2006 Annual 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? This Project is aligned with NP 302, Plant Biological and Molecular Processes. The utilization of soybeans is limited by their chemical composition. Research is needed to determine optimal strategies for modification of soybean seed composition. Seed composition will be modified so that nutritional quality is improved in food or feed applications, or, alternatively, to provide starting materials for novel uses. Modification of the biochemical composition of soybeans through the use of contemporary, molecular methods will serve to improve performance in current uses, and at the same time, open new avenues for utilization. Modification of seed composition through classic plant breeding has been limited by the relatively low genetic variability within soybeans. Improved performance of soybean meal in feed applications is a direct and immediate benefit of the research. Additionally, new markets for use as feed or in the food industry could be created through modifications of the quality of the raw material to be employed in subsequent processing. 2.List by year the currently approved milestones (indicators of research progress) Milestone 1 (12 months, 2007) - Extend molecular description of the stages of soybean seed development. - Conduct comparative analyses of transgenic soybean plants expressing methionine-rich protein. - Complete study of the sequence and expression of the soybean myo-inositol phosphate synthase (MIPS) gene family members in Williams 82 and low phytate line CX1834. - Complete second year of field study characterizing the effects of pod temperature on seed composition, germination, and seedling growth. Initiate short and long duration temperature stress treatments at R5 – R8 stages of seed development and initiate analysis of the composition of developing and mature seeds. Conduct germination, and seedling growth analyses of seeds from study of maternal effects on seed composition. Milestone 2 (24 months, 2008) - Refine conditions for in vitro culture of developing soybean embryos, and establish conditions to be used for chemical genomics screening. - Extend our biochemical understanding of the cysteine synthase enzyme complex. - Complete study of the sequence and expression of soybean FAD2 gene family members in Williams 82 and plant introductions with elevated oleic acid. - Complete germination and seedling growth analyses of seeds derived from the pod temperature study. Repeat and refine short and long duration temperature stress treatments at R5 – R8 stages of seed development and continue compositional analyses of developing and mature seeds. Complete chemical analyses of seeds from study of maternal effects on seed composition. Conduct compositional analyses of seeds from study on SCN effects on seed composition. Milestone 3 (36 months, 2009) - Clone, sequence, and characterize promoter regions for genes encoding the plastidial subunits of the pyruvate dehydrogenase complex. - Develop plasmid constructs for constitutive expression of O-acetylserine (thiol) lyase in soybean. - Complete study on the use of allele-specific molecular markers to combine mid-oleic and low linolenic acid traits in a backcross breeding program. - Conduct germination and seedling growth analyses of seeds exposed to short and long duration temperature stress. Collate data sets from two years of pod temperature/seed composition studies. Complete analyses of seeds from study on SCN effects on seed composition. Milestone 4 (48 months, 2010) - Extend use of in vitro embryo culture to define conditions optimal for increasing seed oil levels at the expense of NSC, without decreasing protein quantity. - Extend studies on transgenic soybean plants over-expressing O-acetylserine (thiol) lyase. - Complete study on identification of soybean raffinose synthase genes and analysis of their expression in Williams 82 and low raffinose line PI200508. - Complete composition analyses of seed exposed to short and long duration temperature stress. Conduct compositional analyses of seeds from flood stressed plants. Milestone 5 (60 months, 2011) - Define components of plastidial PDC subunit gene promoters that can be modified to yield increased expression at specific periods during seed development. - Elaborate on analysis of transgenic soybeans simultaneously over-expressing O-acetylserine (thiol) lyase and expressing the Met-rich protein delta-zein. - Complete study evaluating backcross breeding strategies to combine as many as seven genes in a single line. - Complete performance analyses of seeds exposed to short and long duration temperature stress during development. Complete germination and seedling vigor analyses of seeds from flood stressed plants. 4a.List the single most significant research accomplishment during FY 2006. A sensitive, non-invasive method for biochemical analysis of soybean seeds was developed. The method is based upon near-infrared reflectance (NIR) spectroscopy, and can be used to quantify total oil as well as complete fatty acid profiling. With further refinement, it can also be used to quantify total protein and carbohydrate levels. The analyses were achieved using a NIR spectrophotometer exclusively. No capability in the visible spectrum is needed. Accurate analyses were achieved using samples as small as 250 mg, which is one-tenth the normal sample size used for compositional analysis. The NIR spectroscopy-based method will allow rapid analysis of seed fatty acid catabolism during post-germinative seedling growth. 4b.List other significant research accomplishment(s), if any. None. 4c.List significant activities that support special target populations. None. 4d.Progress report. This CRIS project was approved and implemented on March 20, 2006, replacing 3622-21000-020-00D. The molecular description of soybean seed development was undertaken. A staging platform was developed based upon the results of physical and biochemical analyses. A two-dimensional proteomic map was established for each of the developmental stages. Transgenic soybean plants that express a methionine-rich protein were developed and comparative analyses of wild-type and modified plants were initiated. The sequences of myo-inositol phosphate synthase gene family members were determined, and studies of the expression of gene family members in Williams 82 and the low phytate line CX1834 were designed and initiated. The second year of a field study characterizing the effects of pod temperature on seed composition, germination, and seedling growth was initiated. Analyses will include short and long duration temperature stress treatments at the R5 – R8 stages of seed development. Compositional analyses of the developing and mature seeds will be conducted, as will studies of germination and seedling growth. 5.Describe the major accomplishments to date and their predicted or actual impact. This CRIS project was approved and implemented on March 20, 2006, replacing 3622-21000-020-00D. The molecular description of soybean seed development was undertaken. A staging platform was developed based upon the results of physical and biochemical analyses. A two-dimensional proteomic map was established for each of the developmental stages. Transgenic soybean plants that express a methionine-rich protein were developed, and comparative analyses of wild-type and modified plants were initiated. The sequences of myo-inositol phosphate synthase gene family members were determined, and studies of the expression of gene family members in Williams 82 and the low phytate line CX1834 were designed and initiated. The second year of a field study characterizing the effects of pod temperature on seed composition, germination, and seedling growth was initiated. Analyses will include short and long duration temperature stress treatments at the R5 – R8 stages of seed development. Compositional analyses of the developing and mature seeds will be conducted, as will studies of germination and seedling growth. 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? None.