Proteome Approach to the Characterisation of Protein Composition in the Developing and Mature Wheat-grain Endosperm

Abstract

A proteomic approach was used for the analysis and characterisation of wheat-grain endosperm proteins at a developmental stage (17 days post-anthesis) of the wheat cultivar Wyuna. This involved the extraction, solubilisation and subsequent two-dimensional separation of total wheat-grain endosperm proteins. About 1300 polypeptides were resolved. Separation in the first dimension was performed using isoelectric focusing across two pH ranges: pH 4·0–7·0 and pH 6·0–11·0. Proteins were blotted to PVDF, excised and characterised using conventional N -terminal Edman degradation microsequencing. Sequences were submitted to SWISS-PROT and TrEMBL databases via FASTA algorithm. In total, 321 proteins were submitted for post-separation characterisation; 177 (55%) proteins were identified from database matches, 55 (17%) proteins were not matched and 89 (28%) proteins did not yield any N -terminal sequence data. Protein expression within the endosperm at both the developmental (17 days post-anthesis) and mature (45 days post-anthesis) stages of growth were then compared using Melanie II 2-D PAGE image analysis software. This approach has provided an insight into the complex nature of endosperm protein heterogeneity and provides a basis for the future examination of the effects of environmental variation on protein composition.