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...

Metabolic flux analysis (MFA) is a powerful approach for quantifying plant central carbon metabolism based upon a combination of extracellular flux measurements and intracellular isotope labeling measurements. In this chapter, we present the method of isotopically nonstationary (13)C MFA (INST-MFA), which is applicable to autotrophic systems that are at metabolic steady state but are sampled during the transient period prior to achieving isotopic steady state following the introduction of (13)CO2. We describe protocols for performing the necessary isotope labeling experiments, sample collection and quenching, nonaqueous fractionation and extraction of intracellular metabolites, and mass spectrometry (MS) analysis of metabolite labeling. We also outline the steps required to perform computational flux estimation using INST-MFA. By combining several recently developed experimental and computational techniques, INST-MFA provides an important new platform for mapping carbon fluxes that is especially applicable to autotrophic organisms, which are not amenable to steady-state (13)C MFA experiments.