RING (Really Interesting New Gene) finger proteins play crucial roles in abiotic stress responses in plants. We report the RING finger E3 ligase gene, an Oryza sativa salt, ABA and drought stress-induced RING finger protein 1 gene (OsSADR1). We demonstrated that although OsSAR1 possesses E3 ligase activity, a single amino acid substitution (OsSADR1C168A) in the RING domain resulted in no E3 ligase activity, suggesting that the activity of most E3s is specified by the RING domain. Additional assays substantiated that OsSADR1 interacts with three substrates-no E3 ligase acti and OsPIRIN, and mediates their proteolysis via the 26S proteasome pathway. For OsSADR1, approximately 62% of the transient signals were in the cytosol and 38% in the nucleus. However, transiently expressed OsSADR1 was primarily expressed in the nucleus (70%) in 200 mM salt-treated rice protoplasts. The two nucleus-localized proteins (OsSNAC2 and OsGRAS44) interacted with OsSADR1 in the cytosol and nucleus. Heterogeneous overexpression of OsSADR1 in Arabidopsis resulted in sensitive phenotypes for salt- and mannitol-responsive seed germination and seedling growth. With ABA, OsSADR1 overexpression in plants produced highly tolerant phenotypes, with morphological changes in root length and stomatal closure. The ABA-tolerant transgenic plants also showed hypersensitivity phenotypes under severe water deficit conditions. Taken together, OsSADR1 may act as a regulator in abiotic stress responses by modulating target protein levels.

Our results suggest that a rice E3 ligase, OsMAR1, physically interacts with a cytosolic protein OCPI2 and may play an important role under salinity stress. Salt is an important abiotic stressor that negatively affects plant growth phases and alters development. Herein, we found that a rice gene, OsMAR1 (Oryza sativa microtubule-associated RING finger protein 1), encoding the RING E3 ligase was highly expressed in response to high salinity, water deficit, and ABA treatment. Fluorescence signals of its recombinant proteins were clearly associated with the microtubules in rice protoplasts. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) showed that OsMAR1 interacted with a cytosolic protein OCPI2 (O. sativa chymotrypsin protease inhibitor 2) and led to its degradation via the 26S proteasome. Heterogeneous overexpression of OsMAR1 in Arabidopsis showed retarded root growth compared with that of control plants, and then led to hypersensitivity phenotypes under high salinity stress. Taken together, OsMAR1 negatively regulates the salt-stress response via the regulation of the OCPI2 protein in rice.

Soil alkalinity is an important stressor that impairs crop growth and development, resulting in reduced crop productivity. Unlike salinity stress, research efforts to understand the mechanism of plant adaptation to alkaline stress is limited in rice, a major staple food for the world population. We evaluated a population of 193 recombinant inbred lines (RIL) developed from a cross between Cocodrie and N22 under alkaline stress at the seedling stage. Using a linkage map consisting of 4849 SNP markers, 42 additive QTLs were identified. There were seven genomic regions where two or more QTLs for multiple traits colocalized. Three important QTL clusters were targeted, and several candidate genes were identified based on high impact variants using whole genome sequences (WGS) of both parents and differential expression in response to alkalinity stress. These genes included two expressed protein genes, the glucan endo-1,3-beta-glucosidase precursor, F-box domain-containing proteins, double-stranded RNA-binding motif-containing protein, aquaporin protein, receptor kinase-like protein, semialdehyde hydrogenase, and NAD-binding domain-containing protein genes. Tolerance to alkaline stress in Cocodrie was most likely due to the low Na+/K+ ratio resulting from reduced accumulation of Na+ ions and higher accumulation of K+ in roots and shoots. Our study demonstrated the utility of integrating QTL mapping with WGS to identify the candidate genes in the QTL regions. The QTLs and candidate genes originating from the tolerant parent Cocodrie should be targeted for introgression to improve alkalinity tolerance in rice and to elucidate the molecular basis of alkali tolerance.

A rice gene (OsSIRP2) encoding the RING Ub E3 ligase was highly induced under salinity stress and physically interacted with a transketolase (OsTKL1). Overexpression of OsSIRP2 conferred salinity and osmotic stress tolerance in plants. The RING E3 ligases play a vital role in post transitional modification through ubiquitination-mediated protein degradation that mediate plants responses during abiotic stresses and signal transduction. In this study, we report an Oryza sativa salt induced Really Interesting New Gene (RING) finger protein 2 gene (OsSIRP2) and elucidate its role under salinity and osmotic stress. The transcript levels of OsSIRP2 in rice leaves were induced in response to different abiotic stresses, such as salt, drought, heat, and abscisic acid (ABA) exposure. In vitro ubiquitination revealed that the OsSIRP2 protein formed poly-ubiquitin products, whereas a single amino acid substitution in OsSIRP2 (OsSIRP2) in the RING domain did not form ubiquitinated substrates, supporting the hypothesis that E3 ligase activity requires the functional RING domain. Using the yeast two-hybrid (Y2H) assay, O. sativa transketolase 1 (OsTKL1) was identified as an interacting partner. OsSIRP2 was localized in the nucleus, whereas its interacting partner (OsTKL1) was localized in the cytosol and plastids in the rice protoplasts. Fluorescence signals between OsSIRP2 and OsTKL1 were observed in the cytosol. The pull-down assay confirmed the physical interaction between OsSIRP2 and OsTKL1. In vitro ubiquitination assay and in vitro protein degradation assay revealed that OsSIRP2 ubiquitinates OsTKL1 and enhances the degradation of OsTKL1 through the 26S proteasomal pathway. Heterogeneous overexpression of OsSIRP2 resulted in conferring tolerance against salinity and osmotic stress. Overall, our findings suggest that OsSIRP2 may be associated with plant responses to abiotic stresses and act as a positive regulator of salt and osmotic stress tolerance.

Arsenic (As) accumulation adversely affects the growth and productivity of plants and poses a serious threat to human health and food security. In this study, we identified one As-responsive Really Interesting New Gene (RING) E3 ubiquitin ligase gene from rice root tissues during As stress. We named it Oryza sativa As-Induced RING E3 ligase 2 (OsAIR2). Expression of OsAIR2 was induced under various abiotic stress conditions, including heat, salt, drought and As exposure. Results of an in vitro ubiquitination assay showed that OsAIR2 possesses an E3 ligase activity. Within the cell, OsAIR2 was found to be localized to the Golgi apparatus. Using yeast two-hybrid (Y2H) assay, the 3-ketoacyl-CoA thiolase (KAT) protein was identified as an interaction partner. We found that the O. sativa KAT1 (OsKAT1) is localized to the cytosol and peroxisomes. Moreover, in vitro pull-down assay verified the physical interaction between OsAIR2 and OsKAT1. Interestingly, in vitro ubiquitination assay and in vivo proteasomal degradation assay revealed that OsAIR2 ubiquitinates OsKAT1 and promotes the degradation of OsKAT1 via the 26S proteasome degradation pathway. Heterogeneous overexpression of OsAIR2 in Arabidopsis improved the seed germination and increased the root length under arsenate stress conditions. Therefore, these results suggest that OsAIR2 may be associated with the plant response to As stress and acts as a positive regulator of As stress tolerance.