Abstract
GATA transcription factors play a crucial role in regulating plant growth and development as well as stress responses. However, systematic analysis of GATA genes in barley remains uncharacterized, and their functional roles in salt and drought stress responses are poorly understood. This study conducted genome-wide identification and gene expression analysis of the GATA transcription factor family in barley through bioinformatics approaches. A total of 27 HvGATA genes were identified and divided into 4 subfamilies (I-IV), which were unevenly distributed on 7 chromosomes. Overall tertiary structural similarity of GATA proteins existed with differences, but similarity within the same subfamily was higher. Fragment repetition has been identified as the main driving factor for the expansion of the HvGATA family. Members of the same subfamily exhibit highly conserved exon-intron structures and motif compositions, indicating strong functional conservation among subfamilies. Cis-element analysis of the HvGATA promoter reveals potential regulatory complexity and enrichment of stress-responsive elements related to hormone signals (such as abscisic acid) and drought stress responses. qRT-PCR analysis showed that at 6 h after salt treatment, HvGATA1, HvGATA19, and HvGATA25, were significantly up-regulated (P < 0.05). Under drought treatment, at least three HvGATA genes (HvGATA1, HvGATA19, and HvGATA25) showed dynamic expression patterns, which were down-regulated and up-regulated at 2 h, 24 h, and 12 h, respectively, indicating that they have potential roles in regulating salt tolerance and drought tolerance of barley. This research provides new insights into the molecular mechanisms of salt and drought tolerance in barley and offers potential targets for enhancing the crop’s stress resistance under abiotic stresses.
1 Introduction
Transcription factors (TFs) play indispensable roles in plant growth and development, regulating processes such as cell differentiation (), physiological metabolism (), signal transduction (), stress responses (; ), and other biological functions. 64 transcription factor families have been identified in plants (), including WRKY (; ), MYB (), GRAS (), bHLH (), and GATA (). Among them, GATA is a transcription factor widely present in eukaryotes and an important regulatory protein that specifically binds to the DNA sequence WGATAR (W is T or A, R is G or A), thereby regulating the expression of downstream genes (; ) and modulating plant growth and development. For instance, it plays a crucial role in regulating chlorophyll synthesis () and stress responses (). The GATA protein domain contains a class IV zinc finger structure characterized by CX2CX17-20CX2C, followed by a conserved region. Some members of this zinc finger structure can specifically bind to the DNA sequence (A/T) GATA (A/G) in the gene regulatory region, thereby modulating gene transcription level (; ). Most GATA transcription factors in fungi and animals contain zinc finger domains characterized by CX2CX17CX2C or CX2CX18CX2C (; ), while plant GATA transcription factors typically contain 18–20 residues. The zinc finger domain in plants is typically CX2CX18CX2C or CX2CX20CX2C, and it contains a single zinc finger motif (; ).
The GATA transcription factor NTL1, cloned from tobacco firstly, has been reported to participate in nitrogen metabolism pathways (). In plants, GATA transcription factors play crucial regulatory roles in nitrogen metabolism, stress responses, growth, development, and hormone signaling pathways (). In Arabidopsis thaliana, GATA transcription factor family members, GNC and its homologous gene GNLI, have been shown to involve in chlorophyll biosynthesis, signal transduction, and regulation of developmental processes. Additionally, during plant growth, GNC and GNL regulate brassinosteroids, while GATA2 regulates brassinosteroids during Arabidopsis photomorphogenesis (; ). In maize, GATA transcription factor family members GATA7 and GATA33 regulate the growth and development of the shoot apical meristem (). In rice, the GATA transcription factor family members NECKLEAF1 () and Cga1 () have been found to regulate rice organ formation, chlorophyll synthesis, and chloroplast development. In wheat (Triticum aestivum), the transcription factor TaGATA1 enhances seed dormancy by directly regulating TaABI5 expression, thereby improving resistance to pre-harvest sprouting (). To date, genome-wide analyses of GATA transcription factor families have been conducted in various crops. For instance, 30, 28, and 33 members have been identified in Arabidopsis thaliana (), Oryza sativa (), and Sorghum bicolor (), respectively, providing valuable insights into their structure and biological functions in other crops.
Barley (Hordeum vulgare L.) is the fourth most important cereal crop globally, cultivated extensively across diverse agroecological systems. Barley is rich in protein, vitamins, trace elements, and dietary fiber and exhibits agronomically valuable traits, including a short growth cycle, high tolerance to drought and cold, and broad adaptability (). The complete genome sequence of barley has been fully established, enabling comprehensive molecular biology studies on this species. However, systematic identification of barley GATA family genes and their functional roles in abiotic stress responses remains limited. In this study, systematically identified barley GATA family genes through bioinformatics approaches and analyzed their phylogeny, gene structure, conserved motifs, cis-acting elements, and chromosomal distribution. Furthermore, putative orthologs of barley GATA family genes in Arabidopsis and rice were identified for collinearity analysis to infer evolutionary relationships. Additionally, the barley cultivar Morex were subjected to salt stress (150 mM NaCl) and drought stress (20% PEG-6000) to evaluate abiotic stress responses. The transcriptional levels of four HvGATA genes were quantified using quantitative real-time PCR to abiotic stress provide insights into the functional characterization of barley GATA transcription factors.
2 Materials and methods
2.1 Identification of GATA members in barley
To identify members of the barley GATA family, the whole genome sequence, protein sequence, and gene annotation file of barley were obtained from the publicly released Ensembl Plant database (https://plants.ensembl.org/Hordeum_vulgare/Info/Index), and protein sequences of Arabidopsis and rice were retrieved from previously published data (). Putative HvGATA proteins were identified by performing a BLASTp search against the barley proteome (score ≥100, E-value ≤1e−10) (). Subsequently, the Pfam database (https://www.ebi.ac.uk/Tools/hmmer/) was used to screen candidate proteins for the presence of the GATA zinc finger domain (PF00320) using its corresponding hidden Markov model (HMM) profile with a significance threshold of 0.01 (, ). To further validate conserved domains, CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and SMART (http://smart.emblheidelberg.de/) were employed (; ). Proteins lacking the GATA domain were excluded from further analysis.
2.2 Bioinformatics analysis of HvGATA members
ExPASy (https://web.expasy.org/) was used to predict the amino acid length (aa), molecular weight (MW), isoelectric point (pI), instability index (II), aliphatic index, and grand average of hydropathicity (GRAVY). And use BUSCA (http://busca.biocomp.unibo.it/) for subcellular localization prediction.
2.3 Phylogenetic analysis of HvGATA proteins
Based on studies of GATA family genes in Arabidopsis and rice (; ), zinc finger domain sequences of 30 Arabidopsis and 28 rice GATA proteins were downloaded. Multiple sequence alignment of barley, Arabidopsis, and rice GATA proteins was performed using Clustal W () with default parameters in MEGA11 (). A phylogenetic tree was constructed using the maximum likelihood method with 1,000 bootstrap replicates.
2.4 Analysis of conserved motifs, gene structures, and Cis-acting elements
Conserved domains in barley GATA proteins were analyzed with DNAMAN. The MEME suite (https://meme-suite.org/meme/tools/meme) was employed to identify protein motifs in the full-length sequences of 27 HvGATAs (). Gene structures of HvGATAs were visualized using TBtools by aligning coding sequences (CDS) with their corresponding genomic DNA sequence (). Cis-regulatory elements in the promoter regions (2,000 bp upstream) of HvGATA genes were predicted using PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
2.5 Detection and homology analysis of paralogous gene pairs
Homologous gene pairs and syntenic relationships in barley GATA genes were identified using the Multiple Collinearity Scan toolkit (MCScanX) with default parameters (). To predict the function of barley GATA family genes, syntenic relationships of homologous GATA genes among barley, Arabidopsis, and rice were analyzed. Circos software () was utilized to visualize the syntenic relationships of GATA genes across these three species.
2.6 Analysis of HvGATA genes expression profiles in barley transcriptome data
The expression profiles of barley GATA genes in different tissues and under salt/drought stress conditions were obtained from the BarleyExpDB database (http://barleyexp.com/index.html). These data were visualized using TBtools software to generate expression heatmaps of barley GATA genes.
2.7 Plant materials, stress treatment, and qRT-PCR analysis of 4 HvGATA genes
The barley variety Morex was used in this study. Seeds were placed on filter paper in germination boxes, germinated in darkness at 24°C for 48 h, transferred to Hoagland medium in 96-well plates, and cultivated in a growth chamber at 24°C (day) and 18°C (night) with a 14 h/10 h day/night cycle. When seedlings reached the three-leaf stage, they were subjected to two abiotic stress treatments, salt stress (150 mM NaCl) and drought stress (20% PEG-6000) (). Three biological replicates were performed for each sample, and leaves were collected at 0, 2, 6, 12, and 24 h following the initiation of each stress treatment. All samples were frozen in liquid nitrogen and stored at -80°C for RNA extraction.
Total RNA was extracted using the SteadyPure Plant RNA Extraction Kit (Accurate Biotechnology Co., Ltd., Hunan, China). RNA integrity was verified by 1% agarose gel electrophoresis, and concentration was measured using a UV-Vis spectrophotometer. First-strand cDNA was synthesized from total RNA with the Evo M-MLV RT Premix (Accurate Biotechnology, Hunan, China) for qPCR. Gene-specific primers for four HvGATA genes were designed with Primer 5.0 software (Supplementary Table S1), yielding amplicons of 80–150 bp. HvActin (HORVU6Hr1G054520) was used as the internal reference gene. qRT-PCR was performed on a Roche LightCycler 96 system using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology Co., Ltd., Hunan, China) with the following reaction components: 1.0 µL cDNA, 10.0 µL 2× SYBR mixture, 0.8 µL primer mix (forward/reverse), and 8.2 µL ddH2O. Thermal cycling conditions were 95°C for 3 min; 40 cycles of 95°C for 5 s and 58°C for 30 s. Gene expression levels were calculated using the 2−ΔΔCT method.
2.8 Prediction of structural features and protein interaction networks in HvGATA transcription factors
Secondary structure prediction for barley GATA proteins was performed using SOPMA (https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html). Tertiary structure modeling of identified GATA protein sequences was conducted via the SWISS-MODEL web server (https://swissmodel.expasy.org/interactive/). Protein-protein interaction (PPI) network analysis of barley GATA proteins was executed using the STRING database (https://cn.string-db.org/).
3 Results
3.1 Identification and physicochemical properties of HvGATA members
The hidden Markov model (HMM) of the GATA domain andthe BLASTp method were employed to identify 27 HvGATA genes in the barley genome, designated as HvGATA1-HvGATA27 according to their chromosomal locations (Supplementary Table S2). The encoded proteins ranged from 155 to 775 amino acids in length, with molecular weights ranging from 17343.27 Da to 88608.50 Da. Their isoelectric points varied from 4.66 to 10.22, with 13 members classified as acidic (pI < 7) and 14 as basic (pI > 7), indicating a mixture of acidic and basic proteins. Instability coefficient analysis showed that 26 HvGATA proteins were unstable (values > 40), with HvGATA21 protein exhibiting the highest instability coefficient (86.57). In contrast, HvGATA27 protein was relatively stable, with an instability index below 40. All HvGATA proteins displayed negative mean hydrophobicity coefficients, confirming their hydrophilic nature, though the extent of HvGATA proteins was localized to the nucleus, 3 to extracellular space, and HvGATA7 protein and HvGATA24 protein to chloroplasts.
3.2 Phylogeny, classification, and multiple sequence alignment of HvGATA proteins
Using MEGA11, a phylogenetic tree was constructed from 27 HvGATA, 28 OsGATA, and 30 AtGATA proteins (Figure 1, Supplementary Table S3). The analysis revealed four subfamilies. Subfamily I contained the highest number of HvGATA members (11), followed by Subfamily II (8), while Subfamilies III and IV each comprised four members, respectively. 10 HvGATA proteins clustered closely with AtGATA protein and OsGATA protein orthologs, suggesting conserved functions through evolution.
Figure 1
Multiple sequence alignment of conserved regions (Figure 2) indicated that all barley GATA proteins harbor a single plant-specific GATA domain, Consistent with rice and Arabidopsis, 21 HvGATA proteins (from Subfamilies I, II, and IV) displayed the motif CX2CX18CX2C, whereas 6 HvGATA proteins (four from Subfamily III and two from Subfamily IV) featured an extended variant (CX2CX20CX2C). All subfamilies shared conserved sequences (e.g., T-7, P-8, G-13, P-14), highlighting both conservation and diversification within GATA domains.
Figure 2
3.3 Conserved motifs, gene structures, and Cis-acting elements of HvGATA genes
TBtools was used to construct a phylogenetic tree of 27 HvGATA proteins (Figure 3A), while the MEME online program was employed to analyze their conserved motifs (Figure 3B, Supplementary Table S4). Analysis revealed that the number of motifs among HvGATA members ranged from 1 to 8. Specifically, HvGATA1 contained the lowest motifs, whereas HvGATA6 and HvGATA16 had the highest number (8 motifs each). Notably, Motif 1 was conserved across all barley GATA proteins, suggesting its critical role in the evolution of this gene family. Subgroup-specific patterns were observed: Motif 2 and Motif 6 were exclusive to Subfamily I; Motif 4 was absent solely in HvGATA1; Motif 5 was restricted to Subfamily III; and Motif 7 was unique to Subfamily IV. Additionally, HvGATA6 and HvGATA16 uniquely contained Motifs 8-10, while HvGATA16 shared Motif 3 with all Subfamily III members. The widespread conservation of motifs among most HvGATA members implies functional similarities within this gene family.
Figure 3
Gene structure analysis (Figure 3C), revealed that HvGATA genes contain 0–7 introns. Among the 27 genes, two (HvGATA3 and HvGATA18) lacked introns, 10 harbored a single intron, seven contained two introns, one exhibited four introns, three possessed five introns, and two displayed six or seven introns, respectively. Subfamilies I and II exhibited minimal intron content (0–2 introns). In contrast, Subfamilies III and IV showed marked intron expansion: Subfamily III genes contained 6–7 introns, while Subfamily IV genes had 4–5 introns. These results highlight a significant increase in intron number in Subfamilies III and IV compared to Subfamilies I and II, suggesting divergent evolutionary dynamics among subfamilies.
To investigate the regulatory potential of promoters in the barley HvGATA gene family, cis-acting elements were computationally identified within the 2,000 bp upstream promoter regions of 27 HvGATA genes (Figure 4, Supplementary Table S5). Photoresponsive elements (187 total) and methyl jasmonate (MeJA)-responsive elements (140 total) were the most abundant, with Sp1 (36 occurrences) and I-box (29 occurrences) dominating the light-responsive category. Among hormone-related elements, MeJA-responsive motifs (CGTCA-motif and TGACG-motif) were present in 20 promoters, followed by gibberellin (GA)-responsive elements (31 total). Notably, 26 promoters harbored multiple hormone-responsive motifs, including MeJA, abscisic acid (ABA; ABRE, DRE1), auxin (IAA; TGA-element), and GA-responsive elements (GARE-motif, P-box, TATC-box), while HvGATA13 lacked these motifs.
Figure 4
Stress-responsive cis-elements were also identified: drought-related motifs (as-1, MYC, and MBS) totaled 36, with as-1 restricted to HvGATA9 and HvGATA19, MYC to HvGATA1, HvGATA3, HvGATA5, HvGATA9, and HvGATA21; and MBS present in all 16 promoters analyzed. Additionally, 12 defense/stress motifs (e.g., STRE, CARE, and TC-rich repeats) were detected, with STRE in HvGATA9 and HvGATA25; and CARE in a single promoter. Elements linked to hypoxia (ARE, GC-motif), pathogens (WRE3, WUN), and drought (DRE core) occurred in 3, 1, and 19 promoters, respectively. These findings suggest that HvGATA genes may regulate not only plant growth but also hormone signaling and abiotic stress responses, particularly drought adaptation.
3.4 Chromosome distribution, gene replication, and collinearity analysis of HvGATA genes
To elucidate the chromosomal distribution of GATA genes in barley, 27 HvGATA genes were mapped to seven chromosomes (Figure 5A). The distribution frequency varied significantly across chromosomes: Chromosome 1 harbored the highest number of genes (5 genes, 18.52%), followed by Chromosome 4 (6 genes, 22.22%). Chromosomes 2 and 6 each contained 4 genes (14.81% per chromosome), while Chromosomes 3, 5, and 7 showed lower densities with 3 genes (11.11%) on Chromosome 3 (HvGATA10-HvGATA12), 3 genes (11.11%) on Chromosome 5 (HvGATA19-HvGATA21), and 2 genes (7.41%) on Chromosome 7 (HvGATA26 and HvGATA27).
Figure 5
MCScanX based segmental duplication analysis of the 27 HvGATA genes identified seven duplication events involving 14 genes (Figure 5B, Supplementary Table S6), accounting for 51.85% (14/27) of the gene family. These duplicated pairs included HvGATA2/HvGATA18, HvGATA4/HvGATA11, HvGATA5/HvGATA10, HvGATA6/HvGATA16, HvGATA7/HvGATA24, HvGATA22/HvGATA27, and HvGATA23/HvGATA26. Subfamily classification revealed that four duplication pairs (57.14%) belonged to Subfamily I, two pairs (28.57%) to Subfamily II, and one pair each (14.29%) to Subfamilies III and IV. This uneven distribution suggests that segmental duplications may have been a major driver of HvGATA gene family expansion. Furthermore, HvGATA genes were asymmetrically distributed across seven barley linkage groups (LGs). LG1 and LG6 harbored the highest number of duplicated genes (3 genes each, 21.43% of total duplications), followed by LG2, LG3, LG4, and LG7 (2 genes each, 14.29% per LG).
To elucidate the evolutionary dynamics of HvGATA genes, synteny analysis was conducted between two monocotyledonous species (barley and rice) and one dicotyledonous species (Arabidopsis). The results revealed (Figure 5C, Supplementary Table S7), 23 OsGATA genes exhibiting orthologous relationships with 21 HvGATA genes, while 3 AtGATA genes showed orthology with 3 HvGATA genes. These findings indicate that barley, as a monocotyledonous poaceae species, shares a closer phylogenetic relationship with rice than with the dicotyledonous Arabidopsis. Furthermore, collinear gene pairs are often functionally conserved, suggesting that the 33 barley-rice collinear GATA gene pairs and 4 barley-Arabidopsis collinear GATA gene pairs may perform analogous roles in their respective species. This syntenic conservation underscores potential functional similarities driven by shared evolutionary origins.
3.5 Expression profile of HvGATA genes in different tissues and abiotic stresses
To investigate the tissue-specific expression patterns of HvGATA genes, transcript levels of 27 HvGATA genes were analyzed across eight barley tissues (Figure 6A, Supplementary Table S8). 15 genes (HvGATA1, HvGATA2, HvGATA4, HvGATA6-HvGATA9, HvGATA12, HvGATA13, HvGATA19-HvGATA22, HvGATA25, and HvGATA27) exhibiting ubiquitous and/or high expression across multiple tissues, suggesting their potential roles in barley growth and development, 6 genes (HvGATA3, HvGATA5, HvGATA10, HvGATA11, HvGATA15, HvGATA17) with low or undetectable expression in all tested tissues, and 6 genes (HvGATA14, HvGATA16, HvGATA18, HvGATA23, HvGATA24, HvGATA26) showing preferential expression in specific tissues. For instance, HvGATA2 was absent in senescent leaves and showed reduced expression in seeds at 15 days post-fertilization but was highly expressed in other tissues. In contrast, HvGATA26 displayed elevated expression in senescent leaves compared to other tissues, implicating its potential role in regulating leaf senescence during seedling development.
Figure 6
To further explore the expression patterns of HvGATA genes in barley under abiotic stress, expression profiles were analyzed to characterize their responses to salt and drought treatments. The results revealed that under salt stress, HvGATA1 and HvGATA25 expression slightly decreased (Figures 6B, C), while HvGATA19 and HvGATA21 expression increased. Under normal and drought conditions, HvGATA1 and HvGATA19 expression declined, whereas HvGATA21 and HvGATA25 expression was up-regulated. A notable exception occurred under the SBC073D drought treatment, where HvGATA1, HvGATA21, and HvGATA25 expression decreased, contrasting with the elevated expression of the HvGATA19 gene.
3.6 HvGATA genes expression under salt and drought stress by qRT-PCR
In this study, qRT-PCR was used to detect the expression changes of HvGATAs under salt stress and drought stress conditions. Based on the expression profile of barley treated with salt and drought, four genes with high expression levels and significant changes in expression levels under salt and drought treatments (HvGATA1, HvGATA19, HvGATA21, and HvGATA25) were selected for qRT-PCR analysis. The results showed (Figure 7A, Supplementary Table S9), that under salt stress, the HvGATA1, HvGATA21, and HvGATA25 genes were significantly down-regulated at 2 h, reaching approximately 4.17 times, 2.63 times, and 1.51 times, respectively, while the HvGATA19 gene was significantly up-regulated by approximately 2.21 times. The HvGATA1, HvGATA19, and HvGATA25 genes were significantly up-regulated at 6 h under salt treatment, reaching 2.78 times, 3.15 times, and 1.44 times, respectively. Furthermore, the HvGATA21 gene was significantly down-regulated by approximately 2 times at 12 h under salt treatment. However, with the increase of salt treatment time, the HvGATA1, HvGATA19, HvGATA21, and HvGATA25 genes were up-regulated by 1.64 times, 1.40 times, 1.65 times, and 2.29 times, respectively, at 24 h under salt treatment. However, significant differences emerged in the HvGATA25 gene. The results show that these genes respond to salt stress.
Figure 7
Under drought stress (Figure 7B), with the increase of 20% PEG solution treatment time, the HvGATA1, HvGATA19, and HvGATA21 genes were significantly down-regulated at 2 h, reaching approximately 1.47 times, 1.96 times, and 4.67 times, respectively. HvGATA21 gene was significantly up-regulated by 2.02 times at 6 h, and the HvGATA1 and HvGATA25 genes were significantly up-regulated at 12 h, reaching 1.86 times and 1.52 times, respectively. At 24 h of PEG treatment, the HvGATA19, HvGATA21, and HvGATA25 genes were significantly down-regulated, among which the expression level of HvGATA21 gene was down-regulated by approximately 5.6 times.
3.7 Structural prediction and protein interaction correlation analysis of HvGATA transcription factors
Secondary structure prediction of 27 barley GATA proteins was performed using the SOPMA online tool (Table 1). The results demonstrate that all 27 HvGATA proteins contain α-helices, β-turns, extended strands, and random coils in their secondary structures. Random coils constitute the predominant structural component among these proteins, with HvGATA5 protein exhibiting the highest proportion (91.24%) and HvGATA16 protein the lowest (40.93%). Within subfamily IV, HvGATA6 and HvGATA16 proteins displayed notably higher α-helix contents of 40.77% and 43.41%, respectively. This elevated α-helical composition may contribute to conformational stability in these proteins.
Table 1
| Gene name | Alpha helix (%) | Beta turn (%) | Random coil (%) | Extended strand (%) | Protein Secondary Structure Schematic |
|---|---|---|---|---|---|
| HvGATA1 | 32.26 | 1.28 | 63.23 | 3.23 |  |
| HvGATA2 | 12.22 | 1.39 | 83.89 | 2.50 |  |
| HvGATA3 | 10.74 | 2.76 | 82.82 | 3.68 |  |
| HvGATA4 | 18.04 | 1.06 | 79.58 | 1.33 |  |
| HvGATA5 | 1.84 | 1.84 | 91.24 | 5.07 |  |
| HvGATA6 | 40.77 | 3.23 | 45.03 | 10.97 |  |
| HvGATA7 | 16.01 | 1.16 | 80.28 | 2.55 |  |
| HvGATA8 | 18.14 | 1.36 | 74.60 | 5.90 |  |
| HvGATA9 | 23.03 | 1.10 | 68.20 | 7.68 |  |
| HvGATA10 | 7.33 | 0.86 | 88.79 | 3.02 |  |
| HvGATA11 | 8.42 | 1.52 | 86.73 | 3.32 |  |
| HvGATA12 | 17.68 | 2.21 | 75.69 | 4.42 |  |
| HvGATA13 | 16.46 | 0.95 | 79.75 | 2.85 |  |
| HvGATA14 | 16.85 | 1.87 | 75.28 | 5.99 |  |
| HvGATA15 | 7.69 | 1.14 | 89.74 | 1.42 |  |
| HvGATA16 | 43.41 | 3.71 | 40.93 | 11.95 |  |
| HvGATA17 | 22.18 | 2.42 | 73.39 | 2.02 |  |
| HvGATA18 | 14.85 | 1.12 | 81.79 | 2.24 |  |
| HvGATA19 | 10.89 | 1.43 | 86.25 | 1.43 |  |
| HvGATA20 | 6.95 | 1.35 | 89.46 | 2.24 |  |
| HvGATA21 | 21.60 | 2.47 | 70.99 | 2.47 |  |
| HvGATA22 | 21.96 | 1.78 | 69.14 | 7.12 |  |
| HvGATA23 | 15.25 | 1.17 | 80.06 | 3.52 |  |
| HvGATA24 | 19.01 | 1.49 | 76.79 | 2.72 |  |
| HvGATA25 | 9.88 | 1.23 | 86.42 | 2.47 |  |
| HvGATA26 | 15.00 | 1.11 | 81.39 | 2.50 |  |
| HvGATA27 | 15.44 | 0.74 | 78.68 | 5.15 |  |
Secondary structure analysis of barley GATA transcription factors.
Protein Secondary Structure Schematic: Blue indicates α-helix; Green indicates β-turn; purple indicates extended strand; yellow indicates random coil.
Tertiary structure prediction of barley GATA proteins revealed that these proteins adopt spatial conformations consisting primarily of α-helices and random coils (Figure 8). Comparative analysis demonstrated moderate structural complexity with significant variation in overall structural similarity. Notably, proteins within the same subfamily exhibited higher structural conservation corresponding to their phylogenetic relationships. For instance, in subfamily III, HvGATA13, HvGATA14, HvGATA22, and HvGATA27 proteins displayed strikingly similar tertiary architectures, suggesting evolutionary conservation of structural features within conserved clades.
Figure 8
Protein interaction network analysis of barley GATA transcription factors (Figure 9) revealed that 10 GATA proteins (HvGATA5, HvGATA10, HvGATA11, HvGATA15, HvGATA17, HvGATA18, HvGATA19, HvGATA23, HvGATA26, and HvGATA27) form a densely interconnected cluster. This topological configuration suggests potential functional homology among these proteins, indicating their cooperative regulation in physiological and developmental processes. Conversely, 17 GATA proteins (HvGATA1-4, HvGATA6-9, HvGATA12-14, HvGATA16, HvGATA20-22, and HvGATA24-25) showed no detectable interactions, revealing significant functional diversification within this transcription factor family.
Figure 9
4 Discussion
4.1 Conservation of HvGATA genes during evolution
GATA transcription factors are a class of DNA-binding proteins ubiquitous in eukaryotes, playing pivotal roles in plant biological processes such as chlorophyll biosynthesis, photomorphogenesis, and stress adaptation. In this study, 27 HvGATA genes were systematically identified from the barley genome through bioinformatic analysis. Among these, 21 HvGATA proteins harbor the canonical CX2CX18CX2C zinc finger domain, while the remaining six possess a CX2CX20CX2C zinc finger domain. This domain architecture aligns with reported GATA families in Arabidopsis (), rice (), and Brassica napus (), suggesting evolutionary conservation. Notably, the variation in the number of amino acid residues (18 or 20) within the CX2CX18-20CX2C zinc finger domains of some GATA proteins may result from structural deletions or evolutionary divergence. Substantial variation was observed in HvGATA protein properties, including amino acid length (ranging from 155 to 775 aa), molecular weight (17343.27 Da-88608.50 Da), and isoelectric point (pI 4.66-10.22), potentially reflecting functional diversification. Prediction analysis of barley GATA protein structures and interaction relationships demonstrated that secondary structures were predominantly composed of random coils. Overall similarity of tertiary structures existed with differences, but the same subfamily exhibited higher similarity, indicating that members within the same subfamily possess high structural homology during evolution. Protein interaction correlation analysis also indicated functional similarity among family members, suggesting cooperative regulatory functions in certain plant growth and developmental processes. Phylogenetic clustering, supported by conserved motif and gene structure analyses, classified barley GATA members into four subfamilies, consistent with classifications in Arabidopsis (), rice (), and Sorghum bicolor (). Functional predictions for barley GATA genes were inferred based on orthologous subfamily functions in Arabidopsis. Notably, members within the same subfamily exhibited highly similar motif compositions and exon-intron architectures, indicating strong evolutionary conservation and potential functional redundancy. Conversely, structural divergence across subfamilies likely arose from lineage-specific evolutionary pressures, such as domain shuffling or selective gene loss.
In higher plants, the GATA gene family exhibits significant variation in member numbers across species, even among closely related taxa. For instance, wheat harbors 79 GATA members (), compared to 30 in Arabidopsis (dicot) () and 28 in rice (monocot) (). Such divergence likely reflects lineage-specific evolutionary trajectories, where gene duplication events-key drivers of family expansion play pivotal roles (). In barley, we identified seven segmental duplication events among 27 HvGATA genes, with no tandem duplications observed. Notably, two duplication pairs localized to Subfamilies I and II, suggesting subfunctionalization or neofunctionalization within these clades. Synteny analysis further revealed stronger conservation between barley and rice than with Arabidopsis, corroborating closer phylogenetic relationships within the poaceae family.
4.2 HvGATA genes are involved in hormone signaling and abiotic stress response
Cis-regulatory elements are non-coding DNA sequences within gene promoters that orchestrate transcriptional regulation. Analyzing these elements in HvGATA promoters provides critical insights into their functional diversification in barley. Previous studies demonstrate that plant GATA transcription factors mediate light signaling by binding to GATA motifs in photoresponsive gene promoters (), exemplified by GATA2’s dual role in modulating light and brassinosteroid signaling pathways (). This functional interplay suggests that GATA genes may both regulate and be regulated by light-responsive genes, underscoring the importance of HvGATA promoter analysis in barley. Plant GATA factors are also implicated in stress adaptation, developmental regulation, and hormone signaling (; ), consistent with our findings in the barley HvGATA family. Promoter analysis revealed an enrichment of stress-responsive cis-elements (e.g., drought-inducible MBS MYC) and hormone-related motifs (ABRE for abscisic acid, GARE for gibberellins, TGA-element for auxin, and CGTCA-motif for jasmonate) in HvGATA promoters. Additionally, specific HvGATA genes harbor elements associated with meristem expression (e.g., CAT-box), endosperm specificity (e.g., GCN4-motif), and hypoxia response (e.g., GC-motif), indicating their potential roles in diverse physiological processes. However, while these cis-element profiles suggest environmental sensitivity and multifunctional regulation, direct experimental evidence is required to confirm their binding efficacy and regulatory polarity (activation or repression) under specific conditions.
4.3 HvGATA genes play an important role in the response mechanism of barley salt and drought stress
Gene expression patterns across tissues often reflect functional specialization. For instance, OsGATA23a in rice exhibits broad stress responsiveness, with significant up-regulation under salt and drought conditions (), underscoring the conserved regulatory role of GATA transcription factors in stress signaling. In barley, tissue-specific expression profiling of four HvGATA genes revealed conserved expression patterns within subfamilies, indicative of functional conservation. qRT-PCR analysis further demonstrated that HvGATA genes are dynamically regulated under abiotic stress. Under salt stress, at 2 h, only HvGATA19 was significantly up-regulated, while the other three genes were significantly down-regulated. HvGATA1 and HvGATA19 were transiently up-regulated at 6 h post-treatment, whereas HvGATA21 and HvGATA25 peaked at 12 h. Conversely, drought stress triggered pronounced down-regulation of HvGATA1, HvGATA19, and HvGATA21 at 2h. At 12 h, HvGATA1 and HvGATA25 genes were significantly up-regulated. The HvGATA19, HvGATA21, and HvGATA25 genes were significantly down-regulated by approximately 2.63 times, 5.56 times, and 3.57 times, respectively, at 24 h. These temporal and stress-specific expression dynamics suggest functional diversification within the HvGATA family, enabling coordinated regulation of developmental processes and stress adaptation.
5 Conclusion
In this study, 27 GATA genes were identified in the barley genome using bioinformatics approaches and classified into four subfamilies. Members within the same GATA subfamily exhibit higher sequence similarity and conservation, suggesting potential functional similarities. Analysis of promoter regions indicated that the cis-acting elements associated with HvGATA genes may be subject to complex regulation, particularly elements responsive to hormone signaling pathways and abiotic stress. Furthermore, the expression patterns of HvGATA1, HvGATA19, HvGATA21, and HvGATA25 genes differed following salt and drought stress treatments applied over different time courses, indicating their potential involvement in barley’s salt and drought tolerance mechanisms.
Statements
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
Author contributions
HZ: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. PY: Conceptualization, Data curation, Formal Analysis, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. HX: Data curation, Formal Analysis, Writing – original draft. CW: Methodology, Software, Writing – review & editing. XY: Software, Writing – review & editing, Formal Analysis. XZ: Writing – review & editing, Data curation, Supervision.
Funding
The author(s) declare that financial support was received for the research and/or publication of this article. This research was supported by the Interdisciplinary Research Project of Hangzhou Normal University (2025JCXK01) and the HZNU scientific research and innovation team project (TD2025005).
Acknowledgments
We thank the public database for the downloaded raw data. We are very grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declare that no Generative AI was used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1661591/full#supplementary-material
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Summary
Keywords
barley, GATA transcription factor, bioinformatics, salt and drought stress, expression analysis
Citation
Zhang H, Yu P, Xiao H, Wang C, Yan X and Zhang X (2025) Genome-wide characterization and expression analysis of the GATA transcription factor family in response to salt and drought stress in barley (Hordeum vulgare L.). Front. Plant Sci. 16:1661591. doi: 10.3389/fpls.2025.1661591
Received
08 July 2025
Accepted
29 August 2025
Published
17 September 2025
Volume
16 - 2025
Edited by
Alla Yemets, National Academy of Sciences of Ukraine (NAN Ukraine), Ukraine
Reviewed by
Feifei Wang, Yangzhou University, China
Mangi Kim, Sangmyung University, Republic of Korea
Updates
Copyright
© 2025 Zhang, Yu, Xiao, Wang, Yan and Zhang.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Xiaoqin Zhang, zxq@hznu.edu.cn; Haitao Zhang, tao@ahau.edu.cn
†These authors have contributed equally to this work and share first authorship
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.