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Research Project: Soybean Seed Improvement Through Quantitative Analysis of Phenotypic Diversity in Response to Environmental Fluctuations

Location: Plant Genetics Research

Title: Isotopically nonstationary 13C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation

item MA, FANGFANG - Danforth Plant Science Center
item JAZMIN, LARA - Vanderbilt University
item YOUNG, JAMEY - Vanderbilt University
item Allen, Douglas - Doug

Submitted to: Proceedings of the National Academy of Sciences(PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/9/2014
Publication Date: 11/25/2014
Publication URL:
Citation: Ma, F., Jazmin, L.J., Young, J.D., Allen, D.K. 2014. Isotopically nonstationary 13C flux analysis of changes in Arabidopsis thaliana leaf metabolism due to high light acclimation. Proceedings of the National Academy of Sciences. 111(47):16967-16972.

Interpretive Summary: Transformative gains in photosynthetic capacity, including the ability to fix carbon and produce biomass in plants are critical to feeding a growing population and to generate much-needed renewable chemical feedstocks for fuels, plastics and other common products currently made from fossil fuels. Quantitative Systems Biology methods such as isotopic labeling-based metabolic flux analysis (using chemical isotopes to track carbon and nitrogen into metabolites) provide a combined experimental and computational approach to assess and test metabolism, but are not sufficiently developed for plant systems. We developed computational and experimental methods for these analyses in plants and tested them by assessing the photosynthetic metabolism of Arabidopsis thaliana, a model plant species with a well-studied genome. We documented the flow of metabolites through cellular pathways in plant leaves exposed to different levels of light. This is the first time that 13C (isotopic) flux analysis has been successfully applied to map photosynthetically driven carbon flow through leaf metabolism in a terrestrial plant system. Our analysis revealed significant rerouting of photosynthetic carbon flux to sucrose biosynthetic pathways in response to increasing light intensity. We also observed an inverse relationship between metabolite pool sizes and pathway fluxes. This study establishes a comprehensive approach to map the flow and fate of carbon within plant metabolic networks. These tools can be leveraged to guide rational metabolic engineering of important crops like soybeans.

Technical Abstract: Improving plant productivity is an important aim for metabolic engineering. There are few comprehensive methods that quantitatively describe the primary metabolism of leaves, though such information would be valuable for improving photosynthetic capacity, increasing 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 13C labeling patterns of intracellular metabolites. However, experimental and computational difficulties have hindered its application to terrestrial plant systems. Having overcome these limitations, we performed in vivo isotopic labeling of Arabidopsis thaliana rosettes with 13CO2, measured the transient labeling of 30 metabolite fragment ions using mass spectrometry, and estimated fluxes throughout leaf photosynthetic metabolism by INST-MFA. Leaves were exposed to either 200 or 500 µmol m-2s-1 light, with or without prior acclimation. Approximately 1,200 independent mass isotopomer measurements were regressed to estimate 110 fluxes under each condition. The results suggest distinct carbon precursors for starch and sucrose biosynthesis and similar relative partitioning of 3-phosphoglycerate to ribulose-1,5-bisphosphate regeneration under all light conditions. Photorespiration flux increased from 12% to 36% of net CO2 assimilation with increasing light, despite concomitant increases in carboxylation flux that led to more sucrose production. Modeling indicated some exchange of hexose phosphate or neutral sugar across the chloroplast membrane though net carbon export was not required in acclimated conditions and may reflect the short-term response to light. Also, measured Calvin cycle intermediates were inversely tied to fluxes as light was increased.