The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating) and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, OsUPS, from rice (Oryza sativa).The cDNA encoding the O.sativa U-box protein(OsUPS) comprises 1338bp, with an open reading frame of 445 amino acids. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/ arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (Pi). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. Suppression of OsUPS resulted in servre signs of toxicity caused by the over-accumulation of Pi. These results support the notion that OsUPS plays an important role in the Pi signaling pathway through the ubiquitin-26S proteasome system.

E3 ubiquitin ligase plays a central role in determining specificity of the ubiquitination system by selecting appropriate candidate proteins. Compared with other eukaryotic species there are significantly more U-box protein-encoding genes in plant genome. The rice genome contain 77 U-box-type E3 encoding genes. The 77 members of the rice plant U-box (PUB) E3 family can be placed into 8 major groups based on their domain organizations. In this study, we generate and analyze phylogenomic data integrating anatomical expression patterns consisting of anatomical and stress responsive expression patterns base on 1150 affymetrix arrays to the phylogenic tree of rice plant U-box E3 family. We further developed functional gene network meditated by E3 ligases and refined the components in network by integrating gene expression patterns in response to drought stresses. We expect that our analysis will be a useful platform to facilitate the functional identification for each of U-box E3 family.

Rice is a staple food crop in the world. A number of agronomically important traits including enhancement of stress tolerance, quality improvement, and nutrition value increases have been introduced to rice. In this study, an Oryza sativa cDNA containing a U-box motif was cloned; its deduced amino acid sequence was compared to that of other U-box genes and indicated that encodes a U-box-containing E3 ligase. E3 ligases are structurally divided into three groups. We isolated the OsUPS gene from rice (Oryza sativa). The OsUPS protein has domain which is a single~70-amino acid region of the protein and GKL domain containing conserved Glycine, Lysine/ Araginine residues and leucine-rich feature. A full-length expression of OsUPS was up-regulated in the rice plant and in cell culture in the absence of phosphate. To express the OsUPS cDNA, it was inserted into the pGEX-2T vector. And the gene was expressed in E.coli strain BL21 (DE3). Induced after 3h of IPTG treatment and was isolated by affinity chromatography. Using the GUS reporter genes regulated by the OsUPS promoter, we have carried out the analysis of transcriptional and spatial regulation of gene expression. To investigate the function of these genes, the CaMV 35S promoter-driven these genes were introduced into Arabidopsis and rice via Agrobacterium tumefaciens-mediated gene transformation. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (Pi). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the Pi signaling pathway through the ubiquitin-26S proteasome system.

E3 ubiquitin ligase plays a central role in determining specificity of the ubiquitination system by selecting appropriate candidate proteins. Compared with other eukaryotic species there are significantly more U-box protein-encoding genes in plant genome. The rice genome contain 77 U-box-type E3 encoding genes. The 77 members of the rice plant U-box (PUB) E3 family can be placed into 8 major groups based on their domain organizations. In this study, we generate and analyze phylogenomic data integrating anatomical expression patterns consisting of anatomical and stress responsive expression patterns base on 1150 affymetrix arrays to the phylogenic tree of rice plant U-box E3 family. Then, we highlighted 8 genes in subfamilies II (5) and VII (3) of E3 ligases showing significant upregulation in water stress conditions such as drought and high concentration of salt stress. We further developed functional gene network meditated by E3 ligases and refined the components in network by integrating gene expression patterns in response to drought and salt stresses. We expect that our analysis will be a useful platform to facilitate the functional identification for each of U-box E3 family.

The experimental design and response surface methodology (RSM) have been applied to the investigation of the electro-UV complex process for the disinfection of E. coli in the water. The disinfection reactions of electro-UV process were mathematically described as a function of parameters power (X1), NaCl dosage (X2), initial pH (X3) and disinfection time (X4) being modeled by use of the Box-Behnken technique. The application of RSM using the Box-Behnken technique yielded the following regression equation, which is an empirical relationship between the residual E. coli number and test variables in actual variables: Ln (CFU) = 23.57 - 0.87․power - 1.87․NaCl dosage - 2.13․pH - 2.84․time - 0.09․powe r․time - 0.07․NaCl dosage․pH + 0.14․pH․time + 0.03․power 2 + 0.47․NaCl dosage 2 + 0.20․pH 2 + 0.33․time 2 . The model predictions agreed well with the experimentally observed result (R 2 = 0.9987). Graphical response surface and contour plots were used to locate the optimum point. The estimated ridge of maximum response and optimal conditions for the E. coli disinfection using canonical analysis was Ln 1.06 CFU (power, 15.40 W; NaCl dosage, 1.95 g/L, pH, 5.94 and time, 4.67 min). To confirm this optimum condition, the obtained number of the residual E. coli after three additional experiments were Ln 1.05, 1.10 and Ln 1.12. These values were within range of 0.62 (95% PI low)～1.50 (95% PI high), which indicated that conforming the reproducibility of the model.