Molecular Characterization of Chilling-induced Rice RING Finger Ligase 1 (OsCRF1): Expression Pattern, In vitro Ubiquitination Assay, and Subcellular Localization

Chilling (sub-optimal temperature) stress adversely affects plant growth and productivity during rice cultivation period, especially at seedling and reproductive stages. We selected the Oryza Sativa chilling-induced RING E3 ligase gene (OsCRF1) and examined its expression pattern in different tissues during vegetative and reproductive stages. In addition, we evaluated the phenotypes and electrolyte leakages from two rice cultivars, under chilling stress, that revealed that Donganbyeo as a chilling-insensitive type and Satbyeolbyeo as a sensitive type. qRT- PCR was employed to evaluate OsCRF1 expression patterns in both plants. Results showed that OsCTR1 is highly induced in both cultivars during chilling stress, although the patterns differ. In vitro ubiquitination assay showed clear polyubiquitin chains of OsCRF1, confirming its E3 ligase activity. Furthermore, we determined the subcellular localization of OsCRF1 through EYFP vector fusion and observed that OsCRF1 is localized within the cell nucleus. Our findings indicate that nucleus localized OsCRF1 may play a role in the modulation of responses to chilling stress.