2007 Annual Report 1a.Objectives (from AD-416) Identify and characterize genes or gene products that control metabolic expression of protein and oil accumulation during legume seed development; Develop genomic resources to characterize the function of genes or gene products that mediate genetic variation in composition and allergenicity of seed storage proteins in soybean and comparable legumes; Develop technology to quantitatively measure immune responses by swine challenged with food and feed derived from legume seeds. 1b.Approach (from AD-416) The cDNA suppression subtraction libraries will be analyzed for differentially expressed genes that are predicted to be important in controlling the onset of seed fill, including primarily those encoding proteins with DNA binding activity, regulatory proteins, or involved in regulatory cascades. The influence of over-expression, mutation, and silencing of selected genes on seed development will be determined by transformation of soybean and Arabidopsis. The existing soybean TILLInG populations with high mutation frequencies will be screened with several genes, including those encoding allergenic storage proteins and enzymes involved in seed development and fatty acid synthesis. The populations can be screened for mutations in genes of interest to other investigators and M3 seed distributed. Monoclonal antibodies against swine IgE will be developed by (1) generating antibodies that recognize the swine heavy chain region of the epsilon chain and (2) expression in the baculovirus-insect vector system. The line of monoclonals with least cross-activity will be used to create an immunoassay to quantify IgE produced by pigs in response to legume storage proteins. 3.Progress Report The scientist position for this project was vacant for the beginning of the 2007 fiscal year (October 2006-February 2007) and a new scientist started in March 2007. The new scientist in the position has chosen to focus on the first two objectives at this time: to characterize the genes and gene products involved in the accumulation of protein and oil during seed development, and to develop genomic resources to study soybean gene function. TILLInG (Targeting Induced Local Lesions In Genomes) is a method that permits the selection of mutations in a gene of choice based only on sequence information for the gene, when no information about the phenotypic effects of the mutation is known. A chemically mutagenized TILLInG population was created in this laboratory several years ago and consists of approximately 9000 identity-preserved tissue samples in the Williams-82 background and seeds from M2 plants. This year we have grown 1100 of these lines in the field to increase the seed supply and observe the plants for morphological abnormalities. In addition, we have started to extract DNA from the archived tissue samples for use in reverse-genetic screening for mutations in genes that are involved in soybean seed composition. Much is known about the proteins that control seed oil and protein content in the model plant Arabidopsis thaliana, and while these factors are conserved in soybean, they have not been studied in soybean. The WRINKLED1 (WRI1) gene encodes a transcription factor required for storage oil biosynthesis in Arabidopsis. As soybean has undergone a genome duplication with respect to Arabidopsis, soybean possesses several closely related genes encoding orthologs of WRI1. We have obtained full-length cDNAs for several WRINKLED-like genes from developing soybean seeds. The transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) is known to regulate the accumulation of seed storage proteins in Arabidopsis by directly binding to an element in the promoter of the seed storage protein genes. We have determined that both copies of the soybean ABI3 gene (GmABI3a and GmABI3b) are expressed, and we have isolated full-length cDNAs for both. We will use the full-length cDNAs in constructs to identify interactors and determine the effects of overexpression of GmWRI1 and GmABI3 on soybean seed development. We have also isolated RNA from staged soybean embryos and subdomains of soybean cotyledons in order to characterize the timing and pattern of expression of these and other genes involved in seed protein and oil accumulation. Impact: We anticipate that when DNA extractions for the soybean TILLING population are complete, that this will be a valuable resource for our own investigations in seed composition as well as for functional genomic efforts for the rest of the soybean research community. A better understanding of the expression of important regulatory factors during soybean seed development may permit improvements in soybean protein and oil content. 4.Accomplishments Genes involved in soybean seed protein and oil content We have obtained DNA sequences that will allow us to monitor the expression of and test for interactions between proteins that are important regulators of soybean seed composition. An understanding of the expression and function of these genes could lead to improvement of seed protein and oil content. This addresses Problem 2A, Understanding Growth & Development of the NP302 (Plant Biological and Molecular Processes) action plan.