Identification and analysis of the NAC gene family in barley (Hordeum vulgare L.) and functional validation of HvNAC38 in salt tolerance

The NAC gene family is a key group of plant-specific transcription factors that play essential roles in regulating plant growth, development, and responses to abiotic stress. In this study, we focused on the salt-tolerance candidate gene HORVU3Hr1G014090, identified through previous multi-omics screening in our laboratory. A genome-wide identification and bioinformatics analysis of the NAC gene family in barley were conducted, followed by expression profiling and qRT-PCR validation of all members under salt stress. The subcellular localization of the candidate gene was also determined. Finally, the function of HORVU3Hr1G014090 was validated through overexpression in Arabidopsis thaliana. A total of 116 HvNACs genes were identified in barley, distributed across seven chromosomes. The candidate gene HORVU3Hr1G014090 was designated as HvNAC38. Phylogenetic analysis classified the HvNACs into six subclades, and three gene pairs were identified as segmental duplications: HvNAC19/HvNAC57, HvNAC32/HvNAC85, and HvNAC14/HvNAC58. Physicochemical property analysis showed that HvNAC proteins ranged from 112 to 894 amino acids in length, with molecular weights ranging from 12,782.78 to 99,779.03 Da, isoelectric points from 4.20 to 10.54, instability indices from 21.9 to 70.6, and average hydrophilicity values from -1.029 to -0.264. Several stress-related binding motifs were identified, and five conserved domains or superfamilies were found: the NAM domain, PHA03378, PTZ00449, PHA03052, and PHA03247 superfamilies. A total of 41 cis-acting elements were detected across the HvNACs, and 18 members were found to lack introns. Under salt stress, 107 HvNACs gene were responsive, and the expression patterns of eight selected genes were validated by qRT-PCR, showing general consistency with transcriptome data. Subcellular localization analysis confirmed that HvNAC38 is localized in the nucleus, consistent with in silico predictions. Overexpression of HvNAC38 in Arabidopsis thaliana enhanced salt tolerance, as evidenced by phenotypic and physiological analyses. These findings provide valuable insights into the molecular mechanisms of salt tolerance in barley and offer a theoretical basis for improving.