The T-cell receptor (TCR) engages with an antigen and initiates a signaling cascade that leads to the activation of transcription factors. Roquin, a protein encoded by the RC- 3H1 gene and characterized as an immune regulator, was recently identified as a novel RING-type ubiquitin ligase family member, but the mechanisms by which Roquin regulates T-cell responses are unclear. We used the EL-4 murine lymphoma cell line to elucidate the role of Roquin in vitro. Roquin-overexpressing EL-4 cells became hyper-responsive after anti-CD3/CD28 stimulation in vitro and were a major source of the cytokines IL-2 and TNF-α. Upon activation, these cells showed particularly enhanced production of IL-2 and TNF-α. To clarify the important role played by Roquin in T-cell responses ex vivo, we generated T-cell-specific Roquin transgenic (Tg) mice. Roquin-Tg CD4+ T-cells showed enhanced production of IL-2 and TNF-α in response to TCR stimulation with anti-CD28 co-stimulation. Further studies are necessary to investigate the role of Roquin in the regulation of primary T-cell activation, survival, and differentiation.

Several E3 ubiquitin ligases have been associated with the response to abiotic and biotic stresses in higher plants. Here, we report that the hot pepper (Capsicumannuum) abscisic acid (ABA)-InsensitiveRINGprotein1gene(CaAIR1) is essential for a hypersensitive response to drought stress. CaAIR1 contains a C3HC4-type RING finger motif, which plays a role for attachment of ubiquitins to the target protein, and a putative transmembrane domain. The expression levels of CaAIR1 are upregulated in pepper leaves by ABA treatments, drought, and NaCl, suggesting its role in the response to abiotic stress. Our analysis showed that CaAIR1 displays self-ubiquitination and localized in the nucleus. We generated CaAIR1-silenced peppers via virus-induced gene silencing (VIGS) and CaAIR1-overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to ABA and drought. VIGS of CaAIR1 in pepper plants conferred an enhanced tolerance to drought stress, which was accompanied by low levels of transpirational water loss in the drought-treated leaves. CaAIR1-OX plants displayed an impaired sensitivity to ABA during seed germination, seedling, and adult stages. Moreover, these plants showed enhanced sensitivity to drought stress because of reduced stomatal closure and decreased expression of stress-responsive genes. Thus, our data indicate that CaAIR1 is a negative regulator of the ABA-mediated drought-stress tolerance mechanism.

The wild relatives of soybean [Glycine soja Sieb. and Zucc.] have curly/wavy nature whereas cultivated varieties are upright. Such morphological characteristics have agronomic importance too. To investigate the molecular mechanism of development contributing to coiled morphology, screening was carried out to look for Arabidopsis mutants in activation tagging lines obtained by activation T-DNA treatment that have curly/wavy morphology. A mutant named Coiled Branch 1 (cbr1), is found to have a wavy and curly morphology with coiling branches. Plasmid rescue and genomic southern blot analysis revealed the site of T-DNA insertion in the genome. RT-PCR was performed to monitor expression levels of the genes adjacent to the T-DNA integration sites, and showed the activation of an E3 ubiquitin ligase gene. Database search showed that the gene with the RING domain belongs to a family of E3 ubiquitin ligases. Complementation test by overexpression and RNA interference of the gene was also carried out. The complementation test results showed that the novel gene activation tagging affected the cbr1 mutant phenotypes. Ubiquitylation has been linked virtually to every cellular process including plant development. E3 ubiquitin ligase has been reported to recognize target proteins that are to be ubiquinated for further degradation by the proteasome complex. Further, more detailed studies are needed to identify the specific substrate(s) of the novel E3 ubiquitin ligase gene.

Arabidopsis E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development including responses to nutrient deficiency and environment stresses. Here, we analyzed the effect of AtSIZ1 on the stability and amount of seed proteins. Proteomic analysis showed that the amount of three major nutrient reservoir proteins, CRUCIFERIN (CRU) 1, 2 and 3, were decreased in siz1-2 mutants. However, quantitative real-time RT-PCR showed that transcript levels of CRU1, 2 and 3 genes were rather significantly higher in siz1-2 mutants than wild-type plants. Yeast two hybrid analysis revealed that AtSIZ1 interacts with CRU1, CRU2 and CRU3, strongly suggesting that CRU1, 2 and 3 proteins are sumoylated by AtSIZ1. In addition, the analysis of amino acid composition by HPLC showed that the contents of amino acids were a bit high in siz1-2 mutants. Our data indicate that AtSIZ1 plays an important function for accumulation of seed storage proteins through its ligase activity.

Floral transition is influenced by environmental factors such as light and temperature. Plants are capable of integrating photoperiod and ambient temperature signaling into their developmental program. Despite extensive investigations on individual genetic pathways, little is known about the molecular components that integrate both pathways. Here, we demonstrate that the RING finger–containing E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) acts as an integrator of photoperiod and ambient temperature signaling. In addition to the role in photoperiodic destabilization of CONSTANS (CO), COP1 also regulates temperature sensitivity by controlling the degradation of GIGANTEA (GI). COP1-impaired mutants showed reduced sensitivity to low ambient temperature. Notably, COP1 is more stabilized at low temperature and accelerates GI turnover in a 26S proteasome-dependent manner. The direct association of GI with the promoter of FLOWERING LOCUS T (FT) depends on ambient temperature, and thus COP1-triggered GI turnover delays flowering at low temperatures via a CO-independent pathway. Taken together, our findings indicate that environmental conditions regulate the stability of COP1, and conditional specificity of its target selection stimulates proper developmental responses and ensures reproductive success.

Gene expression is regulated by DNA and histone methylation by DNA and histone methyltransferases, respectively. In animal system, DNA methyltransferase with CG methylation activity is modified by SUMO conjugation and then its activity was increased, which means that the activity of DNA methyltransferase is modulated by posttranslational modification. so Chromatin remodeling is a new concept for expression of controlling of gene function. We thus analyzed the effect of E3 SUMO ligase AtSIZ1 in CMT3 (chromometnylase 3)-mediated genome methylation by next-generation sequencing (NGS), methyl binding domain MeDIP-sequencing and gene analysis using siz1-2 and cmt3 mutants. we carried out CG-enrich analysis by MeDIP sequencing revealed that the methylation level of the genome including transposons was significantly low in siz1-2 mutants compared to wild-type. Result showed the genes regulated by methylation, that genes related of embryo and root development, cellulose metabolism, and post-translational modifications. All of our data indicate that the methyltransfearse activity of CMT3 may be able to be regulated by AtSIZ1 and thereby CMT3-mediated gene expression and plant development also can be controlled by E3 SUMO ligase activity. Besides, our data also suggest that ammonium (NH4+) can stimulate AtSIZ1- and CMT3- mediated DNA methylation.

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.

Soybean (Glycine max (L.) Merr) is a short day plant and has been adapted to various climates and environments during cultivation. However, the cultivation area is restricted to a very narrow range of latitudes. To date, nine major genes (E1 to E8 and J) have been reported to control the flowering time and maturity. Here, we evaluated the role of E2, E3, E4, and their paralogue genes in late flowering soybean cultivars under long day (LD) conditions using Soybean yellow common mosaic virus (SYCMV)-based virus-induced gene silencing (VIGS) system. A total of nine VIGS constructs were infiltrated into two fully expanded cotyledons and primary leaves. After inoculation with these VIGS constructs on Jangyeobkong, which is a late-flowering cultivar, phenotypic traits were evaluated for the first flowering dates (FFDs) and pod maturities under LD conditions. The FFDs of the silenced plants occurred 50-56 days after sowing (das), while the non-silenced plants bloomed on 60-61 days. We found that the E3 paralogue-silenced plants flowered the fastest and responsive genes were identified to be associated with the promotion of flowering time. As the knock-down of E3 paralogue, expression of E1 was up-regulated, E2 was no difference, E3 and E4 genes were down-regulated in the silenced plants. Expression of GmFT2a and GmFT5a is known to be controlled by E3 and E4. Interestingly, GmFT5a were highly expressed in SYCMV:E3 paralogue-silenced plants, whereas the expression of GmFT2a was not significant. These results support that GmFT5a is able to independently promote flowering under LD conditions.

Population growth and increasing consumption of resources in the process of the industrial development has caused environmental pollution, climate change, and resource exhaustion. Therefore 'sustainable development' has become the important issue for the future. The sustainable development aims at effective resource use, less environmental impacts, and higher social security. Generally the rural area including agricultural fields and forest has various and plentiful natural resources which could make future development sustainable. To develop potential rural resources, the values for energy, environment and economy should be assessed considering the life-cycle of resources. The purposes of this study are to suggest the E3 (Energy, Environment, and Economy) assessment model for rural biomass considering life-cycle of resource and to apply the model to rice, the major agricultural product. As the results of this study, it turned out through E3 assessment that economic gain of rice cultivation is 578,374 won/10a, carbon absorption is 1,530kgCO2/10a, carbon emission is 926.65kgCO2/10a, and bio-energy potential of by-product is 394,028 kcal/10a. When E3 assessment was applied to by province, the results varied by regions because of the amount of input during cultivation. These results would be useful to realize the rural biomass and design regional resources plan in integrated E3 perspective.

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.

UGT72E3/2 gene encodes UDP-glycosyltransferase shown to glucosylate several phenylpropanoids such as syringin and coniferin. Syringin has effect of anti-stress and anti-fatigue. Korean soybean variety Kwangan was transformed with UGT72E3/2 gene. This gene was transformed into Kwangan using highly efficient soybean transformation system. This study used two promoters, beta-conglycinin promoter for seed-specific expression and 35s promoter for total expression. Transgenic plants were confirmed for gene introduction and their expression using PCR and RT-PCR. The analysis of syringin in transgenic plants was performed using HPLC. Currently, the confirmation of stable gene introduction with UGT72E3/2 gene is also performing by Southern blot analysis.

The metalloid arsenic (As) and the hevy metal cadmium (Cd) are ubiquitously found at low concentrations in the earth, while high concentrations of the both elements in soil and crop are severe dangerous to human health. We have tried to retrieve RING E3 ligase gene, which is believed to regulate substrate proteins in As or Cd uptake via ubiquitin 26S proteasome pathway, related to inhibit metal ion transport system. A total of 48 rice RING E3 ligases were randomly selected and then conducted semi-quantitative RT-PCR for their expression patterns as exposed to As and Cd treatments. We discovered one gene, Oryza sativa heavy metal induced RING E3 ligase 1 (OsHIR1) that was significantly up-regulated against both treatments. A total of 31 positive interaction clones with OsHIR1 were screened depending on their strong α-galactosidase activity via yeast-two hybrid screen. Bimolecular fluorescence complementation analysis evidenced that the OsHIR1 protein was clearly interacted with each of six partner protein including aquaporin tonoplast intrinsic protein 4;1 (OsTIP4;1) in the plasma membrane. Protein degradation assay showed that OsHIR1 strongly degraded the protein level of OsTIP4;1 via ubiquitin 26S proteasome system. Heterogeneous overexpression of OsHIR1 in Arabidopsis showed As- and Cd-insensitive phenotype. In addition, the transgenic plant showed low levels of As and Cd accumulation than the control plant in leaf and root. Here, we report the novel finding that OsHIR1 E3 ligase positively regulates OsTIP4;1 related to As and Cd uptake.

Plant growth under water-deficit conditions adversely affects many key processes. Efforts to understand drought stress-related defense mechanisms have revealed a host of plant genes using molecular approaches in rice. Here, we report the novel finding that OsCTR1 E3 ligase regulates both chloroplast-localized chloroplast protein 12 (OsCP12) and ribosomal protein 1 (OsRP1) in protein levels and subcellular localization. The results of a yeast-two hybrid assay, bimolecular fluorescence complementation assay, ubiquitination assay, subcellular localization, and a protein degradation assay support the hypothesis that OsCTR1 functions in trafficking inhibition and proteolysis of OsCP12 and OsRP1 via the ubiquitin 26S proteasome pathway. Heterogeneous overexpression of OsCTR1 in Arabidopsis showed ABA-hypersensitive phenotype in seed germination, seedling growth, and stomatal closure. The transgenic plants also exhibited improvement of water-deficit tolerance with an accumulation of hydrogen peroxide production. These results demonstrate that the OsCTR1 E3 ligase might positively regulate the cellular functions of OsCP12 and RP1 related to photosynthesis under drought stress conditions in rice.

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.

Plants are known to have homeostatic cellular mechanisms to control the concentration of heavy metal inside the cell. We tried to retrieve rice RING finger protein genes, which are believed to regulate substrates via ubiqitinations, related to metal ions detoxification mechanisms. A total of 48 rice RING finger proteins were randomly selected and then examined for their expression patterns as exposed to cadmium and arsenic treatments. We discovered a RING finger protein gene that was significant up-regulated against both treatments and then named Oryza sativa heavy metal induced 1 (OsHMI1). We tested subsequently OsHMI1 expression patterns against to salinity, dehydration, cold, heat stress and phytohormones treatments. In addition, we evaluated its subcellular localization and determined E3 ligase activity. The interaction partner proteins were screened via yeast-two hybridization. These results might shed further light on the understanding of homeostatic cellular mechanisms to control heavy metal detoxification via protein degradation in plants.

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.