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United States Department of Agriculture

Agricultural Research Service

2010 Annual Report

1a.Objectives (from AD-416)
1. The long-term goal of this Objective is to develop soybean seeds that have increased oil levels derived at the expense of non-structural carbohydrates. 2. Molecular biology techniques will be used to improve the nutritional quality of soybean seed proteins. 3. To develop the molecular basis for modification of the fatty acid components of soybean oil and anti-nutritional components in soybean meal to use in accelerated breeding programs. 4. Identify effects on key performance determinants of crop seed quality resulting from modified seed composition using traditional or non-traditional genetic methods.

1b.Approach (from AD-416)
To reach the overriding objective of the modification of soybean seed composition for food, feed, and industrial uses requires a team approach that spans the complete range from basic biochemical assessment of possible target sites to the evaluation of the agronomic properties and value of modified soybeans. Basic biochemical approaches will be used to assess the effect of manipulating the expression of a key enzyme complex that is at the interface of carbon partitioning into oil or carbohydrates. A proteomic approach to the analysis of soybean seed development will allow for the discovery of other key regulatory events that offer possibilities for manipulation. Transgenic approaches will be used to modify the protein content and constitution of the soybean seed such that the nutritional quality as feed can be improved. A similar approach combined with classical molecular genetic approaches to plant breeding will be directed at altering the fatty acid components and anti-nutritional compounds of soybean seed to improve not only the nutritive value of the seed but also the health aspects of soybean consumption. A classical physiological approach serves to address the efficacy of the targeted modifications as they relate to agronomic concerns of yield, seed quality, and storage.

3.Progress Report
The extant systems biology platform for analysis of soybean seed development is based upon the results of proteomic plus ionomic profiling studies. It has been further elaborated by adding results from lipidomic profiling. These results include quantitative analysis of the molecular species of the major glyco- and phospholipids plus the neutral glycerides. Pattern analyses were conducted to identify coincident changes. The model was dissected, and exhaustively focused on pyruvate dehydrogenase. Levels of the transcripts, proteins, and catalytic activities were quantified for the subunits of both plastidial and mitochondrial complexes, along with levels of regulatory phosphorylation of the latter. It was determined that the current computational model of seed development is not sufficiently robust to accommodate the additional data. Preliminary studies have included application of principal component analysis, general linear modeling, and multidimensional scaling. These results will be useful in designing modified composition seeds for food, feed, and industrial applications. Sulfur is incorporated into the amino acids cysteine and methionine as well as into small antioxidants. While soybeans are an excellent source of protein, they do not contain high levels of the sulfur-containing amino acids. Several attempts have been made to increase the levels of sulfur-containing amino acids. A commonly used approach involves expression of heterologous seed proteins rich in codons for sulfur-containing amino acids. However, the overall increase in the sulfur-containing amino acids using this approach is not nutritionally sufficient for monogastric animals. We have generated transgenic plants expressing a cytosolic O-acetylserine sulfhydrylase gene. These plants accumulate increased levels of cysteine in seeds. Thus, the prospects for increasing the sulfur-containing amino acid content of soybean appear promising. A thorough understanding of the fundamental steps regulating thiol metabolism in soybean along with the information from the soybean genome will facilitate improvement of seed protein quality. Research continued to address the molecular genetic basis for soybean seed composition traits which enhance the nutritional aspects of the oil and protein meal. Variant alleles in genes controlling different seed composition traits were identified and used in genetic experiments to confirm their impact on the seed phenotypes. Research focused on altered fatty acid profiles in soybean oil such as the low linolenic acid trait, reduced saturates trait, high oleic acid trait, and high linolenic acid trait. In addition several soybean meal traits were investigated including reducing the anti-nutritional factors raffinose and stachyose, P34 allergen, phytate, and lipoxygenases. Different variant alleles controlling the oil, meal, or both sets of traits were assembled in different combinations to evaluate their collateral effects on seed composition.

1. A new method was developed to determine the location and organization of oil and protein in soybean seeds. The method involves freezing the seeds so that they can be cut into thin slices, and then studied with a microscope. Different methods to detect oil and protein were compared, but none of the methods tested had a large advantage over the others. The fact that different methods gave essentially the same result is a good indicator of the effectiveness of the method. The method was further used to see if there were any differences in the location of several different proteins. Surprisingly, none of the individual proteins was restricted to only a single location in the beans. Instead, all of the proteins tested were spread uniformly throughout the seeds. These results can have important implications for developing either biotechnology or classical breeding-based methods for modifying soybean seed chemical composition.

2. A protein believed to be important in controlling the nutritional composition of soybean seeds was isolated and studied for the first time. The gene for this protein was also isolated and studied. A method was developed for analyzing the protein in the laboratory. The chemical properties of the protein were measured and the results were compared with studies of a similar protein from different sources. Information gained from studying the protein and gene will potentially be useful in developing a biotechnology-based strategy to alter the chemical composition of soybean seeds for feed, food, and industrial applications.

3. Development of high oleic acid soybeans using conventional breeding techniques. Soybean oil can be improved for the nutritional value in human foods and for industrial biodiesel use through altering the fatty acid composition. Vegetable oils with high oleic acid contents are desirable for the health benefits of the monounsaturated fatty acids, which have recently made olive and canola oils very popular. High oleic acid content also dramatically improves oxidative and temperature stability of the oil, and improves cold flow properties in diesel engines. Identifying and combining mutant alleles of two soybean fatty acid desaturase genes resulted in high oleic acid-soybean oil. Scientists of the Plant Genetics Research Unit in Columbia, MO developed technology to directly select the genes conferring the desired fatty acid profile, thus accelerating the rate at which new soybean varieties containing this important trait can be made available to producers. Development of high oleic acid soybean varieties has the potential to deliver the highly desired nutritional and industrial qualities in a commodity oil. Wide adoption by the food industry is therefore likely, and will seamlessly improve the nutritional quality of the American diet and make the U.S. soybean industry more competitive.

4. Development of a method for selecting soybeans with reduced levels of a major protein allergen. Allergenic proteins can reduce the nutritional qualities of soybeans used in food and feed applications. Scientists of the Plant Genetic Research Unit in Columbia, MO discovered that some soybean lines lack a major allergen because of a specific mutation in the gene encoding the protein. A method was developed to directly select for the mutant gene. Adoption of this method will accelerate the rate at which new soybean varieties containing this trait can be evaluated for improved nutritional properties and human health.

5. Discovery of the two genes responsible for the soybean low phytate trait. Much of the phosphate in soybean seeds is present as a component of phytic acid, an anti-nutritional factor which impedes the bio-availability of inorganic elements in food and feed. Studies of the molecular genetic basis of a soybean line containing the low phytate trait by scientists in the Plant Genetics Research Unit in Columbia, MO, resulted in the discovery of two mutant genes that lower phytate by acting in combination. An additional low phytate soybean line was also characterized and found to contain a dramatic mutation in one of the genes. A method was developed to directly select for any/all of the mutations discovered. Application of this method should simplify development and release of soybean varieties with improved nutritional quality as a trait important for feed and food applications. Such varieties would have a significant impact on the livestock finishing industry.

Review Publications
Hajduch, M., Hearne, L.B., Miernyk, J.A., Casteel, J.E., Joshi, T., Agrawal, G.K., Song, Z., Zhou, M., Xu, D., Thalen, J.J. 2010. Systems Analysis of Seed Filling in Arabidopsis Thaliana: Using General Linear Modeling to Assess Concordance of Transcript and Protein Expression. Plant Physiology. 152:2078-2087.

Huang, Y., Houston, N.L., Tovar-Mendez, A., Stevenson, S.E., Miernyk, J.A., Randall, D.D., Thelen, J.J. 2010. A Quantitative Mass Spectrometry-based Approach for Identifying Protein Kinase-Clients and Quantifying Kinase Activity. Analytical Biochemistry. 402(2010):69-76.

Schechter, L.M., Guenther, J., Olcay, E.A., Jang, S., Krishnan, H.B. 2010. Sinorhizobium fredii USDA257 Translocates NopP into Vigna unguiculata Root Nodules. Applied and Environmental Microbiology. 76(11):3758-3761.

Lenis, J., Gillman, J.D., Lee, J., Shannon, J., Bilyeu, K.D. 2010. Soybean Seed Lipoxygenase Genes: Molecular Characterization and Development of Molecular Marker Assays. Theoretical and Applied Genetics. 120(6):1139-1149.

Last Modified: 11/29/2015
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