Plant Physiol. Journal of Pharmacology and Experimental Therapeutics
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Published on October 13, 2006; 10.1104/pp.106.087965

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Received August 3, 2006
Accepted October 5, 2006

Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-box E3 Ubiquitin Ligase Homolog

Seok Keun Cho , Hoo Sun Chung , Moon Young Ryu , Mi Jin Park , Myeong Min Lee , Young-Yil Bahk , Jungmook Kim , Hyun Sook Pai , and Woo Taek Kim *

Department of Biology, College of Science, Yonsei University, Seoul 120-749, Korea
Department of Plant Biotechnology/Agricultural Plant Stress Research Center, Chonnam National University, Gwangju 500-712, Korea
Protein Network Research Center, Yonsei University, Seoul 120-749, Korea

* Corresponding author; email: wtkim{at}yonsei.ac.kr.

The U-box motif is a conserved domain found in the diverse isoforms of E3 Ub ligase in eukaryotes. From water-stressed hot pepper plants, we isolated CaPUB1 (Capsicum annuum putative U-box protein 1) that 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 Ub 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, ABA, 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 over-expressed CaPUB1 in Arabidopsis 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 CaMV 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 over-expressing 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.




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