A novel R3H protein, OsDIP1, confers ABA-mediated adaptation to drought and salinity stress in rice

Aims: Dehydration responsive element-binding factors (DBFs) have recently emerged as essential components of stress tolerance mechanisms in plants. In this work, we investigated the role of OsDIP1 that operate upstream of DBFs as a regulator of drought and salinity tolerance in rice. Methods: Experiments were conducted on rice plants (Oryza sativa) and included combination of physiological (plant phenotyping) and molecular methods. The latter involved real-time PCR (RT-qPCR); yeast two-hybrid, BiFC assay, GST pull-down, CoIP and firefly luciferase (LUC)/ renilla (REN) assays. Results: OsDIP1 expression was induced by hydrogen peroxide (H₂O₂), ABA (abscisic acid), drought (polyethylene glycol treatment), and salt stress. Overexpression of OsDIP1 in rice enhanced drought and salinity tolerance while knocking out OsDIP1 by CRISPR-Cas9 editing resulted in drought and salt sensitive phenotype. The activity and gene expression of antioxidant defense enzymes, superoxide dismutase (SOD), catalase (CAT), was increased in OsDIP1-overexpressed transgenic rice plants, while the content of malondialdehyde (MDA) was decreased. In contrast, the content and gene expression of SODCc2 and CatB, decreased, and the content of MDA was increased in knockout of OsDIP1 rice plants, suggesting that overexpression of OsDIP1 enhances the antioxidant capacity of rice plants. The yeast two-hybrid screening test revealed that OsDIP1 interacted with ZFP36, a key zinc finger transcription factor involved in ABA-induced antioxidant defense. Moreover, OsDIP1 could modulate some key ABA-responsive genes via interacting with ZFP36. Conclusions: In this work, we show that DIP1 plays a central role in modulating drought and salinity stress tolerance in rice.