CaPUB1, a Hot Pepper U-box E3 Ubiquitin Ligase, Confers Enhanced Cold Stress Tolerance and Decreased Drought Stress Tolerance in Transgenic Rice (Oryza sativa L.)

  • Min, Hye Jo (Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University) ;
  • Jung, Ye Jin (Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University) ;
  • Kang, Bin Goo (ReSEAT Program, Korea Institute of Science and Technology Information) ;
  • Kim, Woo Taek (Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University)
  • Received : 2015.10.20
  • Accepted : 2015.11.11
  • Published : 2016.03.31


Abiotic stresses such as drought and low temperature critically restrict plant growth, reproduction, and productivity. Higher plants have developed various defense strategies against these unfavorable conditions. CaPUB1 (Capsicum annuum Putative U-box protein 1) is a hot pepper U-box E3 Ub ligase. Transgenic Arabidopsis plants that constitutively expressed CaPUB1 exhibited drought-sensitive phenotypes, suggesting that it functions as a negative regulator of the drought stress response. In this study, CaPUB1 was over-expressed in rice (Oryza sativa L.), and the phenotypic properties of transgenic rice plants were examined in terms of their drought and cold stress tolerance. Ubi:CaPUB1 T3 transgenic rice plants displayed phenotypes hypersensitive to dehydration, suggesting that its role in the negative regulation of drought stress response is conserved in dicot Arabidopsis and monocot rice plants. In contrast, Ubi:CaPUB1 progeny exhibited phenotypes markedly tolerant to prolonged low temperature ($4^{\circ}C$) treatment, compared to those of wild-type plants, as determined by survival rates, electrolyte leakage, and total chlorophyll content. Cold stress-induced marker genes, including DREB1A, DREB1B, DREB1C, and Cytochrome P450, were more up-regulated by cold treatment in Ubi:CaPUB1 plants than in wild-type plants. These results suggest that CaPUB1 serves as both a negative regulator of the drought stress response and a positive regulator of the cold stress response in transgenic rice plants. This raises the possibility that CaPUB1 participates in the cross-talk between drought and low-temperature signaling pathways.


Supported by : Rural Development Administration, National Research Foundation, Korea Institute of Science and Technology Information


  1. Yee, D., and Goring, D.R. (2009). The diversity of plant U-box E3 ubiquitin ligases: from upstream activators to downstream target substrates. J. Exp. Bot. 60, 1109-1121
  2. Zeng, L.R., Qu, S., Bordeos, A., Yang, C., Baraoidan, M., Yan, H., Xie, Q., Nahm, B.H., Leung, H., and Wang, G.L. (2004). Spotted leaf11, a negative regulator of plant cell death and defense, encodes a U-box/armadillo repeat protein endowed with E3 ubiquitin ligase activity. Plant Cell 16, 2795-2808
  3. Zeng, D-E., Hou, P., Xiao, F., and Liu, Y. (2014). Overexpressing a novel RING-H2 finger protein gene, OsRHP1, enhances drought and salt tolerance in rice (Oryza sativa L.). J. Plant Biol. 57, 357-365
  4. Zhang, Q., Jiang, N., Wang, G.L., Hong, Y., and Wang, Z. (2013). Advances in understanding cold sensing and the coldresponsive network in rice. Adv. Crop Sci. Tech. 1, 104
  5. Su, C.F., Wang, Y.C., Hsieh, T.H., Tseng, T.H., Lu, C.A., Tseng, T.H., and Yu, S.M. (2010). A novel MYBS3-dependent pathway confers cold tolerance in rice. Plant Physiol. 153, 145-158
  6. Thomashow, M.F. (2010). Molecular basis of plant cold acclimation: insights gained from studying the CBF cold response pathway. Plant Physiol. 154, 571-577
  7. Tripathi, R.D., Tripathi, P., Dwivedi, S., Dubey, S., Chatterjee, S., Chakrabarty, D., and Trivedi, P.K. (2012). Arsenomics: omics of arsenic metabolism in plants. Front. Physiol. 3, 275
  8. Trujillo, M., Ichimura, K., Casais, C., and Shirasu, K. (2008). Negative regulation of PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis. Curr. Biol. 18, 1396-1401
  9. Vierstra, R.D. (2009). The ubiquitin-26S proteasome system at the nexus of plant biology. Nat. Rev. Mol. Cell. Biol. 110, 385-397
  10. Wang, Q., Guan, Y., Wu, Y., Chen, H., Chen, F., and Chu, C. (2008). Overexpression of a rice OsDREB1F gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice. Plant Mol. Biol. 67, 589-602
  11. Wang, C., Wei, Q., Zhang, K., Wang, L., Liu, F., Zhao, L., Tan, Y., Di, C., Yan, H., Yu, J., et al. (2013). Down-regulation of OsSPX1 causes high sensitivity to cold and oxidative stresses in rice seedlings. PLoS One 8, e81849
  12. Xu, M., Li, L., Fan, Y., Wan, J., and Wang, L. (2011). ZmCBF3 overexpression improves tolerance to abiotic stress in transgenic rice (Oryza sativa) without yield penalty. Plant Cell Rep. 30, 1949-1957
  13. Yang, C.W., Gonzalez-Lamothe, R., Ewan, R.A., Rowland, O., Yoshioka, H., Shenton, M., Ye, H., O'Donnell, E., Jones, J.D., and Sadanandom, A. (2006). The E3 ubiquitin ligase activity of Arabidopsis PLANT U-BOX17 and its functional tobacco homolog ACRE276 are required for cell death and defense. Plant Cell 18, 1084-1098
  14. Agarwal, P.K., Agarwal, P., Reddy, M., and Sopory, S.K. (2006). Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep. 25, 1263-1274
  15. Benjamin, J.G., and Nielsen, D.C. (2006). Water deficit effects on root distribution of soybean, field pea and chickpea. Field Crops Res. 97, 248-253
  16. Bergler, J., and Hoth, S. (2011). Plant U-box armadillo repeat proteins AtPUB18 and AtPUB19 are involved in salt inhibition of germination in Arabidopsis. Plant Biol. (Stuttg) 13, 725-730
  17. Byun, M.Y., Lee, J., Cui, L.H., Kang, Y., Oh, T.K., P, H., Lee, H., and Kim, W.T. (2015). Constitutive expression of DaCBF7, an Antarctic vascular plant, Deschampsia antarctica CBF homolog, resulted in improved cold tolerance in transgenic rice plants. Plant Science 236, 61-74
  18. Cho, S.K., Chung, H.S., Ryu, M.Y., Park, M.J., Lee, M.M., Bahk, Y.Y., Kim, J., Pai, H.S., and Kim, W.T. (2006). Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-Box E3 ubiquitin ligase homolog. Plant Physiol. 142, 1664-1682
  19. Cho, S.K., Ryu, M.Y., Song, C., Kwak, J.M., and Kim, W.T. (2008). Arabidopsis PUB22 and PUB23 are homologous U-Box E3 ubiquitin ligases that play combinatory roles in response to drought stress. Plant Cell 20, 1899-1914
  20. Cho, S.K., Ryu, M.Y., Seo, D.H., Kang, B.G., and Kim, W.T. (2011). The Arabidopsis RING E3 ubiquitin ligase AtAIRP2 plays combinatory roles with AtAIRP1 in abscisic acid-mediated drought stress responses. Plant physiol. 157, 2240-2257
  21. Dubouzet, J.G., Sakuma, Y., Ito, Y., Kasuga, M., Dubouzet, E.G., Miura, S., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2003). OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and coldresponsive gene expression. Plant J. 33, 751-763
  22. Gilmour, S.J., Sebolt, A.M., Salazar, M.P., Everard, J.D., and Thomashow, M.F. (2000). Over-expression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol. 124, 1854-1865
  23. Han, M., Kim, C.-Y., Lee, J., Lee, S.-K., and Jeon, J.-S. (2014). OsWRKY42 represses OsMT1d and induces reactive oxygen species and leaf senescence in rice. Mol. Cells 37, 532-539
  24. Ito, Y., Katsura, K., Maruyama, K., Taji, T., Kobayashi, M., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2006). Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol. 47, 141-153
  25. Kim, S.I., and Tai, T.H. (2011). Evaluation of seedling cold tolerance in rice cultivars: a comparison of visual ratings and quantitative indicators of physiological changes. Euphytica 178, 437-447
  26. Kim, S.J., and Kim, W.T. (2013). Suppression of Arabidopsis RING E3 ubiquitin ligase AtATL78 increases tolerance to cold stress and decreases tolerance to drought stress. FEBS Lett. 587, 2584-2590
  27. Lee, J-H., and Kim, W.T. (2011). Regulation of abiotic stress signal transduction by E3 ubiquitin ligases in Arabidopsis. Mol. Cells 31, 201-208
  28. Lichtenthaler, H.K. (1987). Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350-382
  29. Liu, F., Xu, W., Wei, Q., Zhang, Z., Xing, Z., Tan, L., Di, C., Yao, D., Wang, C., Tan, Y., et al. (2010). Gene expression profiles deciphering rice phenotypic variation between Nipponbare (Japonica) and 93-11 (Indica) during oxidative stress. PLoS One 5, e8632
  30. Liu, Y.C., Wu, Y.R., Huang, X.H., Sun, J., and Xie, Q. (2011). AtPUB19, a U-box E3 ubiquitin ligase, negatively regulates abscisic acid and drought responses in Arabidopsis thaliana. Mol. Plant 4, 938-946
  31. Lyzenga, W.J., and Stone, S.L. (2012). Abiotic stress tolerance mediated by protein ubiquitination. J. Exp. Bot. 63, 599-616
  32. Mackill, D.J., and Lei, X. (1997). Genetic variation for traits related to temperature adaptation of rice cultivars. Crop Sci. 37, 1340-1346
  33. Park, J.J., Yi, J., Yoon, J., Cho, L.H., Ping, J., Jeong, H.J., Cho, S.K., Kim, W.T., and An, G. (2011). OsPUB15, an E3 ubiquitin ligase, functions to reduce cellular oxidative stress during seedling establishment. Plant J. 65, 194-205
  34. Praba, M.L., Cairns, J.E., Babu, R.C., and Lafitte, H.R. (2009). Identification of physiological traits underlying cultivar differences in drought tolerance in rice and wheat. J. Agron. Crop Sci. 195, 30-46
  35. Seo, D.H., Ryu, M.Y., Jammes, F., Hwang, J.H., Turek, M., Kang, B.G., Kwak, J.M., and Kim, W.T. (2012). Roles of four Arabidopsis U-box E3 ubiquitin ligases in negative regulation of abscisic acid-mediated drought stress responses. Plant Physiol. 160, 556-568
  36. Shaw, C.H. (1995). Introduction of cloning plasmids into Agrobacterium tumefaciens. In Plant gene transfer and expression protocols, Springer New York, 49, 33-37
  37. Shen, C., Li, D., He, R., Fang, Z., Xia, Y., Gao, J., Shen, H., and Cao, M. (2014). Comparative transcriptome analysis of RNAseq data for cold-tolerant and cold-sensitive rice genotypes under cold stress. J. Plant Biol. 56, 337-348
  38. Shinozaki, K., and Yamaguchi-Shinozaki, K. (2000). Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr. Opin. Plant Biol. 3, 217-223
  39. Stegmann, M., Anderson, R.G., Ichimura, K., Pecenkova, T., Reuter, P., Zarsky, V., McDowell, J.M., Shirasu, K., and Trujillo, M. (2012). The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis. Plant Cell 24, 4703-4716

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