Plant Growth Regulation

, Volume 15, Issue 3, pp 271–279

Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts

  • Robert P. Walker
  • Wanda M. Waterworth
  • Michael H. Beale
  • Richard Hooley
Article

DOI: 10.1007/BF00029900

Cite this article as:
Walker, R.P., Waterworth, W.M., Beale, M.H. et al. Plant Growth Regul (1994) 15: 271. doi:10.1007/BF00029900

Abstract

Aleurone protoplasts of wild oat (Avena fatua L.), and subcellular fractions isolated from them, were photoaffinity labeled using the synthetic gibberellin (GA) derivative GA4-17-yl-1′-(1′-thia)propan-3′-ol-4-azido-5-[125I]iodosalicylate. Labeled polypeptides were identified by electrophoresis under denaturing conditions followed by autoradiography. GA-photoaffinity labeling of both intact protoplasts and isolated subcellular fractions led to the covalent attachment of the reagent to many polypeptides. A 50 kD polypeptide in the soluble fraction of homogenates of aleurone protoplasts GA-photoaffinity labeled in vivo showed specific binding. The biologically active GA1, GA4 and GA4-17-yl-1′(1′-thia)propan-3′-ol-4-azidosalicylate completed for binding whereas the biologically inactive GA8 and GA34 did not. The GA-photoaffinity labeling characteristics of this polypeptide suggested that it might interact specifically with biologically active GAs in vivo. Attempts to detect specific GA-binding in in vitro GA-photoaffinity labeling experiments met with only limited success perhaps indicating the labile nature of specific binding observed in vivo. The potential of GA-photoaffinity labeling for identifying GA-binding proteins in aleurone and other GA-responsive tissues is discussed.

Key words

α-amylase Avena fatua gibberellin-binding proteins gibberellins Gramineae photoaffinity wild oat 

Abbreviations

azido IAA =

5-azido-7-[3H]indole-3-acetic acid

azido NPA =

5′-azido-[3,6-3H]1-N-napthylpthalamic acid

BTP =

1,3-bis(Tris(hydroxymethyl)methylamino)-propane

GA4-O-ASA =

GA4-17-yl-1′-(1′-thia)propane-3′-ol-4-azidosalicylate

[125I]GA4-O-ASA =

GA4-17-yl-1′-(1′-thia)propan-3′-ol-4-azido-5-[125I]iodosalicylate

NPA =

1-Naphthylphthalmic acid

PAGE =

Polyacrylamide gel electrophoresis

PMSF =

phenylmethylsulfonyl fluoride

SDS =

Sodium dodecyl sulphate

TLCK =

L-1-Chloro-3-(4-tosylamido)-7-amino-2-heptanone-HCl

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Robert P. Walker
    • 1
  • Wanda M. Waterworth
    • 1
  • Michael H. Beale
    • 1
  • Richard Hooley
    • 1
  1. 1.Department of Agricultural SciencesUniversity of Bristol, 1 ACR Long Ashton, Long Ashton Research StationBristolUK
  2. 2.Department of Plant and Animal SciencesRobert Hill InstituteSheffieldUK
  3. 3.School of Biological Sciences, Department of Biochemistry and Molecular BiologyUniversity of ManchesterManchesterUK

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