Long-Term Salt Exposure Reprograms the Nicotiana tabacum BY-2 Suspension Proteome and Metabolome Toward Stabilization of the Core Metabolic Pathways, Protein Turnover Machinery Modifications, and Protective Metabolome Adjustments.

In this study, we analyzed a unique Nicotiana tabacum BY-2 line that was gradually adapted to and subsequently maintained in 190 mM NaCl for over 15 years. Years of continuous high salinity shaped a stable "new homeostasis" in BY-2 suspension cells. Salt-adapted cells were smaller and formed tighter clusters. Metabolomics revealed constitutive enrichment of osmoprotectants and antioxidant-associated metabolites-including proline, GABA, and selected purine derivatives-and a marked increase in β-sitosterol, together pointing to osmoadaptation, membrane stabilization, and ROS buffering without broad induction of classical antioxidant enzymes. Proteomics showed modest changes dominated by information-processing layers: higher abundance of histone deacetylases, RNA-binding and splicing-related factors, and enzymes linked to mRNA polyadenylation/decapping. In contrast, many 60S ribosomal proteins were less abundant, indicating restrained translation. Despite persistent osmotic pressure, enzymes of central metabolism changed little overall, whereas lipid-associated shifts and sterol enrichment suggest ongoing membrane remodeling. Collectively, these multi-omics data indicate that long-term salt adaptation in BY-2 cells prioritizes small-molecule osmolytes and post-transcriptional control over costly protein turnover, supporting sustained function under high osmolarity. This work provides novel insights into the molecular features underlying the reprogramming of the metabolome and proteome, enabling plant cells to survive long-term in high-salinity conditions.