Positive regulation of rice RING E3 ligase OsHIR1 in arsenic and cadmium uptakes

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.