Integrative Astral-DIA proteomics and transcriptomics reveal candidate salt-tolerance genes in the halophyte Hordeum...

Sea barleygrass (Hordeum marinum) is a halophytic wild barley with exceptional salt tolerance. However, the molecular genetic basis that confers its super salt tolerance remains to be elucidated. Here, we applied high-resolution Astral-DIA quantitative proteomics using root samples of sea barleygrass accession H559 exposed to 300 mM NaCl for 2 and 7 days. Among 9032 quantified proteins, 1875 and 2052 proteins showed significant changes in abundance after 2 and 7 days of salt treatment, respectively. KEGG enrichment indicated that proteins with increased abundance were predominantly associated with carbon metabolism, MAPK signaling and calcium signaling, whereas proteins with decreased abundance were enriched for protein processing in the endoplasmic reticulum, protein folding and vesicle-mediated transport. Subcellular-localization analysis indicated coordinated activity of key ion transporters (e.g., SOS1, V-ATPase subunits and KEA2) involved in Na⁺ extrusion, vacuolar sequestration and K⁺ uptake. Integration with the root transcriptomes of H559 revealed convergent enrichment of the glutathione-metabolism pathway, highlighting its pivotal role in maintaining redox homeostasis and regulating ions. Collectively, our results suggest that H. marinum maintains a low root Na⁺/K⁺ ratio through synergistic ion-homeostasis and antioxidant defenses, a strategy that constitutes the key determinant of its superior salt tolerance. These findings provide mechanistic insights and candidate genes for developing salt-tolerant crops. SIGNIFICANCE: Soil salinization is a major global threat to agriculture, yet the molecular mechanisms enabling extreme salt tolerance in halophytic species remain poorly understood. Hordeum marinum, a close wild relative of cultivated barley, represents an invaluable genetic reservoir for improving salt tolerance in crops. By integrating high-resolution Orbitrap Astral-DIA proteomics with transcriptome profiling, this study uncovers the temporal ion-homeostasis and antioxidant mechanisms that underpin the exceptional salt tolerance of halophyte H. marinum. We identify key early-acting Na⁺-detoxification components (SOS1, V-ATPases), prolonged K⁺-retention regulators (e.g., KEA2), and glutathione-centered redox pathways as core determinants of its stress tolerance. These findings significantly advance our understanding of halophyte stress biology and provide high-confidence candidate genes and pathways that can be exploited for engineering or breeding salt-tolerant cereal crops.