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

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.

Keywords: TeNACs; Th. elongatum; Expression pattern; Salt stress.

Fig. 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

Fig. 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

Fig. 3

Phylogenetic tree of TeNACs and the A. thaliana NAC family

Fig. 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

Fig. 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)