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Identification and localization of an active cutinase in the pollen of Brassica napus L.

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Abstract

Polyclonal antiserum and monoclonal antibodies raised to a purified cutinase from Fusarium solani f. sp. pisi have been used to identify an active cutinase in the pollen of Brassica napus. These antibodies recognized a polypeptide with an estimated molecular weight of 22kDa — a molecular weight indentical to that of the Fusarium cutinase — and localized this polypeptide to the intine of the pollen wall. Enzyme assays on the renatured 22kDa polypeptide after electroelution from a preparative SDS-PAGE gel revealed the polypeptide to be an enzyme capable of catalysing the hydrolysis of tritiated apple cutin and the synthetic substrate p-nitrophenyl butyrate. The molecular weight, immunological properties and substrate specificity of the Brassica cutinase suggest that this enzyme resembles more closely fungal cutinases than it does the cutinase from the pollen of Nasturtium (Tropaeolum majus) — the only angiosperm cutinase so far characterized (Maiti et al., 1979, Arch. Biochem. Biophys. 196, 412–423). These differences between the pollen cutinases from two members of the Dicotyledoneae are unexpected and predict a diversity of this class of pollen enzyme within the angiosperms.

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Abbreviations

PNB:

p-nitrophenyl butyrate

PNBase:

PNB esterase

PNP:

p-nitrophenyl palmitate

ELISA:

enzyme-linked immunosorbent assay

FITC:

fluorescein isothiocyanate

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Authors and Affiliations

  1. Department of Plant Sciences, University of Oxford, South Parks Road, OX1 3RB, Oxford, UK

    Simon J. Hiscock, Frances M. Dewey & Hugh G. Dickinson

  2. Biochemistry and Physiology Department, Rothamsted Experimental Station, AL5 2JQ, Harpenden, Herts., UK

    Julian O. D. Coleman

Authors
  1. Simon J. Hiscock
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  2. Frances M. Dewey
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  3. Julian O. D. Coleman
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  4. Hugh G. Dickinson
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Additional information

We are indebted to Professor P.E. Kolattukudy for the gift of the Fusarium cutinase. We thank Dr. C. Thornton for his help with antibody detection and informative discussions, Miss M. Davis for technical assistance and Mrs A. Rogers for typing the manuscript. This work was supported through an Agricultural and Food Research Council studentship to S.J.H.

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Hiscock, S.J., Dewey, F.M., Coleman, J.O.D. et al. Identification and localization of an active cutinase in the pollen of Brassica napus L.. Planta 193, 377–384 (1994). https://doi.org/10.1007/BF00201816

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  • DOI: https://doi.org/10.1007/BF00201816

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