Rice E3 ligase OsRFPH2‐16 acts as a negative regulator to mediate the degradation of OsPIP1;1 under salt stress

SUMMARY Soil salinity has a significant negative effect on rice productivity. We characterized the Oryza sativa RING Finger Protein H2‐type‐16 gene ( OsRFPH2‐16 ), which plays a negative role in response to salinity. The transcript levels of OsRFPH2‐16 decreased under saline conditions. OsRFPH2‐16 was expressed in the ER and tonoplasts of rice protoplasts. In addition, OsRFPH2‐16 exhibited E3 ligase activity in an in vitro ubiquitination assay, whereas the mutant OsRFPH2‐16 C188A E3 ligase did not exhibit any activity. We constructed OsRFPH2‐16 ‐overexpressing (OX‐2 and OX‐4) and CRISPR/Cas9‐mediated OsRFPH2‐16 ‐knockout (KO‐4 and KO‐16) plants and evaluated their salt responses. Under salt stress, OsRFPH2‐16‐ knockout plants exhibited improved salt tolerance, characterized by low Na + accumulation, high non‐antioxidant content, and dynamic changes in the expression levels of Na + transporter genes, compared with wild‐type and OsRFPH2‐16 ‐overexpression plants. The aquaporin OsPIP1;1, an interacting partner, was identified using yeast two‐hybridization, bimolecular fluorescence complementation, and pull‐down assays. Degradation of OsPIP1;1 by the E3 ligase OsRFPH2‐16 via the 26S proteasome system was confirmed through an in vitro degradation assay with the inhibitor MG132. These findings support that the E3 ligase functions as a negative regulator, leading to reduced Na + accumulation in salt stress responses.