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Genome-wide identification, tissue expression pattern, and salt stress response analysis of the NAC gene family in Thinopyrum elongatum

BMC Plant Biology volume 25, Article number: 643 (2025) Cite this article

Abstract

Background

The NAC (NAM, ATAF1/2, and CUC2) gene family plays a critical role in regulating plant growth, developmental processes, and stress response mechanisms. While NAC genes have been systematically characterized in multiple plant species, this study focused on genome-wide identification of NAC family members in Thinopyrum elongatum (designated as TeNACs) through integrated bioinformatics approaches. Comprehensive analyses were conducted to determine the physicochemical characteristics, conserved motifs, gene structure, phylogenetic relationships, chromosomal collinearity, and expression profiles of the identified TeNACs. This multi-dimensional characterization provides fundamental insights into the structural and functional diversity of NAC transcription factors in Th. elongatum.

Results

A total of 116 NAC transcription factors were systematically identified in Th. elongatum, distributed across all seven chromosomes. Comprehensive physicochemical characterization revealed substantial variation among TeNAC proteins: amino acid length (162–718 aa), molecular weight (18.33–78.25 kDa), isoelectric point (6.5–7.5), instability index (30.34–63.32), and aliphatic index (50.92–80.68). Hydropathicity analysis confirmed the hydrophilic nature of all TeNACs, with grand average values consistently below zero. Conserved motif profiling demonstrated a highly conserved architecture in TeNACs, featuring ordered arrangements of motifs 3, 4, 1, 5, 6, 2, and 7. Phylogenetic reconstruction classified TeNACs into 14 distinct clades through comparative analysis with Arabidopsis thaliana NAC genes, notably lacking ANAC001 and OsNAC8 homologs. Comparative genomic analysis identified significant syntenic conservation between TeNACs and wheat NAC genes. Protein interaction network prediction indicated intricate functional associations among TeNAC proteins. Computational predictions coupled with experimental validation of TeNAC021 confirmed exclusive nuclear localization for all family members. Differential expression analysis across a salt gradient (0–300 mM) identified 14 TeNACs with progressive up-regulation and 5 showing consistent down-regulation. RT-qPCR confirmed salt-responsive expression patterns for eight TeNACs demonstrating marked transcriptional changes.

Conclusions

This systematic investigation establishes a robust theoretical framework for subsequent structural and functional characterization of TeNACs in Th. elongatum.

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Introduction

The NAC family represents a crucial plant-specific gene family, deriving its name from the first letters of three foundational subfamilies: non-apical meristem (NAM) initially characterized in Petunia, along with transcription factor ATAF1/2 and CUC2 (cup-shaped cotyledon) subsequently identified in Arabidopsis. These evolutionarily conserved transcription factors share a highly conserved N-terminal DNA-binding domain while maintaining divergent C-terminal regions [1, 2]. The NAM domain (150–160 amino acids), situated at the N-terminal region of NAC transcription factors, consists of a highly conserved protein sequence. This sequence serves as a critical binding site for NAC transcription factors to interact with the promoters of target genes, forming a fundamental basis for identifying the NAC gene family. In contrast, the C-terminal domain of NAC transcription factors contains diverse protein sequences, which is involved in regulating the transcription of downstream target genes [3]. The N-terminal conserved domain of NAC transcription factors can be divided into five distinct subdomains: A, B, C, D, and E. Among these subdomains, A, C, and D show high conservation, while B and E display moderate conservation. Subdomain A is mainly involved in dimer binding and formation, whereas subdomains C and D are responsible for DNA sequence recognition. In contrast, subdomains B and E play crucial roles in determining the functional diversity of the NAC gene family [4].

The NAC gene family plays essential roles in plant growth and development, including apical meristem formation [5], floral organ development [6], fruit maturation [7], leaf senescence [8], secondary cell wall formation [9] and root development [10]. Furthermore, NAC transcription factors positively or negatively regulate responses to abiotic stresses, including salinity [11], low temperature [12], and drought [13], that plants face during growth and development. Previous studies have demonstrated that overexpression of ONAC066 and TwNAC01 improved drought stress responses in rice (Oryza sativa) and Triticale, respectively. Conversely, suppression of TwNAC01 reduced the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate in Triticcoscale wittmack leaves [14, 15]. The role of LlNAC2 in Lilium lancifolium under abiotic stress is not limited to low temperature, drought, and salt stress responses, as its expression is also induced upon exposure to abscisic acid (ABA) signaling molecules [16]. In Triticum aestivum, TaNAC29 is a key regulator of abiotic stress signaling pathways and antioxidant systems, decreasing H2O2 accumulation and membrane damage for enhanced salt tolerance [17]. In O. sativa, OsNAC3 plays a crucial role in ABA responses and salt tolerance through regulating the expression of stress-related genes and maintaining Na+ homeostasis in rice stems. Additionally, ONAC127 and ONAC129 control grain filling by influencing sugar transport and abiotic stress responses [18, 19]. Furthermore, overexpression of GmNAC06 markedly improves salt tolerance in soybean (Glycine max) hairy roots, while GmNAC12 functions as a key regulator that positively modulates drought tolerance [20].

Thinopyrum elongatum (Host) D.R.Dewey (EE, 2n = 2x = 14) is a perennial plant in the tribe Triticeae with excellent characteristics including disease resistance, high fertility, and multiple stress tolerances, particularly to drought, low temperature, and salinity. Moreover, Th. elongatum is phylogenetically closely related to common wheat and harbors numerous valuable genes that have been effectively utilized in wheat genetic improvement [21, 22]. To investigate the tissue-specific expression patterns and elucidate the salt tolerance mechanisms of the NAC genes in Th. elongatum, a comprehensive study was conducted. In this study, we utilized bioinformatics approaches to perform genome-wide identification of the TeNACs. Furthermore, we systematically analyzed the physicochemical properties, conserved motifs, gene structure, evolutionary relationships, collinearity, protein–protein interactions, and other relevant characteristics. Additionally, we examined the expression patterns under salt stress and across different tissues. Our findings establish a theoretical foundation for further research on the biological functions and molecular mechanisms of the TeNACs.

Materials and methods

Identification and physicochemical property analysis of the TeNACs family

The protein sequences of NAC genes in A. thaliana and the genome/annotation files of Th. elongatum were retrieved from TAIR (https://www.arabidopsis.org/) and Genome Warehouse (https://ngdc.cncb.ac.cn/gwh/Assembly/965/show), respectively. Two complementary methods were employed to identify TeNAC members. The first method involved performing BLASTP (v2.13.0) analysis using the A. thaliana NAC protein sequences as queries with an e-value cutoff of 1e−20 to screen and classify Th. elongatum protein sequences. The second method utilized HMMER (v3.3.2) to build a hidden Markov model profile with the same e-value threshold (1e−20), enabling identification of candidate sequences in Th. elongatum using the NAM (PF02365) domain from Pfam (http://pfam-legacy.xfam.org/). The candidate sequences obtained from both methods were then submitted to NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and InterPro (https://www.ebi.ac.uk/interpro/search/sequence/) databases for precise NAC domain verification. Following stringent screening and validation procedures, the final gene set was obtained and systematically named according to their Latin nomenclature and genomic locations. The fundamental physicochemical parameters of the identified TeNAC proteins, including protein length, molecular weight, theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY), were analyzed using ExPASy (https://web.ExPASy.org/protparam/) [23].

Genomic distribution and collinearity analysis

TBtools (v1.120) software was employed to visualize the chromosomal distribution of TeNACs [24]. To examine the collinearity relationships of TeNACs both within and across species, genome sequences of A. thaliana, O. sativa, and T. aestivum were obtained from TAIR, RGAP (http://rice.plantbiology.msu.edu/), and IWGSC (https://wheat-urgi.versailles.inra.fr/Projects/IWGSC), respectively. Interspecies and intraspecies collinearity analyses were conducted using MCscanX, with the resulting collinearity networks visualized through TBtools.

Conserved motif, cis-acting elements, and gene structure analyses

The conserved motifs and gene structures of TeNACs were systematically characterized using MEME (v5.5.3) (https://meme-suite.org/meme/tools/meme) and TBtools, respectively [25]. Subsequently, the 2,000 bp upstream sequences of TeNACs were extracted and submitted to PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for cis-actingelement prediction. TBtools was employed to visualize the conserved motifs, cis-acting elements, and gene structures. The maximum number of motifs was set to 10, while default parameters were maintained for other settings.

Prediction of protein-protein interactions in TeNACs

STRING (https://cn.string-db.org/) was used to predict the interactions of the TeNAC proteins, with T. aestivum as the selected organism and 116 TeNAC proteins as a reference for predicting protein–protein interactions [26].

Evolutionary analysis

MAFFT (v7.520) was used to compare the amino acid sequences of the identified TeNACs with those of A. thaliana NAC genes [27]. The resulting data were used to construct a phylogenetic tree using IQ-TREE (v2.2.2.7) with maximum likelihood alrorithm and 1000 bootstrap replicates. The online website iTOL (https://itol.embl.de/) was used to visualize the phylogenetic tree [28].

Subcellular localization prediction and TeNAC021 validation

The subcellular localization of TeNACs was determined using Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) [29]. It was predicted that TeNAC021 might be located in both chloroplasts and nucleus, so experimental validation was employed. The coding region of TeNAC021 was amplified using the following primers: Forward primer (5'−3') ATGGCATCGGCAGCGTTA and Reverse primer (5'−3') GTTTTTTTTTTTAGCAAACGTGATG. The PCR product was then cloned into the pCAMBIA1305-eGFP vector, which was fused to a GFP protein in the C terminus using the KpnI and BamHI restriction sites by the In-Fusion reaction. Then, the fusion vector was transformed to the p19 containing Agrobacterium strain GV3101 (pSoup-p19). The Ubi::TeNAC21-GFP was expressed in Nicotiana benthamiana leaves by agro-infiltration. Images were collected on a confocal microscope (LSM900, Zeiss, Germany). GFP was detected at 488 nm laser excitation and collection bandwidth was 510–550 nm.

Sequencing and analyses of the transcriptome of Th. elongatum

The seeds of Th. elongatum (PI 531718) were obtained from United States Department of Agriculture (USDA) National Plant Germplasm System (https://npgsweb.ars-grin.gov/gringlobal/accessiondetail?id=1426654). The plants were cultivated in a growth chamber at Shandong Agricultural University (Tai’an, China), under conditions of 25 °C and a 16 h/8 h (light/dark) photoperiod. Thereafter, the plants were treated with salt at different concentrations (0, 100, 200, and 300 mM NaCl). After one week treatment, leaf tissue samples of Th. elongatum were collected and stored at − 80 °C in liquid nitrogen. The RNAprep Pure Plant Kit (TIANGEN, Beijing, China) was used to extract the RNA from all samples. The NanoDrop 1000 spectrophotometer was used to measure its quantity. In total, four salt treatment groups with 3 biological replicates for each, were subjected to transcriptome sequencing, and 372 Gb datasets were generated. To analyze the expression pattern of TeNACs, total RNA was extracted from the seedling, root, internode, and flag leaf tissues. Three biological replicates were performed for each tissue. RNA extraction was conducted using the TRIzol® reagent (Invitrogen). Sequencing was performed on the Illumina HiSeq 4000 platform. In addition, the transcriptome data of six tissues (half-grain, grain, young spike, flowering, ovary expansion, and pre-flowering) of Th. elongatum we previously produced, were downloaded from NCBI database (accession number PRJNA540081). About 932 Gb datasets were used to perform the tissue expression analyses.

RNA-seq data was then subjected to quality control using fastqc (v0.12.1) with the default parameters [30]. Subsequently, HISAT2 (v2.2.1) was used to align RNA-seq reads onto the reference genome of Th. elongatum. Finally, HTseq-count (v2.0.2) was used to generate per-gene read count for each sample [31, 32]. The transcripts per million (TPM) value for each gene was calculated based on its length and expressed as log2(TPM + 1) using a custom R script. The resulting tables were then visualized using the HeatMap module of TBtools.

RT-qPCR analysis of the TeNACs

The 7-day seedlings of Th. elongatum were exposed to 0, 100, 200, and 300 mM NaCl for 7-day, respectively. These seedlings were then collected and stored at −80 °C. The Beacon Designer tool was used to design specific primers based on the sequences of the coding regions of 8 TeNACs. Subsequently, the total RNA was extracted using RNA prep Pure Plant Plus Kit (DP441, TIANGEN, Beijing, Chain) from seedlings. For the first-strand cDNA synthesis, 1 μg of total RNA was used with the PrimeScript™ II 1 st Strand cDNA Synthesis Kit (6210 A,TaKaRa, Beijing, Chain). Using the three-step program on the Archimed Real-time PCR Determination System (ROCGENE, Beijing, Chain), the RT-qPCR analysis was carried out using the qPCR Super mix SYBRgreen (MF013-01, Mei5bio, Beijing, Chain). As an internal control, the TeTublin was employed (Forward primer (5'−3') GTGGAACTGGCTCTGGC and Reverse primer (5'−3') CGCTCAATGTCAAGGGA). Three biological replicates of the qPCR data were analyzed using the 2-ΔΔCT technique. A one-way ANOVA was used to determine statistical significance at a significance level of P < 0.05 (The sequences of specific primers are listed in Supplementary Table 1).

Results

Identification of TeNAC members, genomic distribution and analysis of physicochemical properties

A total of 116 NAC transcription factors were identified in Th. elongatum and designated as TeNAC001-TeNAC116 based on their Latin nomenclature and genomic positions across seven chromosomes (Supplementary Table 2). Chromosomal distribution analysis showed balanced allocation of all 116 TeNAC members among the seven chromosomes. Chromosome 7E contained the highest number (28 TeNACs), while chromosome 1E had the fewest (7 genes). The distribution pattern was as follows: chromosome 2E (24), chromosome 3E (14), chromosome 4E (13), chromosome 5E (15), and chromosome 6E (16). Multiple tandem duplication events were observed among TeNAC members, indicating their crucial role in gene family expansion (Fig. 1A).

Fig. 1
figure 1

The distribution, physicochemical properties, and collinearity analysis of TeNACs. A The distribution of TeNACs on seven chromosomes 1E to 7E of Th. elongatum. B, C The box plot of protein length, isoelectric point (PI), molecular weight, instability index, aliphatic index, and grand average of hydropathicity in TeNACs. D The intraspecific collinearity of TeNACs in Th. elongatum. E The interspecies collinearity between A. thaliana, O. sativa, T. aestivum, and Th. elongatum, respectively

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The TeNAC proteins varied in length from 162 to 718 amino acids, with molecular weights ranging between 18.33–78.25 kDa and theoretical pIs spanning 4.44–9.84. Based on pI values: 47 acidic proteins (pI < 6.5), 49 basic proteins (pI > 7.5), and 20 neutral proteins (pI 6.5–7.5) were identified, showing balanced distribution between acidic and basic groups (Fig. 1B). Instability indices ranged from 30.34 to 63.32, with 90/116 (77.6%) exceeding 40. Aliphatic indices (50.92–80.68) reflected significant variation in thermal stability. All grand average of hydropathicity (GRAVY) values were negative, confirming strong hydrophilic characteristics (Fig. 1C, Supplementary Table 3).

Collinearity analyses of TeNACs

The collinearity analysis of TeNACs identified seven intra-family duplicated gene pairs (Fig. 1D), specifically between TeNAC004-TeNAC043, TeNAC007-TeNAC041, TeNAC013-TeNAC051, TeNAC014-TeNAC050, TeNAC011-TeNAC060, TeNAC024-TeNAC082, and TeNAC021-TeNAC080. These findings strongly support the crucial contribution of gene duplication events to TeNAC family expansion. Interspecies collinearity analysis showed limited synteny with A. thaliana (4 collinear blocks) but extensive conservation with O. sativa (76 collinear blocks), revealing closer evolutionary relationships with rice. We further identified 201 collinear genes across wheat A, B, and D subgenomes corresponding to 79 TeNACs, with detailed distribution as follows: 70 in subgenome A, 65 in subgenome B, and 66 in subgenome D, confirming strong orthologous relationships between TeNACs and wheat genomes (Fig. 1E).

Conserved motif, cis-acting elements, and gene structure analyses

Ten conserved motifs were identified in the TeNACs (Fig. 2A). Among these, motif 3 represents NAC subdomain A, motif 4 represents subdomain B, motif 1 represents subdomain C, motif 2 represents subdomain D, and motif 7 represents subdomain E (Supplementary Fig. 1). 102 out of all 116 TeNACs contained motifs 1–7. Notably, the seven conserved motifs were consistently arranged in the sequences of motifs 3, 4, 1, 5, 6, 2, and 7. This arrangement underscores the high conservation of motifs 1–7 throughout the evolutionary history of TeNACs. Among the remaining 14 members, TeNAC039 does not contain motif 3; TeNAC083 does not contain motif 4; TeNAC018, TeNAC045, and TeNAC114 do not contain motif 1; TeNAC005, TeNAC044, and TeNAC048 do not contain motif 6; TeNAC021 and TeNAC045 do not contain motif 2; and TeNAC027 and TeNAC074 do not contain motif 7. Motifs 8–10 were only identified in a subset of members with similar gene structure. All TeNAC members contain the NAM (PF02365) domain (Fig. 2B).

Fig. 2
figure 2

Conserved motifs, domain, cis-acting elements, gene structure, and protein–protein interaction networks of TeNACs. A Evolutionary trees and conserved motifs among TeNACs. Different colored blocks in the figure represent the locations of different motifs. B The location of the NAM domain on TeNACs. C The location distribution of homeopathic elements at 2000 bp upstream of TeNACs. D The structural features of CDS and UTR genes were distributed on TeNACs. (E–H) Four networks of protein–protein interaction in TeNACs based on T. aestivum. The lines between balls of different colors are evidence of an interaction between the two TeNACs

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By identifying and screening the cis-acting elements in the 2,000 bp upstream sequence of TeNACs, a total of 15 elements related to hormones, stress and development were discovered (Fig. 2C). The hormone response-related elements include auxin-responsive element (TGA-element, AuxRR-core), gibberellin-responsive element (TATC-box, P-box, GARE-motif), MeJA-responsiveness (TGACG-motif, CGTCA-motif), salicylic acid responsiveness (TCA-element), and abscisic acid responsiveness (ABRE). The stress response-related elements include defense and stress responsiveness (TC-rich repeats), low-temperature responsiveness (LTR), and drought-inducibility (MBS). This indicates that the upstream region of TeNACs is rich and diverse in cis-acting elements. The TeNACs with similar gene structures had a similar number of exons and gene structures (Fig. 2D).

Protein–protein interactions

Protein–protein interaction analysis revealed four distinct interaction networks among 32 TeNAC members, comprising 19, 7, 4, and 2 proteins respectively (Fig. 2E-H). Notably, six central members (TeNAC027, TeNAC085, TeNAC086, TeNAC087, TeNAC088, and TeNAC103) within the 19-member network demonstrated interactions with all 11 peripheral members, indicating intricate regulatory relationships within the TeNAC family (Supplementary Table 4).

Evolutionary analysis

To elucidate the evolutionary relationships of TeNAC members, a phylogenetic tree was generated using 105 Arabidopsis NAC genes along with all 116 TeNAC proteins (Fig. 3). Classification based on Arabidopsis NAC protein domains grouped the TeNACs into 14 distinct clades. Notably, two clades (ANAC001 and OsNAC8) lacked TeNAC representatives, implying potential gene loss events in Th. elongatum during evolution. The remaining 12 clades contained both A. thaliana and Th. elongatum NAC genes, demonstrating strong evolutionary conservation across these NAC family members. Subsequent collinearity analysis further confirmed that these 11 members lacked syntenic counterparts in the Th. elongatum genome assembly (Supplementary Fig. 2).

Fig. 3
figure 3

Phylogenetic tree of TeNACs and the A. thaliana NAC family

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Subcellular localization

Subcellular localization prediction suggests that all 116 TeNAC proteins, with the exception of TeNAC021, which may be localized in either the chloroplast or the nucleus, are predominantly nuclear-localized. To confirm the subcellular localization of TeNAC021, we conducted a experimental validation. The results confirmed that TeNAC021 is indeed localized in the nucleus (Fig. 4A).

Fig. 4
figure 4

The analysis of Subcellular Localization and Expression Patterns in Tissues. A Subcellular localization experiment, GFP represents the position of the protein under green fluorescence, Bright represents the normal light, DAPI stands for DNA stain, and Merge Indicates the result of merging GFP, Bright and DAPI (B) TeNACs in seedling, root, internode, flag leaf, young spike, pre-flowering, flowering, ovary expansion, half-grain, grain log2 TPM in ten parts of organism and developmental period by z-score normalization

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TeNACs expression patterns in different tissues

Thirty-five TeNAC transcription factors exhibited constitutive expression across all examined tissues, while 69 displayed tissue-preferential expression profiles, with TeNAC073, TeNAC048, TeNAC044, and TeNAC045 showing consistently high transcript abundance in all tissues (Fig. 4B, Supplementary Table 5), suggesting their fundamental regulatory roles in developmental processes in Th. elongatum. In contrast, twelve TeNAC family members (TeNAC034, TeNAC038, TeNAC049, TeNAC052, TeNAC062, TeNAC063, TeNAC078, TeNAC079, TeNAC081, TeNAC084, TeNAC102, and TeNAC112) remained transcriptionally silent across all ten investigated tissues, indicating various functions of these TeNACs..

Expression analysis and RT-qPCR of TeNACs under salt stress

RNA-seq analysis detected expression of 91 out of 116 TeNACs under stress conditions (Fig. 5A, Supplementary Table 6). Specifically, TeNAC006, TeNAC105, TeNAC103, TeNAC080, TeNAC100, TeNAC039, TeNAC033, TeNAC007, TeNAC025, TeNAC013, TeNAC029, TeNAC083, TeNAC042, and TeNAC098 showed sustained up-regulation. At the same time, TeNAC005, TeNAC061, TeNAC114, TeNAC021, and TeNAC020 exhibited progressive down-regulation (Fig. 5B). These differential expression patterns demonstrate that TeNACs employ distinct transcriptional responses to salt stress to maintain essential physiological processes in plants. Notably, TeNAC073, TeNAC048, TeNAC044, and TeNAC045 were highly expressed in all tissues and under salt stress conditions. This suggests that they play vital regulatory roles in tissue development and salt stress responses in Th. elongatum. Based on the expression patterns of TeNACs under varying salt concentrations, we selected 4 up-regulation continuous, 2 down-regulation continuous, and 2 non-regulation continuous genes for RT-qPCR validation (Supplementary Table 7). Quantitative real-time PCR (RT-qPCR) investigations were performed to evaluate the expression patterns of these genes under various salt stress conditions. The RT-qPCR results showed consistent variations to the transcriptome data for the specific genes analyzed (Fig. 5C-J). A high degree of similarities among these eight genes, the salt-responsive gene TaNAC29 (TraesCS2A02G102000) and the ABA signaling pathway gene TaNAC48 (TraesCS1B02G274300) in wheat, indicating similar functions in Th. elongatum (Supplementary Table 8) [33, 34].

Fig. 5
figure 5

Expression Analysis and RT-qPCR of TeNACs under Salt Stress. A The log2 TPM of TeNACs in 0–300 mM four salt concentrations by z-score normalization. B Expression characteristics of TeNACs in four salt concentration gradients from 0 to 300 mM NaCl. C-J The RT-qPCR verification of 8 TeNACs in different salt concentrations (CK stands for 0 mM NaCl and 100–300 stands for 100 mM, 200 mM, and 300 mM NaCl respectively)

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Discussion

As plant-specific transcription factors, NAC proteins play crucial roles in regulating plant growth, development, and abiotic stress responses. Currently, genome-wide identification of NAC transcription factors has been completed in multiple species including A. thaliana, O. sativa, and T. aestivum; nevertheless, the NAC gene family in Th. elongatum has not yet been characterized [35, 36]. In this study, 116 TeNACs, each containing a conserved NAM domain (PF02365), were identified through comprehensive bioinformatics analysis in Th. elongatum. Conserved motif examination demonstrated that the seven characteristic motifs in TeNACs were predominantly clustered at the N-terminus following a specific arrangement (motifs 3, 4, 1, 5, 6, 2, and 7), while showing limited distribution at the C-terminus; this pattern aligns with the established NAC family features of highly conserved N-terminal domains and variable C-terminal regions [37]. Furthermore, motifs 3, 4, 1, 2, and 7 were found to correspond precisely to NAC subdomains A-E, confirming the conserved domain architecture characteristic of NAC transcription factors [38]. In addition, gene structure analysis revealed that the number of exons in TeNACs ranged from 2 to 7, with large differences in the number of members, indicating gene structure changes with the environment during plant growth and development [39].

Collinearity analysis identified 70, 65, and 66 orthologous gene pairs between TeNACs and wheat (T. aestivum) subgenomes A, B, and D, respectively, demonstrating high evolutionary conservation between TeNACs and the wheat genome. Intraspecific collinearity analysis indicated that gene duplication events significantly contributed to TeNAC family expansion. Chromosomal distribution analysis revealed multiple tandemly duplicated gene pairs among TeNAC members, highlighting the crucial role of tandem duplications in TeNACs family evolution and diversification. In this study, the TeNACs and Arabidopsis NAC gene families were phylogenetically classified into 14 clusters based on conserved NAC sequences, and collinearity analysis revealed the absence of syntenic relationships between 11 AtNACs and Th. elongatum genomic fragments, suggesting evolutionary loss of two clusters. The TeNAC members in the same cluster shared similar gene structures and conserved motifs, among which two clusters, ANAC001 and OsNAC8, were absent in Th. elongatum. Protein interaction network prediction analysis based on T.asetivum revealed that TeNACs have a complex interaction network. Subcellular localization prediction revealed that except for TeNAC021, which may be localized in the chloroplasts or nucleus, 115 TeNAC members were localized in the nucleus. We experimentally verified TeNAC021 and found that it is localized in the nucleus, so the combination of prediction and validation experiments can prove that TeNACs021 mainly plays a major role in the nucleus.

Salt stress is a major limiting factor for plant growth [40], and several NAC gene families, including MusaNAC042 in bananas (Musa nana), AvNAC030 in kiwifruit (Actinidia chinensis Planch), and TaNAC29 in T. aestivum, are well characterized for salt stress resistance [41, 42]. In total, 14 consistently upregulated and 5 consistently downregulated TeNAC members were identified when analyzing the expression pattern of TeNAC transcription factors in different salt stress environments; at the same time, 111 upstream TeNACs gene members harbored 477 ABRE cis-acting elements, and 75 members contained 116 MBS and salt stress-responsive cis-acting elements; this confirms the positive role of the NAC family in responding to salt stress and provides a reference for determining the function of NAC transcription factors and the mechanism underlying salt tolerance [43, 44]. In addition, several genes with significantly different expression genes were identified under varying salt stress conditions. Furthermore, these genes show a certain degree of orthologs with the wheat genes TaNAC29 and TaNAC48 [33, 34]. Combined with the abovementioned results, we hypothesized that NAC transcription factors are responsive to salt stress in Th. elongatum. The molecular implications of salt-responsive TeNACs have been expanded to address conserved regulatory mechanisms, including ROS detoxification cascades (e.g., peroxidase activation), ion transporter modulation (SOS1/NHX1 coordination), and ABA-mediated stress signaling cross-talk. Testable hypotheses regarding cytoskeletal reorganization and cell wall remodeling under osmotic stress are proposed, with suggested validation pathways such as heterologous expression assays in Arabidopsis mutants or CRISPR-Cas9-mediated knockouts [45]. Expression pattern analysis of TeNAC members across various tissues revealed distinct expression profiles for each member in different tissue types. Sixty-nine TeNAC members showed detectable expression in ten examined tissues of Th. elongatum—including seedling, root, internode, flag leaf, young spike, pre-flowering spike, flowering stage, ovary expansion phase, half-grain, and grain—demonstrating that NAC transcription factors actively participate in plant growth and developmental processes [46]. In this study, three genes, namely, TeNAC073, TeNAC044, and TeNAC048, were significantly expressed under both salt stress conditions and during plant growth and development, suggesting the functional diversity of NAC transcription factors [47].

Conclusions

In this study, we employed comprehensive bioinformatics approaches to systematically investigate and predict various characteristics of the TeNACs family, including physicochemical properties, gene structure, conserved motifs, evolutionary relationships, collinearity patterns, protein–protein interaction networks, and expression profiles. Our analysis revealed that TeNAC proteins display diverse physicochemical features, contain the five characteristic NAC subdomains (A-E), and cluster into 12 distinct subfamilies that show strong collinearity with wheat NAC genes. Notably, TeNACs demonstrate both significant up-regulation and down-regulation under salt stress and exhibit widespread expression across different Th. elongatum tissues. These findings establish a solid foundation for further exploration of the biological functions and molecular mechanisms of the TeNAC gene family.

Data availability

The transcriptome data used in this study have been uploaded to NCBI under BioProjectID PRJNA540081 (https://www.ncbi.nlm.nih.gov/sra/PRJNA540081).

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Acknowledgements

We thank China Perennial Wheat Wild Relatives Germplasm Field Repository for providing the Th. elongatum germplasm resources.

Funding

This work was supported by the Key R&D Program of Shandong Province, China (2023LZGC022).

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Author notes

  1. Jilin Sun and Xiaokuo Cui contributed equally to this work.

Authors and Affiliations

  1. National Key Laboratory of Wheat Improvement, College of Agronomy, Shandong Agricultural University, Tai ‘an, Shandong, 271018, China

    Jilin Sun, Jiaqi Zhang, Fansen Meng, Jiangong Li & Silong Sun

  2. National Key Laboratory of Wheat Improvement, Shandong Engineering Research Center of Plant-Microbial Restoration for Saline-Alkali Land, College of Life Sciences, Shandong Agricultural University, Tai ‘an, 271018, China

    Xiaokuo Cui & Changai Wu

  3. National Key Facility of Crop Gene Resources and Breeding, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Shenghui Zhou & Peng Zhou

  4. Tai ‘an Academy of Agricultural Sciences, Tai ‘an, Shandong, 271000, China

    Long Han

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Contributions

L.H and S.L.S designed the research and experiment; J.L.S, X.C.C, J.Q.Z, F.S.M, and J.G.L performed the experiment and analyzed the data; S.H.Z provided materials; J.L.S and X.C.C prepared the original draft; S.L.S, P.Z, C.A.W and L.H revised the manuscript.All authors contributed to the article and approved the submitted version.

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Correspondence to Long Han.

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Supplementary Information

12870_2025_6696_MOESM1_ESM.xlsx

Supplementary Material 1: Supplementary Table 1. Primers used in RT-qPCR experiments. Supplementary Table 2. The protein sequences of TeNACs. Supplementary Table 3. The physicochemical properties of TeNACs. Supplementary Table 4. The protein-protein interactions of TeNACs. Supplementary Table 5. The log2 TPM of TeNACs expression in different tissues. Supplementary Table 6. The log2 TPM of TeNACs expression under salt stress. Supplementary Table 7. The p-values of differentially expressed TeNACs under salt stress conditions. Supplementary Table 8. The blast output of TeNACs in combination with TaNAC29 and TaNAC48.

Supplementary Material 2: Supplementary Fig. 1. The10 conserved motifs sequence of TeNACs.

Supplementary Material 3: Supplementary Fig. 2. The collinearity of 11 ANACs between Th. elongatum and A. thaliana.

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Sun, J., Cui, X., Zhang, J. et al. Genome-wide identification, tissue expression pattern, and salt stress response analysis of the NAC gene family in Thinopyrum elongatum. BMC Plant Biol 25, 643 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12870-025-06696-3

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BMC Plant Biology

ISSN: 1471-2229

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