2008 Annual Report
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
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: http://springerlink.com/content/432379137526k10x/?p=ae33e422efb94387b5899d4f6e577a23&pi=29.
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.