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 types of white blood cells, such as T cells, B cells, and macrophages, are involved in the immune response. In particular, the processes of T-cell activation play a crucial role in an adaptive immune response, whereby the T-cell receptor (TCR) engages with an antigen and signals a cascade that leads to the activation of transcription factors (AP-1, NF-κB, and NFAT) that are critically involved in cytokine production. Roquin, encoded by the RC3H1 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 proteins regulate T-cell responses are unclear. To elucidate the role of Roquin in vitro, murine lymphoma EL-4 cells were used. Roquin overexpressing Tcells became hyper-responsive upon anti-CD3/CD28 stimulation in vitro and were a major source of cytokines such as IL-2, TNF-α, IL-6, and IL-10. Upon activation, these cells showed preferentially enhanced production of IL-2 and TNF-α, but not IFN-γ. To clarify the important role of Roquin in the T-cell response ex vivo, we generated T-cell-specific Roquin-transgenic (Tg) mice having a higher expression of Roquin in T cells as compared to wild-type mice. Using Roquin-Tg mice, we studied whether immune responses are affected ex vivo. Roquin-Tg CD4+ T cells showed enhanced production of IL-2 or TNF-α to TCR stimulation with anti-CD28 costimulation via up-regulation of CD28. T-cell proliferation also increased in Roquin-Tg CD4+ T cells after anti-CD3/CD28 treatment. Further studies on the role of Roquin in the regulation of primary T-cell activation, survival, and differentiation may be anticipated.

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.

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.

The ubiquitin conjugating enzyme E2 (UBC E2) mediates selective ubiquitination, acting with E1 and E3 enzymes to designate specific proteins for subsequent degradation. In the present study, we characterized the function of the mung bean VrUBC1 gene (Vigna radiata UBC 1). RNA gel-blot analysis showed that VrUBC1 mRNA expression was induced by either dehydration, high salinity or by the exogenous abscisic acid (ABA), but not by low temperature or wounding. Biochemical studies of VrUBC1 recombinant protein and complementation of yeast ubc4/5 by VrUBC1 revealed that VrUBC1 encodes a functional UBC E2. To understand the function of this gene in development and plant responses to osmotic stresses, we overexpressed VrUBC1 in Arabidopsis (Arabidopsis thaliana). The VrUBC1-overexpressing plants displayed highly sensitive responses to ABA and osmotic stress during germination, enhanced ABA- or salt-induced stomatal closing, and increased drought stress tolerance. The expression levels of a number of key ABA signaling genes were increased in VrUBC1-overexpressing plants compared to the wild-type plants. Yeast two-hybrid and bimolecular fluorescence complementation demonstrated that VrUBC1 interacts with AtVBP1 (A. thaliana VrUBC1 Binding Partner 1), a C3HC4-type RING E3 ligase. Overall, these results demonstrate that VrUBC1 plays a positive role in osmotic stress tolerance through transcriptional regulation of ABA-related genes and possibly through interaction with a novel RING E3 ligase.

A low temperature-inducible cDNA designated as VrUBC1 from mungbean (Vigna radiata) was isolated by subtractive hybridization method. By rapid amplification of cDNA end technique, the full-length cDNA of VrUBC1 was obtained. The full-length cDNA of VrUBC1 contains an open reading frame of 444 nucleotides in length and capable of specifying a 16.5-kDa protein of 148 amino acids (aa) with an isoelectric point of 7.72. VrUBC1 mRNA was induced by NaCl and ABA, but not by wounding and low temperature stress. It was shown that VrUBC1-GFP was localized to the cytoplasm in tobacco cell. To examine the function of VrUBC1, VrUBC1 was expressed in Escherichia coli as His-fusion protein. Purified VrUBC1-His recombinant protein was shown to have ubiquitination activity in vitro. For the in vivo functional analysis of VrUBC1, VrUBC1 was expressed in yeast ubc4/5 double mutant. Stress tolerance was tested in the VrUBC1 overexpressing Arabidopsis transgenic plants. We propose that VrUBC1 play an important role in protein degradation processes during abiotic stress in plants.

Post-translational covalent modifications by small molecules or peptides remodel target proteins. One such modification, made by ubiquitin or small ubiquitin-related modifier (SUMO), is a rapidly expanding field in cell signaling pathways. Ubiquitin attachment controls the turnover and degradation of target proteins while SUMO conjugation regulates their activity and function. Recent studies report many examples of cross-talk between ubiquitin and SUMO pathways, indicating that the boundary is no longer clear. Here, we review recent progress concerning how ubiquitin and SUMO participate in new regulatory roles in plant cell, and how ubiquitination and sumoylation control plant growth and development.