Genome-wide identification and functional characterization of the DREB gene family in barley (Hordeum vulgare L.) reveal its role in drought and salinity responses

Abstract

Background

Dehydration response element-binding (DREB) proteins are crucial for plant responses to abiotic stress, particularly in molecular responses to drought and high-salt stress. However, the understanding of the role of DREB proteins in barley, an important and widespread crop, remains limited.

Results

In this study, bioinformatics-based genome-wide analysis revealed 39 DREB genes in barley. These genes are distributed on all barley chromosomes; chr6H has the highest density, with five pairs of segmentally duplicated genes. Moreover, synteny analyses revealed a relatively conserved evolutionary process shared by some HvDREB genes in barley and four other species. Moreover, promoter analysis revealed that HvDREB genes are associated with stress-, drought-, low-temperature- and hormone-responsive cis-acting elements. MicroRNA target site prediction revealed that 14 types of miRNAs regulate 16 HvDREB genes. qRT–PCR analysis verified that upon exposure to distinct stressors, the HvDREB genes presented diverse expression trends in barley roots, stems, and leaves.

Conclusion

These findings indicate that HvDREB genes may regulate plant growth and stress tolerance. On the basis of the bioinformatics and qRT–PCR results, we hypothesized that HvDREB gene expression is closely related to salt and drought stress in barley, providing a basis for further understanding the genetic underpinnings of these key stress adaptations.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-12433-9.

Background

Abiotic stress factors, such as drought, salinity, high temperature, and chilling conditions, frequently interfere with the normal growth and development of crops. Under severe circumstances, these factors can result in a substantial reduction in crop production over large areas. Among these environmental stressors, both drought stress and salinity have significant negative effects on agricultural productivity, encompassing both the overall yield and the qualitative attributes of crop products [1]. When plants encounter harsh conditions such as drought, gene expression can be either induced or suppressed, facilitating plant survival and normal physiological functions. Transcription factors play crucial roles in the regulatory network for downstream stress-related gene transcription [2, 3].

Dehydration response element-binding (DREB) transcription factors are unique to plants. As a subfamily within the AP2/EREBP transcription factor superfamily, DREB transcription factors are beneficial for plant growth and development and can positively contribute to responses to adverse environmental conditions [4]. In the field of molecular breeding, DREB transcription factors have been recognized as among the most pivotal and influential gene families involved in the enhancement of plant stress tolerance [5]. Typically, DREB transcription factors possess a highly conserved AP2/ERF domain whose function was first revealed in Arabidopsis [6]. All DREB proteins possess a single conserved AP2 domain of approximately 60–70 amino acids, characterized by the presence of critical valine (V) and glutamic acid (E) residues at positions 14 and 19, respectively. These residues, located within the β-sheet of the AP2/ERF domain, are essential for the binding of the domain to the DRE [7]. Moreover, members of the DREB subfamily of the AP2/ERF superfamily specifically bind to the ACCGAC/GCCGAC core motif of dehydration-responsive element/C-repeat (DRE/CRT) cis-acting elements in promoter regions [8].

Recent evidence from genome sequencing and gene family studies has demonstrated the presence of DREB homologs across diverse plant species, including Arabidopsis [8], maize (Zea mays) [9], rice (Oryza sativa) [10], foxtail millet (Setaria italica) [11], wheat (Triticum aestivum) [12], cotton (Gossypium spp.) [13], soybean (Glycine max) [14], and Moso bamboo (Phyllostachys edulis) [7]. These studies have consistently demonstrated that DREB proteins play pivotal regulatory roles in plant responses to abiotic stresses, particularly drought and high salinity, by binding to DRE/CRT cis-elements in promoters to activate downstream stress-responsive genes.

Increased expression of DREB transcription factors can increase the resistance of a wide range of plant species (such as Arabidopsis, rice, wheat, maize, tomato, and soybean) to abiotic stress [15]. Notably, increased expression of AtDREB1 significantly increased the tolerance of Arabidopsis plants to drought stress. Additionally, increased expression of AtDREB2 markedly increased the survival rate of Arabidopsis plants exposed to cold stress [8]. Increased tolerance to drought and freezing stress results from the constitutive expression of OsDREB1 in Arabidopsis [16]. Moreover, overexpression of the Arabidopsis DREB1A gene can increase tolerance to abiotic stress in crops such as soybean, wheat, peanut and rice, and the underlying mechanism has been revealed through transcriptome and metabolome analyses, indicating that this gene has potential for agricultural application [17]. However, as the overexpression of this gene gives rise to dwarf phenotypes and postpones flowering in plants, additional studies are necessary prior to its application in improving the efficiency of agricultural production [17].

In rice, ARAG1 is a DREB gene whose expression is upregulated by abscisic acid (ABA) or drought and that acts on the drought resistance of plants and the germination of seeds [18]. The overexpression of OsDREB2B (upregulation) negatively regulates rice plant height by interacting with OsAP2-9 and OsWRKY21 to affect the expression of genes related to gibberellic acid (GA) metabolism, thereby causing dwarf phenotypes, while its knockout mutants (downregulation) also exhibit similar phenotypes [19]. Transgenic expression of the sorghum DREB2 gene in rice effectively increased tolerance and yield under water-limited conditions. Notably, compared with wild-type plants, transgenic SbDREB2-overexpressing plants under drought stress presented a slightly greater number of panicles [20]. Research on banana plants has demonstrated that, under low temperatures, osmotic stress, or salt stress, the expression of MaDREB1F is strongly induced. Furthermore, overexpression of MaDREB1F effectively increased the resistance of banana plants to cold and drought stress via a series of mechanisms, including via joint modulation of the levels of protective metabolites such as soluble sugars and proline, activation of the antioxidant system, and stimulation of the synthesis of jasmonate and ethylene [21]. In maize, overexpression of the ZmDREB2A gene can increase tolerance to heat stress [22]. Additionally, the expression of ZmDREB2.9-S, ZmDREB2.2 and ZmDREB2.1/2A tends to increase under cold stress, drought stress and ABA treatment, and these genes might have dual functions in strengthening stress resistance in maize [23]. Investigations into the DREB genes of soybean have shown that natural variations in the expression of DREB3a and DREB3b are significantly correlated with differences in the salt tolerance of soybean germplasms. In addition, the absence of the DREB3b^39Del allele during the domestication of cultivated soybean is potentially linked to a decrease in salt tolerance [14]. In tomato, the expression levels of SlDREB2 increased significantly in both roots and young leaves when they were exposed to NaCl, and further analysis demonstrated that this gene could also be activated in response to KCl treatment and drought conditions [24]. In addition, a study of Moso bamboo indicated that PeDREB expression could be triggered by various abiotic stresses, including salt, drought, and cold stress. Moreover, overexpression of PeDREB28 was shown to increase the tolerance of plants to abiotic stress [7]. In brief, the functions of different DREB genes in the response to abiotic stress vary across species. Therefore, conducting genome-wide and systematic analyses of DREB family members is crucial for understanding the mechanisms underlying their role in abiotic stress tolerance.

Barley (Hordeum vulgare L.), a diploid self-pollinating crop with a genome size of 5.1 Gb, is a member of the genus Hordeum of the family Poaceae [25]. It ranks fourth globally in terms of both the planting area and yield among cereal crops and has a diverse range of uses, including applications in animal feed, food, and medicine [26]. The DREB gene family has been successfully identified and intensively investigated in a diverse array of plants, including Arabidopsis, maize, rice, wheat, soybean, and Moso bamboo. However, research on the DREB gene family in barley remains relatively limited. Considering the latent value of DREB genes in enhancing stress tolerance, performing an elaborate and comprehensive in-depth analysis of the DREB gene family in barley is highly important. Therefore, in the present study, a comprehensive analysis of the gene structure, motif composition, chromosome location, gene duplication, and transcriptomic expression patterns of a total of 39 HvDREB gene family members was conducted. Additionally, the expression of HvDREB genes in root, stem, and leaf tissues under drought and salt stress was explored in detail. The findings from this research offer valuable insights and serve as an essential foundation for future functional investigations on DREB genes in barley.

Results

Identification of the barley DREB gene family

A hidden Markov model (HMM) search and a BLASTP search of the whole-genome-based protein sequences of H. vulgare were conducted by utilizing the AP2 domain (PF00847) and DREB protein sequences of Arabidopsis thaliana. On the basis of the amino acid sequence variations outlined by Hu et al. [8], additional comprehensive studies were conducted to evaluate and infer the reliability and overall integrity of HvDREB candidates. Following the removal of redundant and unnecessary gene sequences, a total of 39 HvDREBs from the H. vulgare genome were successfully identified and subsequently named on the basis of their corresponding chromosomal positions (ordered from top to bottom of each chromosome) as HvDREB1–HvDREB39 (Table 1). By conducting multiple sequence alignments of the DREB domains in HvDREB proteins, this study aimed to examine the conserved domains present within these proteins. Furthermore, the phylogenetic relationships among the HvDREB proteins were analyzed to elucidate their evolutionary connections. In this study, all 39 identified HvDREB proteins contained a single conserved DNA-binding AP2 domain and exhibited complete conservation of the characteristic valine (V) and glutamate (E) residues, as indicated by asterisks in the sequence alignment chart (Fig. 1A). The SWISS-MODEL prediction results demonstrated the three-dimensional structure of the AP2 domains of all the HvDREB proteins. This structure consisted of a three-strand antiparallel β-sheet1, β-sheet2 and β-sheet3, along with an α-helix that was approximately parallel to the β-sheet (Fig. 1B, C). All the above outcomes demonstrated the reliability of the HvDREB members screened from barley. An analysis of the physicochemical properties of the proteins encoded by the 39 identified DREB family genes was conducted (Table 1). The findings revealed that the coding sequence (CDS) lengths varied from 180 to 1662 bp, the amino acid sequences ranged from 60 to 553 aa in length, and the predicted protein molecular weights ranged between 6.68 and 59.94 kDa. Among these proteins, HvDREB29 had the highest molecular weight, whereas HvDREB11 had the lowest. The theoretical isoelectric point of HvDREB21 was the highest, reaching 12.14, and that of HvDREB7 was the lowest, at 4.74. There were 25 proteins whose isoelectric points were less than 7, which are acidic in nature. The remaining 14 HvDREB proteins had isoelectric points greater than 7 and were alkaline. Additionally, the instability index (II) of the identified HvDREB proteins ranged from 25.92 (for HvDREB13) to 86.81 (for HvDREB21); the aliphatic index (AI) ranged from 41.13 (for HvDREB13) to 81.57 (for HvDREB18); and the grand average of hydropathicity (GRAVY) values ranged from − 0.95 (for HvDREB21) to -0.08 (for HvDREB24). Given that the GRAVY values of all HvDREB proteins were less than 0, the HvDREB proteins were hydrophobic. Moreover, among the 39 proteins, only HvDREB8 possessed two transmembrane domains, and the other proteins lacked transmembrane domains.

Table 1.

Information about the HvDREB genes in barley

Gene name	Gene locus	CDS Length (bp)	AAa	MWb	pIc	IId	A.l.e	GRAVYf	TMDg	SLPh
HvDREB1	HORVU0Hr1G016370.1	750	250	26.17	5.72	55.88	60	-0.56	0	nucleus
HvDREB2	HORVU0Hr1G016540.1	654	217	23.14	5.27	60.01	58.62	-0.44	0	nucleus
HvDREB3	HORVU0Hr1G018240.1	660	219	22.99	5.74	43.21	60.05	-0.47	0	nucleus
HvDREB4	HORVU0Hr1G018280.1	657	218	23.72	5.48	51.76	63.39	-0.56	0	nucleus
HvDREB5	HORVU0Hr1G018290.1	750	249	26.05	5.63	56.71	59.08	-0.55	0	nucleus
HvDREB6	HORVU0Hr1G030500.1	660	219	22.99	5.74	43.21	60.05	-0.47	0	nucleus
HvDREB7	HORVU1Hr1G056090.1	831	276	29.39	4.74	70.54	61.38	-0.60	0	chloroplast
HvDREB8	HORVU1Hr1G060490.5	1359	452	49.44	4.76	64.65	79.16	-0.26	2	nucleus
HvDREB9	HORVU1Hr1G070410.1	1164	387	41.98	8.68	45.54	67.34	-0.40	0	chloroplast
HvDREB10	HORVU1Hr1G090250.1	891	296	32.02	9.47	58.75	68.48	-0.39	0	chloroplast outer membrane
HvDREB11	HORVU2Hr1G094810.1	180	60	6.68	10.89	57.65	60.33	-0.48	0	nucleus
HvDREB12	HORVU2Hr1G097850.4	747	249	22.91	11.65	37.07	69.04	-0.28	0	chloroplast
HvDREB13	HORVU2Hr1G113940.3	882	293	19.36	9.52	25.92	41.13	-0.72	0	nucleus
HvDREB14	HORVU3Hr1G017950.12	648	215	23.35	5.02	38.26	59.53	-0.63	0	nucleus
HvDREB15	HORVU3Hr1G026810.1	666	221	23.82	4.83	61.38	54.89	-0.50	0	nucleus
HvDREB16	HORVU3Hr1G026950.1	405	135	14.19	9.94	69.98	51.7	-0.60	0	nucleus
HvDREB17	HORVU3Hr1G090740.2	627	209	21.55	9.75	54.79	62.78	-0.40	0	chloroplast
HvDREB18	HORVU4Hr1G015350.1	654	217	23.83	5.5	46.92	81.57	-0.31	0	chloroplast
HvDREB19	HORVU4Hr1G061340.1	690	229	24.13	5.68	61.92	63.28	-0.30	0	nucleus
HvDREB20	HORVU4Hr1G075810.1	948	315	33.04	6.31	52.68	62.51	-0.44	0	nucleus
HvDREB21	HORVU5Hr1G041600.1	405	134	14.90	12.14	86.81	51.87	-0.95	0	nucleus
HvDREB22	HORVU5Hr1G058270.1	852	283	30.47	6.45	52.83	63.39	-0.55	0	nucleus
HvDREB23	HORVU5Hr1G080310.1	666	221	24.58	4.97	58.34	61.49	-0.52	0	nucleus
HvDREB24	HORVU5Hr1G080340.1	702	233	24.90	5.12	56.32	77.17	-0.08	0	nucleus
HvDREB25	HORVU5Hr1G094730.1	573	190	19.75	6.19	52.86	61.53	-0.31	0	nucleus
HvDREB26	HORVU6Hr1G038120.3	621	206	22.26	9.75	68.24	63.98	-0.57	0	nucleus
HvDREB27	HORVU6Hr1G050500.2	903	300	32.61	9.62	69.32	54.03	-0.81	0	nucleus
HvDREB28	HORVU6Hr1G050520.2	714	237	25.39	6.33	63.72	59.75	-0.57	0	nucleus
HvDREB29	HORVU6Hr1G060140.1	1662	553	59.94	9.81	75.74	68.32	-0.49	0	mitochondrion
HvDREB30	HORVU6Hr1G060220.1	744	247	23.17	5.25	40.84	53.12	-0.60	0	nucleus
HvDREB31	HORVU6Hr1G062940.1	660	219	22.99	5.74	43.21	60.05	-0.47	0	nucleus
HvDREB32	HORVU6Hr1G065430.3	813	270	28.62	6.22	54.18	55.63	-0.54	0	chloroplast outer membrane
HvDREB33	HORVU6Hr1G065580.4	678	225	21.23	5.64	49.4	50.4	-0.47	0	nucleus
HvDREB34	HORVU6Hr1G074970.1	993	330	35.54	6.23	62.25	64.39	-0.45	0	nucleus
HvDREB35	HORVU6Hr1G082770.1	1170	389	43.88	8.94	46.81	76.66	-0.50	0	chloroplast
HvDREB36	HORVU7Hr1G026940.1	549	182	19.30	10.5	62.97	65.11	-0.62	0	chloroplast
HvDREB37	HORVU7Hr1G029870.3	492	163	17.78	6.29	45.36	61.23	-0.45	0	nucleus
HvDREB38	HORVU7Hr1G037180.1	1116	371	39.65	6.4	68.81	59.68	-0.53	0	chloroplast outer membrane
HvDREB39	HORVU7Hr1G089930.5	684	227	24.16	9.3	71.07	74.49	-0.37	0	nucleus

^aLength of the amino acid sequence

^bMolecular weight of the amino acid sequence

^cIsoelectric point of the HvDREB proteins

^dInstability Index

^eAliphatic Index

^fGRAVY (Grand Average of Hydropathicity) the hydrophilic or hydrophobic nature of the protein; positive values indicate hydrophobic proteins, while negative values indicate hydrophilic proteins

^gNumber of transmembrane domains, as predicted by the TMHMM server

^hProtein subcellular localization prediction by the BUSCA web server (https://busca.biocomp.unibo.it/)

Fig. 1.

Identification of HvDREBs in barley. A Multiple sequence alignment and sequence feature analysis of the conserved domain proteins of the barley DREB gene family. B 3-D structural models of the AP2 domain of barley DREB proteins. C AP2 domain sequence logo produced by TBtools II. The sequence logo was acquired by means of multiple alignments of the AP2 domain within the HvDREB protein sequences of barley. The heights of the symbols are directly proportional to the frequency of occurrence of the corresponding amino acid residue at the specific position. D The phylogenetic tree of DREB proteins from barley (Hv, Hordeum vulgare), Arabidopsis (At, Arabidopsis thaliana), Moso bamboo (Pe, Phyllostachys edulis) rice (Os, Oryza sativa) and tomato (Sl, Solanum lycopersicum) was constructed using the maximum likelihood (ML) method with 1000 bootstrap replicates. The blue circles located on the branches of the phylogenetic tree indicate the magnitude of the bootstrap values

To investigate the evolutionary relationships within the DREB gene family across different plant species, we used the amino acid sequences of 39 HvDREBs from barley (Table S1), 44 PeDREBs from Moso bamboo (Phyllostachys edulis), 56 AtDREBs from Arabidopsis, 57 OsDREBs from rice (Oryza sativa), and 57 SlDREBs from tomato (Solanum lycopersicum) to construct an ML phylogenetic tree (Fig. 1D). On the basis of the classification of DREB proteins from A. thaliana into subgroups A1–A6 (Table S2), we found that the barley DREB proteins could be roughly divided into five subgroups, namely, A1, A2/A3, A4, A5, and A6. Among them, A1 included 5 HvREB proteins, A2/A3 included 11 HvREB proteins, A4 included 13 HvREB proteins, A5 included 5 HvREB proteins, and A6 included 5 HvREB proteins.

Conserved motif, domain and gene structure analysis of HvDREBs

To characterize the specific functional regions of members of the barley DREB gene family, the conserved motifs of barley DREB proteins were systematically detected and analyzed using the online software MEME. Twelve motifs, characterized by widths ranging from 6 to 200 aa, were identified within the 39 proteins. These motifs were then analyzed using the MEME tool to investigate their structural properties and distribution patterns across the proteins. The conserved AP2 domain of the identified DREB proteins, comprising motifs 1, 2, 3, and 4, contains key DNA-binding sites within motif 2 and motif 4. These two motifs are thus proposed to form the functional core unit responsible for the activity of the AP2 domain. In addition to having common motifs, certain members within specific branches of the DREB proteins in group 1 presented additional motifs 6, 7, and 10 (HvDREB6). Moreover, other members from different branches of the same group had extra motifs 7, 10, and 11 (HvDREB32). Notably, all the proteins in group 1 were classified under subgroups A4 and A5. Members of the DREB proteins belonging to group 2 were found to possess an additional motif 5, as well as motif 9, 10, or 12. The third group had an additional motif 7, 9 or 10, and the branches containing motif 9 all belonged to subgroup A1 (Fig. 2). Additionally, a conserved domain analysis revealed that all the HvDREB proteins possessed a conserved AP2 domain. The results of the gene structure analysis demonstrated that the HvDREB proteins harbored 1–3 exons. Moreover, a majority of the HvDREB proteins harbored UTRs. Nevertheless, several proteins that belonged to subgroup A1 and contained motif 9, specifically HvDREB2, 23, 24, and 33, lacked UTRs (Fig. 2). As shown in Fig. 2, the conserved motifs within each subfamily were essentially identical, and the gene structures across these subfamilies also exhibited notable similarities. These findings suggest that genes within a given subfamily are likely to have similar functions.

Fig. 2.

Conserved motifs, conserved domains and gene structure of HvDREB proteins in barley. Various colors indicate distinct motifs, conserved domains, CDSs and UTRs both in the figure and in the upper right corner. The black lines separately denote nonconserved sequences in the MEME results and introns within the exon–intron structure. The phylogenetic tree was constructed in the same way as that in Fig. 1D

Analysis of the chromosomal location and collinearity of HvDREBs

As shown in Fig. 3, the HvDREB genes are distributed unequally across the seven chromosomes of barley. Chromosome chr6H harbors the greatest number of genes, with a total of ten genes, followed by chrUn with six genes and chr5H with 5 genes. Moreover, chromosomes chr1H, chr2H, chr3H, chr4H, and chr7H possess 4, 3, 4, 3, and 4 genes, respectively. The expansion of gene families during evolution is a common phenomenon that enables organisms to acquire more genetic material and functional diversity. Tandem repeats and segmental duplications are two important and common molecular mechanisms associated with the expansion of gene families. To improve our understanding of the evolution of HvDREB genes, we examined five sets of segmental duplication gene pairs within the HvDREB gene family (Fig. 3). Among these pairs, two were composed of HvDREB gene family members (blue lines), whereas the remaining three pairs (yellow lines) included an HvDREB gene and a transcript encoding proteins corresponding to these 39 members. These five pairs of genes were as follows: HvDREB12 paired with HvDREB31, HvDREB34 with HvDREB38, HvDREB17 with HORVU1Hr1G064030.1, HvDREB29 with HORVU2Hr1G090970.4, and HvDREB33 with HORVU2Hr1G094780.2 (Table S3). Furthermore, we performed a detailed analysis of the values associated with synonymous substitutions (Ks) and nonsynonymous substitutions (Ka) to explore the selective pressures underlying the duplication events of HvDREB genes, with the analysis covering all the nucleotide sequences. The consistently low Ka/Ks values (< 1) observed for these five paralogous gene pairs demonstrate their evolutionary conservation under purifying selection pressure (Table S4).

Fig. 3.

Genome-wide syntenic and localization analysis of the HvDREB genes in barley. The circles from the inside to the outside in the Circos plot represent the chromosome architecture, GC ratio, GC skew, N ratio and gene density. The HvDREB genes in barley were positioned on diverse chromosomes. The chromosome numbers are presented in black text on the chromosomes. The HvDREB gene pairs with syntenic connections are linked by blue lines. The gene pairs on which one side was the identified HvDREB gene are denoted by a red line. The names of the HvDREB genes are positioned at the outermost part of the Circos plot. The gray lines in the background represent other gene pairs within barley

Collinearity analysis of the DREB gene family of barley and other species

To further explore the origin and evolutionary trajectory of the HvDREB gene family in barley, H. vulgare, we constructed collinearity maps of barley along with Brachypodium distachyon, Oryza sativa, A. thaliana, and Solanum lycopersicum (Fig. 4). Barley shares seven orthologous genes with A. thaliana and eleven orthologous genes with S. lycopersicum. Among them, five genes (highlighted with yellow lines) are orthologous among barley, (A) thaliana, and S. lycopersicum (Fig. 4A). As shown in Fig. 4B, the quantity of orthologous genes identified in the monocotyledonous species O. sativa and (B) distachyon was significantly greater than that in the dicotyledonous species (A) thaliana and S. lycopersicum. Barley shares 25 orthologous genes with O. sativa and shares 25 orthologous genes with (B) distachyon. Among these orthologous genes, 20 genes are concurrently present in all three species. Only a handful of genes in barley have no orthologs in the other two species. Regardless of whether the plants were monocotyledonous or dicotyledonous, the distribution of orthologous genes was not even. Most of the orthologous genes were present on the chr6H chromosome, with this phenomenon being particularly pronounced in monocotyledonous plants. Details regarding the information above are presented in Table S5.

Fig. 4.

Synteny analysis of HvDREB genes between barley and four other plant species. A Synteny analysis of barley HvDREB genes in (A) thaliana and S. lycopersicum. The HvDREB genes that are syntenic among all three species are connected by yellow lines, whereas the HvDREB genes that are syntenic in only two of the species are connected by blue lines. B Synteny analysis of barley HvDREB genes in O. sativa and (B) distachyon. The syntenic HvDREB genes are connected by blue lines. The gray lines in the background of (A) and (B) represent the syntenic blocks between the barley genome and the genomes of the corresponding species. Chromosomes are colored differently in different species. The species names “A. thaliana”, “H. vulgare”, “S. lycopersicum”, “B. distachyon”, and “O. sativa” correspond to Arabidopsis thaliana, Hordeum vulgare, Solanum lycopersicum, Brachypodium distachyon, and Oryza sativa, respectively

Cis-acting element analysis

To identify the factors capable of influencing the expression of HvDREB genes and the regulatory pathways in which DREB genes might be involved, we extracted the regulatory sequence within the 2-kb region upstream of the translation initiation site (ATG) and carried out a cis-acting element analysis using PlantCARE and TBtools software. A variety of cis-acting elements were identified from barley DREBs. Beyond the conventional promoter elements (TATA box and CAAT box), five other elements were either detected in a majority of the members or held particular relevance in terms of functionality. These five promoter elements can be grouped into two primary categories: the hormone-responsive group, encompassing MeJA-responsive (CGTCA motif, TGACG motif) and ABA-responsive (ABRE) elements, and the stress-responsive group, including low-temperature-responsive (LTR) elements and the MYBHv1 binding site (CCAAT box). As illustrated in the Fig. 5, the findings indicate that barley DREB genes might contribute to the response of barley to hormones and stress conditions and that the expression of these DREB genes could be influenced by relevant hormones as well as stress-related cis-acting elements. Furthermore, typically, the genes belonging to the same branch of the phylogenetic tree, such as HvDREB3, HvDREB6, and HvDREB31, harbor identical cis-regulatory elements. Nevertheless, certain disparities in cis-acting elements are observable among genes of different branches (HvDREB27 and HvDREB32), for example, with respect to the quantity of elements. This suggests that among the diverse HvDREB genes in barley, some exhibit similar expression patterns because they are regulated by the same factors, whereas others present relatively significant differences in regulatory and expression modalities, which might also account for their capacity to function in multiple physiological processes (Fig. 5; Table S6).

Fig. 5.

Predicted cis-acting regulatory elements in barley DREB promoters. Putative cis-elements were detected in 2-kb promoters upstream of barley DREB genes. The phylogenetic tree is the same as that shown in Fig. 1D

Identification of miRNAs targeting HvDREB genes in barley

To more thoroughly investigate the role of microRNAs (miRNAs) in the posttranscriptional modification of barley HvDREB members, we performed a comparative analysis with psRNATarget software against all the mature barley miRNAs archived in the miRbase database. Ultimately, we accurately identified 14 varieties of miRNAs that are capable of targeting 16 genes (Fig. 6A). To present the information in a more visually intuitive manner, we have illustrated the miRNA targeting sites of HvDREB8 and HvDREB27 in Fig. 6B and C. Moreover, all the relevant miRNA targeting sites and corresponding genes are listed in the Supplementary Table. A gene may be under the regulatory influence of multiple miRNAs, or a single miRNA may simultaneously direct its targeting effect toward multiple genes. For example, five miRNAs specifically target and regulate the HvDREB8 gene through cleavage-based inhibition mechanisms (Fig. 6B). Similarly, hvu-miR5049a exerts regulatory effects on two genes (HvDREB27 and HvDREB28) via cleavage-based inhibition mechanisms (Fig. 6C). This phenomenon highlights the complex and multiplex regulatory network formed by microRNAs within the genomic environment. Furthermore, the quantitative correlation between genes and miRNAs, along with the inhibition mechanisms mediated by translation (7) and cleavage (17), are shown in Fig. 7.

Fig. 6.

miRNAs targeting HvDREB genes in barley. A The miRNA target network mapping for HvDREB genes is presented, where blue squares symbolize HvDREB genes and yellow circles represent predicted miRNAs. B The graphical illustration clearly shows that the HvDREB8 gene is targeted by miRNAs such as hvu-miR156a, hvu-miR156b, hvu-miR444a, hvu-miR5049b, and hvu-miR5049e. C A single miRNA, hvu-miR5049a, targets two genes, namely, HvDREB27 and HvDREB28. chr1H and chr6H are indicative of chromosomes

Fig. 7.

Sankey diagram for the relationships of miRNAs targeting HvDREB genes. The three columns represent the miRNA, gene, and inhibition effects

Analysis and prediction of regulatory interactions among genes on the basis of transcription factor-binding motifs

By employing the binding motifs for transcription factors to search for binding sites located within the promoter regions of the gene set, we constructed a preliminary transcriptional regulatory network among genes, relying on mutual regulatory mechanisms. The results demonstrated that HvDREB14 (HORVU3Hr1G017950.12), HvDREB8 (HORVU1Hr1G060490.5), and HvDREB4 (HORVU0Hr1G018280.1) occupied crucial and central positions within the entire regulatory network (Figure S1).

Tissue expression analysis of the HvDREB gene family in barley

The barley transcriptomic data were obtained, and the expression levels of the different members of the HvDREB gene family in various tissues, such as germinating embryos, seedling roots, stem internodes, shoots, 5-mm and 5-cm inflorescences, and 5-day and 15-day caryopses, were analyzed (Fig. 8 and Table S7). A total of 37 HvDREB genes with transcripts per million (TPM) expression data were obtained. The phylogenetic tree from the expression heatmap shows that these genes can be divided into three main branches. Members in the first branch generally exhibit low relative expression levels in these barley tissues, except for a few, such as HvDREB37 and HvDREB12, whose expression is relatively high in developing grains. The expression of members of the second branch is relatively high in some tissues, especially in 5-mm inflorescences, lodicules, rachises, and palea. The expression of members of the third branch is relatively high in nearly all the tissues, with HvDREB22 having the highest expression. These results suggest that the HvDREB genes have both similar and differential expression patterns in different tissues, highlighting the crucial role played by this gene family in barley growth and development, as well as the possible functional divergence among its members.

Fig. 8.

Differential expression of representative HvDREB genes in diverse barley tissues revealed by barley-reported RNA-seq data. The legend depicts the logarithmic values of transcripts per kilobase million (TPM). The transcriptome expression outcomes are visualized in the form of a heatmap with blue, yellow, and magenta colors, where distinct colors indicate clusters of low and high expression

Protein–protein interaction network analysis of HvDREBs using STRING 12.0

To investigate the relationships among the entire set of HvDREBs, we constructed a protein interaction network by querying the 39 protein sequences to the Arabidopsis Information Resource (TAIR) database in STRING for k-means clustering (Figure S2). The constructed protein–protein interaction network revealed that the proteins associated with AP2 (an integrase-type DNA-binding superfamily protein), ZAT6 (zinc finger of Arabidopsis thaliana 6), and MYB15 as the cores could be divided into three clusters. These three clusters were named “ethylene response correlation” (blue), “AP2 transcription factor-related” (red), and “stress-related” (green).

Analysis of barley HvDREB gene expression via qRT–PCR

Examination of the cis-acting elements revealed that HvDREB genes participate in the response to abiotic stress. To characterize the response of HvDREB genes to the influence of abiotic stress, qRT–PCR was used to explore the changes in the expression levels of four genes (HvDREB7, 14, 22, 38) in response to treatment with 10% PEG6000 and 150 mM NaCl. Compared with the control seedlings, the seedlings subjected to one week of stress presented distinct phenotypic differences (Figure S3). Specifically, compared with the control seedlings, the seedlings under NaCl stress and PEG6000 stress were clearly shorter in stature and presented shorter root systems. Moreover, the leaves of the plants under PEG6000 stress were clearly curled. Compared with those in the control, the expression levels of the four genes in the roots and stems tended to be high under NaCl stress, and the differences were significant or extremely significant (Fig. 9). The expression levels of the HvDREB7 and HvDREB14 genes in NaCl-stressed leaves differed significantly from those in the control group, whereas the expression of HvDREB22 and HvDREB38 did not significantly differ (Fig. 9A–D). Interestingly, the expression trends in the roots, stems and leaves in response to PEG6000 stress differed considerably from those observed under NaCl stress. With the exception of HvDREB14, the expression of the other three genes was substantially downregulated in the roots of plants exposed to PEG6000 stress compared with those in the control. Specifically, HvDREB7, HvDREB22, and HvDREB38 were downregulated, with fold changes (FCs) of 4.69, 1.88, and 3.43, respectively (Fig. 9A, C, D). The expression of HvDREB7, HvDREB14 and HvDREB22 did not significantly differ between the stems of the control group and those of the stressed group. However, compared with that in the control group, the expression of HvDREB38 in the stems of the stressed group was extremely significantly upregulated (2.25-FC, P < 0.01). The expression of HvDREB7 and HvDREB14 was significantly upregulated (2.67-FC, P < 0.01 and 2.70-FC, P < 0.01, respectively) in the leaves of plants subjected to NaCl stress compared with the control leaves, whereas the expression of the other two genes did not significantly differ (Fig. 9A–D). In the PEG-stressed leaves, HvDREB7, HvDREB22 and HvDREB38 expression was downregulated, with the latter two genes exhibiting a highly significant difference (P < 0.01). In comparison with the expression levels observed in the control leaves, HvDREB14 expression was extremely significantly upregulated (2.27-fold change, P < 0.01) in the stressed leaves (Fig. 9A-D and Table S8).

Fig. 9.

Comparison of the relative expression levels of four HvDREB genes in the roots, stems and leaves of barley under normal conditions and under stress. The x-axis represents the root, stem and leaf tissues under normal conditions, salt stress and drought stress. The y-axis represents the relative gene expression levels. The capped lines indicate the standard error. * P < 0.05; ** P < 0.01

Discussion

DREBs are a class of important transcription factors that can induce the expression of genes associated with abiotic stress and endow plants with the ability to resist stress. Owing to their essential roles in plant growth and the regulation of responses to multiple abiotic stressors, DREB transcription factors have been extensively investigated across a broad spectrum of plant species, especially with respect to their role in the response to drought and salt stress [27]. Barley, which is a crucial agricultural crop globally, currently ranks fifth in terms of production volume (in tons) among grain crops, trailing behind maize, wheat, rice, and soybean [28]. Its nuclear genome is rich in genetic diversity, which makes it quite attractive for stress tolerance breeding and makes it an important model crop for the study of stress responses, as it has great potential for application in crop improvement [29]. However, there is still very limited systematic research information regarding barley HvDREB genes. Therefore, further in-depth research and a comprehensive understanding of the sequence features of DREB transcription factors as well as their response to drought and salt stress are essential.

Here, a total of 39 DREB genes in barley were identified, and they exhibited an uneven distribution pattern across the 7 chromosomes of barley. On the basis of their structural characteristics, members of the DREB family are classified into six subgroups, designated A1 to A6. Among these, subgroups A1 and A2 are considered typical DREB proteins [30]. In barley, the five HvDREB members within subgroup A1 are potentially involved in the response to low-temperature stress [31]. The eleven HvDREB members of subgroup A2/A3 are associated with drought and salt stress, as well as abscisic acid (ABA) and sugar signaling pathways [30, 32]. The thirteen HvDREB proteins from the A4 subgroup may function in drought and cold stress responses and are also involved in ethylene and methyl jasmonate signaling [33]. The five HvDREB members of the A5 subgroup likely contribute to the regulation of drought and freezing tolerance [34]. Finally, the five HvDREB proteins in subgroup A6 may participate in responses to drought and salt stress through mechanisms involving light- and ethylene-mediated developmental processes [35]. Furthermore, the results of the phylogenetic tree indicated that all HvDREB members clustered closely with the DREB members from rice or Moso bamboo, which may reflect a closer genetic relationship among these monocotyledonous plants belonging to the Poaceae family (Fig. 1). Notably, the DREB gene family in barley characteristically displays conserved domains and motifs (Fig. 1). It is highly likely that genes exhibiting similar motif distributions or gene structures may be evolutionarily conserved and, thus, serve equivalent functions. Members of the A2/A3 subfamily cluster together on the same clade of the barley phylogenetic tree and exhibit high similarity in both motif composition and gene structure, such as HvDREB8, 14, 20, 26, 27, 28, 35, and 39 (Fig. 2). The cis-acting elements associated with these genes are related to stress response and ABA signaling pathways (Fig. 5).

Gene structure analysis, which involves examination of the quantity and arrangement of exons and introns, plays a pivotal role in providing an in-depth understanding of gene functions [36]. Most members of the HvDREB gene family are devoid of introns. This structural idiosyncrasy potentially endows the transcription process with enhanced efficiency, obviating the need for intricate intron splicing procedures and thereby permitting the expeditious synthesis of corresponding transcripts and a more rapid enactment of their biological functions [37]. Furthermore, structural analysis revealed that 64% (25/39) of the DREB gene family constituents lack introns. Among the members of the HvDREB gene family that possess introns, all but HvDREB8 possess only one or two exons. These findings are consistent with those of previous reports on species such as grape (Vitis vinifera) [38], Chinese jujube (Ziziphus jujuba) [39], pineapple (Ananas comosus) [40] and maize [9]. The relatively limited number of exons implies that the gene has a relatively simple and direct mechanism of expression regulation and protein coding and is likely influenced by a relatively small number of regulatory elements, thus rendering its functional manifestation more stable and specific.

Chromosomal or genomic duplications, which are crucial to evolution, generate novel gene functions via mutations and alter expression patterns under diverse regulatory elements, providing a genetic impetus for biological evolution [41, 42]. Typically, segmental duplications expand genes, regulate gene expression, and evolve genome structure and chromosomes, whereas tandem duplications contribute to gene expression, genetic diversity, and environmental adaptation [43]. The occurrence of purifying selection pressure within the duplicated HvDREB genes in barley aligns with a broader pattern observed across various plant species. Specifically, this pattern reflects the important evolutionary influence of purifying selection on duplicated genes within relevant gene families. Similar trends have been documented in species such as wheat [44], quinoa [45], peanut [46] and lettuce [47], where duplicated genes also appear to be subject to strong purifying selection forces. In our research, no tandem duplications were detected. However, five pairs of segmental repeat genes were discovered, three of which were located on chr2H and chr6H (Fig. 3). These findings suggest that segmental duplications are likely to contribute substantially to the proliferation of DREB family members in barley. Furthermore, synteny analysis was employed to evaluate the presence of orthologous genes among different species. The number of collinear gene pairs between barley and monocotyledonous plants such as rice and B. distachyon is substantially greater than that between barley and dicotyledonous plants such as Arabidopsis and tomato. This difference may be associated with the degree of evolutionary relatedness or with distinct selective pressures. Barley shares closer phylogenetic affinity and analogous evolutionary trajectories with rice and related monocotyledonous species. Congruence in genomic replication and rearrangement events has resulted in a greater number of collinear gene pairs. In contrast, barley diverged from the dicotyledonous species Arabidopsis and tomato at an earlier evolutionary juncture. This has led to pronounced differences in gene function divergence, exposure to disparate selective pressures, and considerable differences in the dynamic alterations of genomic structure, thereby accounting for the paucity of collinear gene pairs in the latter comparison. Furthermore, certain genes (e.g., HvDREB34 and HvDREB38) of barley exhibit synteny in both monocotyledonous and dicotyledonous plants (Fig. 4; Table S5). These genes were highly likely inherited from a common ancestor. During the processes of speciation and differentiation, the genomic regions in which these genes are located maintained relative stability and were not readily modified by evolutionary pressures.

Cis-acting elements perform essential and irreplaceable functions in the processes of plant growth and development, plant hormone responses, and stress responses [48]. In barley, DREB genes harbor a diverse range of cis-acting elements. In addition to the fundamental response elements that are crucial for basic plant life activities, DREB genes harbor several elements that participate in the regulation of adverse stress conditions and plant hormones. The intricate regulatory networks controlling the expression of HvDREB genes were revealed by the identification of multiple cis-acting elements associated with hormone responses (MeJA and ABA) and stress signals within the promoters. Comparable findings have been documented in other plant species [49, 50]. Within the above species, cis-acting elements are likely to actively regulate genes under abiotic stress. Studies have shown that DREB proteins can activate stress responses in an ABA-independent manner through DREBs [51]. ABA integrates a diverse range of stress signals and modulates downstream stress responses, thereby facilitating the ability of plants to acclimate to various stress environments via important regulatory mechanisms [52]. In addition, cis-acting elements related to MeJA responsiveness were detected in most of the members (Fig. 5). A recent study demonstrated that in Arabidopsis, DEAR4, a member of the DREB1/CBF family, is linked to leaf senescence and can be induced by ABA, JA, darkness, drought, and salt stress [53]. Notably, 59% of the members contained LTR cis-acting elements, indicating that these HvDREB genes could be involved in the regulation of responses to low-temperature stress. Similar findings have also been reported in relevant studies on quinoa [54].

In this study, a total of 14 putative barley miRNAs (hvu-miRNAs) were successfully identified, with 16 HvDREB genes identified as the targets of these miRNAs. miRNAs, which are widely distributed in plant cells, constitute a class of small nuclear regulatory molecules that are capable of directly regulating the expression of target genes through posttranscriptional repression [55]. Our prediction results revealed that miRNAs and target genes formed an intricate and interconnected regulatory network (Fig. 6A). The expression of a single gene could be finely tuned by the combination of multiple miRNAs, or one miRNA could regulate several genes. For example, five miRNAs target HvDREB8, and hvu-miR5049a regulates both HvDREB27 and HvDREB28 (Fig. 6B, C). These phenomena are consistent with the findings regarding miRNAs obtained by previous investigators [56].

According to the qRT–PCR results, the expression of four HvDREB genes in the roots, stems, and leaves of barley plants exposed to NaCl stress and PEG stress differed from that in normal plants (Fig. 9). On the basis of the RNA-seq data, in the high-expression clustering modules of the barley DREB family, most members presented high expression in inflorescences (5 mm), lodicules (6 weeks), rachises (5 weeks), palea (6 weeks) and seedling roots, with HvDREB22 exhibiting the highest expression (Fig. 8). Furthermore, the expression levels of these genes were observed across diverse tissues across 16 different developmental stages. We selected four representative genes for in-depth analysis: HvDREB7, which was moderately expressed according to the RNA-seq data; HvDREB14, a core gene identified through transcription factor binding motif-based regulatory network analysis (Fig S1); HvDREB22, the most highly expressed gene according to the RNA-seq data; and HvDREB38, whose collinearity was confirmed by intraspecific synteny analysis. Using qRT–PCR, we analyzed the expression of these genes in the root, stem, and leaf tissues of barley under salt and drought stress conditions. Elucidating the expression dynamics of four representative HvDREB genes under salt and drought stress revealed differential expression patterns. These results are consistent with those of previous studies showing that barley DREB genes exhibit differential expression patterns under abiotic stress and phytohormone treatments [57]. These differences likely reflect the distinct adaptive mechanisms barley uses to counteract ionic versus osmotic stress. The consistent and significant upregulation of HvDREB genes across roots, stems, and leaves under NaCl stress suggests a coordinated whole-plant response to ionic imbalance. This widespread activation is characteristic of a generalized salt overly sensitive signaling pathway, which is rapidly triggered to reestablish cellular ion homeostasis through the regulation of ion transporters and the synthesis of protective osmolytes [58]. The pronounced upregulation in roots and stems, in particular, underscores the critical roles these organs play as the primary sites of ion exclusion and the central conduits for ion and solute transport, respectively [59]. In contrast, the expression trends in different tissues under PEG stress significantly differed, indicating that barley may employ more targeted and organ-specific strategies to cope with osmotic stress and water deficit. The broad downregulation of HvDREB7 across all tissues may indicate a strategic shutdown of certain metabolic pathways to conserve energy under severe water scarcity [60]. The expression of the genes HvDREB22 and HvDREB38 significantly decreased in roots and leaves but increased in stems, with HvDREB38 showing an especially pronounced and extremely significant increase in expression (Fig. 9A, C, D). This difference in expression may reflect distinct adaptation priorities among various tissues. The downregulated expression in roots and leaves could be part of a resource reallocation program, which prioritizes survival by suppressing nonessential functions [61]. The significantly upregulated expression of HvDREB38 in stems suggests that this transcription factor may play a specific role in maintaining hydraulic integrity under drought stress to ensure the continuous transport of limited available water from roots to leaves [62].

NaCl stress and PEG stress resulted in distinct expression trends. This disparity might have resulted from differences in stress perception and signal transduction pathways. NaCl stress induces specific signaling pathways to regulate genes in response to a cellular ion imbalance, whereas PEG stress, triggered by a cellular water deficit, affects signal transduction via ABA and other mediators. This finding is also consistent with the presence of ABA-responsive elements in the HvDREB members of the A2/A3 and A6 subclades (Fig. 5). Additionally, the specific physiological functions and adaptive demands of various tissues differ. Roots upregulate ion transport under NaCl stress and shift to water absorption and root structure modulation under PEG stress. Stems ensure substance transport and increase tolerance under NaCl stress and coordinate root–leaf communication under PEG stress. Leaves maintain photosynthesis and resist damage under NaCl stress, regulate stomata and adapt to the substance–energy balance under PEG stress, which may contribute to the differential gene expression trends.

In this study, the DREB gene family in barley was comprehensively characterized using bioinformatics approaches, and its association with osmotic and salt stress responses was investigated through qRT–PCR experiments. However, the specific functions of these genes in stress adaptation remain to be validated through transgenic methods, such as overexpression or CRISPR-mediated gene knockout, which will be the focus of subsequent research. Additionally, the miRNA and PPI networks proposed in this study are based on predictions and require experimental validation. For example, RACE assays could be applied to verify miRNA–target interactions, while yeast two-hybrid or bimolecular fluorescence complementation assays could be used to confirm protein interactions.

Materials and methods

Identification and analysis of DREB genes in barley

Using HMMER 3.0 and the conserved AP2/ERF domain sequence (PF00847) from Pfam (https://www.ebi.ac.uk/interpro/), the DREB genes in barley (Reference genome: Hordeum vulgare r1) were identified. The E value threshold for the HMMER search was established at 0.01 to obtain possible DREB proteins. The DREB protein of Arabidopsis served as the foundational seed sequence utilized to perform a BLAST search against the barley genome. The intersection of the results from the two methods was subsequently obtained and forwarded to the SMART database (http://smart.embl-heidelberg.de/) for verification. A total of 86 DREB proteins were screened. After the proteins from multiple transcripts of the same gene were removed (taking the protein of the longest transcript sequence as the representative), 39 barley DREB proteins were ultimately obtained (Table S1). All 39 HvDREB proteins contained a conserved DNA-binding AP2 domain with the sequence pattern [A-Z]*WV[A-Z]E[A-Z]R[A-Z]*WLG[A-Z](7)A[A-Z]. ExPASy (http://cn.expasy.org/tools) and the BUSCA server (http://busca.biocomp.unibo.it) were used to predict the isoelectric point (pI), theoretical molecular weight (Mw), and protein subcellular localization of the predicted HvDREBs. Visual analysis of sequence alignment was conducted by using the Simple MSA Viewer plug-in of TBtools. The three-dimensional structure of the proteins was estimated through SWISS-MODEL (https://swissmodel.expasy.org/), with HvDREB1 serving as the template.

Phylogenetic analysis

Multiple-sequence alignment of all predicted DREB protein sequences from the genomes of various species, which were selected on the basis of their well-annotated DREB gene families and representation of evolutionarily distinct lineages such as monocots and dicots, was performed using ClustalX 2.1 (http://clustalx.software.informer.com/2.1/). An unrooted phylogenetic tree was subsequently generated by employing the maximum likelihood (ML) method, which was executed by using the Jones–Taylor–Thornton (MEGA11) model with a gamma distribution [63]. The bootstrap resampling number was set to 1000 to evaluate the reliability of the interior branches. Afterward, the resulting phylogenetic tree was imported into the Interactive Tree of Life (iTOL: https://itol.embl.de/) program for further optimization and refinement.

Gene structure analysis and conserved motif detection

The genomic sequences, gff3 files and coding sequences (CDSs) of the HvDREB genes were retrieved from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html). The positions of the introns, exons and conserved domains of the HvDREBs were utilized to construct DREB exon–intron structures via TBtools [64]. The MEME motifs of the predicted HvDREB proteins were detected through MEME suite 5.5.7 (https://meme-suite.org/meme/) on the basis of the following parameters: site distribution, zero or one occurrence per sequence; number of motifs, 12; and motif width, 6–200. These files were subsequently merged and optimized using TBtools [64].

Chromosomal distribution, homologous gene pair and synteny analysis

In accordance with the initial position and length of the chromosomes of the HvDREB genes, the chromosomal distribution was mapped by the Advanced Circos plug-in of TBtools. To analyze the duplication pattern for each HvDREB gene, the BLASTP and MCScanX (Multiple Collinearity Scan toolkit) programs were used in the Bio-Linux system and TBtools [64, 65]. The Ka/Ks ratios of the gene pairs in the duplication blocks were determined through the Simple KaKs Calculator (NG) software plug-in of TBtools. The synteny analysis maps of the three species were generated by utilizing the Dual Synteny Plotter software plug-in of TBtools.

Expression profiles of the HvDREB genes in the RNA-Seq data

The RNA-Seq data were obtained from the H. vulgare Expression Database on the WheatOmics 1.0 website [66]. The transcripts per million (TPM) values of the HvDREB genes were subsequently selected and log² transformed, and a heatmap was created using TBtools.

Analysis of cis-acting elements in HvDREB gene promoters

The 2.0-kilobase sequence upstream of the barley gene CDS was retrieved through the Phytozome database and then uploaded to PlantCARE [67] for the examination of cis-acting elements, which were subsequently visualized using TBtools software.

Prediction of intergenic regulatory relationships on the basis of the binding motifs of transcription factors

TBtools software was used to predict whether binding sites were present in the promoter regions of the gene set on the basis of the binding motifs of the transcription factors. The Fimo binding site scan plug-in was used to scan the promoter sequences of the HvDREBs. Subsequently, Cytoscape_v3.9.1 was used for visualization [64, 68].

Prediction of putative MiRNAs targeting HvDREB genes

The psRNATarget website [69] was used, with default parameters, to predict miRNA target sites for all the HvDREB CDSs. Subsequently, Cytoscape-v3.9.1 was used for mapping operations, and the HvDREB gene was used as the creation object to construct its miRNA target network diagram.

Quantitative real-time-PCR (qRT–PCR)-based examination of HvDREB genes

The barley variety Zhu 3 was propagated and grown under controlled conditions in the Zhoukou Normal University tissue culture room using Hoagland’s nutrient solution. The light and temperature conditions were alternated between 12 h of darkness and 12 h of light at 25 °C. When the germinated seed sprouts reached a length of 2 cm, they were subjected to treatment with 10% PEG6000 solution or 150 mmol/L NaCl. Additionally, a group of untreated sprouts was designated the control (CK), and three replicates were established for each treatment. One week after treatment, diverse seedling tissues, including leaves, stems, and roots, were collected to prepare a cDNA template for HvDREB gene cloning. All the freshly obtained materials were immediately submerged in liquid nitrogen to halt their physiological responses and subsequently stored in a -80 °C freezer until total RNA extraction. Specific primers for qRT–PCR were designed using Primer Premier (version 5.0; Premier, Canada), and the details of the primer sequences are provided in Table S9. The experimental apparatus used was the Bio-Rad CFX fluorescence quantitative PCR detection system (Bio-Rad, USA), and the reagent used in this experiment was Power SYBR^® Green PCR Master Mix (Applied Biosystems). Other operational steps were implemented in strict compliance with the methods and procedures that we have previously published [70]. We chose HvActin (AY145451) as the internal reference gene for comparison. All the treatments were completed three times in accordance with the regulations for biological replication, and the 2^−ΔΔCT method was used to accurately calculate the effective relative gene expression levels [71]. Statistical analyses were performed using SPSS 27.0. Differences were considered statistically significant at P < 0.05 and highly significant at P < 0.01. The data are presented as the mean ± SE.

Conclusions

This study identified 39 HvDREB genes in barley. Through the analysis of sequence patterns, phylogenetic trees, gene structure and protein motifs, chromosomal locations, Ka/Ks ratios, synteny, predicted miRNAs, cis-acting elements, and expression patterns, it was revealed that HvDREB genes exhibit characteristics of both conservation and diversification. Notably, the expansion of the HvDREB gene family is predominantly driven by segmental duplication events. Furthermore, synteny analysis revealed widespread collinearity with B. distachyon and rice, underscoring the evolutionary conservation of these genes among Poaceae species. Additionally, this study identified 14 miRNAs targeting 16 HvDREB genes, laying the groundwork for future in-depth research on the posttranscriptional regulatory mechanisms of stress responses in barley. Furthermore, the promoter regions of the HvDREB gene family possess many hormone-related and stress-responsive cis-acting elements. qRT–PCR analysis revealed that HvDREB genes exhibited distinct expression patterns under drought and high-salt stress induction. Particularly under PEG stress, the significant downregulation of HvDREB22 and HvDREB38 in root and leaf tissues suggests that barley may implement a resource reallocation strategy to prioritize survival under stress conditions. Conversely, the significant upregulation of HvDREB38 in stems indicates the potential specialized function of this gene in maintaining hydraulic conductivity and facilitating water transport during drought periods. In brief, the results of this investigation offer an in-depth and comprehensive understanding of the molecular basis of the function of the DREB family in barley, providing valuable insights and laying a foundation for further in-depth studies.

Authors’ contributions

LH, ZM and HC designed the research and conducted the experiments, and LH, ZM, HS and WH performed the data analysis and wrote the manuscript. LC, WX, and ZJ helped improve the methods and edited the manuscript. HC, FS, and MK revised the manuscript and participated in funding acquisition. All the authors read and approved the manuscript.

Funding

This work was supported by the 2019 Postdoctoral Research Project Start-up Funding of Henan Province (No. 226152), the 2019 Young Master Teacher Funding Project of Zhoukou Normal University (No. ZKNU20190022), the National Natural Science Foundation (No. 32300116), and Henan Provincial Science and Technology R&D Program: Joint Fund (Industrial Category) Project (245101610090).

Data availability

All datasets generated for this study are included in the manuscript/supplementary files. The genomic sequences, gff3 files, protein sequences and coding sequences (CDSs) of the HvDREB genes were retrieved from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html). Genome-wide transcriptome data of different barley tissues were obtained from the H. vulgare Expression Database on the WheatOmics 1.0 website (http://wheatomics.sdau.edu.cn/expression/barley.html). The hidden Markov model (HMM) of the AP2 domain (PF00847) of DREB was obtained from InterPro (https://www.ebi.ac.uk/interpro/).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.