TY - JOUR

T1 - Spatio-temporal profiling and degradation of alpha-amylase isozymes during barley seed germination

AU - Bak-Jensen, Kristian Sass

AU - Laugesen, Sabrina

AU - Østergaard, Ole

AU - Finnie, Christine

AU - Roepstorff, Peter

AU - Svensson, Birte

PY - 2007

Y1 - 2007

N2 - Ten genes from two multigene families encode barley alpha-amylases. To gain insight into the occurrence and fate of individual isoforms during seed germination, the alpha-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 alpha-amylase positive 2D gel spots contained products of one (GenBank accession gi|113765) and two (gi|4699831 and gi|166985) genes encoding alpha-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 alpha-amylase 2 entries co-migrated in five full-length and one fragment spot. The alpha-amylase abundance and the number of fragments increased during germination. Assessing the fragment minimum chain length by peptide mass fingerprinting suggested that alpha-amylase 2 (gi|4699831) initially was cleaved just prior to domain B that protrudes from the (betaalpha)(8)-barrel between beta-strand 3 and alpha-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 alpha-amylase 2 (gi|166985). The 2D gels of dissected tissues showed alpha-amylase degradation to be confined to endosperm. In contrast, the aleurone layer contained essentially only full-length alpha-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.

AB - Ten genes from two multigene families encode barley alpha-amylases. To gain insight into the occurrence and fate of individual isoforms during seed germination, the alpha-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 alpha-amylase positive 2D gel spots contained products of one (GenBank accession gi|113765) and two (gi|4699831 and gi|166985) genes encoding alpha-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 alpha-amylase 2 entries co-migrated in five full-length and one fragment spot. The alpha-amylase abundance and the number of fragments increased during germination. Assessing the fragment minimum chain length by peptide mass fingerprinting suggested that alpha-amylase 2 (gi|4699831) initially was cleaved just prior to domain B that protrudes from the (betaalpha)(8)-barrel between beta-strand 3 and alpha-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 alpha-amylase 2 (gi|166985). The 2D gels of dissected tissues showed alpha-amylase degradation to be confined to endosperm. In contrast, the aleurone layer contained essentially only full-length alpha-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.

KW - Amino Acid Sequence

KW - Blotting, Western

KW - Electrophoresis, Gel, Two-Dimensional

KW - Germination/physiology

KW - Gibberellins/pharmacology

KW - Hordeum/enzymology

KW - Hydrogen-Ion Concentration

KW - Isoenzymes/metabolism

KW - Molecular Sequence Data

KW - Multigene Family

KW - Seeds/enzymology

KW - Sequence Alignment

KW - Tandem Mass Spectrometry

KW - Tissue Distribution

KW - alpha-Amylases/metabolism

U2 - 10.1111/j.1742-4658.2007.05790.x

DO - 10.1111/j.1742-4658.2007.05790.x

M3 - Journal article

C2 - 17437525

VL - 274

SP - 2552

EP - 2565

JO - F E B S Journal

JF - F E B S Journal

SN - 1742-464X

IS - 10

ER -