13C-Metabolic flux analysis of soybean somatic embryos for identification of metabolic control points in developing seed

摘要

Of the world\u27s 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 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 media sucrose concentrations below 88 mM, where carbon limited growth and no starch was found to accumulate in the tissues.This work describes the in-depth studies of zygotic and SHaM embryos under similar culture conditions and carbon and nitrogen sources. There is no significantly different in relative growth rate for both embryos. Both protein and oil content were lower for SHaM embryos than in zygotic embryos; however, starch contents were comparable, and the balance of the biomass differences, which was accounted for by the residual (structural carbohydrate) pool, was higher in the SHaM embryos. Flux analysis in cultured embryos resulted changes in nitrogen uptake and flux into oil biosynthesis, respiratory flux (CO2), glutamine biosynthesis flux, fluxes in the total of plastic and cytosolic of triose phosphate to phosphoenolpyruvate pathway, as well as an increase in tricarboxylic acid cycle activity for zygotic embryos. However, fluxes into structure and non-structure carbohydrates were significantly higher in SHaM embryos. Despite these differences, the NMR relative intensities of proteinogenic amino acids and labeling patterns of protein and starch-related glucosyl units were comparable between the two embryo types. Carbon labeling patterns of SHaM embryos well fitted with the metabolic network model of zygotic embryos with three compartments: cytosol, plastid, and mitochondrion. The observations described here shed light onto metabolic pathways of SHaM embryos, especially as compared to soybean seed.This thesis describes experiments in which we have used Metabolic Flux Analysis to investigate the influence of transgenic perturbations and nutritional status on resource partitioning in a soybean somatic embryo system. SHaM embryos of transgenic cultures with the plastidic phosphoglucomutase (PGM) gene knocked out (PGM-KO), and the control (PGM-null) are cultured in sucrose concentrations ranged from 88 to 234 mM as a carbon source and initial glutamine concentrations ranged from 20 to 60 mM as a nitrogen source. These concentrations correspond to C:N ratios ranging from 8.8 to 70.2. Two C: N mole ratio conditions are further examined through metabolic flux analysis with labeling experiment of U-13C12 sucrose for both PGM culture. The result indicates that: (1) protein and oil of PGM-KO were consistently higher than the PGM-null; (2) content in PGM-KO shows nearly two fold as compared to PGM-null; and (3) for both PGM culture, protein content is strongly correlated with the glutamine uptake rate. Fluxes through cytosolic glucose-6-phosphate isomerase, transketolase, and transaldolase, contributed significantly to the soluble sugar content for PGM-KO culture. These fluxes changed in response to the absence of starch synthesis.