2011 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 overridging 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.
The extant systems biology platform for analysis of soybean seed development has been heavily lipid-centric, aimed toward developing greater insight into the regulation of carbon incorporation into oil. This emphasis has been at the expense of developing a higher-level understanding of the diversion of both carbon and nitrogen into the seed storage proteins (SSP). In order to rectify this discordance, part of the research emphasis has been shifted towards an understanding of amino acid synthesis, breakdown, and incorporation into SSP. The primary initial target has been glutamine synthetase, an enzyme known to have important regulatory roles in non-seed organs. The extant computational model of seed development does sufficiently accommodate the data from analysis of nitrogen metabolism. An expanded model is being developed which includes the 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.
Soybeans are an abundant source of protein. Unfortunately, soybean proteins contain relatively low amounts of two important amino acids (methionine and cysteine) both of which are absolutely essential to both human and animal nutrition. The need to solve this conundrum has led to development of strategies to increase the amount of these two amino acids. Previous strategies for increasing levels of the sulfur-containing amino acids by expression of heterologous methionine-rich proteins met with limited success. As a result, we have proposed an alternative approach involving overexpression of a cytoplasmic form of soybean O-acetylserine sulfhydrylase (OASS), a key enzyme in the synthesis of cysteine. Soybean plants overexpressing OASS contain elevated amounts of the Bowman-Birk protease inhibitor, a cysteine-rich protein. Additionally, amino acid analysis reveals our OASS overproducing transgenic soybean seeds have a 74% increase in protein-cysteine content. The information obtained from this study will help researchers to genetically manipulate sulfur-assimilatory enzyme expression levels in order to improve the overall nutritional quality of soybean seed proteins.
The molecular genetic basis for control of soybean seed composition remain obscure, and must be clarified in order to modify the nutritional and functional aspects of the oil and meal components. Current research has focused on determining the gene combinations necessary to achieve varying levels of linoleic and linolenic acids in high oleic acid oil-background plants. Additionally, several meal traits were investigated including levels of the anti-nutritional raffinosaccharides, the P34 allergen, phytate, lipoxygenases, and total trypsin inhibitor activities. Also important are the interactions among these components and levels of total seed protein. Variant alleles controlling the oil-, meal-, or both-sets of traits were combined in order to evaluate collateral effects. Among the results was a better understanding of the gene combinations necessary to alter soybean fatty acid profiles so that they resemble that of olive oil.
Novel glycinin subunits contribute to high seed protein soybean accessions. Soybeans grown in the United States presently have an average protein content of 40%, however shifts in production to the upper Midwest and continued breeding emphasis on yield rather than quality threaten to lower this value. Lowering the protein content of U.S. soybeans would reduce their competitive value in the global market place and reduce the price that the farmer receives. The development of high-protein soybean lines will facilitate cost-efficient production of seed meal containing at least 48% protein, maintaining and possibly improving market values for U.S. soybeans. Because limited information was available to define the biochemical and genetic mechanisms that regulate seed protein concentration, an ARS scientist in Columbia, MO has developed analytical methods to determine if the high protein lines preferentially accumulate specific protein species. Results using high-resolution two-dimensional gel electrophoresis indicate that the most prominent difference that characterized high-protein accessions was attributed to the 11S seed storage proteins, the glycinins. These data will facilitate efforts to increase both quantity and quality of seed protein, which will increase utilization of soybeans by the food and feed industries.
An alternate means developed to produce soybeans containing high oleic acid oil. Soybeans are a commodity crop that provides a major component of fats and oils in the American diet. Typically around 20% of soybean seed oil is oleic acid, however an increase in oleic acid would be desirable because of the resultant health benefits. High oleic acid also increases oxidative stability and extends the utility of soybean oil at high temperatures. Genetically changing soybean oil from 20% to greater than 55% oleic acid would allow wider use of soybean oil in food, pharmaceuticals, cosmetics, biodiesel and industrial lubricant applications. An ARS scientist in Columbia, MO, developed a genetic strategy for producing soybean varieties with high oleic acid oil using genes from the soybean germplasm collection. This alternative method resulted in oil with over 80% oleic acid content, and is an important complement to existing high oleic acid-soybeans produced using a biotechnology strategy. Soybeans produced in this way will improve the competitiveness and value of U.S. soybeans, directly impacting the soybean and food industries.
Soybean storage protein accumulation is not regulated by glutamine synthase. While glutamine synthatase (GS) is known to be a key regulatory enzyme for nitrogen metabolism and thus protein synthesis in leaves, relatively little is known about the role it plays in seed development and storage protein accumulation. Results developed by an ARS scientist in Columbia, MO from quantitative analyses of GS transcripts, catalytic activity, levels of substrates and products, and organ, tissue, and cellular localization were added to the systems model of soybean seed development that is used to predict how seed composition is altered by environmental conditions or genetic manipulation through breeding. There is a surprising discordance between GS and the abundant seed storage proteins, indicating that this particular enzyme is unlikely to have a regulatory role in seed development and would not be a good target for genetic or biotech-based manipulations aimed at increasing seed protein levels, a key target to improve soybean meal for live stock consumption.
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Bilyeu, K.D., Gillman, J.D., Leroy, A. 2011. Novel FAD3 mutant allele combinations produce soybeans containing 1% linolenic acid in the seed oil. Crop Science. 51:259-264.
Krishnan, H.B., Kerley, M.S., Allee, G.L., Jang, S., Kim, W., Fu, C.J. 2010. Maize 27 kDa gamma zein is a potential allergen for early weaned pigs. Journal of Agricultural and Food Chemistry. 58(12):7323-7328.
Miernyk, J.A., Pret'Ova, A., Olmedilla, A., Obert, B., Hajduch, M. 2010. Using proteomics to study sexual reproduction in angiosperms. Sexual Plant Reproduction. 24:9-22.
Pham, A., Lee, J., Shannon, G., Bilyeu, K.D. 2010. Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait. Biomed Central (BMC) Plant Biology. 10:195.
Hirani, T.A., Tovar-Mendez, A., Miernyk, J.A., Randall, D.D. 2011. Asp295 stabilizes the active-site loop structure of pyruvate dehydrogenase, facilitating phosphorylation of Ser292 by pyruvate dehydrogenase-kinase. Enzyme Research. 2011:1-13. Article 939068. Available: http://www.sage-hindawi.com/journals/er/.
Yang, S., Tang, F., Gao, M., Krishnan, H.B., Zhu, H. 2010. R gene-controlled host specificity in the legume-rhizobia symbiosis. Proceedings of the National Academy of Sciences. 107(43):18735-18740.
Lewandowska-Gnatowsk, E., Johnston, M.L., Antoine, W., Szczegielniak, J., Muszynska, G., Miernyk, J.A. 2011. Using multiplex-staining to study changes in the maize leaf phosphoproteome in response to mechanical wounding. Phytochemistry. 72:1285-1292.
Krishnan, H.B., Jang, S., Kim, W., Kerley, M.S., Oliver, M.J., Trick, H.N. 2011. Biofortification of soybean meal: immunological properties of the 27kDa gamma-zein. Journal of Agricultural and Food Chemistry. 59:1223-1228.
Krishnan, H.B., Natarajan, S.S., Bennett, J.O., Sicher Jr, R.C. 2011. Protein and metabolite composition of xylem sap from field-grown soybeans (Glycine max). Planta. 233:921-931.
Krishnan, H.B., Nelson, R.L. 2011. Proteomic analysis of high protein soybean (Glycine max) accessions demonstrates the contribution of novel glycinin subunits. Journal of Agricultural and Food Chemistry. 59:2432-2439.
Miernyk, J.A., Klubicova, K., Bercak, M., Danchenko, M., Skultey, L., Rashydov, N.M., Berezhna, V.V., Hajduch, M. 2010. Agricultural recovery of a formerly contaminated area: Establishment of a high-resolution quantitative protein map of mature flax seeds harvested from the remediated chernobyl area. Phytochemistry. 72:1308-1315.