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6 November 2025

Transcriptional Regulation of Salt Stress Tolerance in Triticum aestivum (Wheat): NAC Transcription Factors and Their Target Genes

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1
Department of Biological and Environmental Sciences, University of Gothenburg, Box 461, 405 30 Gothenburg, Sweden
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Triticeae Research Institute, Sichuan Agricultural University, Wenjiang 611130, China
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Department of Biochemistry and Molecular Biology, Hajee Mohammad Danesh Science and Technology University (HSTU), Dinajpur 5200, Bangladesh
*
Author to whom correspondence should be addressed.
Crops2025, 5(6), 81;https://doi.org/10.3390/crops5060081
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Abstract

Salinity is one of the key threats to food security and sustainability. To make saline soils productive again, we need to develop salt-tolerant crop varieties. Developing salt-tolerant wheat requires a detailed understanding of the molecular mechanisms underlying salt stress responses. In this study, we analyzed the Chinese Spring genome and identified 559 putative NAC transcription factors (TFs), which are recognized as key regulators of both abiotic and biotic stress. Protein family analysis revealed four distinct domain architectures, with more than 95% of the proteins containing a single NAC domain, consistent with their conserved regulatory role. Through in silico analyses, four salt stress-responsive TFs, NAC_1D, NAC_2D, NAC_4A, and NAC_5A, were highlighted, sharing nine of 13 DNA-binding residues. Promoter analysis of their putative target genes identified seven candidates, which, together with the NAC TFs, were subjected to RT-qPCR expression analysis in BARI Gom-25 plants exposed to 100 mM NaCl. The expression data revealed contrasting regulatory patterns between NAC TFs and their target genes. For example, Hsp70 was strongly upregulated in both shoots and roots, despite opposite patterns of NAC_1D expression between tissues. Similarly, bZIP expression mirrored the downregulation of NAC_2D, whereas HKT8 expression remained stable under salt stress. NAC_4A showed a root-specific pattern suggestive of positive regulation of a Non-specific serine/threonine protein kinase, while NAC_5A upregulation corresponded with downregulation of Plant cadmium resistance 2. Collectively, these results provide functional insights into four NAC TFs and identify potential molecular targets for improving wheat salt tolerance. By targeting key tolerance genes at the DNA level offers greater precision and can significantly reduce breeding time.

1. Introduction

Abiotic stresses such as drought, high temperature, and salinity significantly reduce global food production. If current rates of anthropogenic climate change continue, global mean temperatures could rise by up to 6.4 °C by the end of the century [], accelerating glacier melt and raising sea levels by an estimated 0.9 m by 2100 and up to 2.5 m by 2300 []. Such climatic changes increase the frequency of extreme events—including floods, droughts, storms, and shifts in precipitation patterns—which directly impact agriculture.
Salinity, one of the most severe abiotic stresses, currently affects about 20% of arable land worldwide, and the problem is intensifying due to both climate change and human activities [,]. Salt stress disrupts physiological and biochemical processes, causing substantial yield and quality losses in crops, including Triticum aestivum (wheat) []. The cultivation of wheat is critically important for sustaining food and nutritional security, but it faces a significant global danger from rapidly increasing soil and water salinity []. As global population growth will require a 60% increase in food production by 2050 compared to 2005–2007 levels [], improving the salt tolerance of wheat. a major staple crop and one of the world’s most important cereal for human consumption [], critical for food security.
Salt tolerance in plants is a complex quantitative trait influenced by multiple genetic and environmental factors. Genome-wide association studies (GWAS) have been widely applied to uncover the genetic basis of such traits []. Developing stress-tolerant wheat varieties for saline-prone regions is a major goal of modern breeding, and numerous studies have sought to identify genes that enhance tolerance to abiotic stress [,].
Transcription factors (TFs) are central to regulating plant stress responses, with several families e.g., WRKY, NAC, AP2/EREBP, bZIP, and MYB, playing pivotal roles in stress adaptation [,,,,,,,]. Among these, NAC TFs—named after the first characterized members NAM (no apical meristem), ATAF1/2, and CUC2 [,,]—are broadly conserved and participate in both developmental processes and abiotic stress responses [,]. The highly conserved NAC DNA-binding domain, located at the N-terminus, typically spans 150–160 amino acids and comprises five subdomains (A–E) involved in DNA binding, nuclear localization, and homo-/heterodimerization [,].
NAC TFs have been identified in a wide range of plant species, with over 100 members in Oryza sativa (rice) and Arabidopsis thaliana (Arabidopsis) [], yet only a fraction has been functionally characterized [,]. Early studies linked NAC genes to developmental regulation, such as NAM in Petunia hybrida (petunia) and CUC1_2 in Arabidopsis, which are expressed at the primordial and meristem boundaries and play important roles in directing the development of boundary cells define organ boundaries [,,,]. Other NAC members, such as ATAF1_2 in Arabidopsis and StNAC in Solanum lycopersicum (tomato), are induced by pathogen attack and mechanical injury []. Stress-responsive NAC genes have been reported in several species e.g., Brassica napus (mustard) [], Arabidopsis [,], and rice [], with functions in drought, salt, cold, and hormonal signaling pathways.
In wheat, initially only a few NAC TFs were linked to abiotic stress and plant growth [,,]. GRAB1 and GRAB2 confer resistance to wheat dwarf geminivirus [], and TaNAC4 is associated with fungal defense []. Although more knowledge has been collected in recent years [,,], still given the limited knowledge of wheat NAC TFs in abiotic stress tolerance, comprehensive identification and characterization of these genes is needed. Although NAC TFs are one of the largest TF families in plants, most functional insights so far come from model species such as Arabidopsis and rice [,,]. In contrast, wheat, despite its global importance as a staple food crop, remains comparatively underexplored [,,,]. This gap limits the translation of functional genomics into crop improvement strategies, especially because wheat’s complex polyploid genome makes gene identification and functional validation more difficult. However, with the rapid progress of technologies such as gene editing and multi-omics, more wheat genes will be functionally characterized, and their regulatory networks, interactions with other signaling pathways, and roles in stress adaptation will become better understood. Thus, functional validation studies in wheat are emerging e.g., TaNAC069 linked to resistance to Puccinia triticina (the cause of wheat leaf rust), TaNAC018-7D linked to seed dormancy, and TdNAC8470 linked to grain starch accumulation [,,]. Here, finding new wheat NAC genes should help us to understand how plants defend themselves against abiotic stress like salinity. In our study using genome-wide analysis we identify existing NAC genes, the target genes they regulate, and examine their expression profiles under salt stress.

2. Materials and Methods

2.1. Identification of NAC Sequences

The Hidden Markov Model (HMM) profile for the wheat No apical meristem (NAM) protein (NAC) domain (PF02365) was obtained from the InterPro database (PFAM version 37.4) [] to identify NAC sequences in the wheat genome. The proteome of the wheat cultivar Chinese Spring was downloaded from the Ensembl Plants database (https://plants.ensembl.org/index.html, accessed on 10 August 2023) and used as a reference. The NAC domain HMM profile was used as a query to search the wheat proteome with HMMER software (version 3.1), applying an E-value threshold of 1 × 10−5. An in-house Python >3.8 script was then used to extract amino acid sequences of proteins containing the NAC domain. Redundant protein sequences were removed using CD-HIT (https://github.com/daugherty-lab/CD-hit, accessed on 7 September 2023) with a sequence identity cut-off of 100%. The resulting non-redundant representative sequences were retained for further analysis.

2.2. Transcription Factor Binding Site Prediction

The 2000 bp upstream regions of all identified genes were extracted from the IWGSC Chinese Spring wheat genome, accessed via the Ensembl database. The getfasta function in Bedtools was used to retrieve the upstream sequences for each gene. NAC protein sequences were analyzed with the Profile Inference Tool to identify JASPAR TF binding profiles. An in-house Python >3.8 script (https://github.com/Sameerpython/Transcription-Factors, accessed on 18 December 2023) was used to query the JASPAR database.
Next, Position Weight Matrices (PWMs) for the four most prominent NAC sequences, designated as NAC domain-containing proteins followed by chromosome location (NAC_1D, TraesCS1D02G263800; NAC_2D, TraesCS2D02G100900; NAC_4A, TraesCS4A02G130600; NAC_5A, TraesCS5A02G468300), were downloaded in MEME format from the JASPAR CORE database. The upstream region of each gene was scanned against these PWMs to predict NAC transcription factor binding sites using the FIMO tool 4.11.4 (http://meme-suite.org/index.html, accessed on 8 January 2024) with a significance threshold of p < 1 × 10−5.

2.3. Functional Gene Extraction

The 144 genes previously reported to be involved in salt stress [] were compared against the most significant 1000 target genes identified for each of the NAC_1D, NAC_2D, NAC_4A, and NAC_5A TF family members. Many genes were repeatedly identified for individual NAC genes, but when combining the targets from all FIMO analyses for the NAC family, the total number of unique target genes was just over 3000. Gene IDs obtained from the FIMO output were processed in R to extract matches to the 144 salt stress-related genes, thereby identifying potential NAC-regulated salt stress genes.
The identified NAC functional genes were further analyzed in silico to assess their expression patterns under salt stress, using the dataset available on the Wheatomics 1.0 website (http://202.194.139.32, accessed on 25 January 2024) []. Within the “Online Tools” section, the “Gene Expression” option was used to examine transcriptome responses of two wheat cultivars to salt stress, as listed under the “wheat biotic options” category.

2.4. Mapping of DNA-Binding Residues in NAC Transcription Factors

The NAC protein sequences were searched against the Protein Data Bank (PDB) using the BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 2 February 2024). The experimentally determined structure of a NAC domain bound to DNA (PDB ID: 3SWM) was identified and aligned with the NAC transcription factor domain sequences obtained in this study. DNA-contacting amino acids were identified using MSALigMap [].

2.5. Chromosomal Localization of NAC Genes

The chromosomal positions of the wheat NAC TFs were obtained from the Ensembl Plants database. Their distribution across the wheat chromosomes was visualized using the Triticum aestivum karyotype available in Ensembl Plants (https://plants.ensembl.org/Triticum_aestivum/Location/Genome, accessed on 18 March 2024).

2.6. Protein Structure Prediction

The three-dimensional structures of the four selected NAC sequences were predicted using AlphaFold2 []. NAC domain sequences were extracted and used as input for the AlphaFold pipeline. The highest-ranked model for each protein was selected based on the predicted Local Distance Difference Test (pLDDT) score. Structural models were visualized using ChimeraX [].

2.7. Plant Growth Conditions and Treatments

The BARI Gom-25 wheat variety, developed by the Bangladesh Agricultural Research Institute (BARI) for its moderate salt tolerance and previously used in salt tolerance assays [,,,,], was used in this study. Seeds were germinated on wet filter paper (Munktell filter paper, A1-100-80TM, Fischer Scientific, Mölnlycke, Sweden) for three days in darkness at room temperature. On the fourth day, seedlings were transferred to a hydroponic growth system containing tap water supplemented with Nelson Garden Hydroponic Nutrition™ (2 mL/L) under continuous aeration. After six days of hydroponic growth, 100 mM NaCl was added to one unit of the system to induce salt stress, while another unit was maintained without salt as the control. After six days of treatment, shoot and root tissues were harvested separately 2–3 h after the onset of light, immediately frozen in liquid nitrogen, and stored at −80 °C until further analyses. Each root or shoot sample consisted of a pooled collection from 20 individual plants.

2.8. Reverse Transcription-Quantitative Real-Time PCR (RT-qPCR)

The RT-qPCR assays were performed using shoot and root tissues from 12-day-old BARI Gom-25 wheat plants grown under control or salt stress conditions in the hydroponic system. Frozen tissues were pulverized using a Mixer Mill MM 301 (Retsch GmbH, Haan, Germany) at 15 s × 2 cycles. Total RNA was extracted using the NucleoSpin RNA Plant™ kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. cDNA was synthesized from 100 ng of total RNA using the iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA).
The RT-qPCR reactions were carried out on a Bio-Rad CFX96 Real-Time™ system using the SsoAdvanced Universal SYBR Green Supermix™ (Bio-Rad, Hercules, Sweden) following the manufacturer’s protocol. The BARI Gom-25 actin gene was used as the internal reference. Relative expression levels were calculated using the 2−ΔΔCt method []. Each reaction included three technical replicates, and all experiments were performed with three biological replicates. Primers were designed using the Primer3 tool and are listed in Supplementary Table S1.

3. Results

3.1. Identification of NAC Transcription Factors in the Wheat Genome

To comprehensively identify NAC genes in wheat, a Hidden Markov Model (HMM) profile of the NAC domain (PF02365) was used to search the latest wheat genome database. This analysis identified a total of 559 NAC gene sequences, which included 99 transcript isoforms. To further validate the high sequence conservation among NAC family members, the conserved NAC domain—a defining feature of this family—was specifically analyzed. The length of the NAC domain ranged from 21 to 636 amino acids, while the full-length NAC proteins varied between 104 and 1029 amino acids (https://ftp.ensemblgenomes.ebi.ac.uk/pub/plants/release-57/fasta/triticum_aestivum/pep/Triticum_aestivum.IWGSC.pep.all.fa.gz, accessed 10 August 2023).

3.2. Domain Architecture of NAC Transcription Factors

Protein function can often be inferred from their domain architecture. A PFAM analysis of the 460 identified NAC proteins revealed four distinct domain architectures, involving four different domains (Figure 1). Specifically, 441 sequences contained a single NAC domain, 16 sequences had two NAC domains, two sequences possessed one NAC domain in combination with an AA_kinase domain, and one sequence included a NAC domain together with a C2H2 domain and a DnaJ domain (Figure 1).
Figure 1. Four distinct domain architectures were observed among the NAC transcription factors identified in the Chinese Spring wheat genome. These architectures involved a total of four different domains: NAC (PF02365), AA_kinase (PF00696), C2H2 (PF02892), and DnaJ (PF00226). The number of identified sequences is indicated next to the corresponding domain structure.

3.3. Phylogenetic Analysis of NAC Transcription Factors

To investigate the phylogenetic and evolutionary relationships among NAC TFs in wheat, a maximum-likelihood tree was constructed using RAxML with 377 NAC sequences and 100 bootstrap replicates (Figure 2). Many of the NAC domains among the initially identified 441 sequences shared high sequence similarity. To reduce redundancy and prevent over-representation of nearly identical sequences in the phylogenetic analysis, we used CD-HIT (≥95% similarity) to cluster highly similar sequences and retained only a representative sequence from each cluster. Thus, after this filtering step, 377 sequences were used when constructing the tree (Supplementary Table S2). The resulting tree revealed clear clustering of NAC proteins into multiple distinct clades, indicating evolutionary diversification within the family. Most internal branches showed high bootstrap support, demonstrating the statistical reliability of the inferred relationships. This phylogenetic framework provides a basis for classifying NAC proteins and selecting candidate sequences for further functional characterization.
Figure 2. Phylogenetic analysis of NAC protein sequences from the wheat genome. The evolutionary relationships of 377 wheat NAC sequences were inferred using the maximum-likelihood method with 100 bootstrap replicates. To avoid redundancy and prevent over-representation of identical sequences in the tree, CD-HIT (>95% similarity) was used to cluster highly similar sequences among the initial identified 441 sequences. Thus, in the end 377 sequences were used in the tree.

3.4. Chromosomal Location of NAC Transcription Factors

All identified NAC TFs were mapped to the wheat chromosomes to determine their distribution across the genome. The distribution of NAC TFs across the seven chromosomes in the three wheat subgenomes (A, B, and D) is shown in Figure 3. Chromosome 1 of all three subgenomes contained the fewest NAC sequences, with 6, 8, and 7 genes in the A, B, and D genomes, respectively. In contrast, chromosome 2 of the A subgenome had the highest number of NAC genes, with a total of 39 sequences (Supplementary Table S3, Figure 3).
Figure 3. Chromosomal distribution of all identified NAC transcription factors in the wheat genome. The four NAC transcription factors associated with salt tolerance are highlighted across the seven chromosomes of the A, B, and D subgenomes in Chinese Spring wheat. “Un” indicates unassigned scaffolds of the wheat chromosomes. The centromere of each chromosome is indicated by a small constriction. Orange, NAC_1D (TraesCS1D02G263800) on chromosome 1D; green NAC_2D (TraesCS2D02G100900) on chromosome 2D; NAC_4A (TraesCS4A02G130600) on chromosome 4A and NAC_5A (TraesCS5A02G468300) on chromosome 5A are both colored red.

3.5. Protein Structure Prediction and DNA Binding Site Analysis

The three-dimensional structures of the four NAC TFs were predicted using AlphaFold. All predicted models exhibited high confidence scores, indicating reliable structural predictions (Figure 4). To identify the DNA-binding residues, the NAC domain sequences of NAC_1D, NAC_2D, NAC_4A, and NAC_5A were aligned with the experimentally determined structure of an Arabidopsis NAC TF bound to DNA (PDB ID: 3SWM) (Figure 5). DNA-binding residues in 3SWM were identified using MSALigMap []. The alignment revealed that the DNA-binding sites are highly conserved among the four NAC proteins. Specifically, amino acids mapped as DNA-binding residues and highlighted in Figure 5 are identical, demonstrating strong conservation at these positions. Overall, nine out of the 13 DNA-interacting residues are identical across all four NAC sequences (Figure 6).
Figure 4. Predicted three-dimensional structures of the four wheat NAC transcription factors generated using AlphaFold. (A) NAC_1D; (B) NAC_2D; (C) NAC_4A; (D) NAC_5A.
Figure 5. Multiple sequence alignment of wheat NAC sequences with the Arabidopsis NAC structure (PDB ID: 3SWM). Amino acids identified as DNA-binding residues in 3SWM are colored according to their different categories: magenta, proline/glycine (conformationally special); blue, polar; red, with positive charge; pink, aliphatic/hydrophobic; green, with negative charge; orange, aromatic. Color coded secondary structures elements are labeled as follows: B, beta-bridge residue; E, extended strand (beta sheet); G, 3/10 helix; H, hydrogen bond; I, Pi-helix; S, bend; T, H-bonded turn. To be noted, no beta-bridge residues were identified in the NAC sequences.
Figure 6. DNA-binding residues of wheat NAC transcription factors. Amino acids that are identical across all four sequences are highlighted in red and positions where the amino acids differ across the sequences are highlighted in blue.

3.6. Identification and Extraction of Target Genes

Target genes were extracted based on gene IDs from the FIMO analysis using R, allowing the identification of potential salt-stress-related functional genes. The extracted genes were further analyzed in relation to the four selected NAC transcription factors: NAC_1D, NAC_2D, NAC_4A, and NAC_5A. Genes were ranked according to the most significant p-values and q-values obtained from the FIMO analysis. These target genes were then compared with publicly available RNA-seq datasets on wheat salt tolerance from NCBI (https://www.ncbi.nlm.nih.gov/bioproject/, accessed on 13 October 2024), enabling the identification of the most prominent salt-responsive target genes regulated by the four NAC TFs (Table 1).
Table 1. The four NAC transcription factors and their predicted target genes involved in salt stress response. Proposed molecular functions and biological processes are based on Gene Ontology annotations. Information retrieved from NCBI (www.ncbi.nlm.nih.gov, accessed on 13 October 2024) and Wheatomics 1.0 (http://202.194.139.32/, accessed on 25 January 2024) [].

3.7. NAC Family Gene Expression Under Salt Stress

In silico analyses of NAC family members using publicly available RNA-seq data revealed that specific NAC genes, NAC_1D, NAC_2D, NAC_4A, and NAC_5A, are expressed under salt stress conditions. All these NAC proteins exhibit DNA-binding TF activity (Table 1). Additionally, the target genes predicted to be regulated by these NAC TFs were evaluated for their responsiveness to salt stress before being selected for further expression analyses.

3.7.1. NAC_1D Family

The RT-qPCR analysis of the gene encoding the NAC_1D (TraesCS5A02G237200) TF revealed upregulation in shoot tissues under NaCl stress compared to control samples (Figure 7A). Its predicted target genes, Calcium-dependent protein kinase (TraesCS2A02G456100) and Hsp70 (TraesCS1D02G284000), also showed upregulation in shoot tissues (Figure 7B,C). In contrast, in root tissues, downregulation of NAC_1D was associated with a similar downregulation of Calcium-dependent protein kinase, whereas Hsp70 exhibited strong upregulation under salt stress (Figure 7).
Figure 7. Expression analysis of NAC_1D and its target genes under salt stress. BARI Gom-25 wheat seedlings were grown hydroponically for six days, followed by treatment with or without 100 mM NaCl for an additional six days. Shoot and root tissues were analyzed for expression of the TF (A) and its predicted target genes (B,C). The actin gene was used as the internal reference. Relative expression levels were calculated using the 2−ΔΔCt method. Values represent the mean ± SEM of three independent biological replicates (n = 3), each comprising three technical replicates. Dots indicate individual sample values.

3.7.2. NAC_2D Family

The expression of the gene encoding NAC_2D (TraesCS2D02G100900) was downregulated in both shoot and root tissues under salt stress. This downregulation corresponded with reduced expression of its predicted target genes, bZIP transcription factor (TraesCS3D02G364900) and HKT8 (TraesCS4D02G361300) (Figure 8). The effect was most pronounced for the bZIP transcription factor.
Figure 8. Expression analysis of NAC_2D and its target genes under salt stress. BARI Gom-25 wheat seedlings were grown hydroponically for six days, followed by treatment with or without 100 mM NaCl for an additional six days. Shoot and root tissues were analyzed for expression of the TF (A) and its predicted target genes (B,C). The actin gene was used as the internal reference. Relative expression levels were calculated using the 2−ΔΔCt method. Values represent the mean ± SEM of three independent biological replicates (n = 3), each comprising three technical replicates. Dots indicate individual sample values.

3.7.3. NAC_4A Family

The expression of NAC_4A (TraesCS4A02G130600) was analyzed by RT-qPCR in shoot and root tissues. NAC_4A showed no significant change in expression in shoot tissues under salt stress but was upregulated in root tissues (Figure 9A). Its predicted target gene, Non-specific serine/threonine protein kinase (TraesCS7B02G279300), exhibited a similar pattern in roots, while showing downregulation in shoot tissues under 100 mM NaCl stress (Figure 9B).
Figure 9. Expression analysis of NAC_4A and its target gene under salt stress. BARI Gom-25 wheat seedlings were grown hydroponically for six days and then treated with or without 100 mM NaCl for another six days. Shoot and root tissues were analyzed for expression of the TF (A) and its predicted target gene (B). The actin gene was used as the internal reference. Relative expression levels were calculated using the 2−ΔΔCt method. Values represent the mean ± SEM of three independent biological replicates (n = 3), each comprising three technical replicates. Dots indicate individual sample values.

3.7.4. NAC_5A Family

The expression of NAC_5A (TraesCS5A02G468300) was upregulated in both shoot and root tissues under NaCl stress, with a more pronounced increase in shoots (Figure 10A). Its predicted target gene, Plant cadmium resistance 2 (TraesCS3B02G214000), showed opposite behavior, being downregulated in both shoot and root tissues (Figure 10B). The other predicted target gene, Plasma membrane-associated cation-binding protein 1 (TraesCS3B02G183300), was downregulated in roots but showed no significant change in shoots under 100 mM NaCl stress (Figure 10C).
Figure 10. Expression analysis of NAC_5A and its target genes under salt stress. BARI Gom-25 wheat seedlings were grown hydroponically for six days and then treated with or without 100 mM NaCl for another six days. Shoot and root tissues were analyzed for expression of the TF (A) and its predicted target genes (B,C). The actin gene was used as the internal reference. Relative expression levels were calculated using the 2−ΔΔCt method. Values represent the mean ± SEM of three independent biological replicates (n = 3), each comprising three technical replicates. Dots indicate individual sample values.

4. Discussion

NAC (NAM, ATAF1/2, and CUC2) TFs are pivotal regulators of plant responses to diverse environmental stresses, including salt stress. These TFs modulate the expression of downstream target genes involved in stress signaling, metabolic processes, and developmental pathways, thereby enhancing stress tolerance mechanisms. In wheat, four NAC family genes have been identified as potential key regulators under salt stress conditions, showing differential expression in various tissues, such as shoots and roots. Salt stress imposes both osmotic and ionic challenges, disrupting cellular homeostasis and metabolic activities. In response, plants activate transcriptional regulators, including NAC TFs, to mitigate stress effects and improve survival under adverse conditions. Understanding the expression dynamics of NAC genes and their target genes is therefore essential for deciphering the molecular mechanisms underlying salt stress responses in wheat.
Using the Triticum_aestivum.IWGSC.54 annotation, we identified 16 wheat genes containing two NAC domains, compared to eight sequences reported previously []. Only one gene contained three additional domains in addition to the NAC domain. The four NAC TFs linked to salt stress in this study belong to subgroup 1, characterized by a single NAC domain. Overall, we characterized 460 NAC TF genes coding for 559 protein sequences, including 99 transcript isoforms. These numbers are largely consistent with prior reports [], although our analysis revealed differences in domain organization (higher number of genes with two NAC domains), likely reflecting improvements in the wheat genome assembly and annotation.
Phylogenetic analysis using maximum-likelihood methods revealed several bootstrap-supported clades, indicating robust evolutionary relationships among the sequences. While these groupings provide preliminary insights into potential functional divergence, further comparative analyses with related species and experimental validation are necessary to determine whether these clades correspond to conserved functional modules. Thus, the hexaploid wheat differs somewhat from the tetraploid Triticum dicoccoides L. (wild emmer wheat) where a total of 200 NAC TFs were identified and classified into twelve groups []. In another study of wild emmer wheat, 249 NAC family members were reported and grouped into seven clades [].
Chromosomal mapping indicated a relatively balanced distribution of NAC genes across the wheat subgenomes, with 157, 149, and 145 genes in the A, B, and D subgenomes, respectively, and only seven unassigned genes. This distribution suggests that no major gene loss or expansion has occurred in the NAC family across the three subgenomes, consistent with previous observations []. The small number of unassigned genes likely reflects gaps or ambiguities in the genome assembly version used.
Structural modeling of the four salt-responsive NAC TFs revealed a high degree of similarity, with RMSD values below 1.4 Å. DNA-binding site analysis against the Arabidopsis NAC-DNA complex (PDB ID: 3SWM) demonstrated that key residues involved in DNA interaction are highly conserved. Previous studies have suggested that structurally similar TFs may share TF binding motifs (TFBMs) [], although small variations in amino acid composition can alter binding specificity []. Among the NAC TFs analyzed here, NAC_1D and NAC_5A share identical DNA-binding residues, as do NAC_2D and NAC_4A. Interestingly, NAC_1D and NAC_5A also exhibit similar expression profiles under salt stress (Figure 7 and Figure 10), supporting the notion that DNA-binding domain (DBD) amino acid sequence similarity may predict TFBM specificity [].
NAC_1D (TraesCS1D02G263800) has been previously classified as a stress-responsive NAC (SNAC) subfamily member, designated TaSNAC1-1D []. TaSNAC1-1D was shown to be upregulated in response to severe drought in both leaves at the three-leaf stage and in seedling roots. In our study, NAC_1D exhibited clear upregulation in shoot tissues under continuous NaCl stress, whereas expression in roots was slightly downregulated (Figure 7A). This differential tissue-specific expression contrasts with its drought response, highlighting how NAC_1D activity varies under different stressors. Among its putative target genes, Calcium-dependent protein kinase (TraesCS2A02G456100) is shown to be upregulated under NaCl stress [] and contributes to disease resistance against rust and sheath blight [,]. Hsp70 (TraesCS1D02G284000), previously reported to confer thermotolerance under heat stress under the name TaHSP70d [,,,], was also upregulated in shoots during NaCl stress (Figure 7C). The effect is observed both during early heat stress and at vegetative stages when examining leaf tissue. It has been reported to be regulated by the sHSP hub gene TraesCS4D02G212300 [], as well as by HVA1. Overexpression of HVA1, which is known to improve drought and heat tolerance, was shown to upregulate Hsp70 []. Thus, Hsp70 appears to respond to several abiotic stressors, and we also observed its upregulation in shoots exposed to NaCl stress (Figure 7C). Given that Ca2+ ions serve as essential secondary messengers in signal transduction during plant defense responses, it is worth considering whether calcium-dependent protein kinases, such as TraesCS2A02G456100, are upregulated in parallel with Hsp70 (TraesCS1D02G284000) in wheat shoot tissues under NaCl stress, thereby amplifying the signal and enhancing salt tolerance.
Public RNA-seq datasets suggested that NAC_2D (TraesCS2D02G100900) is upregulated under salt stress. However, our observations indicate clear downregulation in both shoot and root tissues under 100 mM NaCl stress (Figure 8A). Its putative targets, a suggested ABA-responsive bZIP TF, ABA INSENSITIVE 5 (ABI5; TraesCS3D02G364900) and the HKT8 also known as. as HKT1:5-D (TraesCS4D02G361300)—also exhibited negative regulation under salt stress (Figure 8B,C). While TaABI5 is upregulated under drought and regulated by the TaGW2–TaARR12 module [], its downregulation here suggests a distinct regulatory mechanism under salt stress. Similarly, TaHKT1:5-D did not contribute to salt tolerance in synthetic hexaploid wheat [], aligning with our data. However, it is suggested that the salt tolerance is differentially controlled in synthetic hexaploid wheat than in common wheat where HKT overall is supposed to play a crucial role in salt stress tolerance []. These results indicate that NAC_2D and its target genes play a less prominent role in continuous salt stress response in wheat, though the underlying mechanisms remain to be elucidated.
NAC_4A (TraesCS4A02G130600) genes associated with stress signaling and metabolic processes, showing clear upregulation in roots but no significant changes in shoots under NaCl stress (Figure 9A). Its putative target gene, the Non-specific serine/threonine protein kinase (TraesCS7B02G279300), also known as. as TaSOS2 [], also showed increased expression in roots (Figure 9B). TaSOS2 is part of the salt overly sensitive (SOS) pathway, where SOS3 senses elevated Ca2+ under salt stress and activates SOS2, which phosphorylates SOS1 to export Na+ from the cytoplasm, acting as a Na+/H+ antiporter []. This suggests that NAC_4A may positively regulate TaSOS2 in roots, although direct mechanistic evidence is still required.
NAC_5A (TraesCS5A02G468300) belongs to the SNAC subfamily and has been reported to respond positively to drought under the name TaSNAC5-5A [] and cold stress []. Under 100 mM NaCl, NAC_5A exhibited strong upregulation in shoots and slight upregulation in roots (Figure 10A). Its putative targets, Plant cadmium resistance 2 (TraesCS3B02G214000) and Plasma membrane-associated cation-binding protein 1 (TraesCS3B02G183300), were downregulated in roots during salt stress (Figure 10B,C), indicating potential negative regulatory roles. In shoots, Plant cadmium resistance 2 expression was also reduced, while the Plasma membrane-associated cation-binding protein 1 showed no significant change. In Nicotiana benthamiana (benth), the Plasma membrane-associated cation-binding protein 1 negatively regulates cell-to-cell movement of viruses, thereby protecting the plant from infection []. However, whether such proteins are also involved in regulating processes such as cell-to-cell movement of Na+ ions remains speculative. It has been suggested that the addition of NaCl can alter soil metabolites involved in mediating Cd transport into wheat tissues in arid, Cd-contaminated soils []. However, the underlying mechanisms remain poorly understood. Under salt stress, if NAC_5A no longer maintains its expression level in roots, it may bind less effectively to its putative target gene, Plant Cadmium Resistance 2, thereby reducing that gene’s expression. This could, in turn, lower resistance to Cd, assuming the gene’s true function is to confer Cd tolerance. Consequently, Cd levels in cells might increase under salt stress.
Some of our accumulated results contrast with public RNA-seq datasets, e.g., in the case of NAC_2D. These differences may be due to variations in time points, treatment conditions, or duration of salt stress. This highlights the need for further experiments using multiple time points to better capture the long-term expression patterns of NAC genes. Further studies to clarify the roles and interactions of the genes presented in our study could include the creation of knockouts using gene-editing approaches or the use of individual TILLING lines with confirmed knockouts of specific genes in polyploid wheat []. Overexpression studies of wheat genes have been successfully carried out in other model systems [] but less frequently in wheat []. Although these approaches could help elucidate the functional relevance of both up- and downregulated genes, both gene editing and overexpression strategies are classified as GMO. As such, they continue to face major regulatory hurdles and public acceptance challenges in many regions, creating a less certain pathway for societal implementation of the results.
As an alternative, marker-assisted selection (MAS) for salt tolerance could provide a more practical strategy for applying new findings, as it enables more precise and efficient breeding compared to conventional approaches [,]. However, the polygenic nature of the salt tolerance and the limited understanding of salt tolerance in wheat—particularly the lack of well-defined gene locations and quantitative trait loci (QTLs)—remains a significant challenge for MAS [].
Regarding TFs, many TF families exhibit overlapping functions, and redundancy must therefore be considered when evaluating the specific role of a single gene. This is also relevant for members of the NAC family where functional overlap complicates straightforward interpretation. Moreover, some TF genes can function as either a positive or negative regulator of downstream target genes, with their role determined by several factors. The timing and duration of TF expression can dictate whether it activates or represses gene expression, as seen in various plant systems. Additionally, interactions with co-factors or other proteins can modulate the TF’s activity, influencing its role as a repressor or activator. Post-translational modifications, such as phosphorylation, further contribute to this regulatory flexibility by altering the TF’s stability, localization, or DNA-binding affinity, thereby switching its functional role [,,].
Finally, while laboratory studies provide important first insights, they need to be followed up with validation in field trials to confirm their practical value. This remains a challenge, but it is possible and has been demonstrated [,].

5. Conclusions

Comprehensive evaluation of NAC TFs and their target genes provides critical insights into their regulatory functions in wheat under salinity stress where, in some cases, the expression pattern of NAC was found to be opposite of its putative target gene. Understanding these molecular mechanisms is essential for guiding future strategies to breed stress-tolerant wheat varieties via targeted genetic modifications or MAS. Future research should focus on identifying additional NAC TF members, elucidating their interactions with diverse signaling pathways, and translating these findings into practical agricultural applications. By integrating advanced molecular modeling, multi-omics approaches and gene-editing technologies, the specificity and efficacy of NAC TF-mediated regulation can be enhanced, thereby strengthening wheat resilience to salt stress and supporting sustainable agriculture in saline environments. Overall, the detailed characterization of TaNAC gene families highlights their crucial roles in fortifying stress tolerance mechanisms. Their tissue-specific regulatory diversity underscores the complexity of plant stress responses, and leveraging this knowledge holds significant promise for developing climate-resilient crop varieties—an imperative for ensuring global food security amid escalating environmental challenges.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/crops5060081/s1, Table S1: Primers for the RT-qPCR expression analyses. Table S2. The 377 NAC sequences used to construct the phylogenetic tree. Table S3: Summary of numbers of NAC sequences at each wheat chromosome.

Author Contributions

Conception and design of the experiments, X.L., S.S., S.H. and H.A.; execution of the experiments, X.L., S.S., M.A.S. and S.H.; data analysis, X.L., S.S., T.A., M.A.S., S.H. and H.A.; writing of the paper, X.L., S.S., T.A., M.A.S., S.H. and H.A.; funding acquisition, X.L. and H.A. All authors have read and agreed to the published version of the manuscript.

Funding

The authors gratefully acknowledge financial support from the Carl Tryggers Foundation (grant refs. CTS 15:34, CTS 17:32, CTS 19:22), the Chinese Scholarship Council (File no. 202106910024) and the Swedish Research Council (2021-04265).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments

During the preparation of this manuscript the authors used ChatGPT-5 to polish the English language. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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The Sprayed Application of Recombinantly Expressed CpRap2.4A Confers Protective Activity Against Heat Stress in Nicotiana tabacum
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