Partial purification and kinetic characterization of an auxin-binding activity in cytoplasmic extract of rape seed (Brassica napus. L.) hypocotyls

  • Kirsten Jørgensen
  • Søren V. S. Nielsen
Chapter

Abstract

This paper reports the partial purification by cation exchange chromatography of an auxin-binding activity from etiolated Brassica napus hypocotyls. The activity has a well defined pH optimum at pH 7.2 and is highly specific towards indole-3-acetic acid (IAA) with a Kd of 1.7-2 × 10-8 M at this pH. The Ki for 2,4-dichloro-phenoxyacetic acid (2,4-D) was determined to be 10-5 M, while the activity was not inhibited by 1-naphthaleneacetic acid (1-NAA). The auxin-binding activity showed a broader range of specificity at pH 7.8 where 2,4-D, 1-NAA, 2-NAA, and D-tryptophan were inhibitory to IAA-binding. In addition the Kd for IAA was slightly higher being 5 x 10-8 M at this pH. Affinity column chromatography at pH 7.8 of active fractions and of crude extract resulted in preparations exhibiting a triplet with molecular weights of 53, 58 and 62 kD on SDS-PAGE, the most prominent band being at 58 kD. At pH 7.2 additional bands with molecular weights of 42, 45 and 47 kD were seen.

Key words

affinity chromatography auxin-binding activity Brassica napus IAA proteins 
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Bailey HM, Barker RDJ, Libbenga KR, van der Linde PCG, Mennes AM and Elliott MC (1985) Auxin binding site in tobacco cells. Biologia Plantarum 27: 105–109CrossRefGoogle Scholar
  2. 2.
    Bilang J, Macdonald H, King PJ and Sturm A (1993) A soluble auxin-binding protein from Hyoscyamus muticus is a glutathione S-transferase. Plant Physiol 102: 29–34PubMedCrossRefGoogle Scholar
  3. 3.
    Celis JE, Madsen P, Rasmussen HH, Leffers H, Honoré B, Gesser B, Dejgârd K, Olsen E, Magnusson N, Kiel J, Celis A, Lauridsen JB, Basse B, Ratz GP, Andersen AH, Walbum E, Brandstrup B, Pedersen PS, Brandt NJ, Puype M, Van Damme M J and Vanderkerckhove J (1991) A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: Towards an integrated approach to the study of the cell proliferation, differentiation and skin deseases. Electrophoresis 12: 802–872PubMedCrossRefGoogle Scholar
  4. 4.
    Herber B, Ulbrich B and Jacobsen H-J (1988) Modulation of soluble auxin-binding proteins in soybean cell suspensions. Plant Cell Reports 7: 178–181CrossRefGoogle Scholar
  5. 5.
    Jacobsen H-J (1991) Somatic embryogenesis in seed legumes: The possible role of soluble auxin receptors. Israel J Botany 40: 139–143Google Scholar
  6. 6.
    Jones AM (1990) Do we have the auxin receptor yet? Physiologia Plantarum 80: 154–158CrossRefGoogle Scholar
  7. 7.
    Kikuchi M, Imaseki H and Sakai S (1989) Modulation of gene expression in isolated nuclei by auxin-binding proteins. Plant Cell Physiol 30: 765–773Google Scholar
  8. 8.
    Laemmli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227: 680–685PubMedCrossRefGoogle Scholar
  9. 9.
    Libbenga KR and Mennes AM (1987) Hormone binding and its role in hormone action. In: Davies PJ (ed) Plant hormones and their role in plant growth and development, pp 194-221. Kluwer Academic Publishers, Dordrecht.Google Scholar
  10. 10.
    Napier RM and Venis MA (1991) From auxin-binding protein to plant hormone receptor. TIBS 16: 72–75PubMedGoogle Scholar
  11. 11.
    Oostrom H, Van Loopick-Detmers MA and Libbenga KR (1975) A high affinity receptor for indoleacetic acid in cultured tobacco pith explants. FEBS Letters 59: 194–197CrossRefGoogle Scholar
  12. 12.
    Oostrom H, Kulescha Z, Van Vliet TB and Libbenga KR (1980) Characterization of a cytoplasmic auxin receptor from tobacco-pith callus. Planta 149: 44–47CrossRefGoogle Scholar
  13. 13.
    Polyacrylamide gel electrophoresis. Published by Pharmacia, Laboratory Separation Division. 1984 Uppsala, Sweden.Google Scholar
  14. 14.
    Poulsen GB and Nielsen SVS (1989) Regeneration of plants from hypocotyl protoplasts of rapeseed (Brassica napus L. var Oleifera) cultivars. Plant Cell, Tissue and Organ Culture 17: 153–158CrossRefGoogle Scholar
  15. 15.
    Prasad PV and Jones AM (1991) Putative receptor for the plant growth hormone auxin identified and characterized by anti-idiotypic antibodies. Proc Natl Acad Sci 88: 5479–5483PubMedCrossRefGoogle Scholar
  16. 16.
    Roy P and Biswas BB (1977) A receptor for indoleacetic acid from plant chromatin and its role in transcription. Biochem Biophys Res Comm 74: 1597–1606PubMedCrossRefGoogle Scholar
  17. 17.
    Sakai S (1992) Regulatory functions of soluble auxin-binding proteins. International Review of Cytology 135:239–267PubMedCrossRefGoogle Scholar
  18. 18.
    Sakai S (1985) Auxin-binding protein in etiolated mung bean seedlings: Purification and properties of auxin-binding protein-II. Plant Cell Physiol 26: 185–192Google Scholar
  19. 19.
    Sakai S and Hanagata T (1983) Purification of an auxin-binding protein from etiolated mung bean seedlings by affinity chromatography. Plant Cell Physiol 24: 685–693Google Scholar
  20. 20.
    Schleif RF and Wensink (1981) Practical methods in molecular biology. 62–88. Manor P (ed) Springer Verlag, New York, Heidelberg, Berlin.Google Scholar
  21. 21.
    Van Der Linde PCG, Bouman H, Mennes AM and Libbenga KR (1984) A soluble auxin-binding protein from cultured tobacco tissues stimulates RNA synthesis in vitro. Planta 160: 145–157Google Scholar
  22. 22.
    Van Der Zaal EJ, Mennes AM and Libbenga KR (1987) Auxininduced rapid changes in translatable mRNAs in tobacco cell suspensions. Planta 172: 514–519CrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Kirsten Jørgensen
    • 1
  • Søren V. S. Nielsen
    • 1
  1. 1.The Biotechnology Group, Danish Institute of Plant and Soil ScienceLyngbyDenmark

Personalised recommendations