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First published online October 13, 2006; 10.1104/pp.106.087965 Plant Physiology 142:1664-1682 (2006) © 2006 American Society of Plant Biologists Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog1,[C]Department of Biology, College of Science (S.K.C., H.S.C., M.Y.R., M.M.L., H.S.P., W.T.K.) and Protein Network Research Center (Y.-Y.B.), Yonsei University, Seoul 120–749, Korea; and Department of Plant Biotechnology/Agricultural Plant Stress Research Center, Chonnam National University, Gwangju 500–712, Korea (M.J.P., J.K.)
The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35S::CaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.
1 This work was supported in part by grants from the Basic Research Program of the Korea Science and Engineering Foundation (project no. R01–2004–000–10487–0 to W.T.K. and H.S.P.), the Plant Diversity Research Center (Twenty-First Century Frontier Research Program funded by the Ministry of Science and Technology of the Korean government to W.T.K., H.S.P., and J.K.), and the Plant Metabolism Research Center at Kyung Hee University (Science Research Center project no. R11–2000–081 from the Korea Science and Engineering Foundation to W.T.K.). The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Woo Taek Kim (wtkim{at}yonsei.ac.kr). [C] Some figures in this article are displayed in color online but in black and white in the print edition. www.plantphysiol.org/cgi/doi/10.1104/pp.106.087965 * Corresponding author; e-mail wtkim{at}yonsei.ac.kr; fax 82–2–312–5657. Received August 3, 2006; accepted October 5, 2006; published October 13, 2006. This article has been cited by other articles:
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