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United States Department of Agriculture

Agricultural Research Service

2008 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 quantitative phospho-proteomic (phosphorylated proteins), ionomic (ion profiles), and directed transcriptomic (gene expression) analyses. Additionally, metabolite profiling studies were initiated along with detailed quantitative analysis of the rates of oil accumulation. Studies on the phosphorylation of pyruvate dehydrogenase by the intrinsic kinase were extended to include both knock-out and knock-down plants. A key regulatory feature in cysteine biosynthesis is the physical association of the two enzymes serine acetyltransferase and O-acetylserine sulfhydrylase, forming the cysteine synthase complex. These two enzymes have been purified from soybean, and attempts to crystallize them are in progress. Additionally, this multienzyme complex is being studied as a "molecular sensor" in a regulatory circuit that coordinates sulfur assimilation and modulates cysteine production. Research continued on identification of variant alleles for candidate genes controlling important seed composition traits. Tools were developed to select desired alleles to more efficiently develop new soybean varieties with improved seed composition traits. The phenotypes under investigation are: meal traits (accumulation of raffinose oligosaccharides and phytic acid, production of allergen proteins) and oil traits (low linolenic acid, high linolenic acid, elevated oleic acid). Studies on development and germination of low phytate seeds were continued. The second year of study on field growth analysis of high phytase transgenic soybean versus wild-type plants was completed. A study of the effects of heat stress on soybean seed quality and viability was continued, as was a study of the graft effects on expression of fatty acids in modified soybeans. The study of maternal influences on soybean seed size, and performance of reciprocal F1 hybrids was continued. (NP 302, Component 2A).

1. Promoter analysis of the genes encoding the components of the plastidial pyruvate dehydrogenase complex (PDC).

To alter oil:carbohydrate ratios in soybean seeds for crop improvement the pivotal role of the enzyme complex, pyruvate dehydrogenase (PDC), has to be understood and our ability to manipulate its activity has to be investigated. There are three components that comprise the soybean chloroplastic PDC, E1, E2, and E3. E1 can also be broken down into two subunits E1 alpha and E1 beta. Within the soybean genome the E1 alpha, E1 beta and E3 proteins are derived from the expression of unique single genes. There are two genes that encode two independent forms of the E2 protein component of the chloroplastic PDC. Using DNA primers we isolated the regions of these five genes that control their expression, i.e., the gene promoters. The promoters were cloned and sequenced and using analytical software we have identified regions of each that we predict are critical for the activity of each gene. Each promoter has been used in genetic constructs to place the promoter in front of a sequence encoding a reporter protein (a protein that can be visualized within plant cells) so that the activity of each promoter can be readily monitored. These promoter:reporter constructs are to be used for transformation of soybean embryogenic cultures. An understanding of how the promoters for each of the PDC component protein genes are controlled will ultimately lead to breakthroughs in directing soybean seed composition for value added traits. (NP 302 – Component 2A)

2. Molecular analysis of the soybean sulfur assimilation pathway. The basic knowledge of the soybean sulfur assimilation pathway was not sufficient to develop a molecular strategy for improvement of seed composition. A full-length cDNA clone for 5’-adenylylsulfate reductase (APS reductase) was isolated and characterized. The cDNA clone encodes an open reading frame of 1,414 bp, yielding a 52 kDa protein with an N-terminal transit peptide. Southern analysis revealed that APS reductase in soybean is encoded by small multigene family. Biochemical characterization of the purified recombinant protein demonstrated that clone encodes a functional APS reductase. Gene expression and protein activity were both highest in the early stages of seed development, declining thereafter with seed maturity. Both sulfur and phosphorus deprivation increased expression, while nitrogen starvation repressed APS reductase transcript and protein levels. These results comprise detailed biochemical information on the sulfur assimilation pathway of this nutritionally important legume leading towards better strategies for the improvement of the nutritional quality of soybean meal used in animal feeds. (NP 302 – Component 2A)

3. Development of a perfect molecular marker assay for low allergen soybeans.

Soybean seeds contain allergens that have undesirable consequences when soy products are consumed by people or animals. Previous research led to the discovery of germplasm accessions with low levels of the major allergen, P34. Current research led to the characterization of P34 gene sequences from different soybean accessions and the discovery of the mutation associated with the low allergen trait. Molecular marker assays were developed for direct selection of the low allergen trait. Development of low allergen soybean varieties can now be accelerated by the use of the molecular markers in breeding programs. (NP 302 – Component 2A)

4. Soybean cyst nematode infection affects fatty acid levels of soybean seeds.

The impact of soybean cyst nematode (SCN) on the quality of soybean lines with modified seed composition was unknown. A study using grafted soybean plants was conducted to determine if SCN resistant or susceptible rootstock influenced the composition of seeds from soybean line scions developed for unsaturated fatty acids (FA). In 2004 and 2005 grafted plants were transplanted into field plots verified as infested with SCN. Seeds were harvested at physiological maturity (R8) and analyzed for five unsaturated FA. Seed oleic acid of the mid-oleic line S03-1379-2 was significantly greater when grafted onto a SCN-resistant line than when grafted onto SCN-susceptible lines. The low linolenic trait of line IA3017 appeared insensitive to SCN presence. The level of seed linolenic acid was not different when grafted onto SCN-resistant or -susceptible lines. Thus, SCN can negatively impact seed quality of soybean lines developed for modified FA composition. (NP 302 – Component 2A)

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of the New MTAs (providing only)1
Number of Invention Disclosures Submitted1
Number of Non-Peer Reviewed Presentations and Proceedings10

Review Publications
Phartiyal, P., Kim, W., Cahoon, R., Jez, J.M., Krishnan, H.B. 2008. The role of 5'-adenylylsulfate reductase in the sulfur assimilation pathway of soybean: molecular cloning, kinetic characterization, and gene expression. Phytochemistry. 69(2):356-364.

Bilyeu, K.D., Zeng, P., Coello, P., Zhang, Z.J., Krishnan, H.B., Beuselinck, P.R., Polacco, J.C. 2008. Conversion of seed phytate to utilizable phosphorus in soybean seeds expressing a bacterial phytase. Plant Physiology. 146:468-477.

Kumaran, S., Francois, J.A., Krishnan, H.B., Jez, J. 2008. Regulatory Protein-Protein Interactions in Primary Metabolism: The Case of the Cysteine Synthase Complex. In: Khan, N.A., Singh, R.P., editors. Sulfur Assimilation and Abiotic Stress in Plants. Springer-Verlag, New York. p. 97-109.

Han, S., Kim, C., Lee, J., Park, J., Cho, S., Park, S., Kim, K., Krishnan, H.B., Kim, Y. 2008. Inactivation of pqq Genes of Enterobacter Intermedium 60-2G Reduces Antifungal Activity and Induction of Systemic Resistance. FEMS Microbiology Letters. 282:140-146.

Cooper, J., Till, B., Laport, R., Darlow, M., Kleffner, J., Jamai, A., El-Mellouki, T., Lui, S., Ritchie, R.D., Nielsen, N.C., Bilyeu, K.D., Meksem, K., Comai, L., Henikof, S. 2008. Tilling to detect induced mutations in soybean. Biomed Central (BMC) Plant Biology. 8:9.

Flores, T., Karpova, O., Su, X., Zeng, P., Bilyeu, K.D., Sleper, D.A., Nguyen, H.T., Zhang, Z.J. 2008. Silencing of Gm FAD3 gene by siRNA leads to low a-linolenic acids (18:3) of fad3 -mutant phenotype in soybean [(Glycine max (Merr.)]. Transgenic Research. Available:

Krishnan, H.B., Chronis, D. 2008. Functional nodFE genes are present in Sinorhizobium sp. strain MUS10, a symbiont of tropical legume Sesbania rostrata. Applied and Environmental Microbiology. 74:2921-2923.

Krishnan, H.B. 2008. Preparative Procedures Markedly Influence the Appearance and Structural Integrity of Protein Storage Vacuoles in Soybean Seeds. Journal of Agricultural and Food Chemistry. 56:2907-2912.

Last Modified: 11/27/2015
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