2009 Annual Report 1a.Objectives (from AD-416) 1) To develop soybeans with altered seed coat color to facilitate the segregation and identity preservation of seeds with genetically enhanced compositional traits.. 2)To produce soybeans with enhanced food, feed, and industrial properties by modification of the oil and lipid-soluble antioxidant composition of seeds.. 3)To introduce genes into soybeans that result in high-level accumulation of foreign and engineered proteins valuable for food, feed, and industrial uses.. 4)To develop procedures and methods for analyzing and predicting seed protein allergenicity in food and feed. To develop non-allergenic seeds by suppressing intrinsic allergens and to modify proteins that are potential transgenes for improving biosafety. 1b.Approach (from AD-416) 1) Co-transformation of soybean with a seed coat color-conferring transgene and a trait transgene will yield visually distinct seeds with enhanced composition.. 2)Identify enzymes that are specialized for the metabolism of unusual fatty acids to produce agronomically-viable soybean seeds.. 3)Manipulate protein content of soybean seeds by use of plant promoters and compartmentalization of introduced proteins.. 4)Identify and characterize soybean food and feed allergens. 3.Progress Report Seeds possess a high intrinsic capacity for protein production that makes seeds a desirable bioreactor platform for the manufacture of transgene products. A strategy to enhance foreign protein production is to exchange the capacity to produce intrinsic proteins for the capacity to produce a high level of foreign proteins. Suppression of the alpha/alpha’ subunit of beta-conglycinin storage protein synthesis in soybean has been previously shown to result in the increase of the accumulation of the glycinin storage protein some of which is sequestered as proglycinin in de novo ER-derived protein bodies. The exchange of glycinin for conglycinin is quantitative with the remodeled soybeans possessing normal protein content with an altered proteome. To produce a soybean line with a reporter GFP-kdel was transferred in a construct using the glycinin promoter and terminator to mimic the glycinin gene that when expressed in soybean seeds the GFP-kdel accretes to form ER-derived protein bodies. The introgression of GFP-kdel into the alpha/alpha’ subunit of beta-conglycinin suppression background results in a four-fold enhancement of GFP-kdel accumulation to >7% (w/w) of the total protein in soybean seeds. The resulting seeds accumulate a single population of ER-membrane bounded protein bodies that contain both GFP-kdel and glycinin. These results show that by mimicking the allele of the proteins that compensate for suppressed seed proteins the accumulation of the gene mimic’s product is greatly enhanced. This result shows that the collateral proteome rebalancing that occurs with the suppression of intrinsic proteins in soybean can be exploited to produce an enhanced level of foreign proteins. These results were published in Plant Biotechnology and the subject of a US and international patent application jointly by ARS and the Donald Danforth Plant Science Center. This project is being further extended by suppression of all the major soybean storage protein genes and work in progress is attempting to replicate the enhanced foreign protein yield in single storage protein suppressors with similar expression in double storage protein suppressors. The goal is to exceed 10% of foreign protein production. The significance of this accomplishment is to develop a strategy that will permit the economically viable production of diverse industrially important enzymes and biologicals using the cost-efficient multiplier of agricultural production. 4.Accomplishments 1. The enhancement of foreign protein accumulation by the exchange with intrinsic storage proteins. The use of crop plants to produce foreign proteins to industrial proteins has been limited by the yield of protein produced per unit mass of harvested plant materials. The suppression of seed protein accumulation results in compensatory protein synthesis to maintain content. By expressing the foreign protein to mimic the compensating protein’s allele the accumulation of the foreign protein can be enhanced with more than 8% foreign protein yield. The high-level accumulation of foreign proteins such as industrial and biofuel enzymes in a low cost of production platform can have a positive role in lowering the cost of production of industrial enzyme products. Review Publications Herman, E.M. 2008. Endoplasmic Reticulum Bodies: Solving the Insoluble. Current Opinion in Plant Biology. 11(6):672-679.