Thermotolerance is very important for plant survival when plants are subjected to lethally high temperature. However, thus far little is known about the functions of RING E3 ligase in response to heat shock in plants. This study found that one rice gene encoding the RING finger protein was specifically induced by heat and cold stress treatments but not by salinity or dehydration and named it OsHCI1 (Oryza sativa heat and cold induced 1). Subcellular localization results showed that OsHCI1 was mainly associated with the Golgi apparatus and moved rapidly and extensively along the cytoskeleton. In contrast, OsHCI1 may have accumulated in the nucleus under high temperatures. OsHCI1 physically interacted with nuclear substrate proteins including a basic helix-loop-helix transcription factor. Transient co-overexpression of OsHCI1 and each of three nuclear proteins showed that their fluorescent signals moved into the cytoplasm as punctuate formations. Heterogeneous overexpression of OsHCI1 in Arabidopsis highly increased survival rate through acquired thermotolerance. It is proposed that OsHCI1 mediates nuclear–cytoplasmic trafficking of nuclear substrate proteins via monoubiquitination and drives an inactivation device for the nuclear proteins under heat shock.
The metalloid arsenic (As) and the heavy metal cadmium (Cd) are ubiquitously found at low concentrations in the earth. High concentrations of these elements in the soil and crops are severely dangerous to human health. We attempted to retrieve the RING E3 ubiquitin ligase gene for regulating As and Cd uptakes via the ubiquitin 26S proteasome system. Semi-quantitative reverse transcription polymerase chain reaction was conducted for a total of 47 Oryza sativa RING finger protein (OsRFP) genes to assess their expression patterns when exposed to As and Cd treatments. We identified one gene Oryza sativa heavy metal induced RING E3 ligase 1 (OsHIR1), which was significantly upregulated with both treatments. A yeast hybrid screen and a bimolecular fluorescence complementation assay showed that OsHIR1 clearly interacts with 5 substrate proteins, including tonoplast intrinsic protein 4;1 (OsTIP4;1) in the plasma membrane. In addition, OsHIR1 strongly degraded the protein level of OsTIP4;1 via the ubiquitin 26S proteasome system. Heterogeneous overexpression of OsHIR1 in Arabidopsis exhibited As- and Cd-insensitive phenotypes and resulted in decreased As and Cd accumulation in the shoots and roots, relative to the control. Herein, we report the novel finding that the OsHIR1 E3 ligase positively regulates OsTIP4;1 related to As and Cd uptakes.
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.
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