Comprehensive genome-wide characterization of NAC transcription factors in Barley influence insights into stress tolerance and evolutionary dynamics

Abstract

Barley (Hordeum vulgare L.), a crucial cereal crop known for its resilience to harsh environmental conditions, relies on complex genetic networks to withstand abiotic stressors such as drought, salinity, and extreme temperatures. In this study, a comprehensive genome-wide identification and characterization of the NAC (NAM, ATAF, and CUC) transcription factor family in barley was conducted, revealing 26 HvNAC genes. Detailed analyses included assessments of gene structure, conserved motifs, cis-regulatory elements, chromosomal localization, and evolutionary relationships with other species. The findings demonstrated significant diversity in the physicochemical properties and structural features of HvNAC proteins, with several genes harboring stress-responsive elements linked to Abscisic acid (ABA), Methyl jasmonate (MeJA), auxin, and gibberellin pathways. Phylogenetic analysis revealed six distinct clades of NAC genes, indicating the evolutionary divergence of HvNACs from related species, such as wheat, rice, and Arabidopsis thaliana. Additionally, gene duplication events and synteny analysis highlighted the evolutionary forces shaping this gene family. The investigation of microRNA (miRNA) interactions identified miRNA164 and Hvu-miR156 as key regulators of HvNAC expression under drought stress, underscoring the functional importance of these genes in stress adaptation. Under drought and salt stress, HvNAC2 and HvNAC6 were significantly upregulated in barley roots, highlighting their key roles in stress adaptation, while leaves showed minimal expression changes. Additionally, under temperature stress, HvNAC4, HvNAC5, and HvNAC3 were upregulated in leaves during heat stress, whereas HvNAC6 and HvNAC6-C were more active in roots during cold stress, indicating tissue-specific responses to environmental conditions. This study offers valuable insights into the molecular mechanisms governing stress tolerance in barley and provides a foundation for breeding programs aimed at enhancing barley’s resilience to environmental challenges.

Introduction

Barley (Hordeum vulgare L.), one of the earliest domesticated crops, holds significant agricultural and economic importance worldwide, particularly in regions with harsh environmental conditions^1,2. As the fourth most important cereal globally, barley is cultivated across diverse climatic zones, from the arid regions of the Middle East to the high-altitude plateaus of Tibet³. Its versatility and adaptability to abiotic stresses such as drought, salinity, and extreme temperature fluctuations make it a crucial crop for food security, particularly in the face of climate change⁴. In addition to its role in human nutrition, barley is a vital feed crop for livestock and a key ingredient in the brewing industry, further enhancing its economic value. Its ability to thrive in poor soils and marginal lands, where other cereals may fail, underpins its importance in sustainable agricultural systems¹. Barley’s relatively short growing season allows it to be cultivated in regions with shorter summers or unpredictable weather patterns, adding to its adaptability^1,5,6. Despite this, the crop’s yield potential can be limited under extreme stress conditions, which necessitates continued research into the genetic and molecular bases of its resilience⁷. Identifying the genes and regulatory networks that govern barley’s stress tolerance could lead to the development of improved cultivars, capable of sustaining high productivity in an era of increasing environmental challenges.

One of the key regulatory components involved in plant responses to abiotic stresses is the NAC (NAM, ATAF, and CUC) transcription factor family⁸. NAC proteins are among the largest families of transcription factors in plants and play critical roles in various biological processes, including growth, development, and stress response^{9, 10–11}. In particular, NAC transcription factors have been widely recognized for their involvement in mediating plant responses to abiotic stresses such as drought, salinity, and cold^11,12. These transcription factors regulate the expression of downstream target genes through binding to specific cis-regulatory elements, thereby modulating plant growth and adaptation under stress conditions¹³.

The NAC family has been extensively studied in model plants like Arabidopsis thaliana^14,15 as well as in major crops such as rice^16,17, wheat^{18, 19–20}, and maize^21,22. Genome-wide identification of NAC genes in these species has revealed the importance of these transcription factors in stress signaling and developmental processes²³. In Arabidopsis, NAC transcription factors such as ANAC019 and ANAC055 have been shown to modulate drought-responsive genes and confer drought tolerance^15,24. Similarly, in rice, overexpression of OsNAC6 has been reported to improve tolerance to both drought and salinity stresses²⁵. Despite the substantial research conducted on NAC genes in other species, the functional roles of NAC transcription factors in barley have not been comprehensively explored.

Barley, with its inherent resilience to various abiotic stressors, presents a unique opportunity to study NAC gene function in the context of stress tolerance²⁶. Given the increasing interest in improving crop resilience through genetic approaches, a detailed investigation of barley’s NAC gene family could provide valuable insights into its adaptability²⁷. Such knowledge is crucial for developing barley cultivars with enhanced stress tolerance and productivity, particularly in regions vulnerable to climate-induced stresses. In 2011, Christiansen et al.²⁸ reported the first phylogenetic classification of barley NAC genes, identifying 48 HvNACs and their expression profiles, which highlighted their role in regulating important agronomic traits in both monocots and dicots²⁸. Understanding the precise functions of these NAC genes is crucial for improving cereal crops through breeding; however, at that time, only a few NAC genes in barley (Hordeum vulgare L.) had been investigated²⁸. In this study, we conducted a comprehensive genome-wide analysis of the NAC transcription factor family in Hordeum vulgare. We identified 26 NAC genes (HvNACs) and performed an in-depth characterization of their gene structures, conserved domains, and physicochemical properties. Furthermore, we analyzed the evolutionary relationships of HvNACs with other plant species, explored their chromosomal localization, and investigated their expression patterns under different stress conditions. Additionally, we examined cis-regulatory elements and microRNA (miRNA) interactions to understand the regulatory mechanisms governing HvNAC gene expression, particularly in response to abiotic stresses. Barley leaves exhibited no changes in gene expression, the roots showed significant upregulation of HvNAC2 and HvNAC6 during drought and salt stress, underscoring their crucial roles in stress adaptation. Additionally, tissue-specific responses to temperature stress were observed: HvNAC4, HvNAC5, and HvNAC3 were upregulated in leaves during heat stress, while HvNAC6 and HvNAC6-C were more active in roots during cold stress. This study aims to provide a foundational understanding of the NAC gene family in barley and its potential role in improving stress tolerance through molecular breeding approaches.

This study expands on the work of Christiansen et al., (2011), who discovered 48 HvNACs and offered a first phylogenetic categorization, with a particular emphasis on the activities of NAC genes in response to hormones-related responses. In contrast, our work focuses on 26 stress-responsive HvNAC genes in the presence of abiotic stressors such as drought, salt, and temperature extremes. To guarantee high-confidence selection, these genes were identified using severe criteria (60% sequence identity and an E-value cutoff of 10^–5). We examined their gene architectures, conserved themes, evolutionary links, and expression patterns under abiotic stress. Through this genome-wide analysis, we aim to offer valuable insights into how NAC transcription factors contribute to barley exceptional stress tolerance, which is critical for sustaining crop productivity in a rapidly changing climate. By understanding the molecular mechanisms that underpin barley flexibility, we may progress breeding techniques to create more robust crops capable of surviving in diverse and demanding situations.

Results

Identification of HvNAC Family members in Barley

In this study, we identified and characterized 26 distinct NAC (NAM, ATAF1/2, CUC2) genes within the barley (Hordeum vulgare) genome. The identification process involved comparative genomic analyses using the NAC1 gene from Arabidopsis thaliana as a reference sequence. This approach facilitated a comprehensive Blast search against the genomic data of Hordeum vulgare and other related species. Our investigation included an extensive analysis of coding sequences, genomic locations, and protein sequences. We assembled a dataset comprising NAC proteins from six plant species: Hordeum vulgare (26 proteins), Brachypodium distachyon (14 proteins), Oryza sativa (20 proteins), Zea mays (23 proteins), Arabidopsis thaliana (27 proteins), and Triticum aestivum (19 proteins). The search parameters were set to an e-value threshold of 10^-5 and a minimum sequence identity of 60%. SMART domain analysis confirmed the presence of the NAM domain in these proteins while filtering out redundant sequences. The final dataset included protein sequences featuring the NAM domain, which were subjected to further computational analyses to elucidate their evolutionary relationships and functional roles.

Physiochemical properties

The HvNAC proteins demonstrated considerable variation in terms of peptide length and molecular weight. The amino acid residues ranged from 217 (HvNAC8) to 689 (HvNAC15), with molecular weights varying from 24.44 kDa (HvNAC8) to 75.74 kDa (HvNAC15). The frequency of negatively charged residues (aspartic acid and glutamic acid) ranged from 21 to 89, while positively charged residues (arginine and lysine) ranged from 27 to 77. Subcellular localization predictions indicated that HvNAC1, HvNAC11, and HvNAC18 are localized in the cytoplasm. In contrast, HvNAC2, HvNAC3, HvNAC5, HvNAC7, HvNAC8, HvNAC9, HvNAC13, HvNAC16, HvNAC19, HvNAC20, HvNAC23, HvNAC24, and HvNAC25 are localized in the nucleus. HvNAC4, HvNAC14, and HvNAC22 were found in the chloroplast, HvNAC6, HvNAC10, HvNAC15, and HvNAC17 in the peroxisome, HvNAC12 and HvNAC26 in the mitochondria, and HvNAC21 in the Golgi apparatus. The isoelectric point (pI) values of HvNAC proteins ranged from 4.68 to 9.22, indicating diverse surface charge properties which may affect their solubility, stability, and molecular interactions. The grand average hydropathy (GRAVY) scores varied from − 0.83 (HvNAC16) to – 0.366 (HvNAC6), reflecting differences in hydrophobicity among the proteins (Table S1).

Domain, motif and gene structure analysis

To elucidate the functional importance of the HvNAC gene family, we conducted a detailed domain analysis. Using the SMART server, we confirmed the presence of the NAM superfamily domain across all 26 HvNAC proteins. Notably, HvNAC20 possessed an additional, unidentified domain beyond the NAM domain (Fig. 1A). The NAM domain is critical for the transcriptional regulation and functional diversity of NAC proteins. This analysis ensured that each HvNAC protein contained at least one NAM domain, highlighting its significance in plant development and stress responses. Conserved motifs within the HvNAC proteins were identified using the MEME Suite and TBtools software. A total of ten distinct motifs were detected across the HvNAC proteins, all localized within the NAM domain. The analysis revealed that HvNAC9, HvNAC18, and HvNAC22 contained the full complement of ten motifs in group I, while HvNAC5 and HvNAC11 displayed seven motifs. Other HvNAC proteins (20 in total) exhibited five motifs in groups II through V, with HvNAC16 (group III) showing six motifs. This variation suggests that HvNAC proteins within specific subfamilies may perform similar physiological roles (Fig. 1C).

We performed a comprehensive analysis of the exon-intron organization for HvNAC genes, revealing considerable variation in gene structure among the different members. The number of exons in HvNAC genes ranged from two to six. Specifically, HvNAC15 was found to have the maximum number of six exons, while genes such as HvNAC1, 3, 4, 5, 6, 9, 11, 12, 13, 14, 16, 17, 18, 19, 20, 25, and 26 each contained three exons. In contrast, HvNAC2, 7, 8, 10, 21, 22, 23, and 24 were composed of only two exons. Notably, the most common exon count among HvNAC genes was three, accounting for 65.38% of the analyzed genes. Genes with two exons comprised 30.78%, while genes with only a single exon were the least common, representing 3.84% of the total (Fig. 1B). This variation in gene structure among HvNAC members may reflect differences in functional roles and regulatory mechanisms.

[See PDF for image]

Fig. 1

Domain architecture, gene structures and motif analysis of HvNAC proteins. (A) The structural organization of HvNAC proteins is depicted, showing the distribution of different conserved domains. The color-coding represents specific domains: NAM superfamily (yellow) and DUF4175 superfamily (green). Each bar corresponds to a distinct HvNAC protein, with the domains positioned along the sequence length. The x-axis indicates the amino acid length (from 0 to 700), and the y-axis lists the HvNAC protein identifiers. This illustration highlights the diversity in domain composition among the HvNAC family members. (B) Schematic representation of HvNAC gene structures. The diagram illustrates the arrangement of exons²⁹, introns (black lines), and untranslated regions (UTRs) (blue) in HvNAC genes. The lengths of these elements are proportionally scaled to reflect their relative sizes within the genes. The scale bar at the bottom provides a reference for estimating the sizes of exons and introns. (C) A schematic representation of the HvNAC protein sequences along with the phylogenetic relationships among barley NAC genes. The phylogenetic tree visually categorizes the HvNAC proteins into distinct groups based on their evolutionary relationships. Overlaying this tree is a depiction of the conserved motifs identified through MEME analysis. Each colored box on the motif map represents a unique conserved motif within the HvNAC proteins, with the size of the box proportional to the length of the motif. This visualization highlights the structurally and functionally important regions within the HvNAC proteins, offering insights into their conservation and potential roles in stress responses and plant development.

Phylogenetic analysis

To elucidate the evolutionary relationships among the HvNAC proteins, the Neighbor-Joining phylogenetic tree was constructed using MEGA 11, analyzing complete protein sequences of HvNACs from diverse plant species, with visualization conducted via iTOL. This analysis identified six clades within the NAC gene family, reflecting significant evolutionary diversification. Each clade contains genes exhibiting closer relationships within the group compared to other clades. Clade I (AtNAC-I) is predominantly comprised of Arabidopsis thaliana and Oryza sativa genes, indicating their close evolutionary ties. Clade II (TaNAC-II) includes genes from various plant species, revealing a complex evolutionary history, while clade III is primarily represented by Hordeum vulgare genes, signifying rapid NAC gene diversification in this species. Clade IV (ZmNAC-IV) features genes from Zea mays, suggesting a distinct evolutionary trajectory. Clade V (BdNAC-V) encompasses a diverse array of plant species Brachypodium distachyon, highlighting its intricate evolutionary background, and Clade VI (OsNAC-VI) predominantly consists of Arabidopsis thaliana as well Oryza sativa genes, underscoring close evolutionary affiliations. Bootstrap support values were calculated to evaluate the reliability of the inferred relationships, with values exceeding 70% indicating strong support. The tree exhibited several well-supported clades, particularly involving Hordeum vulgare and Triticum aestivum. Branch lengths on the phylogenetic tree serve as estimates of evolutionary distance between genes, where longer branches imply greater genetic divergence and shorter branches suggest closer relationships. These branch lengths indicate that NAC genes have experienced varying degrees of evolutionary change since diverging from a common ancestor (Fig. 2). For more better understanding of NAC genes in barley, we constructed a rectangular phylogenetic tree. It represents the evolutionary relationships between different HvNAC genes in barley, using branch lengths to show the hierarchy and connections between various gene sequences (Fig. S2).

[See PDF for image]

Fig. 2

Phylogenetic tree of NAC proteins from various plant species, including Arabidopsis thaliana (27), Oryza sativa (20), Hordeum vulgare (26), Triticum aestivum (19), Zea mays (23), and Brachypodium distachyon (14). The tree was constructed using the Neighbor-Joining method implemented in MEGA 11, based on protein sequences aligned with ClustalW. Bootstrap values (1000 replicates) are shown at the nodes to indicate the support for each branching point. Distinct clades are color-coded to highlight the evolutionary relationships and diversification among the NAC proteins from these species.

Stress related Cis elements in the promoters of HvNAC genes

To elucidate the functional roles of HvNAC1 to HvNAC26, a detailed analysis of their 1000 bp upstream promoter sequences was performed using the PlantCARE web server. This investigation aimed to uncover the signal transduction pathways and regulatory mechanisms governing the expression of HvNAC genes by analyzing the distribution of 24 cis-regulatory elements within the promoter regions. Many HvNAC gene promoters exhibited enrichment of cis-regulatory elements associated with light responsiveness, stress responsiveness, and promoter-enhancer activities. The identification of these elements revealed diverse regulatory responses, with 24 elements linked to stress responses, including ABRE (ABA response), AuxRR-core (auxin response), CGTCA-motif (MeJA response), LTR (low temperature response), MBS (binding site for MYB transcription factors related to drought), P-box (gibberellin response), TCA-element (salicylic acid response), TC-rich repeats (defense and stress response), TGA-element (auxin response), AR (abscisic acid response), ME (expression in meristems), PER (promoter and enhancer regions), SSR (seed-specific regulation), PDRE (phytochrome down-regulation expression), and DI (drought inducibility). The presence of these cis-regulatory elements suggests that HvNAC genes are regulated by intricate networks involving multiple signaling pathways (Fig. 3).

[See PDF for image]

Fig. 3

Visualization of cis-regulatory elements in the 1 kb upstream regions of HvNAC genes. The diagram illustrates the distribution of various cis-regulatory elements, with each type represented by a distinct color. This visual representation highlights the potential regulatory mechanisms involved in modulating the expression of HvNAC genes in response to different stress conditions.

The analysis of 1 kb upstream promoter regions of HvNAC genes using the PlantCARE web server uncovered variations in the distribution of cis-regulatory elements (CREs) among the HvNAC gene family. A total of 24 different cis-regulatory elements associated with stress responses and regulatory processes were identified. The distribution of CREs was not uniform across all HvNAC genes. For instance, HvNAC7, HvNAC12, and HvNAC24 exhibited notably higher densities of CREs, with 26, 27, and 26 CREs, respectively. This indicates a potentially higher degree of regulatory control for these genes. In contrast, each HvNAC gene contained at least 10 CREs, with the total number ranging from 10 to 27. Furthermore, specific CREs were found to co-occur frequently within the promoter regions, suggesting potential functional interactions and complex regulatory networks. Notably, several HvNAC genes showed multiple occurrences of the same type of CRE, indicating that these elements may play a significant role in modulating gene expression under stress conditions (Fig. 3).

miRNA interaction network

Among the 156 microRNAs (miRNAs) characterized in Hordeum vulgare, 40 miRNAs were identified to interact with 16 HvNAC transcripts. These interactions primarily involve two mechanisms: mRNA cleavage and translation inhibition, with 36 miRNAs employing mRNA cleavage and 4 miRNAs utilizing translation inhibition. The miRNA interaction network reveals complex regulatory relationships, where each node represents either a miRNA or an HvNAC gene, and the connecting lines indicate potential regulatory interactions. The network can be clustered based on miRNA targeting preferences. For instance, miRNAs targeting genes in Clade I may exhibit distinct regulatory functions compared to those targeting genes in Clade II. Key HvNAC genes, including HvNAC1, HvNAC2, HvNAC3, and HvNAC21, function as central nodes within this network, highlighting their crucial roles in regulating the expression of multiple target genes. Each miRNA influences several HvNAC genes, illustrating the intricate and multifaceted nature of miRNA-mediated regulation. Notably, miRNAs such as miR164 (a, b, c, d, e), miR156 (b, c, d, e, f, g, h), and miRN246 (a, b) are involved in regulating HvNAC1, HvNAC2, HvNAC3, and HvNAC21, indicating a significant level of regulatory control over these genes and their downstream targets. Additionally, HvNAC7, HvNAC15, HvNAC20, and HvNAC23 are targeted by multiple miRNAs, reflecting the complex regulatory dynamics within the network. Of particular note is the specific interaction of HvNAC14 with Hvu-miRN5795, which is unique among the HvNAC genes (Fig. 4).

[See PDF for image]

Fig. 4

This network diagram illustrates the predicted interactions between HvNAC genes and miRNAs in barley. The interactions were identified using the psRNATarget program to locate potential miRNA target sites within HvNAC transcripts. The network, visualized with Cytoscape, displays the connections between miRNAs and their target HvNAC genes, highlighting the direction of miRNA-mediated regulation and showcasing the complex regulatory interactions.

Protein-protein interaction

To elucidate the potential interactions between HvNAC proteins and other Hordeum vulgare proteins, protein-protein interaction networks were constructed using the STRING database. This analysis aimed to reveal the biological functions and regulatory networks associated with HvNAC proteins. Each node in the network represents a protein, with lines connecting nodes indicating potential interactions. The interactions were identified with a moderate confidence level, scoring 0.400. A total of 22 HvNAC proteins were found to interact with five functional proteins, categorized as A0A287T6K5, A0A287HKS8, A0A287NRY0, A0A287LXM8, and A0A287RZ16. Among these, HvNAC proteins 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 22, 23, 24, 25, and 26 were identified to interact with the aforementioned functional proteins. Some of these interacting proteins were uncharacterized, while others, such as A0A287HKS8, were noted for their roles as repressors of early auxin response genes under low auxin conditions and in stress- or development-associated transcription factors. Notably, HvNAC15, HvNAC20, and HvNAC 21 did not show any associations with other proteins, remaining isolated in the network. Additionally, HvNAC6 and HvNAC14 were observed to have a single interaction with the protein A0A287RZ16, whereas the remaining 20 HvNAC genes were linked through multiple interaction pathways. These protein-protein interactions may indicate regulatory relationships, with some HvNAC genes potentially acting as upstream regulators or downstream targets within their respective pathways (Fig. 5).

[See PDF for image]

Fig. 5

The network illustrates the predicted protein-protein interactions among HvNAC proteins, as identified using the STRING V11.5 database. Nodes represent individual proteins, while edges indicate potential interactions. The color of each edge reflects the type of evidence supporting the interaction. The figure emphasizes connections between proteins that were differentially expressed in the study.

Positive selection and purifying selection in HvNAC genes: a Ka/Ks analysis

To elucidate the selection pressures on HvNAC genes, we calculated synonymous substitution rates (Ks), non-synonymous substitution rates (Ka), and Ka/Ks ratios for six paralogous gene pairs. These pairs were further classified into one segmental duplication (HvNAC18-HvNAC22) and five tandem duplications (HvNAC7-HvNAC10, HvNAC16-HvNAC17, HvNAC25-HvNAC26, HvNAC3-HvNAC6, and HvNAC19-HvNAC23). The Ka/Ks ratios ranged from 0.18 to 0.54, with all values below 1, indicating purifying (negative) selection. This suggests that these genes are under evolutionary constraint, preserving essential functions likely related to abiotic stress responses and maintaining structural protein integrity. Tandem duplication emerged as the dominant mode of expansion, highlighting its role in the functional diversification of the HvNAC family. In contrast, segmental duplication contributed to the evolutionary complexity of the gene family (Table S3).

Synteny analysis

Synteny analysis between Hordeum vulgare (barley) and Triticum aestivum³⁰ revealed 78,790 collinear genes out of 143,372 genes used for analysis that display conserved synteny, representing 54.9% of genes. This study identified 77 collinear genes across both species’ chromosomes for 26 target genes from barley, suggesting rich genomic conservation between the two closely related cereal crops. The dual synteny map illustrates these connections, with barley chromosomes presented below and wheat chromosomes above, connected by lines denoting collinear gene pairs. The results will provide a basis for further studies of syntenic gene function and their contributions to stress tolerance as well as other traits of cereals (Fig. 6).

[See PDF for image]

Fig. 6

The dual synteny map illustrates the collinear relationships between Hordeum vulgare (red, bottom) and Triticum aestivum (yellow, top). The extensive synteny is visualized by blue lines representing syntenic gene pairs, with a focus on 77 collinear genes corresponding to 26 barley target genes. This visualization underscores the conserved genomic regions shared by these closely related cereals.

Chromosomal localization and collinear analysis

To map the chromosomal distribution of HvNAC genes in barley, we employed Circos software to visualize their loci across the seven chromosomes of Hordeum vulgare. Our analysis showed that HvNAC genes are dispersed throughout the chromosomes, appearing both at terminal ends and central regions, and are not clustered in any specific chromosomal area. HvNAC genes were found on all seven chromosomes, with positions indicated by colored arcs or bars. The colors may reflect different functional classifications or expression patterns, and lines connecting genes across chromosomes suggest potential regulatory or functional interactions. Notably, only Chromosome 1 contained a single HvNAC gene, HvNAC19, while Chromosome 6 had HvNAC6. Chromosome 2 exhibited the highest gene density with ten HvNAC genes: HvNAC4, HvNAC9, HvNAC11, HvNAC13, HvNAC17, HvNAC18, HvNAC20, HvNAC22, HvNAC24, and HvNAC26. This high concentration on Chromosome 2 might indicate gene duplication events or co-regulation. The distribution of HvNAC genes suggests that while they are spread across all chromosomes, some chromosomes, like Chromosome 2, have a higher density, potentially reflecting specific functional relationships or evolutionary processes (Fig. 7).

[See PDF for image]

Fig. 7

The Circos plot illustrates the chromosomal localization of the 26 HvNAC genes across the seven barley chromosomes. Each chromosome is depicted as a circular segment labeled with its respective number. The genes are represented by colored arcs or bars, with color coding potentially denoting different gene categories or functional groups. The outer scale around the circle indicates the physical distances on the chromosomes, allowing for precise identification of gene locations.

Expression analysis of HvNAC genes in Barley

To validate expression patterns of HvNAC genes under drought, salt, cold and heat stress, seven genes (HvNAC1-7) were analyzed using qRT-PCR and the primers were in supplementary file S4. The heatmap represents the expression patterns of HvNAC genes in barley under drought (A), salt (B), cold (C) and heat (D) stress in both leaves (L) and roots (R) tissues. Under drought stress, HvNAC6 shows strong upregulation in roots (Drought-R), highlighting their crucial roles in root responses to water scarcity. HvNAC2 demonstrates the highest expression, indicating its significant role in managing drought stress at the root level. Overall, the high expression level of HvNAC genes under stress conditions in comparison to the control conditions represent the functional roles of these genes under drought stress (Fig. 8A). Similarly, under salt stress, roots display elevated expression levels for HvNAC2, HvNAC6, and HvNAC3, in comparison to the control conditions, being particularly active in roots (Salt-R). In contrast, leaves show minimal gene expression responses to both drought and salt stress, with genes such as HvNAC1, HvNAC7 exhibiting low expression across all conditions. This suggests that HvNAC2 and HvNAC6 under salt and drought respectively, are essential for root-specific responses to drought and salinity. Comparison of tissue specific expression shows that leaves play a less active role in these stress adaptations in comparison to roots (Fig. 8B).

Under cold and heat stress, HvNAC genes exhibit a clear tissue-specific response. In leaves and roots, HvNAC3, HvNAC5, and HvNAC7 show significant upregulation under heat stress, indicating their key roles in managing heat stress in these tissues. Meanwhile, roots under cold stress exhibit higher expression of HvNAC6 and HvNAC6, highlighting these genes involvement in root adaptation to low temperatures. Moderate expression of HvNAC7 and HvNAC2 is observed in both leaves and roots under heat and cold stress, suggesting a more generalized role for these genes across tissues and stress types (Fig. 8C). In contrast, genes like HvNAC1 and HvNAC2 remain largely inactive under temperature-related stress conditions. This expression pattern underscores the specialization of certain HvNAC genes in handling stress in either leaves or roots, depending on the environmental condition, with heat stress eliciting a stronger response in leaves and cold stress primarily affecting roots (Fig. 8D).

[See PDF for image]

Fig. 8

The figure illustrates the expression levels of various HvNAC genes in response to different abiotic stress conditions, specifically drought (A), salinity (B), cold (C), and heat (D) stress in comparison to the separate control conditions for all treatments, in leaves (L) and roots (R) tissues of barley.

Materials and methods

Identification of HvNAC genes in Barley and sequence acquisition

To conduct a comprehensive genome-wide assessment of NAC genes in barley (Hordeum vulgare), the NAC1 gene from Arabidopsis thaliana (AT1G56010.1), known for its role in abiotic stress resilience, was used as a query for BLAST analysis against the barley genome, as well as genomes of other agricultural species. Following the identification of HvNAC genes, their coding sequences (CDS), genomic sequences, and protein sequences were retrieved from Ensembl Plants (https://plants.ensembl.org/index.html). A BLASTp search was performed against the proteins of Hordeum vulgare, Triticum aestivum, Brachypodium distachyon, Zea mays, Oryza sativa, and Arabidopsis thaliana with parameters set to an E-value threshold of 10^–5 and a minimum identity of 60%. Sequences confirmed to possess the NAM domain were identified using the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/). Redundant sequences were removed, and candidate NAC transcription factor genes were further validated with the Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/cdd/) to ensure the presence of the NAC domain. Only sequences with the NAM domain were retained for subsequent analysis.

Cis-regulatory elements, motif, and domain analysis

To identify cis-regulatory elements and explore the functional roles of HvNAC genes, 1000 bp of upstream promoter sequences were retrieved from Ensembl Plants (https://plants.ensembl.org/index.html). The PlantCARE database was used to identify stress-response cis-regulatory elements, and the results were visualized using TBtools v1.120. Protein motifs were detected using the MEME Suite, and conserved domains were analyzed with the Conserved Domain Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) on NCBI. The MEME Suite v5.5.7 was configured to identify up to ten conserved motifs, with other parameters set to default(https://meme-suite.org/meme/). The default parameters (minimum width: 6, maximum width: 50, and site distribution: zero or one occurrence per sequence) were used to ensure an unbiased and consistent motif discovery.

Gene structure and evolutionary analysis of NAC proteins across crop species

The exon-intron organization of HvNAC genes were analyzed using the Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/). Phylogenetic relationships among NAC proteins from Hordeum vulgare, Triticum aestivum, Brachypodium distachyon, Zea mays, Oryza sativa, and Arabidopsis thaliana were examined by constructing a phylogenetic tree with the neighbor-joining method, using 1000 bootstrap replicates and amino acid sequences from these species. The ClustalW algorithm in MEGA11 software was used for alignment, and the tree was visualized using iTOL software (https://itol.embl.de/).

Chromosomal localization, Synteny analysis and gene duplication

The chromosomal positions of HvNAC genes were determined using coordinates extracted from the Ensembl Plants database and visualized with PhenoGram from(http://visualization.ritchielab.org/phenograms/plot). The Circos software was employed to map the genes to their respective chromosomes, and TBtools v1.120 was used to generate the chromosome position map. To investigate the Synteny relationships between Hordeum vulgar and Triticum aestivum³¹, a synteny analysis was performed using TbTools software³². The analysis was conducted using One step MCScanX and Dual Synteny Plot for MCScanX features toolkit within TbTools v1.120. Synonymous (Ks) and nonsynonymous (Ka) substitution rates were calculated using TBtools v1.120, with Ks values greater than 2.0 excluded to avoid substitution saturation. Divergence times were assessed following Yuan et al.³⁰.

Physicochemical properties

The ProtParam tool on the ExPASY website (https://web.expasy.org/protparam/) and Ensembl Plants were used to analyze the chromosomal number, molecular weight (MW), isoelectric point (PI), total counts of positively and negatively charged residues, grand average of hydropathicity (GRAVY), and aliphatic index of amino acids in HvNAC proteins. The subcellular localization of HvNAC proteins was predicted using the WoLF PSORT online platform (https://wolfpsort.hgc.jp/).

miRNA interaction network and protein-protein interaction

To determine miRNAs interacting with HvNAC genes, previously identified miRNAs from Hordeum vulgare were analyzed using psRNATarget (https://www.zhaolab.org/psRNATarget/). The Cytoscape software Version 3.9.1 was used to visualize the interaction networks among genes, proteins, and other macromolecules. Protein-protein interaction networks were established using the STRING v12.0 database(https://string-db.org/), with interactions analyzed based on protein nomenclature and sequences.

Experimental site and design

Experiments were meticulously designed to evaluate the impact of abiotic stresses on selected barley varieties at National Institute for Genomics and Advanced Biotechnology (NIGAB), NARC, Islamabad. The seeds of barley “Snober” variety, sourced from the Crop Sciences Institute (CSI) at National Agricultural Research Center (NARC), Islamabad, were evaluated through laboratory and greenhouse trials for comprehensive analysis. The selected seeds were surface sterilized for 1 min using 70% ethanol, followed by rinsing with distilled water and then germinating in controlled seedling trays at NIGAB, ensuring optimal growth conditions of 22 °C with an 8-hour dark/16-hour light photoperiod. After a month of careful nurturing, the robust seedlings were transferred into pots with daily irrigation. Plants were grown under the same photoperiod and temperature conditions in a growth chamber until they reached the 3-leaf stage.

Abiotic stress treatment and plant sampling

When the barley plants reached the 2–3 leaf stage, we divided the pots into four equal groups to give drought, salt, cold and heat stress to each group, respectively. Drought stress was applied by stopping the irrigation for 10 days to decrease the soil moisture content up to 30% of field capacity. A moisture meter was used to monitor the soil moisture content regularly. Well-watered plants served as the control group. To induce salt stress plants were watered with a solution of 200 mM NaCl, continuously for seven days. The control plants were irrigated with a standard Hoagland’s solution that did not contain NaCl. For cold stress plants were stored in a refrigerator and to minimize the shock and for acclimatization decrease the temperature by 2 °C every hour until reaching 4 °C. Then, to stimulate the cold stress conditions the plants were stored at this temperature for 48 h. For the control group plants were maintained at 22 °C temperature. For heat stress plants were transferred to the growth chamber with the increase of temperature by 2 °C every hour until reaching 42 °C. The infrared thermometer was used to monitor the leaf temperature and to confirm the stress induction. Then, to maintain the heat stress conditions the plants were stored at this temperature for 48 h. Plants at 22 °C were used as control groups. At the seedling stage the leaf and root tissue were sampled at the end of each stress treatment using liquid nitrogen and then stored at – 80 °C for further analysis. All the stress experiments were conducted in triplicates using the three biological replicates and their control samples for each treatment.

RNA extraction, DNase treatment and quantification

Total RNA was isolated from leaf and root samples of barley varieties using an Invitrogen kit, following the manufacturer’s protocol. Tissue samples were homogenized in TRIzol reagent, followed by chloroform extraction and isopropanol precipitation to isolate total RNA. Genomic DNA contamination was removed through DNase treatment, using DNase I enzyme and appropriate buffer conditions. The quality and quantity of extracted RNA were assessed using a Nanodrop spectrophotometer, measuring absorbance ratios at 260/280 nm and 260/230 nm, and by agarose gel electrophoresis, verifying the integrity of the RNA molecules.

cDNA synthesis, primer design and qPCR

cDNA was synthesized from the extracted RNA using a Thermo Scientific RevertAid kit, following the manufacturer’s instructions. This process involved reverse transcription, using random hexamers or oligo(dT) primers to convert RNA into complementary DNA (cDNA), which serves as a template for subsequent gene expression analysis. Primers targeting HvNAC genes and a housekeeping gene (tubulin) were designed using Primer3 software, ensuring specificity and efficiency for PCR amplification. Real-time quantitative PCR (qPCR) was performed using SYBR Green/ROX qPCR Master Mix on an AB Applied Biosystems instrument. The reaction mixture typically includes cDNA template, primers, SYBR Green dye, and Taq polymerase. Cycling conditions were optimized for efficient amplification and detection of target gene transcripts. Relative gene expression levels were calculated using the 2-ΔΔCt method, with the Tubulin gene serving as an internal control. This analysis allowed for the quantification and comparison of HvNAC genes expression in the different barley cultivars and tissues (7 genes primers listed in Table S4).

Discussion

Plants encounter a range of environmental challenges throughout their life cycles, including abiotic factors like drought, high soil salinity, and extreme temperature variations. To adapt to these external and internal osmotic changes, they have developed complex regulatory networks³¹. Barley (Hordeum vulgare) thrives in diverse environments, from the Dead Sea region where it was first cultivated to the high-altitude areas of the Qinghai-Tibet plateau¹. Its exceptional adaptability allows barley to withstand extreme conditions, making it one of the most resilient staple crops, capable of tolerating salinity, drought, and low temperatures⁶. This resilience positions barley as a valuable genetic resource for breeding stress-tolerant crops. The NAC gene family, present across various plant genomes, has been extensively studied to understand its roles in growth, maturation, and stress response^32,33. NAC genes have been identified in multiple species, including Avena sativa, maize, and Medicago sativa, with varying numbers of NAC transcription factors reported in different plants, 105 in Arabidopsis, 151 in rice, and 168 in wheat^14,16,23. In this study, we identified 26 NAC genes in the barley genome, which likely play critical roles in its development and stress response, though their functions have not been thoroughly explored. Our research aimed to provide a comprehensive analysis of the NAC gene family in barley. We characterized these 26 genes by examining their physicochemical properties, structural domains, and motif features. Additionally, we conducted phylogenetic analyses, explored exon/intron structures, and investigated protein-protein interactions, syteny analysis, and chromosomal mapping. We also assessed their expression patterns under various stress conditions and constructed a network of miRNA interactions, which may regulate these genes during stress responses.

Cis-regulatory elements govern a gene’s expression patterns and influence plant growth, development and interactions with the environment³⁴ and its study is essential for understanding the complex mechanisms governing gene regulation and function³⁵. In HvNAC genes various cis-acting elements were identified associated with different stress responses, including a notable prevalence of CAE-AR in 26 HvNAC genes linked to ABA regulation^15,36, while MeJA-responsive motifs were present in all HvNAC genes except HvNAC16, which influences MYB overexpression to enhance anthocyanin and proanthocyanins levels³⁷. The auxin-responsive elements TGA-element, AuxRR-core, and TGA-box were identified in HvNAC1, HvNAC2, HvNAC4, HvNAC22, HvNAC25, HvNAC26, HvNAC11, HvNAC15, HvNAC16, and HvNAC18 genes. In the HvNAC genes 1, 2, 6, 7, 13, 15, 19, 24, and 25, GARE-motif and TATC-box associated with gibberellin-responsive elements were identified, while elements related to plant growth and development such as MYB binding sites linked to drought-inducibility were present in HvNAC genes 1, 10, 11, 12, 13, 16, 17, and 20.

The HvNACs exhibited significant diversity in the physicochemical characteristics of their corresponding proteins, aligning with NACs from various other plant taxa^{38, 39–40}. By investigating the physicochemical attributes of proteins, such as molecular weight, isoelectric point, and hydrophobicity uncovering the proteins’ characteristics and potential involvement in the unfolded protein response. HvNAC genes elucidated a diverse range of isoelectric points indicating the encoded proteins’ adaptability to various pH environments, while the presence of 26 negatively and 26 positively charged residues implies the protein’s complex charge characteristics. HvNAC proteins lengths and molecular weights are consistent with the findings reported for Dendrobium nobile⁴¹. Previous research has shown that miRNA164 can target NAC gene mRNAs for cleavage, thereby regulating plant growth, development, and responses to abiotic stresses^{42, 43–44}. Our comprehensive analysis identified five high-confidence targets of miRNA164 for multiple HvNAC genes. In Arabidopsis, miRNA164-mediated regulation of CUC1 is crucial for the development of embryonic, vegetative, floral, and shoot apical meristems^45,46. Previous research has demonstrated that miRNA164s can target NAC gene mRNAs for cleavage and may also be involved in regulating HvNAC gene expression^43,47,48. Our study identified Hvu-miR156 as dehydration stress-responsive microRNAs that mediate barley’s genomic response to drought stress⁴⁹. microRNAs are key players in responding to both abiotic and biotic stress factors⁵⁰. Additionally, our study aligned with the Protein interactions that provided valuable insights into gene function at a systems level⁵¹.

The presence of the NAM superfamily domain across all HvNAC proteins highlights the evolutionary stability and functional relevance of HvNAC genes as NAC transcription factors being crucial for orchestrating plant responses to biotic and abiotic stressors^52,53. The conserved NAM domain exhibited the similar regulatory mechanisms of all HvNAC genes as how they affect target genes that are implicated in stress tolerance⁵⁴ and how they interact with DNA and other proteins, which in turn can have an impact on the control of gene expression^55,56. The discovery of the DUF4175 domain in HvNAC20 highlighting the intricacy of transcriptional regulation in plant stress responses and may provide light on its distinct functional activities in relation to other NAC proteins⁵⁷. A thorough analysis of HvNAC transcription factors reveals the presence of 10 conserved motifs within the NAM domain of HvNAC proteins, indicating functional regions critical for barley’s growth and stress response, while variations in motif numbers across subfamilies suggest distinct roles that may be vital for adapting to abiotic stresses like drought and salinity, reflecting the intricate regulatory networks governing these responses²⁸. This knowledge is essential for improving breeding initiatives targeted at improving barley’s resistance to drought. Therefore, understanding HvNAC proteins is essential to create barley cultivars that are productive and resilient to environmental challenges^58,59. NAC gene family, has a range of exon combinations enabling specialized functions in several biological processes⁶⁰. Moreover, the existence of introns in these genes implies that complex regulatory processes might be at play in regulating their expression and functionality, an essential aspect of modulating plant reactions to biotic and abiotic stressors⁶¹. HvNACs genes have more introns than rice, millet, and maize, ranging from 1 to 26. In particular, the intron numbers for millet, maize, and rice are 1–16⁶², 0–14⁶³, and 0–14⁶⁴, respectively. The findings demonstrated that the intron range of the barley crop is larger than that of other crops, indicating a significant degree of structural variability in the HvNAC genes.

The phylogenetic examination of NAC genes, particularly within species such as Hordeum vulgare and Triticum aestivum, unveils an intricate evolutionary narrative defined by six distinct clades. Each clade is linked to a specific collection of plant species, suggesting varied evolutionary trajectories among NAC transcription factors. For example, Clade I primarily consist of genes from Hordeum vulgare and Triticum aestivum, whereas Clade II encompasses a wide array of plant species, indicating a multifaceted evolutionary history. Clade III is distinguished by a high concentration of Oryza sativa genes, reflecting a phase of rapid diversification, while Clade IV is represented by genes from Brachypodium distachyon, showcasing a unique evolutionary path. Clade V includes a broad spectrum of plant species, further highlighting the complex evolutionary background of NAC genes, and Clade VI is largely comprised of Arabidopsis thaliana genes, emphasizing a close evolutionary connection among these species^65,66. Similar clade patterns have been seen in other crops, including Sorghum and Rice, indicating that gene duplication events and functional diversification are the common causes of this NAC gene family diversification^16,22.

HvNAC genes are distributed chromosomally in a variety of ways, with genes found on all seven chromosomes, both centrally and terminally. HvNAC genes, such as HvNAC4, HvNAC9, HvNAC11, HvNAC13, HvNAC17, HvNAC18, HvNAC20, HvNAC22, HvNAC24, and HvNAC26, are notably concentrated on chromosome 2, indicating possible functional links or co-regulation. There are different numbers of HvNAC genes on the remaining chromosomes as well, suggesting a non-random distribution that selection pressures, chromosomal rearrangements, or gene duplication events might impact^67,68. Given that genes close to one another are more likely to be co-expressed or co-regulated, which may allow coordinated expression in related biological processes⁶⁹. Moreover, the presence of gene regulatory elements that affect several genes in the area may be suggested by this clustering⁷⁰. These processes result in the creation of gene clusters with comparable or related functions. Liu et al., (2020) highlight that tandem and segmental duplications significantly influence evolution, functional regulation, and responses to biotic and abiotic stressors⁷¹. Their analysis of orthologous gene pairs revealed diverse evolutionary impacts, assessed through Ka/Ks ratios, which measure selective pressures on genes. Most pairs showed evidence of purifying selection, a process that eliminates harmful alleles and shapes genealogical relationships and genetic variation. These findings illustrate the complex interplay of purifying selection in shaping genomic architectures and driving gene functional diversification.

The genomic relationships between Hordeum vulgare (barley) and Triticum aestivum³⁰ were analyzed, focusing on the NAC gene family in Hordeum vulgare under abiotic stress. Among 1,43,372 genes examined, 78,790 collinear genes were identified, highlighting significant genomic conservation within the Triticeae⁷⁵ and their shared evolutionary history³⁰. Notably, 77 collinear genes corresponding to 26 barley NAC genes were distributed across all wheat chromosomes, likely influenced by wheat whole-genome duplication events³⁰. These results underscore the functional importance and potential selection pressure on these genes in both species.

In cold and heat stress conditions, the heatmap showed a more prominent response in leaf tissues, particularly under heat stress, where genes such as HvNAC4, HvNAC5, and HvNAC3 were highly upregulated, suggesting their involvement in managing heat stress in leaves. Conversely, roots displayed higher expression levels of HvNAC6 and HvNAC6-C under cold stress, indicating their role in root adaptation to low temperatures. Moderate expression of HvNAC7 and HvNAC2 was observed in both leaves and roots under temperature stress, suggesting a more generalized role for these genes across tissues. The results emphasize the specialization of HvNAC genes in handling different abiotic stresses in a tissue-specific manner, with roots being more responsive to water-related stresses and leaves more actively involved in temperature responses (Fig. 8).

This detailed gene expression analysis highlights the complexity of the HvNAC gene family’s role in barley’s stress response mechanisms. The tissue-specific expression patterns suggest that certain HvNAC genes are critical for root survival under drought and salinity, while others contribute to leaf tolerance against extreme temperatures. These findings provide valuable insights into the regulatory mechanisms of HvNAC genes, offering potential targets for genetic improvement and breeding strategies aimed at enhancing barley’s resilience to environmental stressors.

Conclusion

This study provides a comprehensive analysis of the NAC gene family in barley (Hordeum vulgare), identifying 26 members and exploring their roles in plant growth, development, and stress response. The findings reveal that HvNAC genes are integral to barley’s resilience, particularly in its response to abiotic stressors like drought, salinity, and temperature extremes. Through detailed analyses of their physicochemical properties, gene structures, conserved motifs, and chromosomal distribution, this study highlights the diverse functions of HvNAC genes, especially in stress adaptation. The identification of key cis-regulatory elements and microRNA interactions further underscores the complex regulatory networks controlling HvNAC gene expression. The evolutionary analysis, which shows the influence of gene duplication and selective pressures, points to functional diversification within the gene family, helping barley adapt to various environmental challenges. Overall, this research contributes valuable insights into the molecular mechanisms that underlie barley’s stress tolerance. These findings have significant implications for crop improvement programs, providing a genetic foundation for developing barley varieties with enhanced resilience and stability in the face of environmental stress. The characterization of the HvNAC gene family represents a critical step toward improving barley’s productivity and sustainability, which is essential for addressing global agricultural challenges.

Acknowledgements

The authors would like to extend their sincere appreciation to the Ongoing Research Funding Program (ORF-2025-182) King Saud University, Riyadh, Saudi Arabia.

Author contributions

MA and NM: Conceptualization, Writing-original draft, Visualization, Software, Resources, Methodology, Formal analysis. AFN: Formal analysis, Software, Writing-review & editing. MAES: Formal analysis, Software, Writing-review & editing and Funding. SA: Formal analysis, Software, Writing-review & editing and Funding. PA: Conceptualization, Investigation, Validation and Writing-review & editing.

Data availability

All data generated or analyzed during this study are included within the article and its additional files.

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1. Newton, AC et al. Crops that feed the world 4. Barley: a resilient crop? Strengths and weaknesses in the context of food security. Food Secur.; 2011; 3, pp. 141-178. [DOI: https://dx.doi.org/10.1007/s12571-011-0126-3]

2. Dawson, IK et al. Barley: a translational model for adaptation to climate change. New Phytol.; 2015; 206, pp. 913-931. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25605349][DOI: https://dx.doi.org/10.1111/nph.13266]

3. Dai, F. & Zhang, G. In Exploration, Identification and Utilization of Barley Germplasm1–26 (Elsevier, 2016).

4. Munaweera, T; Jayawardana, N; Rajaratnam, R; Dissanayake, N. Modern plant biotechnology as a strategy in addressing climate change and attaining food security. Agric. Food Secur.; 2022; 11, pp. 1-28. [DOI: https://dx.doi.org/10.1186/s40066-022-00369-2]

5. Trnka, M; Dubrovský, M; Žalud, Z. Climate change impacts and adaptation strategies in spring barley production in the Czech Republic. Clim. Change; 2004; 64, pp. 227-255.1:CAS:528:DC%2BD2cXjtlCjur0%3D [DOI: https://dx.doi.org/10.1023/B:CLIM.0000024675.39030.96]

6. Kumar, A; Verma, RPS; Singh, A; Sharma, HK; Devi, G. Barley landraces: ecological heritage for edaphic stress adaptations and sustainable production. Environ. Sustain. Indic.; 2020; 6, 100035.

7. Janni, M et al. Molecular and genetic bases of heat stress responses in crop plants and breeding for increased resilience and productivity. J. Exp. Bot.; 2020; 71, pp. 3780-3802.1:CAS:528:DC%2BB3cXisFKktr7L [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31970395][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316970][DOI: https://dx.doi.org/10.1093/jxb/eraa034]

8. Wang, Z; Dane, F. NAC (NAM/ATAF/CUC) transcription factors in different stresses and their signaling pathway. Acta Physiol. Plant.; 2013; 35, pp. 1397-1408.1:CAS:528:DC%2BC3sXpvFehurc%3D [DOI: https://dx.doi.org/10.1007/s11738-012-1195-4]

9. Nuruzzaman, M; Sharoni, AM; Kikuchi, S. Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants. Front. Microbiol.; 2013; 4, 248. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24058359][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759801][DOI: https://dx.doi.org/10.3389/fmicb.2013.00248]

10. Sun, H et al. Comprehensive analysis of NAC transcription factors uncovers their roles during fiber development and stress response in cotton. BMC Plant Biol.; 2018; 18, pp. 1-15.2018P&SS.158..1S1:CAS:528:DC%2BC1MXitlelsbnP [DOI: https://dx.doi.org/10.1186/s12870-018-1367-5]

11. Diao, P et al. The role of NAC transcription factor in plant cold response. Plant Signal. Behav.; 2020; 15, 1785668. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32662739][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8550289][DOI: https://dx.doi.org/10.1080/15592324.2020.1785668]

12. Tweneboah, S; Oh, SK. Biological roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in solanaceous crops. J. Plant. Biotechnol.; 2017; 44, pp. 1-11. [DOI: https://dx.doi.org/10.5010/JPB.2017.44.1.001]

13. Manna, M et al. Transcription factors as key molecular target to strengthen the drought stress tolerance in plants. Physiol. Plant.; 2021; 172, pp. 847-868.1:CAS:528:DC%2BB3cXitlyjsLbE [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33180329][DOI: https://dx.doi.org/10.1111/ppl.13268]

14. Ooka, H et al. Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana. DNA Res.; 2003; 10, pp. 239-247.1:CAS:528:DC%2BD2cXhtFKmsrk%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15029955][DOI: https://dx.doi.org/10.1093/dnares/10.6.239]

15. Jensen, MK et al. The Arabidopsis thaliana NAC transcription factor family: structure–function relationships and determinants of ANAC019 stress signalling. Biochem. J.; 2010; 426, pp. 183-196.1:CAS:528:DC%2BC3cXhslegurg%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19995345][DOI: https://dx.doi.org/10.1042/BJ20091234]

16. Nuruzzaman, M et al. Genome-wide analysis of NAC transcription factor family in rice. Gene; 2010; 465, pp. 30-44.1:CAS:528:DC%2BC3cXhtVajsLfO [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20600702][DOI: https://dx.doi.org/10.1016/j.gene.2010.06.008]

17. Ray, S; Basnet, A; Bhattacharya, S; Banerjee, A; Biswas, K. A comprehensive analysis of NAC gene family in Oryza sativa japonica: a structural and functional genomics approach. J. Biomol. Struct. Dyn.; 2023; 41, pp. 856-870.1:CAS:528:DC%2BB3MXivVSgt7fJ [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34931596][DOI: https://dx.doi.org/10.1080/07391102.2021.2014968]

18. Borrill, P; Harrington, SA; Uauy, C. Genome-wide sequence and expression analysis of the NAC transcription factor family in polyploid wheat. G3: Genes Genomes Genet.; 2017; 7, pp. 3019-3029.1:CAS:528:DC%2BC1cXisVOqsL7L [DOI: https://dx.doi.org/10.1534/g3.117.043679]

19. Saidi, MN; Mergby, D; Brini, F. Identification and expression analysis of the NAC transcription factor family in durum wheat (Triticum turgidum L. ssp. durum). Plant Physiol. Biochem.; 2017; 112, pp. 117-128.1:CAS:528:DC%2BC2sXltlWgsQ%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28064119][DOI: https://dx.doi.org/10.1016/j.plaphy.2016.12.028]

20. Guerin, C et al. Genome-wide analysis, expansion and expression of the NAC family under drought and heat stresses in bread wheat (T. Aestivum L.). PLoS One; 2019; 14, e0213390. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30840709][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402696][DOI: https://dx.doi.org/10.1371/journal.pone.0213390]

21. Peng, X et al. Genomewide identification, classification and analysis of NAC type gene family in maize. J. Genet.; 2015; 94, pp. 377-390.1:CAS:528:DC%2BC2MXhs1Cns7bI [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26440076][DOI: https://dx.doi.org/10.1007/s12041-015-0526-9]

22. Ramaswamy, M et al. Genome wide analysis of NAC gene family ‘sequences’ in sugarcane and its comparative phylogenetic relationship with rice, sorghum, maize and Arabidopsis for prediction of stress associated NAC genes. Agri Gene; 2017; 3, pp. 1-11. [DOI: https://dx.doi.org/10.1016/j.aggene.2016.10.003]

23. Liu, T et al. Genome-wide analysis and expression patterns of NAC transcription factor family under different developmental stages and abiotic stresses in Chinese cabbage. Plant. Mol. Biol. Rep.; 2014; 32, pp. 1041-1056. [DOI: https://dx.doi.org/10.1007/s11105-014-0712-6]

24. Borràs, D et al. Transcriptome-based identification and functional characterization of NAC transcription factors responsive to drought stress in Capsicum annuum L. Front. Genet.; 2021; 12, 743902. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34745217][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8570119][DOI: https://dx.doi.org/10.3389/fgene.2021.743902]

25. Rachmat, A., Nugroho, S., Sukma, D. & Aswidinnoor, H. Overexpression of OsNAC6 transcription factor from Indonesia rice cultivar enhances drought and salt tolerance. Emirates J. Food Agric. (EJFA)2014, 26 (2014).

26. Singh, S; Koyama, H; Bhati, KK; Alok, A. The biotechnological importance of the plant-specific NAC transcription factor family in crop improvement. J. Plant. Res.; 2021; 134, pp. 475-495.1:CAS:528:DC%2BB3MXhtFKltL%2FM [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33616799][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106581][DOI: https://dx.doi.org/10.1007/s10265-021-01270-y]

27. Marok, MA; Marok-Alim, D; Rey, P. Contribution of functional genomics to identify the genetic basis of water‐deficit tolerance in barley and the related molecular mechanisms. J. Agron. Crop. Sci.; 2021; 207, pp. 913-935.1:CAS:528:DC%2BB3MXhs1yjtLfF [DOI: https://dx.doi.org/10.1111/jac.12526]

28. Christiansen, MW; Holm, PB; Gregersen, PL. Characterization of barley (Hordeum vulgare L.) NAC transcription factors suggests conserved functions compared to both monocots and dicots. BMC Res. Notes; 2011; 4, pp. 1-13. [DOI: https://dx.doi.org/10.1186/1756-0500-4-302]

29. Howard, TP et al. Use of advanced recombinant lines to study the impact and potential of mutations affecting starch synthesis in barley. J. Cereal Sci.; 2014; 59, pp. 196-202.1:CAS:528:DC%2BC2cXhs1Kit7o%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24748716][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3990431][DOI: https://dx.doi.org/10.1016/j.jcs.2013.12.012]

30. Yuan, S et al. Comprehensive analysis of CCCH-type zinc finger family genes facilitates functional gene discovery and reflects recent allopolyploidization event in tetraploid switchgrass. BMC Genom.; 2015; 16, pp. 1-16. [DOI: https://dx.doi.org/10.1186/s12864-015-1328-4]

31. Zhu, JK. Abiotic stress signaling and responses in plants. Cell; 2016; 167, pp. 313-324.1:CAS:528:DC%2BC28Xhs1Cltr%2FK [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27716505][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104190][DOI: https://dx.doi.org/10.1016/j.cell.2016.08.029]

32. Ling, L et al. Genome-wide identification of NAC gene family and expression analysis under abiotic stresses in Avena sativa. Genes; 2023; 14, 1186.1:CAS:528:DC%2BB3sXhsVSisb3J [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37372366][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10298501][DOI: https://dx.doi.org/10.3390/genes14061186]

33. Min, X et al. Genome-wide identification of NAC transcription factor family and functional analysis of the abiotic stress-responsive genes in Medicago sativa L. J. Plant Growth Regul.; 2020; 39, pp. 324-337.1:CAS:528:DC%2BC1MXhtFyisrzE [DOI: https://dx.doi.org/10.1007/s00344-019-09984-z]

34. Priest, HD; Filichkin, SA; Mockler, TC. Cis-regulatory elements in plant cell signaling. Curr. Opin. Plant. Biol.; 2009; 12, pp. 643-649.1:CAS:528:DC%2BD1MXht1ais7rI [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19717332][DOI: https://dx.doi.org/10.1016/j.pbi.2009.07.016]

35. Higo, K; Ugawa, Y; Iwamoto, M; Korenaga, T. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res.; 1999; 27, pp. 297-300.1:CAS:528:DyaK1MXpsVKgug%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/9847208][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC148163][DOI: https://dx.doi.org/10.1093/nar/27.1.297]

36. Guo, J et al. Polyamines regulate strawberry fruit ripening by abscisic acid, auxin, and ethylene. Plant Physiol.; 2018; 177, pp. 339-351.1:CAS:528:DC%2BC1cXhslOmsrjK [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29523717][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933135][DOI: https://dx.doi.org/10.1104/pp.18.00245]

37. An, XH et al. MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples. Plant Cell Physiol.; 2015; 56, pp. 650-662.1:CAS:528:DC%2BC28Xhtlaqt7vL [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25527830][DOI: https://dx.doi.org/10.1093/pcp/pcu205]

38. Fan, K et al. Asymmetric evolution and expansion of the NAC transcription factor in polyploidized cotton. Front. Plant Sci.; 2018; 9, 47.2018E&PSL.493..47F [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29441080][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797638][DOI: https://dx.doi.org/10.3389/fpls.2018.00047]

39. Fan, K et al. Molecular evolution and expansion analysis of the NAC transcription factor in Zea mays. PloS One; 2014; 9, e111837.2014PLoSO..9k1837F [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25369196][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219692][DOI: https://dx.doi.org/10.1371/journal.pone.0111837]

40. Hu, W et al. Genome-wide identification and expression analysis of the NAC transcription factor family in cassava. PLoS One; 2015; 10, e0136993. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26317631][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552662][DOI: https://dx.doi.org/10.1371/journal.pone.0136993]

41. Fu, C; Liu, M. Genome-wide identification and molecular evolution of NAC gene family in Dendrobium nobile. Front. Plant Sci.; 2023; 14, 1232804. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37670854][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10475575][DOI: https://dx.doi.org/10.3389/fpls.2023.1232804]

42. Lee, MH; Jeon, HS; Kim, HG; Park, OK. An Arabidopsis NAC transcription factor NAC4 promotes pathogen-induced cell death under negative regulation by microRNA164. New Phytol.; 2017; 214, pp. 343-360.1:CAS:528:DC%2BC2sXjtlCksb4%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28032643][DOI: https://dx.doi.org/10.1111/nph.14371]

43. Guo, HS; Xie, Q; Fei, JF; Chua, NH. MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant. Cell.; 2005; 17, pp. 1376-1386.1:CAS:528:DC%2BD2MXksVKksrk%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15829603][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1091761][DOI: https://dx.doi.org/10.1105/tpc.105.030841]

44. Fang, Y; Xie, K; Xiong, L. Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice. J. Exp. Bot.; 2014; 65, pp. 2119-2135.1:CAS:528:DC%2BC2cXmsVGkurY%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24604734][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991743][DOI: https://dx.doi.org/10.1093/jxb/eru072]

45. Mallory, AC; Dugas, DV; Bartel, DP; Bartel, B. MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs. Curr. Biol.; 2004; 14, pp. 1035-1046.1:CAS:528:DC%2BD2cXltVOgtLg%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15202996][DOI: https://dx.doi.org/10.1016/j.cub.2004.06.022]

46. Aida, M; Ishida, T; Fukaki, H; Fujisawa, H; Tasaka, M. Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant. cell.; 1997; 9, pp. 841-857.1:CAS:528:DyaK2sXktFCmu70%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/9212461][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC156962][DOI: https://dx.doi.org/10.1105/tpc.9.6.841]

47. Kim, JH et al. Trifurcate feed-forward regulation of age-dependent cell death involving miR164 in Arabidopsis. Science; 2009; 323, pp. 1053-1057.2009Sci..323.1053K1:CAS:528:DC%2BD1MXitVyntr4%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19229035][DOI: https://dx.doi.org/10.1126/science.1166386]

48. Nikovics, K et al. The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis. Plant. Cell.; 2006; 18, pp. 2929-2945.1:CAS:528:DC%2BD2sXit1ClsA%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17098808][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1693934][DOI: https://dx.doi.org/10.1105/tpc.106.045617]

49. Kantar, M; Unver, T; Budak, H. Regulation of barley miRNAs upon dehydration stress correlated with target gene expression. Funct. Integr. Genom.; 2010; 10, pp. 493-507.1:CAS:528:DC%2BC3cXhtlKrsbvL [DOI: https://dx.doi.org/10.1007/s10142-010-0181-4]

50. Kamthan, A; Chaudhuri, A; Kamthan, M; Datta, A. Small RNAs in plants: recent development and application for crop improvement. Front. Plant Sci.; 2015; 6, 208. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25883599][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382981][DOI: https://dx.doi.org/10.3389/fpls.2015.00208]

51. Pellegrini, M; Haynor, D; Johnson, JM. Protein interaction networks. Expert Rev. Proteomics; 2004; 1, pp. 239-249.1:CAS:528:DC%2BD2cXns1CisLs%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15966818][DOI: https://dx.doi.org/10.1586/14789450.1.2.239]

52. Liu Xu, L. X. & Li Ling, L. L. Cloning and characterization of the NAC-like gene AhNAC2 and AhNAC3 in peanut (2009).

53. Podzimska-Sroka, D; O’Shea, C; Gregersen, PL; Skriver, K. NAC transcription factors in senescence: from molecular structure to function in crops. Plants; 2015; 4, pp. 412-448. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27135336][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4844398][DOI: https://dx.doi.org/10.3390/plants4030412]

54. Wärnmark, A; Treuter, E; Wright, AP; Gustafsson, JA. Activation functions 1 and 2 of nuclear receptors: molecular strategies for transcriptional activation. Mol. Endocrinol.; 2003; 17, pp. 1901-1909. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12893880][DOI: https://dx.doi.org/10.1210/me.2002-0384]

55. Todeschini, AL; Georges, A; Veitia, RA. Transcription factors: specific DNA binding and specific gene regulation. Trends Genet.; 2014; 30, pp. 211-219.1:CAS:528:DC%2BC2cXntFCqu70%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24774859][DOI: https://dx.doi.org/10.1016/j.tig.2014.04.002]

56. Szabados, L., Kovács, H., Zilberstein, A. & Bouchereau, A. In Advances in Botanical Research, Vol. 57 105–150 (Elsevier, 2011).

57. Dassi, E. Mapping of Post-Transcriptional Regulatory Networks by Means of Mechanistic and High Throughput Data (University of Trento, 2012).

58. Giancarla, V et al. Assessment of drought tolerance in some barley genotypes cultivated in West part of Romania. J. Hortic. Biotechnol.; 2010; 14, pp. 114-118.

59. Kishor, P. B. K., Rajesh, K., Reddy, P. S., Seiler, C. & Sreenivasulu, N. Drought stress tolerance mechanisms in barley and its relevance to cereals. Biotechnol. Approaches Barley Improv.2014, 161–179 (2014).

60. Kikuchi, K et al. Molecular analysis of the NAC gene family in rice. Mol. Gen. Genet. MGG; 2000; 262, pp. 1047-1051.1:CAS:528:DC%2BD3cXhtlSitLo%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10660065][DOI: https://dx.doi.org/10.1007/PL00008647]

61. Jensen, MK; Skriver, K. NAC transcription factor gene regulatory and protein–protein interaction networks in plant stress responses and senescence. Iubmb Life; 2014; 66, pp. 156-166.1:CAS:528:DC%2BC2cXks1Onsbs%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24659537][DOI: https://dx.doi.org/10.1002/iub.1256]

62. Shan, Z et al. Genome-wide analysis of the NAC transcription factor family in broomcorn millet (Panicum miliaceum L.) and expression analysis under drought stress. BMC Genom.; 2020; 21, pp. 1-13. [DOI: https://dx.doi.org/10.1186/s12864-020-6479-2]

63. Li, W et al. NAC family transcription factors in tobacco and their potential role in regulating leaf senescence. Front. Plant Sci.; 2018; 9, 1900. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30622549][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6308388][DOI: https://dx.doi.org/10.3389/fpls.2018.01900]

64. Shang, H; Li, W; Zou, C; Yuan, Y. Analyses of the NAC transcription factor gene family in Gossypium Raimondii Ulbr.: chromosomal location, structure, phylogeny, and expression patterns. J. Integr. Plant Biol.; 2013; 55, pp. 663-676.1:CAS:528:DC%2BC3sXhsVaqsL7F [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23756542][DOI: https://dx.doi.org/10.1111/jipb.12085]

65. Hao, YJ et al. Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants. Plant J.; 2011; 68, pp. 302-313.1:CAS:528:DC%2BC3MXhsVCksrvM [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21707801][DOI: https://dx.doi.org/10.1111/j.1365-313X.2011.04687.x]

66. Pascual, MB; Cánovas, FM; Ávila, C. The NAC transcription factor family in maritime pine (Pinus Pinaster): molecular regulation of two genes involved in stress responses. BMC Plant Biol.; 2015; 15, pp. 1-15. [DOI: https://dx.doi.org/10.1186/s12870-015-0640-0]

67. Ono, T; Yoshida, MC. Differences in the chromosomal distribution of telomeric (TTAGGG) n sequences in two species of the vespertilionid bats. Chromosome Res.; 1997; 5, pp. 203-205.1:CAS:528:DyaK2sXkslyguro%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/9246415][DOI: https://dx.doi.org/10.1023/A:1018403215999]

68. Kanazin, V. Characterization of diversity among members of multigene families in the barley genome (1993).

69. Jahan, B; Vahidy, AA. N-banding patterns of heterochromatin distribution in Hordeum jubatum chromosomes. Pak J. Bot.; 2009; 41, pp. 1037-1041.

70. Wang, H. Molecular Phylogeny of the genus Hordeum and origins of Hordeum polyploidy species (2011).

71. Liu, X et al. The LRR-RLK protein HSL3 regulates stomatal closure and the drought stress response by modulating hydrogen peroxide homeostasis. Front. Plant Sci.; 2020; 11, 548034. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33329622][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728693][DOI: https://dx.doi.org/10.3389/fpls.2020.548034]