Expression of EuNOD-ARP1 Encoding Auxin-repressed Protein Homolog Is Upregulated by Auxin and Localized to the Fixation Zone in Root Nodules of Elaeagnus umbellata

  • Kim, Ho Bang (School of Biological Sciences, College of Natural Sciences, Seoul National University) ;
  • Lee, Hyoungseok (School of Biological Sciences, College of Natural Sciences, Seoul National University) ;
  • Oh, Chang Jae (School of Biological Sciences, College of Natural Sciences, Seoul National University) ;
  • Lee, Nam Houn (School of Biological Sciences, College of Natural Sciences, Seoul National University) ;
  • An, Chung Sun (School of Biological Sciences, College of Natural Sciences, Seoul National University)
  • Received : 2006.11.01
  • Accepted : 2006.12.12
  • Published : 2007.02.28


Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.


Auxin-repressed Protein Gene;Cellular Localization;Elaeagnus umbellata;Frankia;In Situ Hybridization;Root Nodule


Supported by : Korean Ministry of Science and Technology


  1. Berry, A. M., Kahn, R. K. S., and Booth, M. C. (1989) Identification of indole compounds secreted by Frankia HFPArI3 in defined culture medium. Plant and Soil 118, 205−209
  2. Datta, C. and Basu, P. S. (1998) Production of indole acetic acid in root nodules and culture by a Rhizobium species from root nodules of the fodder legume Melilotus alba DESR. Acta Biotechnologica 18, 53−62
  3. Kim, H. B. and An, C. S. (1999) Isolation and characterization of a cDNA clone encoding polyubiquitin from the root nodule of Elaeagnus umbellata. Can. J. Bot. 77, 1270−1278
  4. Reddy, A. S. N. and Poovaiah, B. W. (1990) Molecular cloning and sequencing of a cDNA for an auxin-repressed mRNA: correlation between fruit growth and repression of the auxinregulated gene. Plant Mol. Biol. 14, 127−136
  5. Stafstrom, J. P., Ripley, B. D., Devitt, M. L., and Drake, B. (1998) Dormancy-associated gene expression in pea axillary buds. Planta 205, 547−552
  6. Stevens, G. A. and Berry, A. M. (1988) Cytokinin secretion by Frankia sp. HFPArI3 in defined medium. Plant Physiol. 87, 15−16
  7. Thimann, K. V. (1936) On the physiology of the formation of nodules on legume roots. Proc. Natl. Acad. Sci. USA 22, 511− 514
  8. Wall, L. G. (2000) The actinorhizal symbiosis. J. Plant Growth Regul. 19, 167−182
  9. Wheeler, C. T., Henson, I. E., and McLaughlin, M. E. (1979) Hormones in plants bearing actinomycete root nodules. Bot. Gaz. 140S, 52−57
  10. Franche, C., Laplaze, L., Duhoux, E., and Bogusz, D. (1998) Actinorhizal symbioses: recent advances in plant molecular and genetic transformation studies. Crit. Rev. Plant Sci. 17, 1−28
  11. McKhann, H. I. and Hirsch, A. M. (1993) In situ localization of specific mRNAs in plant tissues; in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. (eds.), pp. 179−205, CRC Press, Boca Raton
  12. Rinhardt, D., Mandel, T., and Kuhlemeier, C. (2000) Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell 12, 507−518
  13. An, C. S., Kim, H. B., Lee, S. H., Jang, H. J., Oh, C. J., et al. (2005) Gene expression in the root nodules of Elaeagnus umbellata; in Biological Nitrogen Fixation, Sustainable Agriculture and the Environment, Wang, Y. P., Lin, M., Tian, Z. X., and Newton, W. E. (eds.), pp. 207−208, Springer, Dordrecht
  14. Cox, K. H. and Goldberg, R. B. (1988) Analysis of plant gene expression; in Plant Molecular Biology: A Practical Approach, Shaw, C. H. (ed.), pp. 1−35, IRL press, Oxford
  15. Hagen, G. and Guilfoyle, T. (2002) Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Mol. Biol. 49, 373−385
  16. Jeong, Y. M., Mun, J. H., Lee, I., Woo, J. C., Hong, C. B., et al. (2006) Distinct roles of the first introns on the expression of Arabidopsis profilin gene family members. Plant Physiol. 140, 196−209
  17. Abel, S. and Theologis, A. (1996) Early genes and auxin action. Plant Physiol. 111, 9−17
  18. Hirsch, A. M., Fang, Y., Asad, S., and Kapulnik, Y. (1997) The role of phytohormones in plant-microbe symbioses. Plant Soil 194, 171−184
  19. Park, S. and Han, K. H. (2003) An auxin-repressed gene (RpARP) from black locust (Robinia pseudoacacia) is posttranscriptionally regulated and negatively associated with shoot elongation. Tree Physiol. 23, 815–823
  20. Hirsch, A. M. and Fang, Y. (1994) Plant hormones and nodulation: what's the connection? Plant Mol. Biol. 26, 5−9
  21. Steiner, C., Bauer, J., Amrhein, N., and Bucher, M. (2003) Two novel genes are differentially expressed during early germination of the male gametophyte of Nicotiana tabacum. Biochim. Biophys. Acta. 1625, 123–133
  22. Davis, S. J. and Vierstra, R. D. (1998) Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol. Biol. 36, 521−528
  23. Kim, H. B., Lee, S. H., and An, C. S. (1999) Isolation and characterization of a cDNA clone encoding asparagine synthetase from root nodules of Elaeagnus umbellata. Plant Sci. 149, 85− 94
  24. de Billy, F. Grosjean, C., May, S., Bennett, M., and Cullimore, J. V. (2001) Expression studies on AUX1-like genes in Medicago truncatula suggest that auxin is required at two steps in early nodule development. Mol. Plant-Microbe Interact. 14, 267−277
  25. Ferguson, B. J. and Mathesius, U. (2003) Signaling interactions during nodule development. J. Plant Growth Regul. 22, 47−72
  26. Hirsch, A. M. (1992) Developmental biology of legume nodulation. New Phytol. 122, 211−237
  27. Gualtieri, G. and Bisseling, T. (2000) The evolution of nodulation. Plant Mol. Biol. 42, 181−194
  28. Hwang, E. W., Kim, K. A., Park, S. C., Jeong, M. J., Byun, M. O., et al. (2005) Expression profiles of hot pepper (Capsicum annuum) genes under cold stress conditions. J. Biosci. 30, 101− 111
  29. Fedorova, E. E., Zhiznevskaya, G. Y., Kalibernaya, Z. V., Artemenko, E. N., Izmailov, S. F., et al. (2000) IAA metabolism during development of symbiosis between Phaseolus vulgaris and Rhizobium phaseoli. Russian J. Plant Physiol. 47, 203− 206
  30. Mangiarotti, G., Chung, S., Zuker, C., and Lodish, H. F. (1981) Selection and analysis of cloned developmentally-regulated Dictyostelium discoideum genes by hybridization-competition. Nucleic Acids Res. 9, 947−963
  31. Kim, H. B. and An, C. S. (2002) Differential expression patterns of an acidic chitinase and a basic chitinase in the root nodule of Elaeagnus umbellata. Mol. Plant-Microbe Interact. 15, 209−215
  32. Altschul, S. F., Gish, W., Miller, W., Meyers, E. W., and Lipman, D. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403−410
  33. Kim, S. C., Ku, C. D., Park, M. C., Kim, C. H., Song, S. D., et al. (1993) Isolation of symbiotic Frankia EuIK1 strain from root nodule of Elaeagnus umbellata. Kor. J. Bot. 36, 177−182
  34. Mansour, S. R. (1994) Production of growth hormones in Casuarina cunninghamiana root nodules induced by Frankia strain HFPCgI4. Protoplasma 183, 126−130