Progress 01/15/02 to 04/05/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The research to be undertaken falls under National Program 302- Plant Biological and Molecular Processes and addresses component 3a (Improving and Assessing Genetic Engineering Technology) as described in the National Program Action Plan. Soybean is the second most valuable crop in the U.S. with an estimated annual value of $17.7 billion and provides an inexpensive source of protein for humans and animals. Genetic modifications of several nutritional deficiencies in soybean would make soybean a more valuable and nutritious protein source. However, there are consumer concerns and issues about the biosafety of transgenic soybeans, which have caused a loss of export income to the U.S. Genetic and transgenic approaches have been used to eliminate the Kunitz trypsin inhibitor, reduce the
expression of P34, and introduce other transgenes into soybean, but very little information is available regarding collateral or unintended effects of these transgenic modifications. Therefore, the goals of the research project are to select and apply suitable proteomics protocols to quantify any differences between transgenic vs. non-transgenic soybean. In this research, collaboration with scientists active in the production of transgenic soybean containing a variety of novel genes is anticipated. One way for the U.S. to remain competitive in the world soybean market is through the production of high quality soybean using transgenic technology. In 2003, 55% of the 76 million hectares of soybean planted globally were transgenic, up from 51% in 2002. Transgenic soybean produced approximately $1 billion of income through savings in the production costs associated with the use of Roundup Ready soybean. The study of the biosafety of transgenic soybean is important in order to
maintain consumer acceptability of U.S. soybeans and for enhancing the soybean export market. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2003) 1) Develop proteomic protocols for soybean seed protein characterization. 2) Compare four protein extraction methods and select an efficient method to solubilize both abundant and less abundant soybean seed proteins. Year 2 (FY 2004) 1) Compare natural variation of seed proteins in sixteen different soybeans genotypes using proteomics. Year 3 (FY 2005) 1) Define the range of natural variation of seed proteins in a small group of cultivated and wild soybean genotypes. Year 4 (FY 2006) 1) Define the range of natural variation of allergen proteins in cultivated and wild soybean genotypes. 2) Compare protein variation of anti-nutritional proteins in cultivated and wild soybean genotypes. A new Project Plan, 1275-21000-223-00D, was submitted and approved entitled Evaluation of the Quality and
Safety of Transgenic Soybeans with the following milestones: Year 1 (FY 2007) 1) Evaluate protein profiles of early, medium, and late Maturity Group soybean genotypes by 2D-PAGE using samples from three locations, Brookings, SD, Beltsville, MD, and Clemson soybean genotypes planted at two different sowing times in the field. 2) Determine protein variation due to different environmental conditions using greenhouse experiments in Beltsville, MD with medium Maturity Group genotypes. Year 2 (FY 2008) 1) Complete the characterization and identification of proteins of early Maturity Group genotypes using MALDI- TOF and LC-MS and compare the protein variation among the genotypes. 2) Assess the quantity of the seed proteins of early Maturity Group genotypes using Image Quant and complete statistical analysis. 3) Develop database using the results on natural variations of proteins. Year 3 (FY 2009) 1) Characterize and identify proteins of medium and late Maturity Group soybean genotypes using
MALDI-TOF and LC-MS and compare the protein variation among the genotypes. 2) Evaluate protein profiles of medium Maturity Group genotypes (greenhouse and field) using 2D-PAGE. Year 4 (FY 2010) 1) Characterize and identify proteins of medium Maturity Group genotypes (GH &Field) using MALDI-TOF, LC-MS and database searching. 2) Complete statistical analysis of medium Maturity Group genotypes protein variation. 3) Complete protein variation database development 4) Compare and characterize protein profiles of transgenic and non- transgenic soybean seeds by 2D-PAGE and MALDI-TOF-MS. Year 5 (FY 2011) 1) Compare 2D-PAGE analysis of early, medium, and late Maturity Group genotypes at two different sowing times. 2) Characterize and identify proteins using MALDI-TOF, LC-MS and database searching. 3) Compare quantity of the proteins using Image Quant of transgenic and non-transgenic and complete statistical analysis. 4) Complete the statistical analysis of protein variation in transgenic and
non-transgenic soybean seed proteins. 4a List the single most significant research accomplishment during FY 2006. Analysis of Major Allergens and Anti-Nutritional Proteins in Cultivated and Wild Soybean: Comparative analysis of soybean proteins: We have successfully completed a comparative analysis of major allergen and anti- nutritional protein profiles both in wild and cultivated soybean genotypes. To understand the variation of allergenic proteins that could be expected to occur in genetically modified (GM) soybeans, it is important to determine the natural variation of protein composition both in wild and cultivated soybeans that have been used in conventional soybean breeding programs. We combined two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry (MS) tools to separate and identify all allergen and anti-nutritional proteins extracted from both wild and cultivated soybean seeds. We observed that the wild soybean has even more abundant
allergenic and anti-nutritional proteins as well as a higher number of such polypeptides than the cultivated soybeans we examined. These results can be used to determine if the amounts of allergen and anti-nutritional proteins accumulated in GM soybeans exceed the level of these same proteins that occur naturally in non-GM soybean varieties. This accomplishment aligns with National Program 302, Component 3a - Improving and Assessing Genetic Engineering Technology. 5. Describe the major accomplishments to date and their predicted or actual impact. Using integrated proteomic technologies, we have successfully identified most of the storage, allergen and anti-nutritional proteins of both wild and cultivated soybean seeds. These data will serve as a baseline for comparison to help evaluate the quality and safety of transgenic soybean by determining whether the expression of transgenes in soybean results in an unusual accumulation of non-target proteins in the seed. Proteins were
extracted from soybean seeds of wild and cultivated genotypes analyzed, identified using a device called a mass spectrometer. These data demonstrate the range of variation in abundance of proteins in the seed of wild and cultivated soybean germplasm and contributes to baseline data for scientists wishing to evaluate the quality and safety of transgenic plants. These data can be used by scientists as a baseline to which GM soybean can be compared to assure that unwanted levels of allergens and anti-nutritional proteins are not present in soybean cultivars that are released to the grower. This accomplishment aligns with National Program 302, Component 3a - Improving and Assessing Genetic Engineering Technology.
Impacts
(N/A)
Publications
- Bock, C., Parker, P., Gottwald, T.R. 2005. The change in quantity of bacteria of xanthomonas axonopodis pv citri dispersed down wind from canker-infected grapefruit trees during a wind/rain event. International Citrus Canker and Huanglongbing Workshop, Orlando, FL, Nov. 2005. P13,p.78.
- Natarajan, S.S., Cregan, P.B., Xu, C., Caperna, T.J., Garrett, W.M., Sullivan, J. 2005. Proteomic analysis of beta-gonglycinin and glycinin in different soybean genotypes [abstract]. Mid Atlantic Plant Molecular Biology Society Conference. Paper No. 7.
- Xu, C., Garrett, W.M., Sullivan, J., Caperna, T.J., Natarajan, S.S. 2005. Separation and identification of soybean leaf proteins by 2d-page and mass spectrometry [abstract]. Mid Atlantic Plant Molecular Biology Society Conference. Paper No. 16.
- Natarajan, S.S., Xu, C., Caperna, T.J., Cregan, P.B., Bae, H., Garrett, W. M. 2006. Proteomic characterization of allergen proteins in sixteen soybean genotypes [abstract]. Plant Biology Annual Meeting 2006. August 05- 09, Boston, MA. Abstract No. 32025.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Soybean is the second most important cash crop in the US with an estimated value of $15.2 billion (2002/03) and provides an inexpensive source of protein for humans and animals. Genetic modifications of several nutritional deficiencies in soybean would make soybean a more valuable and nutritious protein source. However, there are consumer concerns and issues about the biosafety of transgenic soybeans, which have caused a loss of export income to the U.S. Genetic and transgenic approaches have been used to eliminate the Kunitz trypsin inhibitor, reduce the expression of P34, and introduce other transgenes into soybean, but very little information is available regarding collateral or unintended effects of these transgenic modifications. Therefore, the goals of the research project are to select and
apply suitable proteomics protocols to quantify any differences between transgenic vs. non- transgenic soybean. In this research, collaboration with scientists active in the production of transgenic soybean containing a variety of novel genes is anticipated. One way for the U.S. to remain competitive in the world soybean market is through the production of high quality soybean using transgenic technology. In 2003, 55% of the 76 million hectares of soybean planted globally were transgenic, up from 51% in 2002. Transgenic soybean produced approximately $1 billion of income through savings in the production costs associated with the use of Roundup Ready soybean. The study of the biosafety of transgenic soybean is important in order to maintain consumer acceptability of U.S. soybeans and for enhancing the soybean export market. The research to be undertaken falls under National Program 302 - Plant Biological and Molecular Processes and addresses Program Component 3: Plant
Biotechnology Risk Assessment. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2005) 1) Define the range of natural variation of seed proteins in a small group of cultivated and wild soybean genotypes. Year 2 (FY 2006) 1) Conduct field experiments in three locations, Brookings, SD, Beltsville, MD, and Clemson, SC with early, medium, and late Maturity Group soybean genotypes planted at two different sowing dates at each location. 2) Compare protein profiles of early, medium, and late Maturity Group soybean genotypes by 2D-PAGE. Year 3 (FY 2007) 1) Characterize and identify proteins of early Maturity Group genotypes using MALDI- TOF and LC-MS and compare the protein variation among the cultivated and wild soybean genotypes. 2) Compare quantity of the proteins using Image Quant of early Maturity Group soybean genotypes and complete statistical analysis. Year 4 (FY 2008) 1) Characterize and identify proteins of medium and late Maturity Group soybean
genotypes using MALDI-TOF and LC-MS and compare the protein variation among the genotypes. 2) Conduct field and greenhouse experiments in Beltsville, MD with medium Maturity Group genotypes to determine protein variation due to different environmental conditions. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Compare the natural variation of seed proteins in cultivated and wild soybeans. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006: 1) Conduct field experiments in three locations, Brookings, SD, Beltsville, MD, and Clemson, SC with early, medium, and late Maturity Group soybean genotypes planted at two different sowing dates. 2) Compare protein profiles of early, medium, and late
Maturity Group soybean genotypes by 2D-PAGE. In this milestone, experimental soybean seeds of early, medium and late Maturity Group genotypes grown at three different locations will be obtained for studying the natural variation of seed proteins. We will also obtain data on the comparative protein profiles of early, medium and late Maturity genotypes. These data will provide the basis of a publishable database of comparative natural variation of soybean proteins. FY 2007: 1) Characterize and identify proteins of early Maturity Group genotypes using MALDI-TOF and LC-MS and compare the protein variation among the genotypes. 2) Compare the quantity of the seed proteins of early Maturity Group genotypes using Image Quant and complete statistical analysis. In this milestone, comparative data of protein profiles of early Maturity Group genotypes will be generated and a detailed list of proteins of early Maturity Group genotypes will be accomplished. Image analysis of protein spots will
provide intensity and quantity of each protein spot. Statistical analysis will provide means and variation of spot intensity and number of proteins of early Maturity Group genotypes. FY 2008: 1) Characterize and identify proteins of medium and late genotypes using MALDI-TOF and LC-MS and compare the protein variation among the genotypes. 2) Conduct field and greenhouse experiments in Beltsville, MD with medium Maturity Group genotypes to determine protein variation due to different environmental conditions (early vs. late planting). Comparative data on natural variation of proteins in medium and late genotypes will be obtained. The protein profiles of seeds of medium Maturity Group genotypes produced in the greenhouse and the field will be compared to provide an analysis of how well protein profiles of genotypes grown in the greenhouse relate to those grown in the field. This will provide information relative to the likelihood of obtaining reliable protein profiles of greenhouse
grown transgenic soybean. 4a What was the single most significant accomplishment this past year? Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry (MS), we identified more than one hundred proteins that include storage, allergen and anti-nutritional proteins in both wild and cultivated soybean seeds. These data will serve as a baseline for comparison to help evaluate the quality and safety of transgenic soybean by determining whether the expression of transgenes in soybean results in an unusual accumulation of non-target proteins in the seed. Proteins were extracted from soybean seeds of wild and cultivated genotypes and analyzed by 2-D PAGE and MS. These data demonstrate the range of variation in abundance of proteins in the seed of wild and cultivated soybean germplasm and contributes to baseline data for scientists wishing to evaluate the quality and safety of transgenic plants the variation in abundance of proteins in transgenic plants.
5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This is a new project and established less than two years ago. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? During this period, no technology has been transferred. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Natarajan, S., Cregan, P., Xu, C., Caperna, T.J., Garrett, W., Sullivan, J. 2005. Study the variation of storage proteins in cultivated and wild soybean seeds. Plant Biology 2005, July 16-20, Seattle, Washington, Abstract # 526. Natarajan, S.S., Xu, C., Caperna, T.J., Garrett, W. 2005.
Comparison of protein solubilization methods suitable for proteomic analysis. Analytical Biochemistry 342: 214-220.
Impacts
(N/A)
Publications
- Natarajan, S.S., Xu, C., Caperna, T.J., Garrett, W.M. 2005. Comparison of protein solubilization method suitable for proteomic analysis of soybean seed proteins. Analytical Biochemistry. 342: 214-220.
- Natarajan, S.S., Xu, C., Caperna, T.J., Garrett, W.M. 2004. Selection of an efficient method to characterize soybean seed proteins for 2D and MALDI- TOF analysis [abstract]. American Society of Plant Biologists Annual Meeting. Abstract 710.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Soybean is the second most important cash crop in the US with an estimated value of $15.2 billion (2002/03) and provides an inexpensive source of protein for humans and animals. Genetic modifications of several nutritional deficiencies in soybean would make soybean a more valuable and nutritious protein source. However, there are consumer concerns and issues about the biosafety of transgenic soybeans, which have caused a loss of export income to the US. Genetic and transgenic approaches have been used to eliminate the Kunitz trypsin inhibitor, reduce the expression of P34, and introduce other transgenes into soybean, but very little information is available regarding collateral or unintended effects of these transgenic modifications. Therefore, the goals of the research project are to select and
apply suitable proteomics protocols to quantify any differences between transgenic vs. non- transgenic soybean. In this research, collaboration with scientists active in the production of transgenic soybean containing a variety of novel genes is anticipated. One way for the US to remain competitive in the world soybean market is through the production of high quality soybean using transgenic technology. In 2003, 55% of the 76 million hectares of soybean planted globally were transgenic, up from 51% in 2002. Transgenic soybean produced approximately $1 billion of income through savings in the production costs associated with the use of Roundup Ready soybean. The study of biosafety of transgenic soybean is important in order to maintain consumer acceptability of U.S soybeans and for enhancing the soybean export market. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2004) Develop proteomic protocols for soybean seed protein characterization.
Compare natural variation of seed proteins in sixteen different varieties of soybeans. Year 2 (FY 2005) Study the homogeneity of collateral effects associated with the Roundup Ready gene in a number of different genetic backgrounds. Year 3 (FY 2006) Apply proteomics tools to study the predictability of collateral effects resulting from different introductions of the same transgene. Year 4 (FY 2007) Identify and characterize highly expressed genes in transgenic soybean seeds during seed development using DNA microarrays. 3. Milestones: All milestones were fully met: Develop proteomic protocols for soybean seed protein characterization. Compare natural variation of seed proteins in sixteen different varieties of soybeans: B. List the milestones (from the list in Question #2) that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2005: Study the homogeneity of collateral
effects associated with the Roundup Ready gene in a number of different genetic backgrounds: We will focus on the expression of proteins in normal soybean versus Roundup Ready soybean with a number of different genetic backgrounds. Roundup Ready cultivars will include those adapted to a range of latitudes from Maturity Groups 00 to IX thus insuring a range of genetic backgrounds. In these experiments, three proteomics techniques will be applied: (i) two-dimensional gel electrophoresis (2-DE), (ii) Matrix- assisted laser desorption ionization (MALDI) and /or (iii) ElectrosprayIonization (ESI). These approaches will be an efficient way to examine the expression of proteins in transgenic and non-transgenic seeds. FY 2006: Apply proteomics tools to study the predictability of collateral effects resulting from different introductions of the same transgene: Our primary focus will be to examine the collateral effects resulting from different introductions of the same transgene. These
studies will be conducted in collaboration with Dr. Clemente, University of Nebraska, Lincoln. In this milestone, proteins will be separated by isoelectric point and molecular weights. Measurable differences in seed proteins would be anticipated. FY 2007: Identify and characterize highly expressed genes in transgenic soybean seeds during seed development using DNA microarrays: In this milestone, differential gene expression of a whole spectrum of genes during different stages of seed development in glyphosate (Roundup Ready) resistant and sensitive soybeans will be examined. First, we will investigate microarray-based expression, profiling differences in gene expression between glyphosate-sensitive and glyophosate resistant cultivars of soybean in early, mid and late stages of pod fill. Specific genes that are highly expressed at specific time points of pod fill will be identified using the Soybean Genomics and Microarray Database (SGMD). This milestone will be completed with a
collaboration of Dr. Benjamin F. Matthews, USDA-ARS, Beltsville, MD. 4. What were the most significant accomplishments this past year? We developed an efficient extraction method for both abundant and non- abundant soybean seed proteins and established comprehensive Standard Operating Procedures (SOP) for seed protein extraction. Though several methods have been reported in the past for the extraction of soybean seed proteins, none was efficient and therefore, this is an important accomplishment that will allow us to meet the objective of quantifying a large variety of protein species present in soybean seeds. In this study, we compared four different extraction methods including (i) lysis buffer containing urea, (ii) lysis buffer containing urea and thiourea, (iii) Tris-HCL buffer (pH 8.8), and (iv) modified TCA/acetone to determine those that increase the solubilization of proteins and also promote their migration from first to second dimension PAGE gels. Among the four methods
tested, the modified TCA/acetone method showed higher solubility of total proteins and also more protein spots on the 2-D gel. The TCA/acetone 2-D gels also displayed more low and high molecular weight proteins with sharper spot definition. This investigation provided an efficient protein extraction protocol, which was not only suitable for soybean seed protein extraction but also for other plant materials such as soybean cotyledons, tobacco leaves, and peach leaves and flower. B. Other significant accomplishment(s), if any. None C. Significant activities that support special target populations. None D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house ("D") projects and the projects listed in Appendix A; mandatory for all other subordinate projects). None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This is a new project and established
less than one year ago. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? During this period, no technology has been transferred. However, research is in progress to develop an innovative technology for assessing the biosafety of transgenic soybeans. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Natarajan, S., Xu, C., Caperna, T., Garrett, W. 2004. Proteome analysis of soybean seed proteins [abstract]. Fifteenth Annual BARC Poster Day. Abstract p.17. Natarajan, S., Xu, C., Caperna, T., Garrett, W. 2004. Selection of an efficient method to Characterize soybean seed proteins for 2D and MALDI- TOF analysis [abstract]. American Society of Plant Biologists Annual
Meeting, Orlando, Florida, USA. Abstract 710. (http://abstracts.aspb. org/pb2004/public/P63/7146.html)
Impacts
(N/A)
Publications
- Natarajan, S.S., Xu, C., Caperna, T.J., Garrett, W.M. 2004. Proteome analysis of soybean proteins [abstract]. BARC Poster Day. Abstract p.17.
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