2004 Annual Report 4.What were the most significant accomplishments this past year? This report serves to document research conducted under Specific Cooperative Agreement No. 58-5325-3-254 between ARS and a University. Additional research details can be found in the report for the parent CRIS 5325-43000-025-00D, Molecular Analysis of Environmental Effect on Wheat Grain Development, Productivity and Quality. Amyloplasts are organelles in non-green plant tissues that specialize in starch biosynthesis. Starch is a major component of the endosperm of wheat grain and is an essential carbohydrate reserve that supports germination and early seedling growth. However, the regulatory mechanisms of the starch biosynthetic pathway, as well as the general metabolic processes of amyloplasts, remain poorly characterized. To enhance our understanding of the functions and metabolic networks of this organelle, we initiated a survey of the amyloplast proteome using 2-D gel electrophoresis and mass spectrometry. Protein extracts of amyloplasts purified from endosperm of wheat grain collected 10 days post-anthesis were separated by 2-D gel electophoresis and the protein spots identified by tandem mass spectrometry. Over 200 polypeptides were identified in this amyloplast preparation. The purification method proved successful in that less than 10% of these proteins appear to be of non-plastid origin. Most of these proteins function in the biosynthesis of amino acids, starch, nucleotides, tetrapyrroles, lipids and isoprenoids or in protein folding and turnover. Interestingly, amino-acid biosynthesis is, with 40 enzymes identified (~20%), by far, the major process of amyloplasts at this stage of development. Thus, these organelles are not only the site of starch production, but provide products for other metabolic processes in the cell. These findings provide new insight into the function of amyloplasts in wheat endosperm and indicate that plastid resources are important in the biosynthetic pathways of other cellular compartments.