Genome‑wide identification of the NHX gene family in Forsythia identifies FsNHX3 and FsNHX6 as potential candidates involved in abiotic stress responses

The Na ⁺ /H⁺ antiporter (NHX) gene family plays a central role in maintaining cellular ion homeostasis and responding to abiotic stress in plants. However, the systematic identification and functional characterization of this gene family in woody plants of the genus Forsythia remain unexplored. To address this, genome-wide identification and systematic comparative analysis of the NHX gene family were performed in three Forsythia species, Forsythia suspensa , F. viridissima , and F. ovata , with a focus on elucidating F. suspensa NHX gene expression under various types of stress: salt, drought (PEG-simulated), and exogenous abscisic acid (ABA). In total, 18 NHX members were identified and were classified into three subclasses based on phylogenetic analysis, resulting in 12 vacuolar membrane-localized ‘Vac’-class members, four plasma-membrane-localized ‘PM’-class members, and two endosomal system-localized ‘Endo’-class members. Those proteins within the same evolutionary clade exhibit highly conserved structural features in terms of motif arrangement and gene structure. Synteny analysis identified whole-genome or segmental duplication as the primary driving force for the expansion of this family, with all of the duplicated gene pairs having undergone strong purifying selection. Based on interspecies synteny analysis, F. suspensa and F. viridissima exhibited the highest degree of NHX gene family homology. Expression analysis revealed that the F. suspensa NHX gene family exhibits distinct and tissue-specific responses to drought, salt, and exogenous ABA stress. Among the NHX members, FsNHX3 and FsNHX6 exhibited the most pronounced and sustained transcriptional upregulation across the three stress treatments, suggesting their potential involvement in stress response. These findings provide valuable insights for the further exploration of the functions and molecular mechanisms of the NHX gene family in Forsythia under salt and drought stress, offering valuable candidate genetic resources for breeding Forsythia varieties with enhanced tolerance to salinity and dehydration.