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.

Genome-wide DNA polymorphism and transcriptome analysis of an early-maturing rice mutant.

In order to develop a rice population with improved important traits such as flowering time, we developed 2,911 M2 targeting-induced local lesions in genomes (TILLING) lines by irradiating rice seeds with γ-rays. In all, 15 M3 lines were obtained from 3 different M2 lines that exhibited an early-maturing phenotype: these plants matured approximately 25 days faster than wild-type (WT) plants. To identify genome-wide DNA polymorphisms, we performed whole-genome resequencing of both the plant types, i.e., WT and early-maturing TILLING 1 (EMT1), and obtained mapped reads of 118,488,245 bp (99.53 %) and 128,489,860 bp (99.72 %), respectively; Nipponbare was used as the reference genome. We obtained 63,648 and 147,728 single nucleotide polymorphisms (SNPs) and 33,474 and 31,082 insertions and deletions (InDels) for the WT and EMT1, respectively. Interestingly, there was a higher number of SNPs (2.6-fold) and slightly lower number of InDels (0.9-fold) in EMT1 than in WT. The expression of at least 202 structurally altered genes was changed in EMT1, and functional enrichment analysis of these genes revealed that their molecular functions were related to flower development. These results might provide a critical insight into the regulatory pathways of rice flowering.

Comprehensive analysis of the rice RING E3 ligase family reveals their functional diversity in response to abiotic stress.

A large number of really interesting new gene (RING) E3 ligases contribute to the post-translational modification of target proteins during plant responses to environmental stresses. However, the physical interactome of RING E3 ligases in rice remains largely unknown. Here, we evaluated the expression patterns of 47 Oryza sativa RING finger protein (OsRFP) genes in response to abiotic stresses via semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and in silico analysis. Subsequently, molecular dissection of nine OsRFPs was performed by the examination of their E3 ubiquitin ligase activity, subcellular localization, and physical interaction with target proteins. Most of the OsRFPs examined possessed E3 ligase activity and showed diverse subcellular localization. Yeast two-hybrid analysis was then employed to construct a physical interaction map of seven OsRFPs with their 120 interacting proteins. The results indicated that these OsRFPs required dynamic translocation and partitioning for their cellular activation. Heterogeneous overexpression of each of the OsRFP genes in Arabidopsis suggested that they have functionally diverse responses to abiotic stresses, which may have been acquired during evolution. This comprehensive study provides insights into the biological functions of OsRFPs, which may be useful in understanding how rice plants adapt to unfavourable environmental conditions.