Spatio-temporal profiling and degradation of α-amylase isozymes during barley seed germination

Authors

  • Kristian S. Bak-Jensen,

    1.  Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark
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    • These two authors contributed equally to this work

  • Sabrina Laugesen,

    1.  Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
    2.  Enzyme and Protein Chemistry, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark
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    • These two authors contributed equally to this work

  • Ole Østergaard,

    1.  Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark
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  • Christine Finnie,

    1.  Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark
    2.  Enzyme and Protein Chemistry, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark
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  • Peter Roepstorff,

    1.  Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark
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  • Birte Svensson

    1.  Carlsberg Laboratory, Department of Chemistry, Copenhagen Valby, Denmark
    2.  Enzyme and Protein Chemistry, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark
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Errata

This article is corrected by:

  1. Errata: Corrigendum Volume 274, Issue 15, 4009, Article first published online: 19 July 2007

B. Svensson
Fax: +45 45 886307
Tel: +45 45 252740
E-mail: bis@biocentrum.dtu.dk
P. Roepstorff, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Fax: +45 65 502467
Tel: +45 65 502404
E-mail: roe@bmb.sdu.dk

Abstract

Ten genes from two multigene families encode barley α-amylases. To gain insight into the occurrence and fate of individual isoforms during seed germination, the α-amylase repertoire was mapped by using a proteomics approach consisting of 2D gel electrophoresis, western blotting, and mass spectrometry. Mass spectrometric analysis confirmed that the 29 α-amylase positive 2D gel spots contained products of one (GenBank accession gi|113765) and two (gi|4699831 and gi|166985) genes encoding α-amylase 1 and 2, respectively, but lacked products from seven other genes. Eleven spots were identified only by immunostaining. Mass spectrometry identified 12 full-length forms and 12 fragments from the cultivar Barke. Products of both α-amylase 2 entries co-migrated in five full-length and one fragment spot. The α-amylase abundance and the number of fragments increased during germination. Assessing the fragment minimum chain length by peptide mass fingerprinting suggested that α-amylase 2 (gi|4699831) initially was cleaved just prior to domain B that protrudes from the (βα)8-barrel between β-strand 3 and α-helix 3, followed by cleavage on the C-terminal side of domain B and near the C-terminus. Only two shorter fragments were identified of the other α-amylase 2 (gi|166985). The 2D gels of dissected tissues showed α-amylase degradation to be confined to endosperm. In contrast, the aleurone layer contained essentially only full-length α-amylase forms. While only products of the above three genes appeared by germination also of 15 other barley cultivars, the cultivars had distinct repertoires of charge and molecular mass variant forms. These patterns appeared not to be correlated with malt quality.

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