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Article

Genome-Wide Identification and Expression Profiling of Dehydration-Responsive Element-Binding Family Genes in Flax (Linum usitatissimum L.)

by
Yan Wang
1,2,
Yanni Qi
2,
Limin Wang
2,
Chenmeng Xu
2,
Wenjuan Li
2,
Zhao Dang
2,
Wei Zhao
2,
Ping Wang
2,
Yaping Xie
2,
Yamin Niu
1,2,
Nan Lu
1,2,
Zuyu Hu
1,2,
Zigang Liu
1,3 and
Jianping Zhang
1,2,*
1
College of Agriculture, Gansu Agricultural University, Lanzhou 730070, China
2
Institute of Crop, Gansu Academy of Agricultural Sciences, Lanzhou 730070, China
3
State Key Laboratory of Aridland Crop Science, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(7), 3074; https://doi.org/10.3390/ijms26073074
Submission received: 3 February 2025 / Revised: 22 March 2025 / Accepted: 24 March 2025 / Published: 27 March 2025
(This article belongs to the Section Molecular Genetics and Genomics)
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Abstract

Dehydration-responsive element-binding (DREB) transcription factors are ubiquitous in plants and regulate plant growth, development, signal transduction, and responses to stress, particularly drought stress. However, DREB genes in flax have not previously been studied. This study conducted a comprehensive and systematic analysis of the DREB gene family in flax (Linum usitatissimum L.). A total of 59 LuDREB genes were identified in Longya-10 (a breeding variety), with an uneven distribution across all 15 chromosomes. Further analysis revealed significant variations among LuDREB members, with predictions indicating that these proteins are hydrophilic and localized in the nucleus and cytoplasm. A phylogenetic analysis classified the LuDREB genes into six subgroups, a classification further supported by gene structure and motif composition. Members within the same subgroup exhibited structural conservation, suggesting functional redundancy. The duplication analysis identified 30 pairs of segmentally duplicated LuDREB genes and one pair of tandemly duplicated genes, indicating that segmental duplication was the primary driver of LuDREB gene expansion. A comparative collinearity analysis revealed that most LuDREB genes had orthologs in other plant species, suggesting that this gene family has remained relatively conserved throughout evolution. Cis-acting element analysis identified numerous hormone- and stress-responsive elements in LuDREB promoters, and the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) results confirmed the role of all LuDREB genes in drought stress response. In addition, transcriptome analysis revealed that LuDREB49 and LuDREB56 exhibited high expression levels in the capsules, whereas LuDREB3 and LuDREB36 showed significantly higher expression levels in the stems, suggesting that these LuDREB genes may have specialized functions in capsule or stem development. Collectively, this study provides a comprehensive overview of LuDREB genes, offering valuable insights into their roles in flax growth, development, and stress responses.

1. Introduction

Drastic changes in the global climate pose substantial challenges to plant growth and development by aggravating abiotic stresses such as extreme temperatures, drought, and salinization, leading to substantial agricultural losses [1,2]. Modifying the expression patterns of stress-responsive genes under these environmental stresses is critical to ensuring plant survival in adverse conditions. These genes are classified into those encoding stress tolerance proteins and those encoding regulatory proteins [3]. Transcription factors (TFs) play a key role in regulating the expression of stress-response genes by specifically binding to cis-acting elements in their promoter regions [4,5]. Several TF families in plants, including WRKY, MYB, and AP2/ERF (APETALA2/ethylene-responsive factor), are induced under abiotic stress [6,7,8]. Based on the number of domains and specific binding sequences, the AP2/ERF family is further divided into four major groups: the AP2 subfamily, the ERF subfamily, the DREB (dehydration-responsive element-binding protein) subfamily, and the RAV subfamily [9,10].
DREB genes play a central role in response to various abiotic stresses, including drought, high salinity, cold, and heat [11,12,13]. Members of the DREB gene family contain a single AP2 domain that interacts specifically with the core motif (A/GCCGAC) of the dehydration-responsive element (DRE) and binds to the C-repeat (CRT) cis-acting element, regulating the expression of downstream genes and enhancing plant tolerance to abiotic stresses (Figure 1) [14,15]. For instance, 5 out of 30 DREB genes in mung bean showed significant upregulation under drought conditions [16]. AtDREB1A and AtDREB1B from Arabidopsis thaliana positively affect drought tolerance in transgenic Salvia miltiorrhiza [17,18]. Similarly, the overexpression of the soybean DREB1 gene in wheat enhances drought tolerance in field conditions [19]. In addition to drought stress, DREB genes are also activated in response to low temperatures, heat, and high salinity conditions [20,21,22,23,24,25,26,27]. The overexpression of TaDREB3 in wheat improves plant tolerance to heat, dehydration, and salt stress [28], and the expression of several DREB genes in barley is significantly upregulated under drought and salt stimulations [29]. In Arabidopsis, DREB2A and DREB2B exhibit positive responses to dehydration and high salinity [30]. Furthermore, SsDREB genes in Saccharum spontaneum have been linked not only to stress responses but also to development and photosynthesis [31]. In rice, the expression of OsDREB1C is induced by both light and low-nitrogen conditions, with its overexpression enhancing grain yield and nitrogen use efficiency and promoting early flowering [32]. Collectively, DREB genes serve as key regulators in plant adaptation to abiotic stresses.
Flax (Linum usitatissimum L.) belongs to the genus Linum within the Linaceae family. It is a self-pollinating, annual, diploid herbaceous dicotyledonous plant (2n = 2x = 30) [33]. Flax serves as a vital oilseed and cash crop in China, exhibiting strong resistance to drought, cold, and nutrient-deficient conditions [34]. Flax has been extensively used in industries such as oil production, textiles, printing, tanning, pharmaceuticals, and food, highlighting its substantial economic value [35]. A bioinformatics analysis of the flax genome identified 43,668 protein-coding genes [36]. Numerous gene families in flax have been identified and characterized to date. For example, the WRKY transcription factor family in flax has been comprehensively identified and analyzed at the whole-genome level, along with its expression patterns [37]. Additionally, 167 R2R3-, 7 3R-, and 1 4R-MYB transcription factors have been identified in flax. Expression analysis indicates that eight R2R3 MYB genes may be involved in lignin biosynthesis [38]. In addition, 50 LuLEA genes were identified, with expression profiles indicating that most of these genes play a role in seed development [39].
Although substantial research has been conducted on the flax genome, the genetic basis underlying its crucial agronomic traits and adaptations to environmental stresses remains poorly understood, and DREB genes within the flax genome have yet to be studied. Given the critical role of DREB genes in mediating responses to environmental stresses across various species, studying the DREB gene family in flax (LuDREB) holds importance. Through a comprehensive genome-wide analysis, this study identified and characterized the LuDREB gene family, focusing on the chromosomal location, gene structure, conserved motifs, cis-acting elements, evolutionary relationships, and expression patterns across different tissues and under drought stress. The findings of this study provide a detailed understanding of the LuDREB gene family and establish a foundation for elucidating the molecular mechanisms underlying drought tolerance in flax.

2. Results

2.1. Identification of DREB Genes in Flax

Orthologous genes of DREB from Arabidopsis were screened in the genomes of Longya-10 and pale flax [40]. A total of 59 and 57 DREB members, designated LuDREB1 to LuDREB59, were identified in Longya-10 and pale flax, respectively (Tables S1 and S2). Further analysis revealed that the protein lengths of DREB members ranged from 123 (LuDREB17) to 973 (LuDREB47) amino acids (aa) in Longya-10 and from 121 to 959 aa in pale flax. The MW ranged from 13.16 (LuDREB17) to 109.95 kDa (LuDREB47) in Longya-10 and from 13.17 to 108.63 kDa in pale flax. The pI ranged from 4.23 to 9.36 in Longya-10 and from 4.71 to 9.52 in pale flax, with most DREB members classified as acidic proteins. Moreover, the GRAVY values for all DREB proteins were less than zero, indicating their hydrophilic nature. A subcellular localization analysis indicated that 23 and 20 DREB members were located in both the cytoplasm and nucleus in Longya-10 and pale flax, respectively, whereas 35 members were exclusively located in the nucleus in both genomes. Only one DREB member in Longya-10 and two in pale flax were found to be located solely in the cytoplasm. The DREB protein sequences are listed in Table S3.

2.2. Phylogenetic Analysis of LuDREB Proteins

To examine the evolutionary relationships of flax DREB genes, a phylogenetic tree was constructed using DREB sequences from the breeding cultivar Longya-10 and its wild ancestor, pale flax (Figure S1). The analysis revealed that most LuDREB genes had orthologs in pale flax, suggesting a high degree of conservation of DREB genes during flax domestication.
To further investigate the evolution of DREB genes across diverse species, a dataset comprising 290 DREB sequences was compiled, including 59, 20, 16, 56, 73, and 66 sequences from flax, maize, rice, Arabidopsis, soybean, and potato, respectively. A comprehensive phylogenetic tree was constructed based on this dataset (Figure 2). The DREB sequences were classified into six subgroups (Groups 1 to 6) following the Arabidopsis classification system [9]. The LuDREB genes were unevenly distributed across these subgroups, with Group 4 containing the highest number (28 members) and Group 3 containing the fewest (2 members). Groups 1, 2, 5, and 6 included 3, 5, 9, and 12 LuDREB members, respectively. Groups 1, 2, and 3 contained DREB genes from both monocotyledonous and dicotyledonous plants, whereas Groups 4, 5, and 6 exclusively comprised DREB genes from dicotyledonous plants. Notably, the Arabidopsis gene AT2G40220.1 (ABI4) exhibited high homology with DREB genes from all five other species in Group 3, indicating the strong conservation of ABI4 across species and highlighting its significant and conserved role in plants.

2.3. Sequence Analysis of Flax DREBs

To evaluate genetic relationships among LuDREB proteins, a phylogenetic tree was constructed based on the amino acid sequences of all LuDREB proteins, which were subsequently classified into six groups (Groups 1 to 6), with most members existing in pairs (Figure 3a). The majority of LuDREB proteins displayed high sequence similarity to another member, forming pairs. The gene structure analysis revealed significant variation in the length of DREB genes. In Longya-10, the lengths ranged from 372 (LuDREB17) to 5636 bp (LuDREB47), while, in pale flax, the lengths varied from 392 bp to 10,637 bp (Figure 3b, Tables S1 and S2). The exon–intron organization of all DREB genes in flax was also examined (Figure 3b). The analysis indicated that most LuDREB family members (78%) contained only one exon. Among the remaining genes, 13 had more than one exon: 5 genes contained two exons (LuDREB11, LuDREB14, LuDREB36, LuDREB51, and LuDREB56), 2 contained four exons (LuDREB49 and LuDREB57), 2 contained seven exons (LuDREB9 and LuDREB52), and 3 had twelve exons (LuDREB25, LuDREB34, and LuDREB47). Notably, LuDREB4 had the highest number of exons (13). In pale flax, 43 out of 57 DREB genes contained only one exon, whereas the remaining genes possessed 2–13 exons.
The MEME program was used to identify conserved motifs within LuDREB proteins (Figure 3c). A total of 80 conserved motifs were identified, ranging in length from 8 to 50 aa. The motif composition varied across subgroups. Motifs 1, 2, and 3 were present in the majority of LuDREB proteins, with a few exceptions, whereas the remaining motifs were specific to certain LuDREB members. Motifs 1 and 2 were found in all members of the five subgroups except Group 4, where 11 and 16 out of the 28 LuDREB members lacked Motifs 1 and 2, respectively. Motif 3 was present in all members of Groups 1, 2, and 6, as well as most members of Groups 4 and 5, but it was absent in Group 3. In addition, Motif 4 was found in Groups 1, 4, and 5, whereas Motif 5 was restricted to Groups 1 and 4. Several motifs were unique to specific subgroups or individual members. For instance, Group 4 contained up to 36 subgroup-specific motifs, whereas Motifs 16, 37, and 55 were exclusive to Groups 6, 5, and 2, respectively. Significant variation in motif composition was observed within Group 4, whereas LuDREB members in the other five subgroups displayed a more consistent motif pattern. For example, all members of Group 1 contained Motifs 1–5, whereas members of Group 3 shared Motifs 1, 2, 27, 30, 31, 47, and 66 (Table S4).

2.4. Chromosomal Location and Synteny Analysis

The genomic chromosomal location analysis of the LuDREB genes revealed an uneven distribution of 56 LuDREB genes across all 15 chromosomes (Figure 4). However, LuDREB57, LuDREB58, and LuDREB59 could not be mapped to any chromosome. Chromosome 10 contained the highest number of LuDREB genes (six genes), whereas chromosome 8 harbored only two genes. Chromosomes 1, 2, 4, 5, 6, 11, and 14 each contained three genes, chromosomes 3, 7, and 9 each harbored four genes, and chromosomes 12, 13, and 15 contained five genes each.
An analysis of duplication events was conducted to elucidate the mechanisms underlying the expansion of the LuDREB gene family during evolution (Figure 4 and Table 1). The results showed that one gene pair, LuDREB42/LuDREB43, located on chromosome 12, underwent tandem duplication (TD). Furthermore, segmental duplication (SD) was observed in 30 gene pairs, involving a total of 51 LuDREB genes, which accounted for 86.4% of the entire LuDREB family. To assess the selective pressure acting on duplicated LuDREB gene pairs, the Ka and Ks substitution rates and Ka/Ks ratios were calculated. Ka/Ks ratios of 1, greater than 1, and less than 1 indicate neutral, positive, and purifying selection, respectively [41]. The Ka/Ks ratios for all duplicated gene pairs were less than 0.5, indicating that purifying selection shaped the evolution of LuDREB genes. The distribution of Ks values can be utilized to identify potential genome duplication events and estimate the timing of such events, as synonymous mutations are generally considered neutral and are not subject to natural selection [42]. By comparing the Ks value distributions of duplicated gene pairs within a genome, whole-genome duplication (WGD) events can be detected, and the frequency and distribution of these WGD events throughout evolutionary history can be inferred. This is because WGD typically results in the simultaneous duplication of a large number of genes, leading to a concentration of Ks values within a relatively narrow range [36,43]. Our study shows that 80% of the collinear genes have Ks values ranging from 0.07 to 0.31, suggesting that the expansion of LuDREB genes likely originated from a recent WGD event in flax (Ks = 0.13). The duplication events of these genes were estimated to have occurred between 6 and 25 Mya, whereas the divergence time of other genes was estimated to be between 78 and 183 Mya.
To further explore the phylogenetic and evolutionary relationships of DREB genes across species, a collinearity analysis was conducted using three dicots (Arabidopsis, potato, and soybean) and two monocots (maize and rice) (Figure 5 and Table S5). The analysis revealed that 39, 33, 44, 9, and 3 out of 56 LuDREB genes had orthologs in Arabidopsis, potato, soybean, maize, and rice, respectively, with 58, 50, 121, 13, and 4 collinear gene pairs identified between flax and these species. These findings indicate that more than half of the LuDREB genes had orthologs in the three dicot plants, and the collinearity between flax and dicots was greater than that observed with monocots. In addition, homologous DREB genes exhibited one-to-many and many-to-one collinear relationships. For instance, LuDREB14, LuDREB35, and LuDREB36 had up to five collinear gene pairs in soybean, whereas LuDREB36 had four pairs in potato, demonstrating a high degree of collinearity. Furthermore, LuDREB30 and LuDREB40 had orthologs in all five species, suggesting that these genes originated from a common ancestor and their functions are conserved in both dicots and monocots. Notably, 20 LuDREB genes had homologs in all dicots but lacked orthologs in monocots, indicating their potential significance in the evolutionary divergence between dicots and monocots.

2.5. Cis-Acting Elements on LuDREB Promoters

To investigate the transcriptional regulation mechanisms and potential functions of LuDREB genes, the 2000-bp upstream sequences of each gene were extracted to predict cis-acting elements in their promoters (Figure 6 and Table S6). The analysis identified seven types of stress-responsive elements on LuDREB promoters, including MBS (drought inducibility), CCAAT-box (heat shock), LTR (low temperature response), GC-motif and ARE (anoxia response), TC-rich repeats (defense and stress responses), WUN-motif (wound response), and AT-rich sequences (other abiotic stress responses). More than half of the LuDREB genes contained elements associated with drought (32 genes), low temperature (38 genes), and anoxia responsiveness (55 genes). The total number of these elements was 43, 57, and 157, respectively. Among the 55 genes, LuDREB58 had the highest number of anoxia-responsive elements. In addition, elements related to abscisic acid (190), methyl jasmonate (MeJA) (119), gibberellin (50), auxin (47), and salicylic acid (41) were abundant in LuDREB promoters, although their distribution was uneven. The number of phytohormone-related elements per gene ranged from 1 (LuDREB36) to 19 (LuDREB6). All LuDREB genes contained at least one phytohormone-responsive element. Specifically, LuDREB10, LuDREB27, LuDREB31, LuDREB34, LuDREB38, LuDREB56, and LuDREB59 contained all five phytohormone-responsive elements, whereas LuDREB36 contained only one type. LuDREB6, LuDREB18, and LuDREB51 had up to 9, 7, and 4 elements associated with abscisic acid, MeJA, and gibberellin, respectively.

2.6. Expression Analysis of LuDREB Genes in Different Tissues of Flax Under Drought Stress

To investigate the potential functions of LuDREB genes in flax development, their expression profiles were analyzed across different cultivars (Longya-10 and Heiya-14) and tissues (stem and capsule), using previously reported transcriptome data (Figure 7) [36]. The analysis revealed the presence of the transcripts of 56 LuDREB genes in at least one organ in one of the two cultivars. Among these, 43 genes were expressed in both the stems and capsules of both cultivars. However, the transcripts of LuDREB17, LuDREB18, and LuDREB38 were not detected. Notably, LuDREB49 and LuDREB56 showed high expression levels in the capsules of both cultivars, with LuDREB56 exhibiting particularly strong expression. By contrast, five genes (LuDREB3, LuDREB10, LuDREB23, LuDREB36, and LuDREB53), especially LuDREB3 and LuDREB36, demonstrated significantly higher expression levels in the stems of both cultivars. The expression levels of other LuDREB genes were relatively low across both cultivars.
To evaluate the potential functions of LuDREB genes in response to drought stress, the expression levels of genes containing MBS (drought-related cis-acting elements) in their promoters were analyzed under drought stress simulated by PEG treatment (Figure 8). The results showed that all analyzed LuDREB genes responded to drought stress to varying degrees, with each displaying distinct spatiotemporal patterns. Most LuDREB genes were upregulated at least at one time point in either the roots or leaves after exposure. Notably, the expression levels of LuDREB23 exhibited an upward regulation trend at all time points in both the roots and leaves following treatment. LuDREB11/51, LuDREB13, LuDREB17, LuDREB22, LuDREB33, LuDREB35, and LuDREB50 exhibited an upward regulation trend in leaves across all time points. Among them, LuDREB11/51 and LuDREB13 reached their peak expression levels at 72 h, indicating their potential involvement in late-phase signaling pathways under drought stress. In contrast, the expression levels of LuDREB1 showed a consistent downward trend at all time points in leaves after treatment, potentially reflecting its negative regulatory role under drought conditions. Based on these findings, it can be inferred that different LuDREB genes regulate drought responses through distinct mechanisms. We also observed distinct expression patterns of LuDREB genes between roots and leaves, which may reflect the complexity of drought response mechanisms and suggest potential functional divergence of LuDREB genes in different tissues. For instance, LuDREB10, LuDREB2/20, and LuDREB27 had lower expression levels in leaves than in roots at all time points after PEG treatment, whereas the expression levels of LuDREB35 and LuDREB50 were significantly higher in leaves than in roots.

3. Discussion

Crop yield and quality are significantly affected by adverse environmental conditions [9]. However, plants have evolved various adaptive mechanisms to resist environmental stresses. Among these, DREB TFs play pivotal roles in mediating abiotic stress responses through both direct and indirect pathways [44,45,46]. The DREB gene family has been identified in numerous plant species. For instance, 56 DREB genes have been identified in Arabidopsis genome [9], whereas the genomes of soybean and potato contain 73 and 66 DREB members, respectively [47,48]. In the present study, 59 and 57 DREB members were identified in the genomes of flax: specifically, in the breeding variety Longya-10 and its ancestor, pale flax. These findings underscore the widespread distribution of the DREB gene family across species. However, variations in the number of DREB members among species, likely driven by gene expansion and deletion events, highlight evolutionary diversity and reflect the specific environmental adaptations required by different plants. Furthermore, the similar number of DREB genes in the two flax genomes suggests that the DREB gene family remained relatively conserved during flax domestication.
To better understand the potential functions of proteins, analyzing their physicochemical properties is essential [49]. DREB proteins in flax vary significantly in terms of length, gene structure, MW, and pI, which suggest their diverse roles in plant development and defense responses. Moreover, most flax DREB genes contained few or no introns. Previous studies have demonstrated that genes with minimal or no introns often exhibit enhanced expression levels in plants [50,51]. In addition, compact gene structures enhance the timely response of plants to various abiotic stresses [52]. Notably, LuDREB23 has a gene structure comprising only one exon, and this compact structure may facilitate its rapid transcription during the early stages of stress. The predicted subcellular localization of flax DREB proteins indicated that they were either distributed in both the nucleus and cytoplasm or restricted to one of these locations. These findings are consistent with those of previous studies on DREB proteins [47,53]. Conserved motifs are crucial for predicting protein functions [7]. Among the 80 motifs identified in LuDREB proteins, Motifs 1, 2, and 3 were present in most members, suggesting that these motifs are conserved within the LuDREB family. Notably, Motif 1, which forms part of the AP2 domain, aligns with previous research findings [31,54]. By contrast, other motifs found exclusively in individual LuDREB members may contribute to functional diversity.
In this study, three phylogenetic trees were constructed to investigate evolutionary relationships among LuDREB members, classifying them into six subgroups with an uneven distribution. Notably, Groups 1, 2, and 3 included DREB genes from both monocotyledonous and dicotyledonous plants, suggesting that the functions of these members are conserved between monocots and dicots. By contrast, Groups 4, 5, and 6 exclusively comprised DREB genes from dicotyledonous plants, indicating their specialized roles in dicots. Moreover, closely related members shared similar gene structures and motif distributions. The analysis revealed that LuDREB members in Groups 1 and 2 exhibited high homology with Arabidopsis genes DREB1A, DREB1B, DREB1C, DREB2A, and DREB2B, which are induced by low temperatures, drought, high salinity, or heat stress [55]. This finding suggests that these LuDREB genes also participate in stress responses. Furthermore, LuDREB45 and LuDREB55 were closely related to AtABI4 and ZmABI4, which are involved in abscisic acid (ABA) signaling, seed maturation, lateral root formation, and sugar signaling [56]. Combined with the presence of ABA-responsive elements in their promoters, LuDREB45 and LuDREB55 are likely involved in ABA signaling.
WGD or polyploidization is a key driver of evolution, generating novel traits and transcriptional regulatory factors that affect the expression patterns of downstream genes [57]. Numerous studies have demonstrated that SD, WGD, and TD contribute significantly to gene expansion and functional diversification within multigene families [58,59]. While SD often leads to functional redundancy, TD can generate novel functions, enabling species to adapt to rapidly changing environments [60]. In this study, we observed that two LuDREB genes underwent TD, whereas SD was observed in 51 genes (86.4%, forming 30 gene pairs). This suggests that functional redundancy within the flax DREB family may serve as a buffering mechanism to cope with fluctuating environments [60]. This finding is consistent with the expansion pattern of DREB genes observed in soybean [19]. Ks analysis further indicated that the expansion of the LuDREB family was primarily driven by a recent WGD event [36]. Moreover, all duplicated LuDREB gene pairs exhibited Ka/Ks ratios of less than 1, suggesting that LuDREB genes have undergone purifying selection throughout their evolution. Collinearity analysis between flax and five other species (three dicots and two monocots) revealed that most LuDREB genes had orthologs in these species, highlighting the evolutionary importance and relative conservation of DREB genes. Furthermore, the number of collinear genes between flax and dicotyledonous plants was significantly higher than that observed between flax and monocotyledonous plants. This finding suggests that DREB genes have undergone widespread duplication events following the divergence of dicots and monocots, potentially contributing to the unique characteristics of these two plant groups.
DREB genes contain numerous hormone- and stress-responsive elements, allowing them to be recognized by upstream signaling molecules, thereby regulating their own expression as well as that of downstream genes. This regulation enables plants to withstand adverse environmental conditions [30,61]. Extensive research has demonstrated that DREB TFs enhance plant resistance to abiotic stresses. For example, transgenic plants overexpressing OsDREB1A, OsDREB1B, and OsDREB1F exhibit increased tolerance to drought stress [62,63,64]. Similarly, Chen et al. (2008) reported that the overexpression of OsDREB1G significantly improves drought tolerance in rice [65]. The heterologous expression of BrDREB2B also enhanced salt, heat, and drought tolerance in Arabidopsis [15]. In addition, DREB genes regulate the expression of stress-responsive genes through both ABA-dependent and ABA-independent pathways [66]. The response of plants to abiotic stresses typically involves complex regulatory networks, where the synergistic interactions between DREB TFs and other TF families (e.g., MYB, WRKY, and NAC), as well as hormone signaling pathways (e.g., ABA and JA), play crucial roles [67]. In this study, an abundance of hormone- and stress-responsive elements was identified in the promoters of LuDREB genes, indicating their potential involvement in stress responses via crosstalk with the ABA or JA signaling pathways. For instance, the presence of ABRE elements suggests that LuDREB genes may be regulated in an ABA-dependent manner, whereas MeJA-responsive elements indicate that jasmonic acid signaling may indirectly regulate downstream defense genes by activating LuDREB expression [44]. Furthermore, the synergistic interactions between DREB and other TFs, such as WRKY or MYB, have been extensively documented in various plants. For example, in Arabidopsis, DREB2A and WRKY18 enhance drought responses through co-binding to the promoter regions of downstream genes [27]. Similarly, the WRKY family members identified in flax may form complex regulatory modules with LuDREBs via analogous mechanisms [37]. Moreover, the synergistic interaction between DREB and NAC TFs also plays a pivotal role in drought signal transduction. For instance, the co-expression of OsDREB1F and OsNAC6 in rice significantly enhances drought tolerance [65]. In the flax genome, multiple NAC family members have been characterized [35], and their potential interactions with LuDREBs merit further investigation. Such multi-factor synergistic interactions may amplify signal transduction pathways or integrate multiple stress signals, thereby enhancing plant adaptability to complex environmental stresses [31].
To further explore their involvement in drought responses, the expression profiles of LuDREB genes containing MBS elements were analyzed under drought stress. The findings indicated that all analyzed LuDREB genes exhibited varying degrees of response to PEG treatment. Previous studies have reported similar findings in other species. For instance, four SsDREB genes (SsDREB1F, SsDREB1L, SsDREB2D, and SsDREB2F) were induced by drought stress in three sugarcane varieties [31], whereas, in sugar beet, nine BvDREB genes showed significant upregulation under drought stress [68]. In Lotus japonicus, three DREB A-2 subgroup genes were rapidly induced by drought stress [54]. These observations suggest the presence of synergistic effects or network regulatory mechanisms that enhance drought resistance in plants [69]. We also observed that the expression patterns of LuDREB genes varied significantly between leaves and roots. Previous studies have demonstrated that the upregulation of DREB gene expression in plant roots is mainly associated with promoting root growth and enhancing water uptake [70], whereas their expression in leaves is linked to stomatal regulation and reduced transpiration [71,72,73]. This implies that the upregulation of LuDREB10, LuDREB2/20, and LuDREB27 in roots may reflect their roles in modulating root growth or improving water uptake efficiency. The upregulation of LuDREB35 and LuDREB50 in leaves could be related to reduced transpiration and stomatal closure, which are essential for water conservation in aerial tissues. Additionally, the concurrent upregulation of LuDREB23 in both roots and leaves suggests its potential involvement in general drought response pathways, such as osmotic adjustment and stress signaling. These findings underscore the complexity of drought response mechanisms and highlight the potential functional divergence of LuDREB genes across different tissues. Notably, most LuDREB genes containing MBS elements also harbored ABRE elements, suggesting that LuDREB genes may mediate drought responses through the ABA signaling pathway, as observed in other plant species [74]. In this study, LuDREB23, LuDREB11, and LuDREB51 were consistently upregulated under drought stress, and their promoter regions were enriched in MBS and ABRE, indicating that they may regulate downstream genes via both ABA-dependent and ABA-independent pathways [66]. In addition, different LuDREB genes exhibited distinct expression patterns in response to drought stress, indicating that they may function through multiple regulatory mechanisms. This study also generated a heatmap using transcriptome data to preliminarily predict the roles of LuDREB genes in organ development. The results indicated that LuDREB56 may be involved in capsule development, whereas LuDREB3 and LuDREB36 may play roles in stem development. However, further functional validation experiments are needed to clarify the specific roles of individual LuDREB genes.
Although this study systematically identified and analyzed the LuDREB family and elucidated its drought response patterns, certain limitations are acknowledged. First, the qRT-PCR analysis was restricted to genes containing MBS elements, and it may have overlooked key members of other regulatory pathways. Second, the functional validation of genes relied solely on expression profile data, lacking direct experimental evidence from transgenic and gene-editing experiments. In future research, we plan to utilize CRISPR-Cas9 to knockout specific genes or heterologously overexpress them in Arabidopsis to clarify their roles in conferring drought tolerance. Additionally, the long-term domestication of flax may have resulted in unique DREB allelic variations, and integrating population genetic analyses (e.g., GWAS) will help to determine their breeding potential.

4. Materials and Methods

4.1. Plant Growth and Drought Treatment

The flax variety Longya-10 was used in this study. To examine the expression patterns of LuDREB genes under drought stress, Longya-10 seeds were germinated in Petri dishes maintained in a climate chamber at 25 °C with a 16:8 light/dark photoperiod. After 14 days of growth, uniform seedlings were selected and transferred to 1/2 Murashige and Skoog (MS) liquid medium for the additional 4 days of cultivation. Then, drought stress was induced by replacing the medium with a fresh medium supplemented with 20% (w/v) polyethylene glycol (PEG). All plants subjected to the PEG treatment originated from the same sowing, and the experiment was conducted in three biological replicates. The leaves and roots were collected at 0, 3, 6, 9, 12, 24, 48, and 72 h during the PEG treatment and at 48 h after recovery in the 1/2 MS liquid medium without PEG supplementation [54,75]. The collected samples were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis.

4.2. Identification of DREB Genes

The genome sequences of Linum usitatissimum (Longya-10) and Linum bienne (pale flax) utilized in this study were obtained from our laboratory and have been deposited in the DDBJ/ENA/GenBank under the accession numbers QMEI00000000 and QMEG00000000 [36]. Their annotation files were downloaded from Figshare (https://figshare.com/ (accessed on 18 December 2024)). The Arabidopsis DREB protein sequences were retrieved from the Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/ (accessed on 18 December 2024)), and DREB protein sequences for Zea mays, Solanum tuberosum, Oryza sativa, and Glycine max were obtained from previous studies [10,47,48,76,77,78]. OrthoFinder 2.5.5 was used to identify orthologs of the Arabidopsis DREB gene family in Longya-10 and pale flax [40]. The molecular weight (MW), isoelectric point (pI), grand average of hydropathicity (GRAVY), and protein length (amino acids) of all LuDREB proteins were calculated using the ExPASy ProParam tool (http://web.expasy.org/protparam/ (accessed on 20 December 2024)) [79]. The subcellular localization of the LuDREB proteins was predicted using Plant-mPloc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ (accessed on 20 December 2024)) [80]. Conserved motifs within LuDREB proteins were identified and analyzed using the MEME suite (https://meme-suite.org/meme/tools/meme (accessed on 21 December 2024)), with default parameter settings [81].

4.3. Chromosomal Mapping and Collinearity Analysis

The chromosomal location information for the LuDREB genes was retrieved from the annotation files of the Longya-10 genome, and visualization was performed using the Map Gene 2 Chromosome (MG2C, http://mg2c.iask.in/mg2c_v2.0/ (accessed on 24 December 2024)) tool. The MCScanX package was used to analyze LuDREB gene duplication events with default parameters, whereas TBtools software (v. 2.146) was used to analyze and visualize collinearity among DREB genes across different species [82,83]. The nonsynonymous (Ka) and synonymous (Ks) substitution rates per site for the duplicated gene pairs were calculated using TBtools software (v. 2.146), and the Ka/Ks ratio was determined to assess the mode and intensity of selective pressure. For each gene pair, the divergence time (million years ago, Mya) was calculated using the following formula [84]:
Mya = Ks/(2 × 6.1 × 10−9) × 10−6.

4.4. Gene Structure and Promoter Analysis

A 2000 bp sequence upstream of the transcription start site was analyzed to identify cis-acting elements within the LuDREB gene family. Cis-acting regulatory elements were predicted using the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 26 December 2024)), and the results were visualized using TBtools software (v2.146) [85]. The exon–intron organization of the LuDREB gene family was determined and analyzed using the Gene Structure Display Server (GSDS, http://gsds.gao-lab.org/ (accessed on 29 December 2024)) [86].

4.5. Phylogenetic Analysis

DREB protein sequences from rice, maize, Arabidopsis, soybean, and potato were used to construct the phylogenetic tree [10,47,48,76,77,78]. The sequence alignment of DREB proteins was performed using ClustalW in MEGA (v. 11.0), with the default parameters. The phylogenetic tree was subsequently constructed based on the sequence alignment using the neighbor-joining method, employing the Poisson model and pairwise deletion, with 1000 bootstrap replicates [87]. Finally, R version 4.1.3 was employed to visualize the phylogenetic tree.

4.6. Gene Expression Analysis

The transcriptome data generated in our lab (PRJNA505721) were used to analyze the expression patterns of LuDREB genes across different genetic backgrounds and tissues [36]. The expression profiles of LuDREB genes under the PEG treatment were further examined using qRT-PCR. Total RNA was extracted using the EZgene Plant Easy Spin RNA Miniprep Kit (BIOMIGA, San Diego, CA, USA), and cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, San Jose, CA, USA). Specific primers for qRT-PCR were designed using Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA) (Table S7). The qRT-PCR experiment was conducted using the Eco Real-Time PCR System (Illumine, San Diego, CA, USA) following the protocol described by Qi et al. (2023) [75], using GAPDH as the reference gene [88]. The relative expression levels of LuDREB genes were calculated using the 2−∆∆Ct method, and the data were subsequently subjected to logarithmic transformation (log10), with the leaves collected at 0 h serving as the control [89]. Because of the high sequence similarity among certain gene pairs (LuDREB16/58, LuDREB17/40, LuDREB18/41, LuDREB46/54, LuDREB12/33, and LuDREB15/38), it was challenging to design specific primers for individual genes. Therefore, a single primer pair was designed for each gene pair.

5. Conclusions

This study identified 59 LuDREB genes in Longya-10 (a breeding variety) and analyzed their protein characteristics, gene structure, evolutionary relationships, cis-acting elements, and expression patterns. The findings revealed that LuDREB promoters contained numerous abiotic-stress- and hormone-responsive cis-acting elements. In addition, the expansion of LuDREB genes was primarily driven by WGD events. The qRT-PCR analysis further demonstrated that the expression of multiple LuDREB genes increased significantly under drought stress. Collectively, these results highlight the critical roles of LuDREB genes in the evolution and drought responses of flax.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26073074/s1.

Author Contributions

Conceptualization, J.Z. and Z.L.; methodology, Y.W.; software, C.X., W.L., Y.X. and Y.N.; validation, N.L., C.X., Z.D., P.W., L.W. and Z.H.; formal analysis, Y.W. and Y.Q.; investigation, Y.W.; resources, J.Z.; data curation, Y.W., C.X. and Y.Q.; writing—original draft preparation, Y.W.; writing—review and editing, J.Z., Z.L. and Y.Q.; visualization, Y.W. and C.X.; funding acquisition, J.Z., W.Z., W.L. and Y.Q. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the National Key Research and Development Program of China (2024YFD600100), the National Industrial Technology System of Characteristics Oil of China, MOF and MARA (CARS-14-1-05), the National Natural Science Foundation of China (32360502), and the key research project of Gansu Province (24YFWA002, 24YFNA003, and 25RCKA006).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are reported in the article and the Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The functions of DREB transcription factors in plants.
Figure 1. The functions of DREB transcription factors in plants.
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Figure 2. Phylogenetic tree of DREB proteins from Longya−10, rice, maize, Arabidopsis, soybean, and potato. The tree is divided into six clades, each represented by a different color and designated as Group 1 to Group 6. Colored circles indicate DREB members from different species.
Figure 2. Phylogenetic tree of DREB proteins from Longya−10, rice, maize, Arabidopsis, soybean, and potato. The tree is divided into six clades, each represented by a different color and designated as Group 1 to Group 6. Colored circles indicate DREB members from different species.
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Figure 3. Phylogenetic relationships, gene structure, and conserved motifs of LuDREB genes. (a) Phylogenetic tree of LuDREB proteins. (b) Gene structure of LuDREB genes. (c) Motif composition of LuDREB proteins identified using MEME. Different colors represent distinct motifs.
Figure 3. Phylogenetic relationships, gene structure, and conserved motifs of LuDREB genes. (a) Phylogenetic tree of LuDREB proteins. (b) Gene structure of LuDREB genes. (c) Motif composition of LuDREB proteins identified using MEME. Different colors represent distinct motifs.
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Figure 4. Chromosomal distribution of LuDREB genes. Red boxes indicate gene pairs that underwent tandem duplication, whereas gray lines represent gene pairs that underwent segmental duplication.
Figure 4. Chromosomal distribution of LuDREB genes. Red boxes indicate gene pairs that underwent tandem duplication, whereas gray lines represent gene pairs that underwent segmental duplication.
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Figure 5. Collinearity analysis of DREB genes between flax and five other plant species (Arabidopsis, soybean, potato, maize, and rice). Gray lines indicate syntenic blocks between the flax genome and those of other species, whereas red lines represent collinear DREB gene pairs.
Figure 5. Collinearity analysis of DREB genes between flax and five other plant species (Arabidopsis, soybean, potato, maize, and rice). Gray lines indicate syntenic blocks between the flax genome and those of other species, whereas red lines represent collinear DREB gene pairs.
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Figure 6. Distribution of cis−acting elements in the promoters of LuDREB genes. Different colored boxes represent distinct cis−acting elements.
Figure 6. Distribution of cis−acting elements in the promoters of LuDREB genes. Different colored boxes represent distinct cis−acting elements.
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Figure 7. Expression analysis of LuDREB genes across different flax varieties and tissues based on transcriptome data (data were log2−transformed).
Figure 7. Expression analysis of LuDREB genes across different flax varieties and tissues based on transcriptome data (data were log2−transformed).
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Figure 8. The expression levels of LuDREB genes in leaves and roots detected by qRT−PCR under PEG treatment. The x-axis represents the time points (0, 3, 6, 9, 12, 24, 48, and 72 h) after the PEG treatment and 48 h after recovery in 1/2 MS liquid medium without PEG supplementation, while the y-axis indicates the gene expression levels. Relative expression levels were calculated using the 2−ΔΔCt method, followed by logarithmic transformation (log10). All results were derived from three biological replicates, with error bars representing ± SD (n = 3).
Figure 8. The expression levels of LuDREB genes in leaves and roots detected by qRT−PCR under PEG treatment. The x-axis represents the time points (0, 3, 6, 9, 12, 24, 48, and 72 h) after the PEG treatment and 48 h after recovery in 1/2 MS liquid medium without PEG supplementation, while the y-axis indicates the gene expression levels. Relative expression levels were calculated using the 2−ΔΔCt method, followed by logarithmic transformation (log10). All results were derived from three biological replicates, with error bars representing ± SD (n = 3).
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Table 1. Duplication events and divergence times of LuDREB genes.
Table 1. Duplication events and divergence times of LuDREB genes.
Seq1Seq2Duplication EventKaKsKa/KsDivergence Time (Mya)
LuDREB1LuDREB14SD0.24771.37970.1795113.0911
LuDREB2LuDREB20SD0.03250.30250.107424.7955
LuDREB2LuDREB26SD0.28241.31080.2154107.4433
LuDREB3LuDREB21SD0.01040.07830.13226.4207
LuDREB4LuDREB25SD0.01610.110.14689.0186
LuDREB6LuDREB16SD0.19061.0690.178387.6266
LuDREB7LuDREB29SD0.01640.25690.06421.0547
LuDREB8LuDREB28SD0.0370.18220.202914.9325
LuDREB10LuDREB27SD0.05290.1170.45259.5896
LuDREB16LuDREB58SD0.03050.27130.112522.2348
LuDREB20LuDREB26SD0.26292.23780.1175183.4266
LuDREB26LuDREB59SD0.02310.07470.30966.1192
LuDREB30LuDREB13SD0.07140.20160.354316.5232
LuDREB31LuDREB6SD0.02910.15330.189812.5668
LuDREB31LuDREB16SD0.19981.02540.194984.0454
LuDREB32LuDREB5SD0.03380.15590.216912.7806
LuDREB33LuDREB12SD0.02970.12480.238410.2259
LuDREB34LuDREB47SD0.03210.08030.46.586
LuDREB35LuDREB48SD0.06280.24610.255420.1684
LuDREB36LuDREB14SD0.01490.14460.10311.8563
LuDREB38LuDREB15SD0.01780.11580.15419.4944
LuDREB39LuDREB24SD0.03410.18390.185715.0722
LuDREB40LuDREB17SD0.0550.2040.269716.7172
LuDREB41LuDREB18SD0.05040.16120.312913.2142
LuDREB44LuDREB56SD0.07590.25690.295421.0547
LuDREB45LuDREB55SD0.09110.31220.291825.5866
LuDREB46LuDREB53SD0.33641.8380.183150.657
LuDREB49LuDREB57SD0.00590.12340.047810.1141
LuDREB51LuDREB11SD0.05930.13280.446710.8847
LuDREB52LuDREB9SD0.01410.12020.1179.8513
LuDREB42LuDREB43TD0.21880.95390.229478.1916
Note: SD represents segmental duplication, and TD represents tandem duplication.
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Wang, Y.; Qi, Y.; Wang, L.; Xu, C.; Li, W.; Dang, Z.; Zhao, W.; Wang, P.; Xie, Y.; Niu, Y.; et al. Genome-Wide Identification and Expression Profiling of Dehydration-Responsive Element-Binding Family Genes in Flax (Linum usitatissimum L.). Int. J. Mol. Sci. 2025, 26, 3074. https://doi.org/10.3390/ijms26073074

AMA Style

Wang Y, Qi Y, Wang L, Xu C, Li W, Dang Z, Zhao W, Wang P, Xie Y, Niu Y, et al. Genome-Wide Identification and Expression Profiling of Dehydration-Responsive Element-Binding Family Genes in Flax (Linum usitatissimum L.). International Journal of Molecular Sciences. 2025; 26(7):3074. https://doi.org/10.3390/ijms26073074

Chicago/Turabian Style

Wang, Yan, Yanni Qi, Limin Wang, Chenmeng Xu, Wenjuan Li, Zhao Dang, Wei Zhao, Ping Wang, Yaping Xie, Yamin Niu, and et al. 2025. "Genome-Wide Identification and Expression Profiling of Dehydration-Responsive Element-Binding Family Genes in Flax (Linum usitatissimum L.)" International Journal of Molecular Sciences 26, no. 7: 3074. https://doi.org/10.3390/ijms26073074

APA Style

Wang, Y., Qi, Y., Wang, L., Xu, C., Li, W., Dang, Z., Zhao, W., Wang, P., Xie, Y., Niu, Y., Lu, N., Hu, Z., Liu, Z., & Zhang, J. (2025). Genome-Wide Identification and Expression Profiling of Dehydration-Responsive Element-Binding Family Genes in Flax (Linum usitatissimum L.). International Journal of Molecular Sciences, 26(7), 3074. https://doi.org/10.3390/ijms26073074

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