Soybeans are a major source of high quality protein, edible oil, food products, and also several industrial non food applications such as paper coating, textiles and plastics. It is difficult to develop soybeans with high protein and high oil, however, due to the inverse relationship between these two components. Consequently, increasing seed protein concentration without adversely affecting the yield and oil content has been difficult to achieve. It has been demonstrated that the temperature and genotype of soybeans influence the protein or lipid composition considerably. However, the interactions between various pathways responsible for the changes in the composition are unknown;Metabolic flux analysis (MFA) quantifies carbon flow in a biological system, which is an important characteristic reflective of the system physiology. The application of MFA to study complex plant metabolic networks has however been recent. Towards this goal, MFA using carbon labeling (13C) experiments, nuclear magnetic resonance spectroscopy (NMR) combined with a generic mathematical framework (NMR2Flux) has been developed in our group. Metabolic flux maps, developed from 13C MFA are effective tools for comparing pathway interactions between genetic or environmental variants of biological systems and identifying possible targets for genetic manipulations;13C MFA has been performed to understand response of central carbon metabolism of developing soybeans to two parameters: temperature and genotype. Experimental treatments were designed to distinguish between temperature effects prior to and during incubation in vitro of Evans genotype. Biomass accumulation increased with temperature as did carbon partitioning into lipid. The flux through the plastidic oxidative pentose phosphate pathway ( pglP) relative to total sucrose intake remained fairly constant (~56 % (+/-24%)) when cotyledons were transferred from an optimum growth temperature in planta to optimum, lower and higher temperatures in in vitro culture. The pglP flux ranged from 57 to 77% of total sucrose intake, however, when growth temperature in planta varied and were cultured in vitro at the same temperature (as the plant). These results indicate that temperature during early stages of cotyledon development has a dominant effect on establishing capacity for flux through certain components of primary metabolism. The flux of carbon through the anaplerotic reactions catalyzed by the plastidic malic enzyme (mep), cytosolic pep carboxylase (ppc) and the malate transporters (malT1 and malT2) between cytosol, mitochondrion and plastid varied considerably with temperature. The redirection of carbon between these cellular compartments had a direct influence on the carbon partitioning into protein and oil from the plastidic pyruvate pool;BC3-128 genotype, a back crossed lined created from High PI and Evans, produced more protein than Evans and less than High PI. 13C MFA was performed on soybeans of the above three genotypes cultured at two stages of development, 21 DAF (Set 2 TP1) and around 32-35 DAF (Set 2 TP2). For 21 DAF, there was an additional replicate (Set 3 TP1). Comparison of metabolic fluxes between High PI and BC3-128 Set 2 TP1 showed that the flux ratio of oxPPPtotal relative to glycolysistotal on a C mol basis was statistically similar (~2.2). The oxPPPtotal relative to glycolysis total ratio in Evans was lower (~1.6) indicating lower carbon flux through the oxPPP. The oxPPP node being an important source of reductant NADPH, the above result could be a direct influence of the higher plastidic NADPH requirement for biosynthetic and glutamine assimilation reactions in High PI and BC3-128 due to higher protein production compared to Evans;Comparison of Set 2 and Set 3 (TP1) showed variations in metabolic fluxes and flux ratios within the genotype for High PI, BC3-128 and Evans. However, there were temperature fluctuations in the growth chamber where Set 3 plants were grown and could have directly influenced the changes. The variation between fluxes between the replicates could be attributed by a combination of factors such as temperature fluctuations in the Set 3 growth chamber, biomass accumulation and composition or a difference in the NMR intensities which directly reflect the intracellular fluxes. For Set 2 TP2, the pglp flux and ratio of the pglP relative to sucrose intake on a C mol basis was statistically similar between all three genotypes. The anaplerotic reactions such as meP, cytosolic ppc and malate transporters (malT1 and malT2 ) between cytosol, plastid and mitochondrion were flexible between genotypes as observed in the temperature study for Set 2 and Set 3. In particular, higher meP flux was observed when protein content was higher. The large variation in meP in the temperature study and higher flux through meP during increased protein production observed in the genotype study suggest that meP could be a possible target for genetic manipulation;Despite differences observed between replicates, certain metabolic trends remained consistent between the genotype study as well as the temperature study. The transketolase and transaldolase of the oxidative pentose phosphate pathway (oxPPP) fluxes were higher in the plastid than the cytosol and agreed with the emerging model of oxPPP in plant systems. The hexose isomerase flux was in the direction of F6P to G6P thereby feeding the oxPPP. There was substantial GABA shunt driving the glutamine assimilation into the system. The net pyruvate kinase flux in the cytosol and plastid was much higher than the me P feeding the plastidic Pyr pool. Thus, 13C MFA has been demonstrated to be an effective tool in understanding pathway interactions and identifying important reactions influencing protein and oil content in developing soybeans. A collaborative study to analyze gene expression data for Set 2 TP1 is under progress. The integration of the flux and transcript data for Set 2 TP1 is proposed to better understand the underlying physiology of developing soybeans.