The genome-wide analysis of the NAC gene family in Pseudoroegneria libanotica highlights the functions of PlNAC17/20 under abiotic stress conditions.

Pseudoroegneria libanotica is a cool-season forage with high drought tolerance. NAC (NAM, ATAF and CUC) plays an important role in plant growth and development and adaptation to a variety of abiotic stresses. To date, no NAC gene family has been reported in Pse. libanotica. The primary objective of this study was to comprehensively characterize the NAC gene family and elucidate its functional involvement in drought stress responses. 217 NAC genes were identified in the Pse. libanotica genome and classified into 11 subfamilies based on phylogenetic analysis with Arabidopsis and rice NAC reference proteins. Collinearity analysis showed stronger syntenic conservation between Pse. libanotica and wheat than between Pse. libanotica and Arabidopsis. Duplication and Ka/Ks analyses suggested that dispersed duplication contributed mainly to PlNAC family expansion and that duplicated PlNAC genes were primarily under purifying selection. Physiological measurements confirmed progressive drought stress, and transcriptome analysis identified 14 drought-responsive PlNAC genes. Among them, PlNAC17 and PlNAC20 showed strong drought-responsive expression patterns. Structural and subcellular localization predictions supported their potential roles as NAC transcription factors. Co-expression analysis identified two LEA-like genes and one U-box domain-containing protein gene as PlNAC17-associated stress-responsive candidates. Yeast heterologous expression assays showed stress-dependent effects of PlNAC17 and PlNAC20 under different abiotic stresses. This study provides the first genome-wide analysis of the NAC gene family in Pse. libanotica and identifies PlNAC17 and PlNAC20 as candidate stress-responsive NAC genes. These findings provide valuable information for understanding NAC-mediated stress regulation and for future improvement of stress tolerance in Triticeae crops.