Proteomic analysis of wheat embryos with 2-DE and liquid-phase chromatography (ProteomeLab PF-2D) - A wider perspective of the proteome

Cereal embryos are a model system to study desiccation tolerance due to their ability to survive extreme water loss during late embryogenesis. To identify proteins accumulating in mature embryos which can be used as potential markers for dehydration tolerance, we compared the embryo proteome from two durum wheat genotypes (Triticum durum Desf.), Mahmoudi (salt and drought sensitive) and Om Rabia3 (salt and drought tolerant). Total protein extracts from wheat embryos were analyzed by using conventional 2-DE and ProteomeLab PF-2D. Analysis using different pH ranges showed that a larger number of fractions were solved by LC, than by conventional 2-DE at extreme technical pHs (pH 4.0-5.0 and pH 6.5-8.0). In contrast, at intermediate pHs (pH 5.0-6.5), resolution was better in 2-DE gels. The two techniques were used in parallel to analyze total protein extracts from embryos of the two wheat varieties. Several proteins belonging to the seed storage family, LEA-type/heat shock proteins, enzyme metabolism and radical scavengers were identified by analysis of trypsin digested peptides via mass spectrometry. These proteins accumulate in different amounts in embryos of tolerant and sensitive wheat varieties. The differences in expression pattern were further validated by enzyme activity, western blotting analysis and correlated with their corresponding mRNA expression by RT-PCR analyses for the corresponding protein. We suggest that the differential expression pattern could be used as a basis for a biochemical screen of tolerance/sensitivity to drought and salt stress in wheat embryos and germplasm.