Soybean [Glycine max (L.) Merr.] seed are valued for their protein and oil content. Soybean somatic embryos cultured in Soybean Histodifferentiation and Maturation (SHaM) medium were examined for their suitability as a model system for developing an understanding of assimilate partitioning and metabolic control points for protein and oil biosynthesis in soybean seed. This report describes the growth dynamics and compositional changes of SHaM embryos in response to change in the carbon to nitrogen ratio of the medium. It was postulated that at media compositions that were sufficient to support maximal growth rates, changes in the C:N ratio are likely to influence the partitioning of resources between the various storage products, especially protein and oil. As postulated, at steady-state growth rates, embryo protein content was strongly correlated with decreasing C:N ratios and increasing glutamine consumption rates. However, oil content remained relatively unchanged across the C:N ratio range tested, and resources were instead directed towards the starch and residual biomass (estimated by mass balance) pools in response to increasing C:N ratios. Protein and oil were inversely related only at concentrations of sucrose in the medium <88mM, where carbon limited growth and no starch was found to accumulate in the tissues. These observations and the high reproducibility in the data indicate that SHaM embryos are an ideal model system for the application of metabolic flux analysis studies designed to test hypotheses regarding assimilate partitioning in developing soybean seeds.

Two-dimensional [(1)H, (13)C] heteronuclear single quantum correlation (HSQC) spectroscopy nuclear magnetic resonance (NMR) is a comprehensive tool in metabolic flux analysis using (13)C-labeling experiments. NMR is particularly relevant when extensive isotopomer measurements are required, such as for plant cells and tissues, which contain multiple cellular compartments. Several isotope isomers (isotopomers) can be detected and their distribution extracted quantitatively from a single 2-D HSQC NMR spectrum. For example, 2-D HSQC detects the labeling patterns of adjacent carbon atoms and provides the enrichment of individual carbon atoms of the amino acids and glucosyl and mannosyl units present in hydrolysates of glycosylated protein. The HSQC analysis can quantitatively distinguish differences between the glucosyl units in the starch hydrolysate and a protein hydrolysate of plant biomass: this specifies crucial information about compartmentalization in the plant system. The peak structures obtained from the HSQC experiment show multiplet patterns that are directly related to the isotopomer abundances. These abundances have a nonlinear relationship to the fluxes via isotopomer balancing. Fluxes are obtained from the numerical solution of these balances and a stoichiometric model that includes biomass composition data as well as consumption rates of carbohydrate and nitrogen sources. Herein, we describe the methods for the experimental measurements for flux analysis, i.e., determination of the biomass composition (lipid, protein, soluble sugar, and starch) as well as detailed procedures of acid hydrolysis of protein and starch samples and NMR sample preparation, using soybean embryo culture as the model plant system. Techniques to obtain the relative intensity of 16 amino acids and glucosyl units for protein hydrolysate and the glucosyl units of starch hydrolysate of soybean embryos in 2-D HSQC NMR spectra also are provided.

Comprehensive analysis of isotopic labeling patterns of metabolites in proteinogenic amino acids and starch for plant systems lay in the powerful tool of 2-Dimensional [(1)H, (13)C] Nuclear Magnetic Resonance (2D NMR) spectroscopy. From (13)C-labeling experiments, 2D NMR provides information on the labeling of particular carbon positions, which contributes to the quantification of positional isotope isomers (isotopomer). 2D Heteronuclear Single Quantum Correlation (HSQC) NMR distinguishes particularly between the labeling patterns of adjacent carbon atoms, and leads to a characteristic enrichment of each carbon atom of amino acids and glucosyl and mannosyl units present in hydrolysates of glycosylated protein. Furthermore, this technique can quantitatively classify differences in glucosyl units of starch hydrolysate and of protein hydrolysate of plant biomass. Therefore, the 2D HSQC NMR method uses proteinogenic amino acids and starch to provide an understanding of carbon distribution of compartmentalization in the plant system. NMR has the advantage of minimal sample handle without separate individual compounds prior to analysis, for example multiple isotopomers can be detected, and their distribution extracted quantitatively from a single 2D HSQC NMR spectrum. The peak structure obtained from the HSQC experiment show multiplet patterns, which are directly related to isotopomer balancing. These abundances can be translated to maximum information on the metabolic flux analysis. Detailed methods for the extractions of protein, oil, soluble sugars, and starch, hydrolysis of proteinogenic amino acid and starch, and NMR preparation using soybean embryos cultured in vitro as a model plant systems are reported in this text. In addition, this chapter includes procedures to obtain the relative intensity of 16 amino acids and glucosyl units from protein hydrolysate and the glucosyl units of starch hydrolysate of soybean embryos in 2D HSQC NMR spectra.

Of the world's major agronomic food crops soybeans rank highest in protein content (~40% Dwt) while also containing significant quantities of oil (~20% Dwt). Based on these unique characteristics soy has become a mainstay in world agriculture, providing a protein source for livestock and human nutrition (68% of global vegetable protein meal consumption in 2011) as well as a primary source of vegetable oil (28% of global vegetable oil consumption in 2011; http://www.soystats.com/2012). The value of soy is therefore found both in its oil and protein content and increasing the content of both is therefore desirable. Research aimed at increasing the oil content, while leaving the protein content unchanged, has exposed a fundamental lack of understanding of resource partitioning and the factors that influence protein and oil content in the soybean seed. Somatic embryos have proven to be a highly productive platform for testing gene combinations designed to change soybean composition and provide a useful model for performing physiological and biochemical studies. Soybean somatic embryos cultured in Soybean Histodifferentiation and Maturation (SHaM) medium were examined for their suitability as a model system for developing an understanding of assimilate partitioning and metabolic control points for protein and oil biosynthesis in soybean seed. It was postulated that at media compositions that were sufficient to support maximal growth rates, changes in the C:N ratio are likely to influence the partitioning of resources between the various storage products, especially protein and oil. As postulated, at steady-state growth rates embryo protein content was strongly correlated to decreasing C: N ratios and