Ubiquitination is a crucial post-translational protein modification and participates in abiotic stress responses in higher plants. In this study, we identified and characterized OsDIRP1 (Oryza sativa Drought-Induced RING Protein 1), a nuclear-localized putative RING E3 ubiquitin (Ub) ligase in rice (Oryza sativa L.). OsDIRP1 expression was induced by drought, high salinity, and abscisic acid (ABA) treatment, but not by low temperature (4°C) stress, suggesting that OsDIRP1 is differentially regulated by different abiotic stresses. To investigate its possible role in abiotic stress responses, OsDIRP1-overexpressing transgenic rice plants (Ubi:OsDIRP1-sGFP) were generated, and their phenotypes were analyzed. The T4 Ubi:OsDIRP1-sGFP lines showed decreased tolerance to drought and salt stress as compared to wild-type rice plants. Moreover, Ubi:OsDIRP1-sGFP progeny were less sensitive to ABA than the wild-type during both germination and post-germination growth. In contrast, Ubi:OsDIRP1-sGFP plants exhibited markedly higher tolerance to prolonged cold (4°C) treatment. These results suggest that OsDIRP1 acts as a negative regulator during drought and salt stress, whereas it functions as a positive factor during the cold stress response in rice.
Identification and characterization of OsDIRP1 in rice. (A) (Upper) Schematic diagram of full-length OsDIRP1 cDNA. The solid bar depicts the coding region. The solid lines represent the 5’- and 3’-untranslated regions. (Lower) Schematic structure of OsDIRP1. The putative beta-ketoacyl synthase active site, nuclear localization sequence (NLS), and RING motif are shown as dark gray bars. (B) RT-PCR analysis of OsDIRP1in different tissues of rice plants. Total RNA was isolated from various rice tissues as indicated and analyzed by RT-PCR. OsUbiquitin was used as an equal loading control. (C) Expression patterns of OsDIRP1 in response to various abiotic stresses in rice plants. Light-grown, 10-day-old wild-type seedlings were subjected to drought, salt, cold, and ABA treatments at different time points as indicated. OsUbiquitin was used as an internal control for all the RT-PCR analyses. OsRab16b was used as a positive control for drought, salt, and ABA treatments, whereas OsDREB1A was used as a positive control for cold stress. (D) Subcellular localization of OsDIRP1. A 35S:OsDIRP1-sGFP fusion construct was transfected into wild-type rice protoplasts, and the fluorescent signals of the expressed proteins were visualized by fluorescence microscopy under dark-field conditions. sGFP and NLS-mRFP were used as cytosol- and nucleus-localized marker proteins, respectively. Bars = 5 μm.