Carbon-13 metabolic flux analysis of soybean central carbon metabolism: Response to temperature and genotype effects

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 gr...