025, 14, 1909. https://doi.org/10.3390/plants14131909

AMA Style

Li J, Zhang Y. The Tissue Expression Divergence of the WUSCHEL-Related Homeobox Gene Family in the Evolution of Nelumbo. Plants. 2025; 14(13):1909. https://doi.org/10.3390/plants14131909

Chicago/Turabian Style

Li, Juanjuan, and Yue Zhang. 2025. "The Tissue Expression Divergence of the WUSCHEL-Related Homeobox Gene Family in the Evolution of Nelumbo" Plants 14, no. 13: 1909. https://doi.org/10.3390/plants14131909

APA Style

Li, J., & Zhang, Y. (2025). The Tissue Expression Divergence of the WUSCHEL-Related Homeobox Gene Family in the Evolution of Nelumbo. Plants, 14(13), 1909. https://doi.org/10.3390/plants14131909

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