The negative association between protein and oil production in soybean (Glycine max) seed is well-documented. However, this inverse relationship is based primarily on the composition of mature seed, which reflects the cumulative result of events over the course of soybean seed development and therefore does not convey information specific to metabolic fluctuations during developmental growth regimes. In this study, we assessed maternal nutrient supply via measurement of seed coat exudates and metabolite levels within the cotyledon throughout development to identify trends in the accumulation of central carbon and nitrogen metabolic intermediates. Active metabolic activity during late seed development was probed through transient labeling with 13C substrates. The results indicated: i) a drop in lipid contents during seed maturation with a concomitant increase in carbohydrates, ii) a transition from seed filling to maturation phases characterized by quantitatively balanced changes in carbon use and CO2 release, iii) changes in measured carbon and nitrogen resources supplied maternally throughout development, iv) 13C metabolite production through gluconeogenic stepsfor sustained carbohydrate accumulation as the maternal nutrient supply diminishes, and v) oligosaccharide biosynthesis within the seed coat during the maturation phase. These results highlight temporal engineering targets for altering final biomass composition to increase the value of soybeans and a path to breaking the inverse correlation between seed protein and oil content.

Protein and oil levels measured at maturity are inversely correlated across soybean lines; however, carbon is in limited supply during maturation resulting in tradeoffs for the production of other reserves including oligosaccharides. During the late stages of seed development, the allocation of carbon for storage reserves changes. Lipid and protein levels decline while concentrations of indigestible raffinose family oligosaccharides (RFOs) increase, leading to a decreased crop value. Since the maternal source of carbon is diminished during seed maturation stages of development, carbon supplied to RFO synthesis likely comes from an internal, turned-over source and may contribute to the reduction in protein and lipid content in mature seeds. In this study, fast neutron (FN) mutagenized soybean populations with deletions in central carbon metabolic genes were examined for trends in oil, protein, sugar, and RFO accumulation leading to an altered final composition. Two lines with concurrent increases in oil and protein, by combined 10%, were identified. A delayed switch in carbon allocation towards RFO biosynthesis resulted in extended lipid accumulation and without compromising protein. Strategies for future soybean improvement using FN resources are described. of 153-12% of biomass is present as sugars and oligosaccharides [4]. Sugars, such as sucrose, represent a metabolizable energy source in animal feed, but raffinose and stachyose that are members of the raffinose family oligosaccharides (RFOs) are indigestible and undesirable for livestock production [5,6]. RFOs have hypothesized roles in seed desiccation tolerance [7-9], stability of liposomes during dehydration, and seed germination [10][11][12], although efforts in breeding and genetic engineering have demonstrated that reduced levels do not impact seed viability [13][14][15]. The RFO and sucrose levels are the result of central carbon metabolism and could adversely impact the levels of oil and protein, which are known to be inversely correlated in mature seeds [16][17][18][19][20][21].Protein and lipid accumulation over the course of seed development is dependent on the supply of amino acids and sugars from the maternal sources (i.e., organic carbon assimilated in the leaf) as well as the metabolism within the developing seed (i.e., cotyledons) [22][23][24][25]. The levels of intermediates of central carbon (C) and nitrogen (N) metabolism that are precursors for storage reserve production, vary depending on the stage of reproductive development and may indicate temporal changes in metabolism [26][27][28]. During maturation, 10-15% of lipids are degraded [28][29][30], coinciding with RFO accumulation, and occurring at the time when there are little to no exogenous resources supplied by the maternal plant. The expression of genes involved in gluconeogenesis and glyoxylate cycle suggest carbon remobilization from lipid may occur. Along with existing sucrose present late in development, carbon from lipid could contribute to central metabolism and enable RFO pro...

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