Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Determine the molecular basis for the regulation of starch biosynthesis in wheat grain with the overall goal of increasing starch production, quality and extractability for ethanol production. Isolate amyloplasts from immature grain, identify and characterize surface and granule- associated proteins. Determine the amounts, activities and mode of regulation of enzymes regulating starch biosynthesis during grain fill. Correlate with starch granule size and composition in plants subjected to different environmental conditions or in transgenic plants expressing modified starch biosynthetic genes. This work will be integrated with ongoing proteomic and gene expression studies. Approach (from AD-416) New knowledge on the regulation of starch biosynthesis could facilitate the use of wheat starch as a feedstock for ethanol production. The goal of the present research is to elucidate the molecular basis of the regulation of starch biosynthesis using proteomics, immunology and enzymology. Amyloplasts will be isolated from immature endosperm. Amyloplast proteins will be separated and individual proteins will be identified by mass spectrometry and N-terminal sequencing. Antibodies will be prepared against amyloplast and starch granule proteins and used to determine the amounts of starch biosynthetic proteins accumulated during grain development. The activities of starch biosynthetic enzymes will be assayed in developing grains and the role of thioredoxin, a protein recently shown to function in regulating starch biosynthesis in amyloplasts, will be explored. Regulation of starch biosynthesis also will be investigated in grain produced under defined environmental regimens with altered starch granule populations and composition and in grain from transgenic plants in which starch biosynthesis has been modified. Documents SCA with UC-Berkeley. Fomerly 5325-43000-025-01S 3/13/06. Significant Activities that Support Special Target Populations This report serves to document research conducted under a specific cooperative agreement between ARS and the University of California. Additional research details can be found in the report for the parent CRIS, 5325-43000-026-00D, Molecular Analysis of Effects of Environment on Wheat Flour Quality and Allergenic Potential. Amyloplasts are specialized organelles that synthesize and store large amounts of starch in the endosperm of wheat grains. Starch is an essential reserve that supports germination and early seedling growth, a major component of wheat yield, a principal source of human nutrition, and a major commodity used for ethanol production. The role of amyloplasts in many other metabolic processes has not been recognized, however. To gain a better understanding of the biochemical processes taking place in amyloplasts, we identified 285 proteins in a proteomics analysis of amyloplasts isolated from developing wheat endosperm. To develop a detailed metabolic map for this organelle, subcellular origins and functions were assigned to these proteins based on a detailed evaluation of genomic and enzyme databases, organelle targeting predictions and scientific literature for each protein type. Of the 285 proteins, 179 were classified as amyloplast specific proteins. These proteins functioned in the following metabolic pathways: glucose metabolism, glycolysis, pentose phosphate, malate and citrate metabolism, starch biosynthesis, folate one-carbon metabolism, aspartate family, branched chain amino acid family, aromatic amino acid family, synthesis of cysteine and other sulfur compounds, synthesis of purines, pyrimidines, isoprenoids, porphyrins, vitamins and fatty acids, and enzymes involved in ion transport, electron transport, energy metabolism, free radical scavenger systems, and the ferredoxin/thioredoxin system. In addition, there were proteins involved in transcription, translation and protein processing, as well as proteins of unknown function. The remaining 96 proteins were classified as endomembrane, mitochondrial, vacuolar or nuclear components, proteins that might easily be included in an amyloplast preparation. These studies indicate that amyloplasts, like chloroplasts, play a major role in plant metabolism. This research indicates that it will be essential to investigate the regulation of the interconnected metabolic pathways within the amyloplast in order to understand the trade-off between producing wheat grains with high starch content or high protein content. The progress of the research was monitored by telephone conferences about once a week, and face to face conferences on a monthly basis. Annual reports are written with cooperator.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the University of California at Berkeley. Additional research details can be found in the report for the parent project 5325-43000-026-00D, "Molecular Analysis of Effects of Environment on Wheat Flour Quality". Amyloplasts are specialized organelles that synthesize and store the large amounts of starch in the endosperm of wheat grain. Starch, an essential reserve that supports germination and early seedling growth, is a major component of wheat yield and a valuable product for ethanol production. Knowledge of the regulation of starch biosynthesis is necessary for improving wheat productivity and starch quality. A mechanism particularly widespread in plants involves thioredoxin, a small disulfide protein that interacts with protein targets to regulate their activities. Fluorescence gel electrophoresis and affinity column
chromatography were used to identify thioredoxin target proteins in amyloplasts isolated from wheat endosperm. The 42 target proteins identified, 13 newly discovered, function in a range of processes, including biosynthesis of lipids, amino acids and nucleotides as well as starch metabolism. Among the proteins identified, all of the components of the thioredoxin regulatory system (ferredoxin, ferredoxin/thioredoxin reductase and thioredoxin), originally described for chloroplasts, were found in amyloplasts. The results suggest that amyloplasts have broader metabolic activities than previously recognized and that they participate in and coordinate the regulation of biosynthetic processes that take place outside as well as within the amyloplast.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under Specific Cooperative Agreement between ARS and the University of California at Berkeley. 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 plant organelles functional in the synthesis and storage of starch in heterotrophic plant tissues. Starch is a major component of the endosperm and is an essential carbohydrate reserve that supports germination and early seedling growth. Aside from pathways leading to the synthesis and breakdown of starch, relatively little is known about the biochemistry of amyloplasts. 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. Our study has led to the identification of 295 proteins that function in a range of processes, including carbohydrate metabolism, cytoskeleton/plastid division, energetics, nitrogen and sulfur metabolism, nucleic acid-related reactions, synthesis of various building blocks, protein-related reactions, transport, signaling, and stress/defense responses. The results highlight the role of the amyloplast as a starch- storing organelle that fulfills a spectrum of biosynthetic needs of the parent tissue. When compared with a recent analysis of whole endosperm, the current study demonstrates the advantage of using isolated organelles in proteomic studies.
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Progress 10/01/03 to 09/30/04
Outputs
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.
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