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Title: Carbon and nitrogen provisions alter the metabolic flux in developing soybean embryos

item Allen, Douglas - Doug
item YOUNG, JAMEY - Vanderbilt University

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/9/2013
Publication Date: 3/1/2013
Citation: Allen, D.K., Young, J.D. 2013. Carbon and nitrogen provisions alter the metabolic flux in developing soybean embryos. Plant Physiology. 161:1458-1475.

Interpretive Summary: This work describes the impact of altered delivery of carbon (primarily as sugars) and organic nitrogen (primarily as amino acids) to developing soybean embryos. It is often reported that if protein goes up in soybean seeds then yield or particular storage reserves such as oil go down but these inverse correlations are not quantitatively understood. Additionally the delivery of different levels of nitrogen to the developing seed can impact storage reserve composition (profile). We systematically probed the response of developing soybean embryos to changes in carbon and nitrogen (provided as sugars and amino acids) to better understand the impact on final storage reserve composition. Young green embryos were dissected and cultured with unlabeled, 13C-labeled or 14C-labeled compounds and the final labeled products were evaluated. The inspection of labeling was used to computationally and quantitatively determine the flow of carbon and nitrogen through the metabolic pathways operating in the seed to generate the final storage reserves. The results indicate that altering the delivery of carbon and nitrogen to the developing seeds generated a dynamic change in protein levels but without changes in total dry weight. This finding indicates that the increase in protein utilizes carbon that would have otherwise been allocated for other storage reserves such as oil. Fatty acids and amino acids are significantly more labeled when more 13C-glutamine was provided to developing embryos, indicating that it plays an important role in providing carbon for both protein and oil production. This work is important because current efforts to develop soybeans with enhanced storage reserve profiles (i.e. more oil without penalty in yield or protein) have been unsuccessful because of our limitated understanding of fundamental metabolic processes in the seed. Our results deepens our knowledge in this area and will ultimately enable more rational efforts and strategies to develop superior soybean seed compositions for U.S. agriculture.

Technical Abstract: Soybeans store approximately 40% of their biomass in the form of protein. Protein concentrations reflect the carbon and nitrogen levels received by the developing embryo. The relationship between carbon and nitrogen supply and seed composition during filling was examined through a series of embryo culturing experiments. Three distinct ratios of carbon to nitrogen supply were further explored through metabolic flux analysis. Labeling experiments utilizing [U-13C5]-glutamine, [U-13C4]-asparagine, and [1,2-13C2]-glucose were performed to assess embryo metabolism under altered feeding conditions and to create corresponding flux maps. Additionally, [U-14C12]-sucrose, [U-14C6]-glucose, [U-14C5]-glutamine, and [U-14C4]-asparagine were used to monitor differences in carbon allocation. The analyses revealed that: i) protein concentration ranged from 15-59% of total soybean embryo biomass; ii) altered nitrogen supply did not dramatically impact relative amino acid or storage protein subunit profiles; and iii) glutamine supply contributed 10-23% of the carbon for biomass production including 9-19% of carbon to fatty acid biosynthesis and 32-46% of carbon to amino acids. The results indicated that seed metabolism can accommodate different levels of protein biosynthesis while maintaining a consistent rate of dry weight accumulation. There is also a significant malic enzyme flux (24-30% of flux to pyruvate) and a conserved flux of carbon through pyruvate, both of which may be important to the inverse correlation between protein and oil. The flux maps that were independently validated by nitrogen balancing highlight the robustness of primary metabolism and are discussed in the context of metabolic engineering.