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Functional analysis of a leucine aminopeptidase fromSolanum tuberosum L.

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Abstract

A protein encoded by a potato cDNA homologous to a leucine aminopeptidase (LAP) from bovine lens (Hildmann et al. 1992) was expressed inEscherichia coli cells and biochemically characterized by hydrolysis of leucinep-nitroanilide. Activity was highest under alkaline conditions with an optimum at about pH 10. Maximal activities were measured at 65° C. Apart from leucinep-nitroanilide the enzyme could also efficiently hydrolyze thep-nitroanilides of arginine and methionine. Complete inhibition of the enzyme was achieved by incubating bacterial extracts with bestatin and EDTA, which classifies the enzyme as a metalloprotease belonging to the same group as the homohexameric LAPs from mammals. Protein blots showed low constitutive expression of the LAP in all organs of potato plants: buds, flowers, tubers, roots and leaves. An increase in steady-state protein that was parallelled by an increase in total LAP activity was observed in leaf extracts after supplying jasmonic acid via the petioles. Plants containing the cDNA in antisense orientation behind the constitutive Cauliflower Mosaic Virus 35S promoter showed nearly complete reduction of the corresponding mRNA in leaves. However, in these plants LAP activities were only decreased by about 20% as compared to non-transgenic potato plants, while after feeding with jasmonic acid the activity of transgenic plants was reduced to about 5% of that of non-transgenic plants also induced by jasmonic acid. There was no phenotypic difference between wild-type and LAP antisense plants.

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Abbreviations

CaMV:

cauliflower mosaic virus

CathInh:

cathepsin D inhibitor

JA:

jasmonic acid

LAP:

leucine aminopeptidase

Leu-NA:

leucinep-nitroanilide

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Author notes

  1. Salomé Prat

    Present address: Centro de Investigacion y Desarrollo, Jordi Girona, 18-26, E-08034, Barcelona, Spain

Authors and Affiliations

  1. Institut für Genbiologische Forschung GmbH, Ihnestr. 63, D-14195, Berlin, Germany

    Salomé Prat & Lothar Willmitzer

  2. Institut für Pflanzengenetik und Kulturpflanzenforschung, Abt. Molekulare Pflanzenphysiologie, Corrensstr. 3, D-06466, Gatersleben, Germany

    Karin Herbers

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  1. Karin Herbers
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  2. Salomé Prat
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Additional information

We are grateful to Birgit Burose, Birgit Hidde and Regina Breitfeld for taking care of the greenhouse plants and Josef Bergstein for the photographic work. We are also indebted to Jessyca Dietze for the potato transformations (Institut für Genbiologische Forschung) and Dr. habil. Uwe Sonnewald for advice in microscopy (Institut für Pflanzengenetik und Kulturpflanzenforschung). We thank Drs. Holger Hesse, Jörg Riesmeier and Bernd Müller-Röber (Institut für Genbiologische Forschung) for helpful comments on the manuscript. The nucleotide sequence of leucine aminopeptidase cDNA is registered in the EMBL Data Library; accession number: X 77015

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Herbers, K., Prat, S. & Willmitzer, L. Functional analysis of a leucine aminopeptidase fromSolanum tuberosum L.. Planta 194, 230–240 (1994). https://doi.org/10.1007/BF01101682

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  • DOI: https://doi.org/10.1007/BF01101682

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