E3 ligase, the Oryza sativa salt-induced RING finger protein 4 (OsSIRP4), negatively regulates salt stress responses via degradation of the OsPEX11-1 protein

Abstract

OsSIRP4 is an E3 ligase that acts as a negative regulator in the plant response to salt stress via the 26S proteasomal system regulation of substrate proteins, OsPEX11-1, which it provides important information for adaptation and regulation in rice. Plants are sessile organisms that can be exposed to environmental stress. Plants alter their cellular processes to survive under potentially unfavorable conditions. Protein ubiquitination is an important post-translational modification that has a crucial role in various cellular signaling processes in abiotic stress response. In this study, we characterized Oryza sativa salt-induced RING finger protein 4, OsSIRP4, a membrane and cytosol-localized RING E3 ligase in rice. OsSIRP4 transcripts were highly induced under salt stress in rice. We found that OsSIRP4 possesses E3 ligase activity; however, no E3 ligase activity was observed with a single amino acid substitution (OsSIRP4^C269A). The results of the yeast two hybrid system, in vitro pull-down assay, BiFC analysis, in vitro ubiquitination assay, and in vitro degradation assay indicate that OsSIRP4 regulates degradation of a substrate protein, OsPEX11-1 (Oryza sativa peroxisomal biogenesis factor 11-1) via the 26S proteasomal system. Phenotypic analysis of OsSIRP4-overexpressing plants demonstrated hypersensitivity to salt response compared to that of the wild type and mutated OsSIRP4^C269A plants. In addition, OsSIRP4-overexpressing plants exhibited significant low enzyme activities of superoxide dismutase, catalase, and peroxidase, and accumulation of proline and soluble sugar, but a high level of H₂O₂. Furthermore, qRT data on transgenic plants suggest that OsSIRP4 acted as a negative regulator of salt response by diminishing the expression of genes related to Na⁺/K⁺ homeostasis (AtSOS1, AtAKT1, AtNHX1, and AtHKT1;1) in transgenic plants under salt stress. These results suggest that OsSIRP4 plays a negative regulatory role in response to salt stress by modulating the target protein levels.

Keywords: OsPEX11-1; Protein degradation; RING E3 ligase; Rice; Salt stress.