Next Article in Journal
The Brucella Effector Protein BspF Crotonylates TRIM38 to Inhibit NF-κB and MAPK Signaling Pathway
Previous Article in Journal
Advances in Metabolomics: A Comprehensive Review of Type 2 Diabetes and Cardiovascular Disease Interactions
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 26
Issue 8
10.3390/ijms26083571
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Identification and Characterization of WOX Gene Family in Flax (Linum usitatissimum L.) and Its Role Under Abiotic Stress

by
Xixia Song
1,†,
Jianyu Lu
2,†,
Hang Wang
2,
Lili Tang
1,
Shuyao Li
2,
Zhenyuan Zang
2,
Guangwen Wu
1,* and
Jian Zhang
2,3,*
1
Heilongjiang Academy of Agricultural Sciences, Harbing 150086, China
2
Faculty of Agronomy, Jilin Agricultural University, Changchun 130118, China
3
Department of Biology, University of British Columbia, Kelowna, BC V1CIV7, Canada
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(8), 3571; https://doi.org/10.3390/ijms26083571
Submission received: 4 March 2025 / Revised: 3 April 2025 / Accepted: 8 April 2025 / Published: 10 April 2025
(This article belongs to the Special Issue Fiber Plants: Genetics and Genomics, Molecular Mechanisms and Potential Applications—Novel Materials, Functional Foods and Beyond)
Browse Figures
Review Reports Versions Notes

Abstract

The WOX (WUSCHEL-related homeobox) gene family plays pivotal roles in plant growth, development, and responses to biotic/abiotic stresses. Flax (Linum usitatissimum L.), a globally important oilseed and fiber crop, lacks a comprehensive characterization of its WOX family. Here, 18 LuWOX genes were systematically identified in the flax genome through bioinformatics analyses. Phylogenetic classification grouped these genes into three clades: Ancient, Intermediate, and WUS Clades, with members within the same clade exhibiting conserved exon–intron structures and motif compositions. Promoter analysis revealed abundant cis-acting elements associated with hormone responses (MeJA, abscisic acid) and abiotic stress adaptation (anaerobic induction, drought, low temperature). Segmental duplication events (nine gene pairs) contributed significantly to LuWOX family expansion. Protein–protein interaction networks implicated several LuWOX proteins in stress-responsive pathways. Expression profiling demonstrated that most LuWOX genes were highly expressed in 5-day-post-anthesis (DPA) flowers and embryonic tissues. qRT-PCR validation further uncovered distinct expression patterns of LuWOX genes under cold, drought, and salt stresses. This study established a foundational framework for leveraging LuWOX genes to enhance stress tolerance in flax breeding and functional genomics.

1. Introduction

The WUSCHEL-related homeobox (WOX) gene family is a plant-specific transcription factor family. Its conserved sequence consists of a homeodomain (HD) formed by approximately 60–66 amino acids arranged in a “helix-loop-helix–turn–helix” structure. WOX transcription factors (TF) bind to specific DNA sequences via their HD to exert biological functions, and they are extensively involved in plant growth and development, the regulation of stem cell division and differentiation, and processes such as embryo and organ formation [1]. Through the phylogenetic analysis of WOX proteins across diverse plant species, the WOX gene family is classified into three clades: the WUS Clade, Intermediate Clade, and Ancient Clade [2].
The WUS gene was first discovered in Arabidopsis, which is mainly expressed in the meristematic tissue of the stem tip and plays an important role in the formation and maintenance of SAM [3]. Genetic and molecular biology studies on WOX genes have shown that WOX members play important roles in various physiological and developmental processes of plants [4,5]. Some studies have found that the WUS gene of maize and the regeneration gene BBM play a key role in the process of plant tissue culture, and the co-transformation of the two genes can significantly improve the transformation efficiency of maize, sorghum, and sugarcane [6]. The WOX1 and WOX3 genes play crucial roles in regulating lateral organ growth across diverse plant species including Arabidopsis, Cucumis sativus, and Panicum virgatum [7,8,9]. The WOX2 transcription factor performs conserved regulatory functions in precursor cell specification during zygotic embryogenesis, with phylogenetic analysis revealing that its ortholog PaWOX2 in Picea abies retains functional equivalence as a molecular marker for monitoring embryogenic competence acquisition in gymnosperm systems [10]. WOX4 can promote the differentiation of vascular cambium in Arabidopsis and tomato [11]. WOX5 is expressed in the root apical meristem (RAM) and maintains the stability of stem cells in RAM [12]. In Arabidopsis, the transcription factor AtWOX7 exhibits constitutive expression during all developmental phases of lateral root organogenesis while functionally demonstrating negative regulation of lateral root primordium formation through carbohydrate-availability-dependent modulation. Furthermore, the overexpression of AtWOX7 significantly reduces the number of lateral root primordia [13]. The WOX11 and WOX12 genes exhibit functional redundancy in regulating root system development. The overexpression of PagWOX11/12a significantly promotes root elongation and enhances biomass accumulation in Populus alba × glandulosa [14]. The WOX13 and WOX14 genes are associated with floral organ formation and development. The overexpression of OsWOX13 in rice advances the flowering time by 7–10 days [15]. The AtWOX14 transcriptional regulator mediates the developmental trajectory of vascular precursor cells in Arabidopsis inflorescence stems, acting through the coordinated activation of protoxylem/metaxylem specification programs and phenylpropanoid biosynthesis networks to drive cellular lignification during secondary cell wall deposition [16]. The evolutionarily conserved PFS2/WOX6 transcriptional regulator in Arabidopsis exhibits spatiotemporal expression specificity synchronized with megasporogenesis and integument morphogenesis, mechanistically orchestrating gametophytic phase transition through the precise modulation of ovule patterning networks [17,18]. In cucumber, the CsWOX9 gene is predominantly expressed in developing fruits, and its overexpression in Arabidopsis resulted in a shortened stem phenotype in a study [19].
The WOX gene family is implicated in dual roles: regulating plant growth and development and mediating stress response mechanisms. Specifically, AtWOX6 plays a critical role in root apical meristem maintenance and root development while also mediating cold stress responses [20]. The GmWOX gene in soybean responds to different abiotic stress treatments (cold and drought) [21]. The overexpression of the rice OsWOX13 gene can improve drought tolerance in rice [15]. Knocking out GhWOX4 in cotton significantly reduces drought tolerance in transgenic cotton [22]. The overexpression of PagWOX11/12a gene significantly enhances drought and salt tolerance in poplar trees [23]. WOX genes play roles in biotic stress; WOX11 restricts cyst nematode parasitism by modulating ROS-mediated cell wall plasticity in syncytia, independent of its known role in adventitious root-mediated tolerance. This dual functionality highlights WOX11 as a key regulator balancing plant survival and pathogen suppression [24].
Flax, a dual-purpose crop domesticated from paleobotanical origins for phloem-derived textile fibers and lipid-rich seeds, maintains contemporary agronomic significance with extensive cultivation spanning temperate latitudes to support global linseed oil production and bast fiber industries [25]. According to their ultimate use, flax plants are divided into three categories: oils, fibers, and dual-use [26]. Flaxseed is rich in essential nutrients, including lignans, dietary fiber, and high-quality protein, with alpha-linolenic acid (ALA), an omega-3 fatty acid, serving as a key component of human dietary requirements [27]. To date, no systematic studies on the WOX gene family in flax have been reported. This study employed bioinformatics approaches to comprehensively investigate flax WOX genes, including their evolutionary relationships, genomic distribution, physicochemical properties, sequence features, phylogeny, promoter cis-regulatory elements, protein interaction networks, and expression patterns. Key WOX genes were identified as responsive to low-temperature and drought stress conditions, providing a foundation for the further exploration of their biological functions in flax. This paper represents the first comprehensive report on WOX genes in flax, offering critical insights into their roles in development and abiotic stress adaptation.

2. Results

2.1. Phylogenetic Characterization and Identification of WOX Family Genes in Flax

A total of 18 WOX genes (LuWOX1–LuWOX18) were identified in the flax genome (longya10) through BLASTP alignment and PF00046 Hidden Markov Model (HMM) analysis (Table 1). The characterization of their encoded proteins revealed significant physicochemical diversity: LuWOX14 exhibited the longest sequence (432 amino acids) whereas LuWOX15 displayed the shortest (192 amino acids), with molecular weights ranging from 21.81 to 47.91 kDa. Notably, 66.67% of LuWOX proteins displayed a pI > 7, indicating a predominance of basic amino acids. Stability predictions classified LuWOX01 as the only stable protein (Instability Index: 39.55–74.39) while Aliphatic Index values (47.40–70.10) and Grand Average of Hydropathicity (GRAVY) analysis confirmed that all LuWOX proteins were hydrophobic. Subcellular localization unanimously predicted nuclear targeting, consistent with these proteins’ putative roles as transcription factors. These findings collectively provide a foundational framework for further functional studies on LuWOX genes in flax development and stress adaptation.
To elucidate the phylogenetic topology of the WOX gene family in flax, a maximum likelihood tree was inferred from aligned amino acid sequences of Arabidopsis (16), Oryza (14), and Linum (18), comprising 48 conserved WOX orthologs (Figure 1) (Table S1). Based on the established Arabidopsis WOX subfamily classification, the 18 LuWOX genes were phylogenetically categorized into three clades: the Ancient Clade, Intermediate Clade, and WUS Clade. The WUS Clade contained the majority of members (12 genes: LuWOX7–LuWOX16) while the Intermediate and Ancient Clades comprised fewer genes. Comparative analysis revealed closer evolutionary relationships between flax and Arabidopsis WOX families than those with rice, suggesting lineage-specific conservation. This phylogenomic framework advances our understanding of WOX gene evolution in flax and provides a basis for cross-species functional studies.

2.2. Analysis of Gene Structure and Conservative Motif of LuWOX

To delineate structural conservation within the flax WOX family, 10 conserved motifs (motif1–motif10) were identified in 18 LuWOX proteins using MEME suite analysis (Figure 2A,B). Phylogenetic reconstruction based on motif alignment mirrored the clade classification in Figure 1, confirming evolutionary coherence. Motif composition analysis revealed that motif1, representing the core WOX domain, was universally present across all LuWOX proteins. Notably, motif8 was detected in all members except LuWOX7 while motif2 was absent solely in LuWOX16. Subfamily-specific motif patterns were evident: within the WUS Clade, motif1, motif2, motif3, and motif8 co-occurred in all members except LuWOX7 and LuWOX16, which lacked specific motifs (Table S2). Motif lengths ranged from comprising nine to fifty amino acids, with positional variability (sites 2–18) reflecting functional diversification. These structural insights underscore the evolutionary conservation and subfunctionalization of LuWOX genes, providing a basis for probing their regulatory roles in flax development. The exon–intron structure analysis of LuWOX genes revealed that exon numbers ranged from two to four while intron numbers varied between one and three (Figure 2C). Six genes (LuWOX5, LuWOX6, LuWOX13, LuWOX14, LuWOX17, and LuWOX18) exhibited the highest structural complexity, containing four exons and three introns. Notably, genes within the same clade shared highly similar exon numbers, suggesting functional conservation across phylogenetically related members.

2.3. Chromosome Localization and Gene Reproduction Events of LuWOX Gene Family

The chromosomal localization analysis of the 18 LuWOX genes within the flax reference genome revealed their uneven distribution across 12 chromosomes (Figure 3). Chromosomes 5 and 11 harbored the highest number of genes (three each, 16.67% of the total), followed by chromosomes 2 and 8 (two genes each, 11.11%). The remaining chromosomes (1, 6, 7, 9, 10, 12, 13, and 15) each contained only one LuWOX gene (5.56% per chromosome). This distribution pattern highlights potential hotspots for gene duplication or evolutionary divergence in flax.
The gene duplication analysis of the LuWOX family using BLAST 2.14.0 and MCScanX revealed no tandem duplication events but identified nine segmentally duplicated LuWOX gene pairs (Figure 4A), suggesting that whole-genome duplication likely drove the expansion of this family. We also found that all collinear gene pairs of LuWOX had Ka/Ks ratios of less than 1, indicating that they had evolved under purifying selection (Table 2). Comparative synteny analysis with Arabidopsis, maize, and rice further uncovered ten, ten, and six collinear gene pairs, respectively (Figure 4B). Specifically, Arabidopsis chromosomes 2, 3, and 5 exhibited collinearity with flax chromosomes 1, 2, 7, 8, 9, 12, and 15; maize chromosomes 3, 6, and 8 aligned with flax chromosomes 2, 5, 8, 11, and 15; and rice WOX genes showed synteny with flax chromosomes—2, 5, 8, 11, and 15. While conserved collinear regions were observed across species, lineage-specific variations in gene pair distribution underscored the dynamic evolutionary trajectory of the LuWOX family. These findings highlight the role of WGD and divergent selection in shaping the structural and functional diversification of WOX genes in flax.

2.4. Analysis of Cis-Acting Elements

To elucidate the regulatory mechanisms of LuWOX genes under abiotic stress, promoter regions (2000 bp upstream) of all 18 LuWOX genes were analyzed, excluding common elements like TATA-box and CAAT-box. A total of 461 cis-acting elements were predicted and categorized into four classes: light-responsive (180 elements, 39.05%), hormone-responsive (132 elements, 28.63%), stress-related (102 elements, 22.13%), and development-related (46 elements, 9.98%) (Figure 5A–C; Table S3). Light-responsive cis-elements predominated, featuring Box 4, G-box, and TCT-motif. Hormone-regulatory elements included MeJA-responsive (TGACG/CGTCA), ABA-associated ABRE, and GA-related motifs (P-box/GARE/TATC), as well as auxin-responsive (TGA-element) and salicylic-acid-responsive (TCA-element) elements, with MeJA-related motifs being the most abundant. Stress-related elements included anaerobic induction (ARE), drought-inducible MYB-binding (MBS), CCAAT-box, TC-rich repeat, and low-temperature-responsive (LTR) elements. Development-related elements featured meristem expression (CAT-box), zein metabolism regulation (O2-site), protein-binding (AT-rich element, HD-Zip 3), and circadian rhythm (MSA-like) motifs. These findings suggest that LuWOX genes are intricately regulated by environmental cues, hormonal signals, and developmental programs, positioning them as key players in flax stress adaptation and growth regulation.

2.5. LuWOX Interactome Network and Gene Ontology Enrichment Profiling

To unravel the biological functions and regulatory networks of the LuWOX family, a protein–protein interaction (PPI) network was constructed using Arabidopsis WOX homologs as reference (Figure 6A). The analysis identified eight LuWOX genes interacting with seven Arabidopsis functional genes: AP2 (involved in dehydration and cold-induced gene expression), LFY, RPL (linked to disease resistance), TFL2, UFO, SEU, and SUP (associated with meristem differentiation and floral development). Within the LuWOX family, only two interaction pairs were detected: LuWOX11–LuWOX5 and LuWOX11–LuWOX17. GO enrichment analysis further revealed that LuWOX genes are predominantly enriched in biological processes such as cell population proliferation, meristem development, anatomical structure formation during morphogenesis, leaf/phyllome development, and embryo development (Figure 6B). These results implicate LuWOX genes in critical developmental pathways and stress adaptation, offering mechanistic insights into their regulatory roles in flax growth and environmental responses.

2.6. Analysis of LuWOX Gene Expression Pattern Based on RNA-Seq Data

To delineate the expression dynamics of the LuWOX gene family in flax development, RNA-seq data were analyzed across diverse tissues (Figure 7). Tissue-specific expression patterns were observed, with 50% of LuWOX genes showing high expression in flowers at 5 days post anthesis (DPA) and 44.44% in flowers at 10 DPA (Figure 7A). Notably, LuWOX05 and LuWOX11 remained highly expressed in flowers at 30 DPA whereas all LuWOX genes exhibited low expression in floral tissues at 20 DPA. These temporally regulated expression profiles suggest the stage-specific functional diversification of LuWOX genes during floral development, potentially linked to their roles in late-phase reproductive processes.
To explore the potential roles of LuWOX genes in abiotic stress responses, their expression profiles under salt and heat stress were analyzed using publicly available transcriptomic data (Figure 7B). Heat stress induced no significant changes in most LuWOX genes, except for LuWOX15, which showed marked upregulation in stem tissues. Conversely, salt stress globally suppressed LuWOX expression compared to untreated controls, with tissue-specific exceptions: LuWOX03, LuWOX11, and LuWOX17 were significantly upregulated in roots while LuWOX02 exhibited enhanced expression in leaves. Strikingly, salt responsiveness varied across tissues: seven genes (LuWOX01, LuWOX04, LuWOX08, LuWOX09, LuWOX11, LuWOX16, LuWOX17, and LuWOX18) were downregulated in roots and five genes (LuWOX01, LuWOX04, LuWOX07, LuWOX13, and LuWOX14) were suppressed in leaves. These findings highlight the salt-sensitive nature of LuWOX genes and their tissue-specific regulatory roles, suggesting their involvement in flax stress adaptation pathways.
The expression profiling of LuWOX genes across 14 flax tissues using published transcriptomic data (Figure 7C) revealed tissue-specific expression patterns. LuWOX02 was highly expressed in torpedo embryos while LuWOX05/LuWOX17 dominated in mature embryos, LuWOX04/LuWOX05 in heart embryos, LuWOX02/LuWOX17 in cotyledon embryos, and LuWOX10/LuWOX11 in anthers. Strikingly, LuWOX01 and LuWOX02 exhibited broad expression in vegetative and reproductive tissues (roots, stems, leaves, ovaries, pistils, stamens, fruits, and seeds) whereas LuWOX08 and LuWOX16 were preferentially expressed in roots and stems. The clustered expression of LuWOX genes during embryonic stages (torpedo, mature, heart, and cotyledon) strongly implies their conserved roles in embryo development and plant morphogenesis. These spatiotemporal expression dynamics underscore the functional versatility of LuWOX genes across flax developmental programs.

2.7. Expression Level of LuWOX Gene in Flax Under Abiotic Stress

To validate the roles of LuWOX genes in abiotic stress responses, qRT-PCR was performed to analyze their expression in leaf tissues under salt, cold, and drought stress at 0, 6, 12, and 24 h (Figure 8, Figure 9 and Figure 10). Under salt stress, 10 genes (LuWOX3, LuWOX5, LuWOX6, LuWOX9, LuWOX11, LuWOX13, LuWOX14, LuWOX16, LuWOX17, and LuWOX18) exhibited significant upregulation at 3 h, peaking with expression levels 3.8- to 14-fold higher than controls. Four genes (LuWOX1, LuWOX2, LuWOX7, and LuWOX8) reached peak upregulation at 12 h (14-, 11-, 6.8-, and 4.5-fold increases, respectively). Conversely, three genes (LuWOX4, LuWOX10, and LuWOX15) were downregulated, with minimal expression at 6–12 h, suggesting potential negative regulatory roles in salt adaptation. These dynamic expression profiles highlight the temporal specificity of LuWOX genes in mediating salt stress responses, offering candidate targets for improving flax salinity tolerance.
Under prolonged cold stress, the expression of LuWOX genes exhibited significant temporal dynamics (Figure 9). Fifteen genes (LuWOX2–LuWOX14, LuWOX16, and LuWOX18) showed rapid upregulation, peaking at 3 h with 6.3- to 35.6-fold increases compared to controls (6.3-, 8.3-, 8.1-, 24.2-, 19.6-, 16.8-, 28.8-, 19.6-, 14.4-, 35.6-, 5.7-, 9.4-, 8.1-, 6.7-, and 4.2-fold, respectively). LuWOX1 and LuWOX15 displayed delayed peaks at 24 h (6.4-fold) and 9 h (11.8-fold), respectively. In contrast, LuWOX17 was downregulated, reaching minimal expression at 12 h (4.2-fold reduction), suggesting its potential role in the negative feedback regulation of cold stress adaptation.
Prolonged drought stress triggered significant temporal shifts in LuWOX gene expression (Figure 10). Four genes (LuWOX3, LuWOX6, LuWOX7, and LuWOX8) were rapidly upregulated, peaking at 3 h with 2.1-, 21.6-, 21.1-, and 9.3-fold increases, respectively. In contrast, 10 genes (LuWOX1, LuWOX2, LuWOX5, LuWOX9, LuWOX11, LuWOX13, LuWOX14, LuWOX16, LuWOX17, and LuWOX18) exhibited delayed upregulation, reaching maximal expression at 24 h with 32.1-, 25.4-, 6.7-, 28.6-, 25.2-, 21.1-, 24.2-, 13.8-, 22.1-, and 7.4-fold increases, respectively, compared to non-stressed controls.

3. Discussion

The WOX genes encode transcription factors (TFs) within the homeodomain superfamily, distinguished by a conserved helix–turn–helix DNA-binding motif. These TFs are integral to orchestrating plant developmental programs and mediating adaptive responses to biotic and abiotic environmental challenges [28,29,30]. Although there have been reports on WOX in other types, there is little information about flax WOX. So far, there has been no study using genomics to identify the LuWOX gene family in flax. The flax genome harbored 18 LuWOX genes, a number significantly smaller than those reported in Camellia sinensis (43) [31] and Glycine max (33) [21]. However, this count was comparable to the numbers in Zea mays (21) [32], Paeonia suffruticosa (22) [33], and Helianthus annuus (18) [34] and slightly higher than in Sorghum bicolor (11) [32], Jatropha curcas (10) [35], Vitis vinifera (12) [36], and Cucumis sativus (11) [37]. These comparative genomic insights highlight the lineage-specific expansion and contraction dynamics of WOX gene families across plant species. Phylogenetic analysis revealed that the LuWOX genes were classified into three clades (Ancient, Intermediate, and WUS Clades) (Figure 1), a categorization consistent with evolutionary patterns observed in WOX families of other plant species [33,34,37].
Motif composition analysis demonstrated that motif1, representing the core WOX domain, was universally conserved across all LuWOX proteins (Figure 2B), a pattern consistent with findings in Melastoma dodecandrum Lour [33]. This further demonstrates that the WOX gene family is highly conserved across plant species. The exon–intron structure analysis of LuWOX genes revealed that exon numbers ranged from two to four while intron numbers varied between one and three (Figure 2C). However, substantial structural divergence was observed among most genes. These structural variations in WOX genes were likely attributed to exon/intron gain/loss mechanisms during evolution [38]. Gene duplication events played pivotal roles in the evolution of WOX genes, with duplicated members potentially acquiring novel functions that enhanced plant adaptive responses to environmental stresses [39]. In this study, the 18 LuWOX genes were unevenly distributed across 12 chromosomes in flax, with no tandem duplication events detected (Figure 3). However, nine pairs of segmentally duplicated collinear relationships were identified (Figure 4A), strongly suggesting that segmental duplication served as the primary driver for the expansion of the LuWOX gene family. This evolutionary mechanism aligns with observations in other plant species, where segmental duplication facilitates functional diversification and adaptive evolution under selective pressures, particularly in stress-responsive gene families [40]. Collinearity analysis proved to be a powerful method for analyzing the evolutionary trajectories of genes [40]. Comparative collinearity analyses with Arabidopsis, Zea mays, and Oryza sativa identified ten, ten, and six collinear gene pairs, respectively (Figure 4B). These conserved syntenic relationships strongly suggest that the homologous gene pairs likely originated from a common ancestral gene predating species divergence. Such evolutionary conservation underscores the critical role of segmental duplication and genome-wide polyploidization events in shaping the functional diversification and adaptive expansion of the WOX family across divergent plant lineages [41]. The results demonstrated that LuWOX genes are highly conserved in flax, and it was proposed that the expansion of the LuWOX family was predominantly attributed to segmental duplication events.
Cis-regulatory elements in promoters mediate and regulate gene expression. The interaction between transcription factors and promoter-binding sites plays a pivotal role in this process [42]. The analysis of WOX gene promoter regions revealed abundant cis-acting regulatory elements, including those associated with light response (G-box), stress adaptation (LTR), and hormone signaling pathways, among which MeJA-responsive motifs (CGTCA and TGACG) and ABA-responsive elements (ABRE) were particularly prominent (Figure 5). Notably, G-box, CGTCA, TGACG, and ABRE emerged as the most enriched motifs in promoter regions. Methyl jasmonate (MeJA), a central mediator of plant responses to biotic and abiotic stresses, and abscisic acid (ABA), which plays critical roles in salinity/drought adaptation, seed dormancy regulation, and developmental plasticity during growth, are likely key hormonal signals orchestrating LuWOX-mediated stress adaptation. These findings highlight the evolutionary conservation of stress-responsive cis-regulatory architectures in WOX promoters and their potential roles in integrating environmental cues with developmental programs [43,44]. These results indicate that the cis-regulatory elements of LuWOX genes are highly conserved and play critical roles in modulating stress-related hormonal pathways and biotic/abiotic stress responses.
Protein–protein interactions are not only critical for maintaining normal protein functionality but also serve as key determinants in predicting the functional diversity of proteins [45]. In this study, eight LuWOX genes were found to interact with seven functional genes from Arabidopsis, with the most prominent interaction observed with AP2 (Figure 6A). AP2, a major class of plant-specific transcription factors, not only regulates abiotic stress responses but also participates in the fine-tuned regulation of developmental processes, highlighting its dual role in coordinating stress adaptation and growth regulation. These interactions suggest conserved regulatory networks between flax and Arabidopsis, where WOX-AP2 modules may serve as critical hubs for integrating environmental and developmental signals [46]. This proves the important role of WOX genes in the plant growth and development network and the stress response network.
RNA-seq data analysis indicated that the expression of LuWOX genes varied across tissues. Most LuWOX genes showed high expression in flowers at 5 days post anthesis (DPA), and LuWOX02 was highly expressed in torpedo embryos, LuWOX05/LuWOX17 in mature embryos, LuWOX04/LuWOX05 in heart embryos, LuWOX02/LuWOX16 in cotyledon embryos, and LuWOX10/LuWOX11 in anthers (Figure 7). Previous studies demonstrated that AtrWUS in Arabidopsis plays a key role in stem cell formation and promotes the transition to embryogenesis [47]. The WOX genes drive embryonic pattern formation by regulating cell fate determination, orchestrating signaling responses, and establishing stem cell niches, underscoring their central role in plant developmental biology [48]. In cotton, the majority of WOX genes were actively expressed during somatic embryogenesis, highlighting their potential roles in regulating cell reprogramming and embryonic patterning [49]. In wheat (Triticum aestivum), the overexpression of TaWOX5 significantly enhanced the transformation and regeneration efficiency of immature embryos while causing no inhibitory effects on shoot differentiation or root development, thereby overcoming the genotype dependency inherent in conventional transformation methods [50]. These results demonstrate that WOX genes play crucial regulatory roles in plant growth and development, with certain members exhibiting distinct functional specializations in specific tissues. Collectively, the tissue-specific expression profiling in flax provided deeper mechanistic insights into the functional diversification of LuWOX genes, advancing our understanding of their roles in orchestrating developmental programs and stress adaptation. The WOX genes, constituting a class of multifunctional regulators widely conserved across plant species, play pivotal roles not only in growth and development but also in stress adaptation. Expression profiling under salt stress revealed that LuWOX03, LuWOX11, and LuWOX17 were significantly upregulated in root tissues while LuWOX02 exhibited enhanced expression in leaves compared to controls (Figure 7). qRT-PCR validation further confirmed that 10 LuWOX genes (LuWOX3, LuWOX5, LuWOX6, LuWOX9, LuWOX11, LuWOX13, LuWOX14, LuWOX16, LuWOX17, and LuWOX18) showed rapid upregulation at 3 h post salt treatment (Figure 8). Under cold stress, 15 genes (LuWOX2–LuWOX14, LuWOX16, and LuWOX18) reached peak expression at 3 h, whereas LuWOX1 and LuWOX15 were maximally induced at 24 h and 9 h, respectively (Figure 9). Similarly, drought stress triggered the transient upregulation of four genes (LuWOX3 and LuWOX6–LuWOX8) at 3 h and delayed the activation of 10 genes (LuWOX1, LuWOX2, LuWOX5, LuWOX9, LuWOX11, LuWOX13, LuWOX14, and LuWOX16–LuWOX18) at 24 h in leaf tissues (Figure 10). These findings underscore the stress-specific temporal dynamics of LuWOX genes, highlighting their roles in mediating early and late responses to abiotic stresses. The distinct expression patterns suggest functional diversification among LuWOX members, enabling flax to fine-tune stress adaptation mechanisms across tissues and developmental stages. A previous study demonstrated that the ectopic expression of JcWOX5 in rice enhanced drought sensitivity, suggesting a potential role of WOX genes in modulating stress tolerance pathways across divergent plant species [51]. PagWOX11/12a was strongly induced in roots under drought and salt stress conditions, and the overexpression of PagWOX genes in poplar significantly enhanced salt and drought tolerance [14]. MdWOX13-1 directly transcriptionally regulated the expression of the reactive oxygen species (ROS) scavenging enzyme MdMnSOD, significantly enhanced the cellular ROS scavenging capacity, and improved drought stress adaptation in plants [52]. These findings provided robust evidence that WOX genes play crucial roles in abiotic stress responses in flax.

4. Materials and Methods

4.1. Plant Material

An experiment was conducted with flax variety “longya10” [53]. First we disinfected the seeds with 75% ethanol for 10 min, then washed them with sterile water and finally transplanted them into the soil. The temperature and lighting duration of the incubator were set to 26/18 °C and 16/8 h. Stress treatments were carried out when flax seedlings grew to 6–7 cm. For the drought and salt treatment groups, we extracted the plants from the soil, thoroughly rinsed them with clean water, and placed them into conical flasks containing 10% polyethylene glycol and 100 mM sodium chloride solutions, respectively. The control group was placed in clean water. Low-temperature treatment (4 °C) was applied with synchronized sampling at 0, 6, 12, and 24 h post-treatment. Triplicate biological replicates were collected concurrently across treatment groups to eliminate circadian rhythm interference. All flax specimens were flash-frozen in liquid nitrogen and cryopreserved at −80 °C.

4.2. Identification of WOX Gene in Flax

The whole flax genome sequence was downloaded from NCBI (entry number QMEI02000000, accessed on 3 October 2024) and the genome annotation was downloaded from Figshare (https://figshare.com/articles/dataset/Annotation_files_for_Longya-10_genome/13614311, accessed on 3 October 2024) [54]. We obtained Arabidopsis WOX protein sequence from Arabidopsis information resource library (https://www.arabidopsis.org/, accessed on 3 October 2024) [54]. Using Arabidopsis WOX protein sequence as query sequence, BLASTp search was performed on all protein sequences of flax (e value was set to 1 × 10−10) [55]. The Hidden Markov Models (PF00046) were obtained from the Pfam database (http://pfam.xfam.org/, accessed on 3 October 2024) [56]. Flax WOX genes were also predicted by hmmsearch program of HMMER3.0 software [57]. The LuWOX genes were filtered and corrected using CDD (https://www.ncbi.nlm.nih.gov/cdd/, accessed on 4 October 2024). A total of 18 LuWOX genes were systematically identified. Computational characterization of their physicochemical parameters—including coding sequence (CDS) length, amino acid residue count, molecular mass (Da), theoretical isoelectric point (pI), and Grand Average of Hydropathicity (GRAVY)—was performed using the ExPASy ProtParam platform (https://web.expasy.org/protparam/; accessed on 4 October 2024) [58]. The WoLF PSORT net station was used to carry out the subcellular location pre-test (https://wolfpsort.hgc.jp/, accessed on 4 October 2024).

4.3. Phylogeny, Chromosome Location, Conserved Domain, and Conserved Motif of LuWOX Gene

WOX protein sequences of Arabidopsis were retrieved from The Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/; accessed 10 October 2024) while the complete proteome of Oryza sativa was acquired from Phytozome v13 (https://phytozome-next.jgi.doe.gov/; accessed 10 October 2024) [58]. ClustalW in MEGA (version 11) uses default parameters to compare the WOX amino acid sequences of Arabidopsis, rice, and flax. Based on the maximum likelihood method (ML), a phylogenetic tree was constructed using MEGA version 11 and default configuration (neighborhood connection; parameter: Bootstrap1000) [59]. The LuWOX gene was located on the chromosome by flax genomic FASTA file and gff3 annotation file. The CD-search website in NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 10 October 2024) predicted its conservative structural region. Using MEME (https://meme-suite.org/meme/tools/meme, accessed on 10 October 2024), motif analysis of LuWOX amino acid motifs [60] and visualization using TBtools (version 2.069) were completed [61].

4.4. Genome-Wide Replication and Collinear Analysis of LuWOX Gene

We downloaded the genome and annotation files of Arabidopsis from the public-database TAIR website (https://www.arabidopsis.org/, accessed on 10 October 2024). Multiple collinear scanning (MCScanX) toolkits were used to predict collinear relationships [62]. The repetitive LuWOX gene was identified as genome-wide repeat (WGD). Tandem repeat genes were two or more homologous genes on chromosomes where the distance was not more than 100 kb and there were no other genes. The fragment repeat gene (Score < 1 × 10−5) was detected by nucleotide BLAST 2.14.0 (BLASTN), which contained a range of 100 kb (upstream and downstream 50 kb) around the coding sequence (CDS). The criteria for identifying repetitive genes included a sequence alignment length of at least 200 bp and a sequence similarity greater than 85% [63].

4.5. Cis-Acting Element Analysis

The genomic sequences upstream (2000 bp) of all LuWOX genes were extracted using TBtools software (version 2.069). Cis-regulatory elements were predicted via the PlantCARE online platform (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 15 October 2024) [64], and the results were visualized using TBtools (version 2.069).

4.6. Construction of Protein Interaction Network and GO Enrichment Analysis

Protein–protein interaction (PPI) network analysis of flax WOX proteins and Arabidopsis stress-responsive genes was performed using the STRING database (https://string-db.org/, accessed on 21 October 2024). The interaction networks were visualized with Cytoscape software (v3.9.1). For GO enrichment analysis of LuWOX genes, the GO-base.ob file was retrieved via TBtools (version 2.069), and eggNOG-mapper (http://eggnog-mapper.embl.de/, accessed on 21 October 2024) [65] was employed to annotate flax genomic protein sequences. Final visualization of GO terms was conducted using TBtools (version 2.069).

4.7. Analysis of Expression Pattern of LuWOX Gene Family

This study sequenced the transcriptomes of five flax species: (I) pistil, stamen, fruit, and stem tip tissues (PRJNA1002756) (https://www.ncbi.nlm.nih.gov/sra/?term=, accessed on 25 October 2024); (ii) flower tissue at 30, 20, 10, and 5 days after flowering (PRJNA833557); (iii) different flax embryo tissues, anthers, and seed tissues (PRJNA663265); (iv) root and leaf tissues after salt stress (PRJNA977728); and (v) stem tissue after heat stress (PRJPA874329). After filtering, the data were aligned with the Longya10 reference genome, and expression quantification was performed using tidyverse [66], Rsubrad [67], limma [68], and edgeR [69] R packages. Finally, a heatmap of log2(FPKM) values was generated using TBtools (version 2.069).

4.8. RNA Extraction and Fluorescence Quantitative PCR Analysis

Total RNA isolation was performed using the SPARKeasy (AC0201) RNA extraction kit (China Shandong Sparkjade Biotechnology Co., Ltd., Shandong, China), followed by reverse transcription into complementary DNA (cDNA). Quantitative reverse-transcription PCR (qRT-PCR) assays were conducted with the TB Green™ Premix Ex Taq™ II system (TaKaRa Bio, Kyoto, Japan), employing 20 μL reaction volumes containing gene-specific primers (Table S4) designed via Primer Premier 5.0 software. Triplicate technical replicates were analyzed per biological sample, normalized against the endogenous GAPDH (glyceraldehyde-3-phosphate dehydrogenase) reference gene. Relative expression quantification was performed using the 2−ΔΔCt method.

5. Conclusions

This paper presents the first comprehensive analysis of the WOX gene family in flax. A total of 18 LuWOX genes were identified in the flax genome and phylogenetically classified into three clades (Ancient, Intermediate, and WUS Clades). Conserved exon–intron structures and motif compositions were observed among members within the same clade, suggesting functional conservation. Collinearity analysis revealed eight segmentally duplicated LuWOX gene pairs, indicating that segmental duplication drove family expansion. Protein–protein interaction networks implicated several LuWOX proteins in abiotic stress-responsive pathways. Promoter analysis demonstrated that most LuWOX genes harbor hormone- (MeJA, ABA) and abiotic stress-related cis-regulatory elements. Expression profiling and qRT-PCR validation revealed that LuWOX genes are dynamically expressed during developmental processes (5-day-post-anthesis flowers and embryos) and in response to hormonal cues and abiotic stresses (cold, drought, and salinity). These findings systematically link LuWOX gene structure, evolution, and stress responsiveness, offering actionable targets for molecular breeding. Beyond flax, the conserved roles of WOX genes in balancing development and stress adaptation could inform resilience breeding in climate-vulnerable crops, contributing to sustainable agriculture under global change.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26083571/s1.

Author Contributions

Conceptualization, X.S. and J.L.; methodology, X.S. and L.T.; validation, H.W.; writing—original draft preparation, X.S., J.L., S.L. and Z.Z.; writing—review and editing, X.S.; visualization, X.S.; supervision, G.W. and J.Z.; project administration, G.W. and J.Z.; funding acquisition, G.W. and J.Z. All authors have read and agreed to the published version of the manuscript.

Funding

We acknowledge the Jilin Agricultural University high-level researcher grant: JLAUHLRG20102006 and Jilin Provincial Department of Human Resources and Social Security Grant: No.201020012.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts 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] [PubMed]
  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] [PubMed]
  3. Laux, T.; Mayer, K.F.X.; 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]
  4. Wang, J.; Tan, M.; Wang, X.; Jia, L.; Wang, M.; Huang, A.; You, L.; Li, C.; Zhang, Y.; Zhao, Y.; et al. WUS-RELATED HOMEOBOX 14 boosts de novo plant shoot regeneration. Plant Physiol. 2023, 192, 748–752. [Google Scholar] [CrossRef]
  5. 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]
  6. Lowe, K.; Wu, E.; Wang, N.; Hoerster, G.; Hastings, C.; Cho, M.-J.; Scelonge, C.; Lenderts, B.; Chamberlin, M.; Cushatt, J.; et al. Morphogenic Regulators Baby Boom and Wuschel Improve Monocot Transformation. Plant Cell 2016, 28, 1998–2015. [Google Scholar] [CrossRef]
  7. Nakata, M.; Okada, K. The three-domain model. Plant Signal. Behav. 2012, 7, 1423–1427. [Google Scholar] [CrossRef]
  8. 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]
  9. Yang, R.; Wu, Z.; Bai, C.; Sun, Z.; Wang, M.; Huo, Y.; Zhang, H.; Wang, Y.; Zhou, H.; Dai, S.; et al. Overexpression of PvWOX3a in switchgrass promotes stem development and increases plant height. Hortic. Res. 2021, 8, 252. [Google Scholar] [CrossRef]
  10. Palovaara, J.; Hakman, I. Conifer WOX-related homeodomain transcription factors, developmental consideration and expression dynamic of WOX2 during Picea abies somatic embryogenesis. Plant Mol. Biol. 2008, 66, 533–549. [Google Scholar] [CrossRef]
  11. Ji, J.; Strable, J.; Shimizu, R.; Koenig, D.; Sinha, N.; Scanlon, M.J. WOX4 promotes procambial development. Plant Physiol. 2010, 152, 1346–1356. [Google Scholar] [CrossRef] [PubMed]
  12. Sarkar, A.K.; Luijten, M.; Miyashima, S.; Lenhard, M.; Hashimoto, T.; Nakajima, K.; Scheres, B.; Heidstra, R.; Laux, T. Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature 2007, 446, 811–814. [Google Scholar] [CrossRef] [PubMed]
  13. Kong, D.; Hao, Y.; Cui, H. The WUSCHEL Related Homeobox Protein WOX7 Regulates the Sugar Response of Lateral Root Development in Arabidopsis thaliana. Mol. Plant 2016, 9, 261–270. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, L.-Q.; Li, Z.; Wen, S.-S.; Wang, J.-N.; Zhao, S.-T.; Lu, M.-Z. WUSCHEL-related homeobox gene PagWOX11/12a responds to drought stress by enhancing root elongation and biomass growth in poplar. J. Exp. Bot. 2020, 71, 1503–1513. [Google Scholar] [CrossRef]
  15. 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]
  16. Denis, E.; Kbiri, N.; Mary, V.; Claisse, G.; e Silva, N.C.; Kreis, M.; Deveaux, Y. WOX14 promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis. Plant J. 2017, 90, 560–572. [Google Scholar] [CrossRef]
  17. Park, S.O.; Zheng, Z.; Oppenheimer, D.G.; Hauser, B.A. The PRETTY FEW SEEDS2 gene encodes an Arabidopsis homeodomain protein that regulates ovule development. Development 2005, 132, 841–849. [Google Scholar] [CrossRef]
  18. Park, S.O.; Hwang, S.; Hauser, B.A. The phenotype of Arabidopsis ovule mutants mimics the morphology of primitive seed plants. R. Soc. Flagship Biol. Res. J. 2004, 271, 311–316. [Google Scholar] [CrossRef]
  19. 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]
  20. Zhu, J.; Shi, H.; Lee, B.H.; Damsz, B.; Cheng, S.; Stirm, V.; Zhu, J.K.; Hasegawa, P.M.; Bressan, R.A. An Arabidopsis homeodomain transcription factor gene, HOS9, mediates cold tolerance through a CBF-independent pathway. Proc. Natl. Acad. Sci. USA 2004, 101, 9873–9878. [Google Scholar] [CrossRef]
  21. Hao, Q.; Zhang, L.; Yang, Y.; Shan, Z.; Zhou, X.-A. Genome-Wide Analysis of the WOX Gene Family and Function Exploration of GmWOX18 in Soybean. Plants 2019, 8, 215. [Google Scholar] [CrossRef] [PubMed]
  22. Sajjad, M.; Wei, X.; Liu, L.; Li, F.; Ge, X. Transcriptome Analysis Revealed GhWOX4 Intercedes Myriad Regulatory Pathways to Modulate Drought Tolerance and Vascular Growth in Cotton. Int. J. Mol. Sci. 2021, 22, 898. [Google Scholar] [CrossRef] [PubMed]
  23. Wang, L.; Wen, S.; Wang, R.; Wang, C.; Gao, B.; Lu, M. PagWOX11/12a activates PagCYP736A12 gene that facilitates salt tolerance in poplar. Plant Biotechnol. J. 2021, 19, 2249–2260. [Google Scholar] [CrossRef] [PubMed]
  24. Guarneri, N.; Willig, J.; Willemsen, V.; Goverse, A.; Sterken, M.G.; Nibbering, P.; Torres, J.L.L.; Smant, G. WOX11-mediated cell size control in Arabidopsis attenuates growth and fecundity of endoparasitic cyst nematodes. Plant J. 2024, 120, 540–551. [Google Scholar] [CrossRef]
  25. Huis, R.; Hawkins, S.; Neutelings, G. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol. 2010, 10, 71. [Google Scholar] [CrossRef]
  26. Chytilová, M.; Mudroňová, D.; Nemcová, R.; Gancarčíková, S.; Buleca, V.; Koščová, J.; Tkáčiková, Ľ. Anti-inflammatory and immunoregulatory effects of flax-seed oil and Lactobacillus plantarum—Biocenol LP96 in gnotobiotic pigs challenged with enterotoxigenic Escherichia coli. Res. Vet. Sci. 2013, 95, 103–109. [Google Scholar] [CrossRef]
  27. Santos, H.O.; Price, J.C.; Bueno, A.A. Beyond Fish Oil Supplementation: The Effects of Alternative Plant Sources of Omega-3 Polyunsaturated Fatty Acids upon Lipid Indexes and Cardiometabolic Biomarkers-An Overview. Nutrients 2020, 12, 3159. [Google Scholar] [CrossRef]
  28. Brandt, R.; Cabedo, M.; Xie, Y.; Wenkel, S. Homeodomain leucine-zipper proteins and their role in synchronizing growth and development with the environment. J. Integr. Plant Biol. 2014, 56, 518–526. [Google Scholar] [CrossRef]
  29. Costanzo, E.; Trehin, C.; Vandenbussche, M. The role of WOX genes in flower development. Ann. Bot. 2014, 114, 1545–1553. [Google Scholar] [CrossRef]
  30. Wang, M.-M.; Liu, M.-M.; Ran, F.; Guo, P.-C.; Ke, Y.-Z.; Wu, Y.-W.; Wen, J.; Li, P.-F.; Li, J.-N.; Du, H. Global Analysis of WOX Transcription Factor Gene Family in Brassica napus Reveals Their Stress-and Hormone-Responsive Patterns. Int. J. Mol. Sci. 2018, 19, 3470. [Google Scholar] [CrossRef]
  31. Rathour, M.; Sharma, A.; Kaur, A.; Upadhyay, S.K. Genome-wide characterization and expression and co-expression analysis suggested diverse functions of WOX genes in bread wheat. Heliyon 2020, 6, e5762. [Google Scholar] [CrossRef] [PubMed]
  32. 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] [PubMed]
  33. Zheng, R.; Peng, Y.; Chen, J.; Zhu, X.; Xie, K.; Ahmad, S.; Zhao, K.; Peng, D.; Liu, Z.-J.; Zhou, Y. The Genome-Level Survey of the WOX Gene Family in Melastoma dodecandrum Lour. Int. J. Mol. Sci. 2023, 24, 17349. [Google Scholar] [CrossRef] [PubMed]
  34. Riccucci, E.; Vanni, C.; Vangelisti, A.; Fambrini, M.; Giordani, T.; Cavallini, A.; Mascagni, F.; Pugliesi, C. Genome-Wide Analysis of WOX Multigene Family in Sunflower (Helianthus annuus L.). Int. J. Mol. Sci. 2023, 24, 3352. [Google Scholar] [CrossRef]
  35. Wang, Z.; Cai, Q.; Xia, H.; Han, B.; Li, M.; Wang, Y.; Jiao, C.; Wang, D.; Zhu, J.; Yuan, W.; et al. Genome-Wide Identification and Comparative Analysis of WOX Genes in Four Euphorbiaceae Species and Their Expression Patterns in Jatropha curcas. Front. Genet. 2022, 13, 878554. [Google Scholar] [CrossRef]
  36. Gambino, G.; Minuto, M.; Boccacci, P.; Perrone, I.; Vallania, R.; Gribaudo, I. Characterization of expression dynamics of WOX homeodomain transcription factors during somatic embryogenesis in Vitis vinifera. J. Exp. Bot. 2011, 62, 1089–1101. [Google Scholar] [CrossRef]
  37. Yin, S.; Zhao, L.; Liu, J.; Sun, Y.; Li, B.; Wang, L.; Ren, Z.; Chen, C. Pan-genome Analysis of WOX Gene Family and Function Exploration of CsWOX9 in Cucumber. Int. J. Mol. Sci. 2023, 24, 17568. [Google Scholar] [CrossRef]
  38. Xu, G.; Guo, C.; Shan, H.; Kong, H. Divergence of duplicate genes in exon-intron structure. Proc. Natl. Acad. Sci. USA 2012, 109, 1187–1192. [Google Scholar] [CrossRef]
  39. Cannon, S.B.; Mitra, A.; Baumgarten, A.; Young, N.D.; May, G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004, 4, 10. [Google Scholar] [CrossRef]
  40. Jiao, Y.; Li, J.; Tang, H.; Paterson, A.H. Integrated syntenic and phylogenomic analyses reveal an ancient genome duplication in monocots. Plant Cell 2014, 26, 2792–2802. [Google Scholar] [CrossRef]
  41. Clark, J.W.; Donoghue, P. Whole-Genome Duplication and Plant Macroevolution. Trends Plant Sci. 2018, 23, 933–945. [Google Scholar] [CrossRef] [PubMed]
  42. Hernandez-Garcia, C.M.; Finer, J.J. Identification and validation of promoters and cis-acting regulatory elements. Plant Sci. 2014, 217, 109–119. [Google Scholar] [CrossRef] [PubMed]
  43. Wang, T.; Zhang, X. Genome-wide dynamic network analysis reveals the potential genes for MeJA-induced growth-to-defense transition. BMC Plant Biol. 2021, 21, 450. [Google Scholar] [CrossRef] [PubMed]
  44. Emenecker, R.J.; Strader, L.C. Auxin-Abscisic Acid Interactions in Plant Growth and Development. Biomolecules 2020, 10, 281. [Google Scholar] [CrossRef]
  45. Nobeli, I.; Favia, A.D.; Thornton, J.M. Protein promiscuity and its implications for biotechnology. Nat. Biotechnol. 2009, 27, 157–167. [Google Scholar] [CrossRef]
  46. Ma, Z.; Hu, L.; Jiang, W. Understanding AP2/ERF Transcription Factor Responses and Tolerance to Various Abiotic Stresses in Plants: A Comprehensive Review. Int. J. Mol. Sci. 2024, 25, 893. [Google Scholar] [CrossRef]
  47. Schoof, H.; Lenhard, M.; Haecker, A.; Mayer, K.F.; Jürgens, G.; Laux, T. The stem cell population of Arabidopsis shoot meristems in maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 2000, 100, 635–644. [Google Scholar] [CrossRef]
  48. 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]
  49. Sun, R.; Zhang, X.; Ma, D.; Liu, C. Identification and Evolutionary Analysis of Cotton (Gossypium hirsutum) WOX Family Genes and Their Potential Function in Somatic Embryogenesis. Int. J. Mol. Sci. 2023, 24, 11077. [Google Scholar] [CrossRef]
  50. Wang, K.; Shi, L.; Liang, X.; Zhao, P.; Wang, W.; Liu, J.; Chang, Y.; Hiei, Y.; Yanagihara, C.; Du, L.; et al. The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation. Nat. Plants 2022, 8, 110–117. [Google Scholar] [CrossRef]
  51. Tang, Y.; Li, H.; Guan, Y.; Li, S.; Xun, C.; Dong, Y.; Huo, R.; Guo, Y.; Bao, X.; Pei, E.; et al. Genome-Wide Identification of the Physic Nut WUSCHEL-Related Homeobox Gene Family and Functional Analysis of the Abiotic Stress Responsive Gene JcWOX5. Front. Genet. 2020, 11, 670. [Google Scholar] [CrossRef] [PubMed]
  52. 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] [PubMed]
  53. Zhang, J.; Qi, Y.; Wang, L.; Wang, L.; Yan, X.; Dang, Z.; Li, W.; Zhao, W.; Pei, X.; Li, X.; et al. Genomic Comparison and Population Diversity Analysis Provide Insights into the Domestication and Improvement of Flax. iScience 2020, 23, 100967. [Google Scholar] [CrossRef] [PubMed]
  54. Garcia-Hernandez, M.; Berardini, T.; Chen, G.; Crist, D.; Doyle, A.; Huala, E.; Knee, E.; Lambrecht, M.; Miller, N.; Mueller, L.A.; et al. TAIR: A resource for integrated Arabidopsis data. Funct. Integr. Genom. 2002, 2, 239–253. [Google Scholar] [CrossRef]
  55. Li, Y.C.; Lu, Y.C. BLASTP-ACC: Parallel Architecture and Hardware Accelerator Design for BLAST-Based Protein Sequence Alignment. IEEE Trans. Biomed. Circuits Syst. 2019, 13, 1771–1782. [Google Scholar] [CrossRef]
  56. Mistry, J.; Chuguransky, S.; Williams, L.; Qureshi, M.; Salazar, G.A.; Sonnhammer, E.L.L.; Tosatto, S.C.; Paladin, L.; Raj, S.; Richardson, L.J.; et al. Pfam: The protein families database in 2021. Nucleic Acids Res 2021, 49, D412–D419. [Google Scholar] [CrossRef]
  57. Potter, S.C.; Luciani, A.; Eddy, S.R.; Park, Y.; López, R.; Finn, R.D. HMMER web server: 2018 update. Nucleic Acids Res. 2018, 46, W200–W204. [Google Scholar] [CrossRef]
  58. Goodstein, D.M.; Shu, S.; Howson, R.; Neupane, R.; Hayes, R.D.; Fazo, J.; Mitros, T.; Dirks, W.; Hellsten, U.; Putnam, N.; et al. Phytozome: A comparative platform for green plant genomics. Nucleic Acids Res. 2012, 40, D1178–D1186. [Google Scholar] [CrossRef]
  59. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  60. Bailey, T.L.; Johnson, J.; Grant, C.E.; Noble, W.S. The MEME Suite. Nucleic Acids Res. 2015, 43, W39–W49. [Google Scholar] [CrossRef]
  61. Chen, C.J.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.H.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef] [PubMed]
  62. Wang, Y.; Tang, H.; DeBarry, J.D.; Tan, X.; Li, J.; Wang, X.; Lee, T.-H.; Jin, H.; Marler, B.; Guo, H.; et al. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40, e49. [Google Scholar] [CrossRef] [PubMed]
  63. Khaja, R.; MacDonald, J.R.; Zhang, J.; Scherer, S.W. Methods for identifying and mapping recent segmental and gene duplications in eukaryotic genomes. Methods Mol. Biol. 2006, 338, 9–20. [Google Scholar] [CrossRef] [PubMed]
  64. 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]
  65. Cantalapiedra, C.P.; Hernández-Plaza, A.; Letunic, I.; Bork, P.; Huerta-Cepas, J. eggNOG-mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the Metagenomic Scale. Mol. Biol. Evol. 2021, 38, 5825–5829. [Google Scholar] [CrossRef]
  66. Carpenter, C.M.; Frank, D.N.; Williamson, K.; Arbet, J.; Wagner, B.D.; Kechris, K.; Kroehl, M.E. tidyMicro: A pipeline for microbiome data analysis and visualization using the tidyverse in R. BMC Bioinform. 2021, 22, 41. [Google Scholar] [CrossRef]
  67. Liao, Y.; Smyth, G.K.; Shi, W. The R package Rsubread is easier, faster, cheaper and better for alignment and quantification of RNA sequencing reads. Nucleic Acids Res. 2019, 47, e47. [Google Scholar] [CrossRef]
  68. Ritchie, M.E.; Phipson, B.; Wu, D.; Hu, Y.; Law, C.W.; Shi, W.; Smyth, G.K. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015, 43, e47. [Google Scholar] [CrossRef]
  69. Robinson, M.D.; McCarthy, D.J.; Smyth, G.K. edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26, 139–140. [Google Scholar] [CrossRef]
Ijms 26 03571 g001
Figure 1. WOX protein phylogenetic tree of three species. The At prefix represents Arabidopsis, the Os prefix represents rice, and the Lu prefix represents flax. The WOX gene can be divided into three subfamilies, represented by different colors.
Figure 1. WOX protein phylogenetic tree of three species. The At prefix represents Arabidopsis, the Os prefix represents rice, and the Lu prefix represents flax. The WOX gene can be divided into three subfamilies, represented by different colors.
Ijms 26 03571 g001
Ijms 26 03571 g002
Figure 2. Structural and evolutionary features of LuWOX genes. (A) Maximum likelihood phylogeny of LuWOX proteins. (B) Conserved motif architecture with 10 identified motifs color-coded. Genomic sequence spans are denoted by gray connectors. (C) Exon–intron organization with coding sequences (CDS, purple), untranslated regions (UTR, orange), and intronic spacers (black).
Figure 2. Structural and evolutionary features of LuWOX genes. (A) Maximum likelihood phylogeny of LuWOX proteins. (B) Conserved motif architecture with 10 identified motifs color-coded. Genomic sequence spans are denoted by gray connectors. (C) Exon–intron organization with coding sequences (CDS, purple), untranslated regions (UTR, orange), and intronic spacers (black).
Ijms 26 03571 g002
Ijms 26 03571 g003
Figure 3. Chromosomal localization of LuWOX genes. Genomic distribution visualized using a 100 kb sliding window, with gene density depicted by a gradient color scale from red (high) to blue (low).
Figure 3. Chromosomal localization of LuWOX genes. Genomic distribution visualized using a 100 kb sliding window, with gene density depicted by a gradient color scale from red (high) to blue (low).
Ijms 26 03571 g003
Ijms 26 03571 g004
Figure 4. Syntenic relationships of LuWOX genes. (A) Intraspecific synteny of LuWOX genes in flax. (B) Interspecific synteny analysis between flax and Arabidopsis, maize, rice. Red connectors designate LuWOX orthologous pairs, gray linkages represent genome-wide syntenic blocks.
Figure 4. Syntenic relationships of LuWOX genes. (A) Intraspecific synteny of LuWOX genes in flax. (B) Interspecific synteny analysis between flax and Arabidopsis, maize, rice. Red connectors designate LuWOX orthologous pairs, gray linkages represent genome-wide syntenic blocks.
Ijms 26 03571 g004
Ijms 26 03571 g005
Figure 5. Cis-regulatory element profiling of LuWOX genes. (A) Element distribution across three LuWOX subfamilies. (B,C) Element quantification categorized by function: yellow (developmental), green (stress-responsive), dark blue (hormonal), and purple (light-related).
Figure 5. Cis-regulatory element profiling of LuWOX genes. (A) Element distribution across three LuWOX subfamilies. (B,C) Element quantification categorized by function: yellow (developmental), green (stress-responsive), dark blue (hormonal), and purple (light-related).
Ijms 26 03571 g005
Ijms 26 03571 g006
Figure 6. LuWOX interactome network and GO enrichment profilin. (A) LuWOX family interactome mapped through Arabidopsis orthologs. Blue represents resistance related genes in Arabidopsis while orange represents the LuWOX gene family. (B) GO enrichment analysis of LuWOX gene family.
Figure 6. LuWOX interactome network and GO enrichment profilin. (A) LuWOX family interactome mapped through Arabidopsis orthologs. Blue represents resistance related genes in Arabidopsis while orange represents the LuWOX gene family. (B) GO enrichment analysis of LuWOX gene family.
Ijms 26 03571 g006
Ijms 26 03571 g007
Figure 7. Transcriptional profiling of LuWOX gene family. (A) Post-anthesis floral expression dynamics. (B) Abiotic stress responses (salt/heat). Col-Stem, Col-root, and Col-leaf represent the stem, root, and leaf tissues of the experimental control group, respectively. (C) Tissue-specific expression atlas in flax. Expression levels are log2(FPKM+1)-transformed, visualized via gradient color scaling from high (orange) to low (blue).
Figure 7. Transcriptional profiling of LuWOX gene family. (A) Post-anthesis floral expression dynamics. (B) Abiotic stress responses (salt/heat). Col-Stem, Col-root, and Col-leaf represent the stem, root, and leaf tissues of the experimental control group, respectively. (C) Tissue-specific expression atlas in flax. Expression levels are log2(FPKM+1)-transformed, visualized via gradient color scaling from high (orange) to low (blue).
Ijms 26 03571 g007
Ijms 26 03571 g008
Figure 8. Salt-stress-induced LuWOX transcriptional dynamics: untreated controls (blue) versus NaCl-treated samples (orange). Statistical significance is denoted by asterisks (Student’s t-test) (** p < 0.01; *** p < 0.001).
Figure 8. Salt-stress-induced LuWOX transcriptional dynamics: untreated controls (blue) versus NaCl-treated samples (orange). Statistical significance is denoted by asterisks (Student’s t-test) (** p < 0.01; *** p < 0.001).
Ijms 26 03571 g008
Ijms 26 03571 g009
Figure 9. Cold-stress-induced LuWOX transcriptional dynamics: untreated controls (blue) versus NaCl-treated samples (orange). Statistical significance denoted by asterisks (Student’s t-test) (** p < 0.01; *** p < 0.001).
Figure 9. Cold-stress-induced LuWOX transcriptional dynamics: untreated controls (blue) versus NaCl-treated samples (orange). Statistical significance denoted by asterisks (Student’s t-test) (** p < 0.01; *** p < 0.001).
Ijms 26 03571 g009
Ijms 26 03571 g010
Figure 10. Drought-stress-induced LuWOX transcriptional dynamics: untreated controls (blue) versus NaCl-treated samples (orange). Statistical significance is denoted by asterisks (Student’s t-test) (** p < 0.01; *** p < 0.001).
Figure 10. Drought-stress-induced LuWOX transcriptional dynamics: untreated controls (blue) versus NaCl-treated samples (orange). Statistical significance is denoted by asterisks (Student’s t-test) (** p < 0.01; *** p < 0.001).
Ijms 26 03571 g010
Table 1. Prediction and characterization of WOX genes in flax.
Table 1. Prediction and characterization of WOX genes in flax.
GeneGene ID in GenomeNumber of Amino AcidsMolecular Weight (KDa)PIInstability IndexAliphatic IndexGrand Average of
Hydropathicity
(GRAVY)		Subcellular
Localization
LuWOX01L.us.o.m.scaffold170.4824526.886.7539.5570.08−0.629Nuclear
LuWOX02L.us.o.m.scaffold255.1523425.945.6355.2165.04−0.832Nuclear
LuWOX03L.us.o.m.scaffold218.729933.448.6954.0763.95−0.638Nuclear
LuWOX04L.us.o.m.scaffold50.4727830.728.7663.1060.72−0.764Nuclear
LuWOX05L.us.o.m.scaffold33.6939543.917.3856.5859.65−0.677Nuclear
LuWOX06L.us.o.m.scaffold107.6136640.697.8660.1359.59−0.763Nuclear
LuWOX07L.us.o.m.scaffold155.9919622.019.6474.3963.72−0.793Nuclear
LuWOX08L.us.o.m.scaffold98.17626629.858.6956.8058.68−0.973Nuclear
LuWOX09L.us.o.m.scaffold8.5419622.588.4251.7356.28−0.825Nuclear
LuWOX10L.us.o.m.scaffold133.8932335.575.6850.3747.43−0.930Nuclear
LuWOX11L.us.o.m.scaffold83.17231534.715.5651.8047.40−0.909Nuclear
LuWOX12L.us.o.m.scaffold2.50323927.738.6963.4251.84−0.954Nuclear
LuWOX13L.us.o.m.scaffold72.5241445.717.1059.5761.55−0.698Nuclear
LuWOX14L.us.o.m.scaffold103.10843247.917.4452.3662.13−0.756Nuclear
LuWOX15L.us.o.m.scaffold258.2619221.819.7154.3370.10−0.741Nuclear
LuWOX16L.us.o.m.scaffold212.3026329.869.3764.6063.80−0.983Nuclear
LuWOX17L.us.o.m.scaffold6.17323124.665.6564.5967.97−0.345Nuclear
LuWOX18L.us.o.m.scaffold141.6729431.135.9661.3866.73−0.366Nuclear
Table 2. Ka, Ks, and Ka/Ks values of replication pairs of WOX gene family in flax.
Table 2. Ka, Ks, and Ka/Ks values of replication pairs of WOX gene family in flax.
Duplicated Gene PairsNon-Synonymous
(Ka)		Synonymous
(Ks)		Ka/Ks
LuWOX01 & LuWOX020.074640.31580.2363
LuWOX04 & LuWOX030.085640.23920.3579
LuWOX06 & LuWOX050.048690.27120.1795
LuWOX15 & LuWOX070.085620.16610.5151
LuWOX08 & LuWOX160.049440.08380.5899
LuWOX09 & LuWOX120.042450.24280.1748
LuWOX10 & LuWOX110.026320.09760.2697
LuWOX13 & LuWOX140.067250.16520.4069
LuWOX17 & LuWOX180.029310.13400.2186
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Song, X.; Lu, J.; Wang, H.; Tang, L.; Li, S.; Zang, Z.; Wu, G.; Zhang, J. Identification and Characterization of WOX Gene Family in Flax (Linum usitatissimum L.) and Its Role Under Abiotic Stress. Int. J. Mol. Sci. 2025, 26, 3571. https://doi.org/10.3390/ijms26083571

AMA Style

Song X, Lu J, Wang H, Tang L, Li S, Zang Z, Wu G, Zhang J. Identification and Characterization of WOX Gene Family in Flax (Linum usitatissimum L.) and Its Role Under Abiotic Stress. International Journal of Molecular Sciences. 2025; 26(8):3571. https://doi.org/10.3390/ijms26083571

Chicago/Turabian Style

Song, Xixia, Jianyu Lu, Hang Wang, Lili Tang, Shuyao Li, Zhenyuan Zang, Guangwen Wu, and Jian Zhang. 2025. "Identification and Characterization of WOX Gene Family in Flax (Linum usitatissimum L.) and Its Role Under Abiotic Stress" International Journal of Molecular Sciences 26, no. 8: 3571. https://doi.org/10.3390/ijms26083571

APA Style

Song, X., Lu, J., Wang, H., Tang, L., Li, S., Zang, Z., Wu, G., & Zhang, J. (2025). Identification and Characterization of WOX Gene Family in Flax (Linum usitatissimum L.) and Its Role Under Abiotic Stress. International Journal of Molecular Sciences, 26(8), 3571. https://doi.org/10.3390/ijms26083571

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop