Kristian Sass Bak-Jensen

Aspects of the barley seed proteome during development and germination

Analysis of the water-soluble barley seed proteome has led to the identification of proteins by MS in the major spots on two-dimensional gels covering the pI ranges 4-7 and 6-11. This provides the basis for in-depth studies of proteome changes during seed development and germination, tissue-specific proteomes, cultivar differences related to quality parameters, analysis of the genetic basis for spot variations and targeted investigations of specific proteins.

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

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 2Du2003gel electrophoresis, western blotting, and mass spectrometry. Mass spectrometric analysis confirmed that the 29 α‐amylase positive 2Du2003gel spots contained products of one (GenBank accession gi|113765) and two (gi|4699831 and gi|166985) genes encoding α‐amylaseu20031 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‐amylaseu20032 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‐amylaseu20032 (gi|4699831) initially was cleaved just prior to domain B that protrudes from the (βα)8‐barrel between β‐strandu20033 and α‐helixu20033, followed by cleavage on the C‐terminal side of domainu2003B and near the C‐terminus. Only two shorter fragments were identified of the other α‐amylaseu20032 (gi|166985). The 2Du2003gels 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.

Integration of the barley genetic and seed proteome maps for chromosome 1H, 2H, 3H, 5H and 7H

Two-dimensional gel electrophoresis was used to screen spring barley cultivars for differences in seed protein profiles. In parallel, 72 microsatellite (simple sequence repeat (SSR)) markers and 11 malting quality parameters were analysed for each cultivar. Over 60 protein spots displayed cultivar variation, including peroxidases, serpins and proteins with unknown functions. Cultivars were clustered based on the spot variation matrix. Cultivars with superior malting quality grouped together, indicating malting quality to be more closely correlated with seed proteomes than with SSR profiles. Mass spectrometry showed that some spot variations were caused by amino acid differences encoded by single nucleotide polymorphisms (SNPs). Coding SNPs were validated by mass spectrometry, expressed sequence tag and 2D gel data. Coding SNPs can alter function of affected proteins and may thus represent a link between cultivar traits, proteome and genome. Proteome analysis of doubled haploid lines derived from a cross between a malting (Scarlett) and a feed cultivar (Meltan) enabled genetic localisation of protein phenotypes represented by 48 spot variations, involving e.g. peroxidases, serpins, α-amylase/trypsin inhibitors, peroxiredoxin and a small heat shock protein, in relation to markers on the chromosome map.

Engineering of Barley α-Amylase

Abstract Protein engineering of barley α-amylase addressed the roles of Ca2+ in activity and inhibition by barley α-amylase/subtilisin inhibitor (BASI), multiple attach in polysaccharide hydrolysis, secondary starch binding sites, and BASI hot spots in AMY2 recognition. AMY1/AMY2 isozyme chimeras faciliatated assignment of function to specific regions of the structure. An AMY1 fusion with starch binding domain and AMY1 mutants in the substrate binding cleft gave degree of multiple attack of 0.9–3.3, compared to 1.9 for wild-type. About 40% of the secondary attacks, succeeding the initial endo-attack, produced DP5-10 maltooligosaccharides in similar proportion for all enzyme variants, whereas shorter products, comprising about 25%, varied depending on the mutation. Secondary binding sites were important in both multiple attack and starch granule hydrolysis. Surface plasmon resonance and inhibition analyses indicated the importance of fully hydrated Ca2+ at the AMY2/BASI interface to strengthen the complex. Engineering of intermolecular contacts in BASI modulated the affinity for AMY2 and the target enzyme specificity.