Nitrogen (N) serves as a macronutrient that is essential to plant growth and development, and significantly influences storage protein and starch biosyntheses and, ultimately, grain yield and quality. In this study, we performed the first comparative proteomic analysis of developing wheat grains under high-N conditions using 2D-DIGE and tandem mass spectrometry. High-N fertilizer application caused significant increases in ear number, ear grain number, and grain yield. 2D-DIGE identified 142 differentially accumulated proteins (DAPs) during grain development in the elite Chinese bread wheat cultivar Zhongmai 175, of which 132 (93%) were identified by MALDI-TOF/TOF-MS, representing 92 unique proteins. These proteins are involved mainly in energy, N and protein metabolism, carbon metabolism, and starch biosynthesis. Subcellular localization prediction and fluorescence confocal microscopic analysis showed that the DAPs identified were localized mainly in the cytosol and chloroplast. Principal component analysis (PCA) revealed a greater proteomic difference among grain developmental periods than between the high-N and control groups. Protein-protein interaction analysis highlighted a complex network centered around enzymes involved in energy, N and protein metabolism, and starch biosynthesis. Six key DAP genes showed expression patterns consistent with their protein accumulation trends during grain development. A putative metabolic pathway was proposed, with synergistic regulatory networks of grain storage protein and starch biosyntheses in response to high-N application.
