Cloning and Functional Verification of Salt Tolerance Gene HbNHX2 in Hordeum brevisubulatum

A high salt environment seriously affects the physiological metabolism and yield of plants. Hordeum brevisubulatum (Trin.) Link has high biomass and important ecological, feeding and economic values, but its growth conditions have serious saline-alkali effects. The NHX gene family plays a vital role in regulating intracellular Na⁺/K⁺ balance, pH homeostasis, and vesicle and protein transport in plants. In this study, the HbNHX2 gene was cloned from Hordeum brevisubulatum and functionally characterized through phenotypic, physiological, and molecular analyses in transgenic tobacco. Expression profiling revealed that HbNHX2 was most abundant in spikes and least abundant in root tips, and the expression level was significantly induced under salt stress. Overexpression of HbNHX2 led to decreased malondialdehyde (MDA) and superoxide anion (O^2-) levels, while it enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Additionally, the levels of glutathione (GSH), soluble proteins, proline, and chlorophyll were also increased. Several stress-responsive genes, including CBL1, ERF2, BI-1, Cu/Zn-SOD, Mn-SOD, POD, GR1, KC1, TPK1, TIP, P5CS, BAS1, STN7 and LTP1, were significantly upregulated, while SERK3B was downregulated. These findings suggest that HbNHX2 enhances plant salt tolerance by maintaining osmotic balance, scavenging reactive oxygen species (ROS), and regulating stress-responsive gene expression. This study provides new insights into the molecular mechanism of salt tolerance in Hordeum brevisubulatum and lays a foundation for breeding salt-tolerant forage crops.