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

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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.

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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

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Author notes

  1. Robert P. Walker

    Present address: Department of Plant and Animal Sciences, Robert Hill Institute, P.O. Box 601, S10 2UQ, Sheffield, UK

  2. Wanda M. Waterworth

    Present address: School of Biological Sciences, Department of Biochemistry and Molecular Biology, University of Manchester, Stopford Building, Oxford Road, M13 9PT, Manchester, UK

Authors and Affiliations

  1. Department of Agricultural Sciences, University of Bristol, 1 ACR Long Ashton, Long Ashton Research Station, BS18 9AF, Bristol, UK

    Robert P. Walker, Wanda M. Waterworth, Michael H. Beale & Richard Hooley

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  1. Robert P. Walker
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  2. Wanda M. Waterworth
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  3. Michael H. Beale
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  4. Richard Hooley
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Walker, R.P., Waterworth, W.M., Beale, M.H. et al. Gibberellin-photoaffinity labeling of wild oat (Avena fatua L.) aleurone protoplasts. Plant Growth Regul 15, 271–279 (1994). https://doi.org/10.1007/BF00029900

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  • DOI: https://doi.org/10.1007/BF00029900

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