Rice RING E3 Ligase OsRFP45 Negatively Regulates Salt Tolerance by Modulating Na⁺/K⁺ Transporter Genes

Salinity stress is a major environmental challenge affecting global rice production by disrupting ion homeostasis and inducing oxidative damage. We characterized Oryza sativa RING Finger Protein 45 (OsRFP45), a RING-v-type E3 ubiquitin ligase, and investigated its role in the salt stress response in rice. OsRFP45-overexpressing (OE) and CRISPR/Cas9-mediated knockout (KO) rice lines were generated to examine their physiological, biochemical, and molecular responses to salt stress. While no significant differences were observed among genotypes under normal conditions, OsRFP45-OE plants exhibited severe growth retardation, high Na⁺ accumulation, low K⁺ retention, increased oxidative stress, and reduced osmotic adaptation under 100 mM NaCl, demonstrating hypersensitivity to salinity. In contrast, OsRFP45-KO plants displayed enhanced salt tolerance, maintained low Na⁺ content, a balanced Na⁺/K⁺ ratio, reduced reactive oxygen species accumulation, and increased proline and soluble sugar levels. Quantitative RT-PCR analysis revealed that the expression of OsRFP45 negatively modulated the expression of key Na⁺ and K⁺ transporters, including OsHKT1;5, OsHKT2;1, OsNHX1, and OsSOS1. In OsRFP45-KO plants, enhanced Na⁺ exclusion and K⁺ retention contributed to improved ionic homeostasis under salt stress. Additionally, in vitro ubiquitination assays confirmed the E3 ligase activity of OsRFP45, indicating its potential role in protein turnover during adaptation to stress. Taken together, our findings suggest that OsRFP45 functions as a negative regulator of salt tolerance by modulating ion transport and oxidative stress response. Understanding the molecular role of OsRFP45 may provide a promising strategy for developing salt-tolerant rice cultivars.