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Improving plant productivity is an important aim for metabolic engineering. There are few comprehensive methods that quantitatively describe leaf metabolism, although such information would be valuable for increasing photosynthetic capacity, enhancing biomass production, and rerouting carbon flux toward desirable end products. Isotopically nonstationary metabolic flux analysis (INST-MFA) has been previously applied to map carbon fluxes in photoautotrophic bacteria, which involves model-based regression of transient 13 C-labeling patterns of intracellular metabolites. However, experimental and computational difficulties have hindered its application to terrestrial plant systems. We performed in vivo isotopic labeling of Arabidopsis thaliana rosettes with . Approximately 1,400 independent mass isotopomer measurements obtained from analysis of 37 metabolite fragment ions were regressed to estimate 136 total fluxes (54 free fluxes) under each condition. The results provide a comprehensive description of changes in carbon partitioning and overall photosynthetic flux after longterm developmental acclimation of leaves to high light. Despite a doubling in the carboxylation rate, the photorespiratory flux increased from 17 to 28% of net CO 2 assimilation with high-light acclimation (Vc/Vo: 3.5:1 vs. 2.3:1, respectively). This study highlights the potential of 13 C INST-MFA to describe emergent flux phenotypes that respond to environmental conditions or plant physiology and cannot be obtained by other complementary approaches.isotopomer modeling | metabolic flux analysis | photosynthesis | 13 C-labeling | primary metabolism P hotosynthetic organisms assimilate more than 100 billion tons of carbon, ∼15% of the atmospheric total, each year and generate organic compounds for food and renewable chemicals (1). However, photosynthesis is a complex process that responds to heterotrophic tissue demands and environmental stimuli such as drought, temperature, and light intensity (2, 3). The light incident on the plant varies with intensities in the range of 0-2,000 μmol photons·m −2 ·s −1 and can change dramatically because of passing clouds, shading, and the position of the sun. Thus, plants adjust light harvesting and carbon assimilation steps to accommodate many fluctuations, resulting in changes in plant morphology, physiology, and metabolism (4).For 95% of all terrestrial plants (i.e., C3 plants), the reductive pentose phosphate (Calvin-Benson-Bassham, or CBB) cycle directly links light and dark reactions and sustains anabolic activities (5). RuBisCO (ribulose-1,5-bisphosphate carboxylase oxygenase) plays a central role in the cycle by carboxylating ribulose-1,5-bisphosphate (RUBP) with CO 2 to form two 3-phosphoglycerate (3PGA) molecules. The other 10 enzymes in the CBB cycle regenerate the RUBP substrate to repeat this process. RuBisCO has a low turnover rate (∼3/s; ref. 6) and also performs a competitive oxygenation side reaction that limits carboxylation activity. The binding of RuBisCO to oxygen produces 2-phosphoglycolat...

SummarySoybean (Glycine max) yields high levels of both protein and oil, making it one of the most versatile and important crops in the world. Light has been implicated in the physiology of developing green seeds including soybeans but its roles are not quantitatively understood. We have determined the light levels reaching growing soybean embryos under field conditions and report detailed redox and energy balance analyses for them. Direct flux measurements and labeling patterns for multiple labeling experiments including [U- CO 2 labeling were performed at different light levels to give further insight into green embryo metabolism during seed filling and to develop and validate a flux map. Labeling patterns (protein amino acids, triacylglycerol fatty acids, starch, cell wall, protein glycan monomers, organic acids), uptake fluxes (glutamine, asparagine, sucrose, glucose), fluxes to biomass (protein amino acids, oil), and respiratory fluxes (CO 2 , O 2 ) were established by a combination of gas chromatography-mass spectrometry, 13 C-and 1 H-NMR, scintillation counting, HPLC, gas chromatography-flame ionization detection, C:N and amino acid analyses, and infrared gas analysis, yielding over 750 measurements of metabolism. Our results show: (i) that developing soybeans receive low but significant light levels that influence growth and metabolism; (ii) a role for light in generating ATP but not net reductant during seed filling; (iii) that flux through Rubisco contributes to carbon conversion efficiency through generation of 3-phosphoglycerate; and (iv) a larger contribution of amino acid carbon to fatty acid synthesis than in other oilseeds analyzed to date.