2007 Annual Report 1a.Objectives (from AD-416) The overall aim of this research project is to evaluate the quality and safety of transgenic soybeans using proteomics technologies. We will determine whether the expression of transgenes in soybeans results in an unusual accumulation of non-target proteins in the seed. The data from these studies would be useful to measure and compare the deviation of protein variation within transgenic soybeans. To achieve this goal, we propose the following two key objectives: Objective 1. Measure the natural variation of seed proteins in a wide range of soybean germplasm using proteomics and establish a baseline for subsequent estimation of collateral effects that may be associated with transgenes. Objective 2. Determine the potential collateral effects in protein expression resulting from tissue culture, transformation protocols, and transgenic events in soybean. 1b.Approach (from AD-416) The experimental approach is to compare and characterize protein variation of a group of twenty-seven soybean genotypes that will include wild, Glycine soja and cultivated Glycine max germplasm accessions. Also, variation in seed protein levels in four different seed samples from plants derived via tissue culture and non-tissue culture methods will be compared. Proteins will be extracted from seeds using the modified TCA/acetone method and separated using 2D-PAGE, analyzed using image analysis and in those cases when unique protein spots appear in any treatment, further characterization using MALDI-TOF-MS will be undertaken. 3.Progress Report Developed collaborations for obtaining seeds from different locations (Brookings, SD and Clemson, SC). Grew soybean seeds at Beltsville, MD, at two different sowing dates for studying natural variation of proteins. Completed 2-dimensional-polyacrylamide gel electrophoresis (2D-PAGE) analysis of all soybean genotypes and scanned the 2D-gels. The 2D-PAGE profiles of the medium Maturity Group genotypes from greenhouse experiments in Beltsville, MD were compared with the same genotypes grown under field conditions. Initiated protein analysis using 2D-PAGE and identification of different types of storage, allergen and anti-nutritional proteins using mass spectrometry, MALDI-TOF-MS (Matrix-assisted Laser Desorption/Ionization Time of Flight Mass Spectrometryand LC-MS (Liquid Chromatography - Mass Spectrometry). 4.Accomplishments Proteomic analysis of major seed allergens of soybeans. Soybean ranks among the eight most significant food allergens. In order to understand the variation of allergen proteins that may occur in genetically modified soybeans, it is critical to determine the natural variation in protein composition among various soybean genotypes. We have characterized profiles of major soybean allergen proteins, Gly m Bd 60K, Gly m Bd 30K and Gly m Bd 28K in seeds of 16 different soybean genotypes. The comparative data of the allergen proteins generated in this study provide a baseline for comparison with new transgenic soybean lines in order to determine if they exceed the threshold level we observed in non-transgenic soybean genotypes. The accomplishment aligns with the National Program 302, Component 3- Plant Biotechnology Risk Assessment; Problem Statement 3A- Improving and Assessing Genetic Engineering Technology. Characterization of storage proteins in soybeans using proteomics and genomics. Data on the natural variation of major soybean storage proteins will be crucial to improve soybean protein composition and its quality. We have reported significant variations in the profiles of glycinin and ß-conglycinin subunits in several soybean genotypes (U.S. commercial and wild, high protein, and genotypes from outside the U.S.). In addition, we observed higher levels of specific amino acids (cysteine and arginine) and total protein in Nepalese soybean as compared to the US commercial cultivars. This novel finding makes Nepalese soybean a potential germplasm resource for the improvement of protein content and quality and also as a preferable alternative for swine and poultry feed. These data will be of vital importance for soy food and biotech industries to develop new value-added soybeans. The accomplishment aligns with the National Program 302, Component 3- Plant Biotechnology Risk Assessment; Problem Statement 3A- Improving and Assessing Genetic Engineering Technology. Proteomic and genomic analysis of anti-nutritional proteins in soybeans. Kunitz trypsin inhibitor (KTI) is one of the anti-nutritional proteins in soybean that inhibits the gastrointestinal enzymes of animals. We investigated the natural variation of KTI in an array of wild and cultivated soybean genotypes and also studied the variation in the genes that code for KTI. We observed that wild soybeans have more KTI proteins than cultivated soybeans. Moreover, our studies of DNA variation revealed remarkable differences in the DNA patterns of the KTI genes between wild and cultivated soybeans. Theses results establish the range of KTI concentrations that naturally occur in soybean and can be used by scientists and industry as the basis of comparison to assess KTI in transgenic soybean that may be developed in the future. KTI levels in transgenic soybean that exceed those that naturally occur in soybean germplasm would be undesirable. The accomplishment aligns with the National Program 302, Component 3- Plant Biotechnology Risk Assessment; Problem Statement 3A- Improving and Assessing Genetic Engineering Technology. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 7 Review Publications Krishnan, H.B., Natarajan, S.S., Mahmoud, A.A., Bennett, J.O., Krishnan, A.H., Prasad, B.N. 2006. Assessment of indigenous nepalese soybean as a potential germplasm resource for improvement of protein in north american cultivars. Plant Science. 54:5489-5497. Natarajan, S.S., Xu, C., Bae, H., Caperna, T.J., Garrett, W.M. 2006. Proteomic analysis of allergen and antinutritional proteins in wild and cultivated soybean. Journal of Plant Biochemistry and Biotechnology. 15: 103-108. Natarajan, S.S., Xu, C., Bae, H., Bailey, B.A. 2006. Proteomic and genomic characterization of kunitz trypsin inhibitors in wild and cultivated soybean genotypes. Journal of Plant Physiology. 164(6):756-763. Kang, S., Jeong, H., Lee, E., Natarajan, S.S. 2007. Characterization of a lipoate-protein ligase A gene of rice (Oryza sativa L.). Gene. 393(1):53-61. Xu, C., Garrett, W.M., Sullivas, J., Caperna, T.J., Natarajan, S.S. 2006. Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry. Phytochemistry. 67: 2431-2440. Dhar, A.K., Lakshman, D.K., Natarajan, Allnutt, F.C.T. and van Beek, N.A.M. 2007. Functional characterization of putative promotor elements from infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp and in insect and fish cell lines. Virus Research. 127(1):1-8. Natarajan, S.S., Xu, C., Bae, H., Bailey, B.A., Cregan, P.B., Caperna, T.J., Garrett, W.M., Luthria, D.L. 2007. Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes. Plant Physiology and Biochemistry. 45(6):436-444.