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United States Department of Agriculture

Agricultural Research Service

2009 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
Data collection was continued for systems study of soybean seed development, including the quantitation of inorganic micro- and macro-elements, and of glycerol-lipids, during the defined stages of seed development. Computational methods are being developed to integrate the various data-sets into a comprehensive model. Several transgenic soybean plants overexpressing O-acetylserine (thiol) lyase have been generated and are currently being characterized. An important regulatory feature in cysteine biosynthesis in soybean is the physical association of the two enzymes involved in cysteine biosynthesis, serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase, (OASS) to form the cysteine synthase complex. We have examined the oligomerization and energetics of formation of the soybean cysteine synthase complex (CSC) using analytical ultracentrifugation, isothermal titration calorimetry, and surface plasmon resonance spectroscopy. Biophysical examination of CSC oligomerization indicates that this macromolecular assembly from soybean consists of a single SAT trimer and three OASS dimers. Our results suggest a new model for the architecture of this regulatory complex as well as additional physiological functions for this macromolecular assembly in modulating plant cysteine biosynthesis. The combining ability of the allele controlling the mid-oleic acid trait and the alleles controlling the low linolenic acid trait were investigated. A backcrossed soybean line was developed that contained both of the oil traits. Investigations were also made into the genes controlling the low raffinose and stachyose trait that can increase the nutritional value of soybean meal. Two independent alleles of the same gene controlling the low raffinose and stachyose trait were identified. One of these alleles was further characterized and shown to have a neutral impact on seed germination. A reverse genetics strategy was used in discovery of one of these alleles as well as an allele controlling the accumulation of oleic acid. New research revealed a novel way to produce soybeans containing high-oleic acid oil. Other seed composition traits were also characterized at the molecular level, and allele-specific molecular marker assays were developed.

1. A method for detecting the location of the pyruvate dehydrogenase complex (PDC) in developing soybean seeds. The seeds are frozen and then cut into thin sections. The sections are pressed on to a membrane so that the proteins are transferred to the membrane surface ("printing"). Specific antibodies were directly labeled with a fluorescent dye, and then incubated with the seed prints. A specific type of microscope was then used to examine each section in order to determine the location of the PDC. The fluorescent images from the microscope were captured, and an image analysis program was used to reconstruct the intact seed. The image displays the location of the PDC within cells, tissues, and organs. The location of the PDC can be compared with the location of the storage lipids in seeds to see if there is a close proximity. By using this method, researchers can study the location of any two seed components for which there are specific detection methods. This will be useful for developing strategies to address modifications of seed composition for food, feed, and industrial applications.

2. A fractionation technique was developed to aid global protein analysis. In an effort to enhance the visualization and characterization of low-abundance proteins, a rapid method for fractionation of seed proteins was developed. Analysis of a total-protein samples from specific organs such as seeds, leaves, or tubers is difficult because the presence of a few very abundant proteins limits the ability to detect inconspicuous low-abundance proteins. The rapid and simple fractionation technique uses calcium to precipitate the soybean seed storage globulins, glycinin and beta-conglycinin. This method is capable of removing as much as 90% of these very abundant proteins, allowing detection of approximately 500 previously inconspicuous proteins. By using this method, researchers will be able to study low-abundance seed proteins that are important for control of nutritional quality, yield potential, and response to environmental stress. It will be particularly useful for the analysis of global changes in the low-abundance proteins prepared from genetically modified soybean cultivars.

3. A new approach to identify soybean lines with desirable seed composition phenotypes. Because soybean has a complicated and somewhat redundant genome, identifying desirable seed composition traits using traditional phenotyping methods is difficult. A reverse genetics strategy has been proposed to identify novel alleles of genes controlling important soybean seed traits. Results from current research indicated that this strategy can be successful for identifying unique sources of seed composition traits. New mutant alleles were identified for genes controlling accumulation of both the anti-nutritional factors in seed meal and the health-promoting oleic acid in the seed oil. In addition to the utility of these new alleles in breeding programs for those seed composition traits, the results provide a new avenue for discovery of other important alleles. The potential impact of this research is the ability to identify new alleles of genes that might not be apparent in traditional genetic screens relying on analysis of phenotype. These new alleles can be rapidly incorporated into other soybean varieties.

6.Technology Transfer

Number of New CRADAS1
Number of Invention Disclosures Submitted3
Number of New Patent Applications Filed1

Review Publications
Krishnan, H.B., Oehrle, N.W., Natarajan, S.S. 2009. A Rapid and Simple Procedure for the Depletion of Abundant Storage Proteins from Legume Seeds to Advance Proteome Analysis: A Case Study Using Glycine Max. Proteomics. 9:3174-3188.

Kumaran, S., Yi, H., Krishnan, H.B., Jez, J.M. 2009. Assembly of the Cysteine Synthase Complex and the Regulatory Role of Protein-Protein Interactions. Journal of Biological Chemistry. 284:10268-10275.

Dierking, E., Bilyeu, K.D. 2009. Raffinose and Stachyose Metabolism are not Required for Efficient Soybean Seed Germination. Journal of Plant Physiology. 166(12):1329-1335.

Dierking, E., Bilyeu, K.D. 2009. New Sources of Soybean Seed Meal and Oil Composition Traits Identified Through TILLING. Biomed Central (BMC) Plant Biology. 9(1):89.

Bilyeu, K.D., Ren, C., Nguyen, H.T., Herman, E.M., Sleper, D.A. 2009. Association of a Four Basepair Insertion in the P34 Gene with the Low Allergen Trait in Soybean. The Plant Genome. 2(2):141-148.

Smehilova, M., Galuszka, P., Bilyeu, K.D., Jaworek, P., Kowalska, M., Sebela, M., Sedlarova, M., English, J.T., Frebort, I. 2009. Subcellular Localization and Biochemical Comparison of Cytosolic and Secreted Cytokinin Dehydrogenase Enzymes from Maize. Journal of Experimental Botany. 60(9):2701-2712.

Ren, C., Bilyeu, K.D., Beuselinck, P.R. 2009. Composition, Vigor, and Proteome of Mature Soybean Seeds Developed under High Temperature. Crop Science. 49:1010-1022.

Gillman, J.D., Pantalone, V.R., Bilyeu, K.D. 2009. The Low Phytic Acid Phenotype in Soybean Line CX1834 is Due to Mutations in Two Homologues of the Maize Low Phytic Acid Gene. The Plant Genome. 2(2):179-190.

Last Modified: 11/29/2015
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