OsHIRP1 is an E3 ligase that acts as a positive regulator in the plant response to heat stress, thus providing important information relating to adaptation and regulation under heat stress in plant. Extreme temperature adversely affects plant growth, development, and productivity. Here, we report the molecular functions of Oryza sativa heat-induced RING finger protein 1 (OsHIRP1), which might play an important role in the response to heat. Transcription of the OsHIRP1 was upregulated in response to heat and drought treatment. We found that the OsHIRP1-EYFP fusion protein was localized to the nucleus after heat treatment (45 °C). Two interacting partners, OsARK4 and OsHRK1, were identified via yeast-two-hybrid screening, which were mainly targeted to the nucleus (OsARK4) and cytosol (OsHRK1), and their interactions with OsHIRP1 were confirmed by biomolecular fluorescence complementation (BiFC). An in vitro ubiquitination assay showed that OsHIRP1 E3 ligase directly ubiquitinates its interacting proteins, OsAKR4 and OsHRK1, as substrates. Using an in vitro cell-free degradation assay, we observed a clear reduction in the levels of the two proteins under high temperature (45 °C), but not under low temperature conditions (4 °C and 30 °C). Seeds of OsHIRP1-overexpressing plants exhibited high germination rates compared with the control under heat stress. The OsHIRP1-overexpressing plants presented high survival rates of approximately 62-68%, whereas control plants displayed a low recovery rate of 34% under condition of acquired thermo-tolerance. Some heat stress-inducible genes (HsfA3, HSP17.3, HSP18.2 and HSP20) were up-regulated in OsHIRP1-overexpressing Arabidopsis than control plants under heat stress conditions. Collectively, these results suggest that OsHIRP1, an E3 ligase, positively regulates plant response to heat stress.
Keywords: E3 ligase; Heat stress; Oryza sativa; Protein degradation; RING finger protein.