Network flux analysis of central metabolism in plants

Thesis

The aim of this thesis was to develop stable-isotope steady-state metabolic flux analysis (MFA) based on ¹³C labeling to quantify intracellular fluxes of central carbon metabolism in plants. The experiments focus on the analysis of a heterotrophic cell suspension culture of Arabidopsis thaliana (L) Heynh. (ecotype Landsberg erecta). The first objective was to develop a robust methodology based on combining high quality steady-state stable labeling data, metabolic modeling and computational analysis. A comprehensive analysis of the factors that influence the outcome of MFA was undertaken and best practice established. This allowed a critical analysis of the subcellular compartmentation of carbohydrate oxidation in the cell culture. The second objective was to apply the methodology to nutritional perturbations of the cell suspension. A comparison of growth on different nitrogen sources revealed that transfer to an ammonium-free medium: (i) increased flux through the oxidative pentose phosphate pathway (oxPPP) by 10% relative to glucose utilisation; (ii) caused a substantial decrease in entry of carbon into the tricarboxylic acid cycle (TCA); and (iii) increased the carbon conversion efficiency from 55% to 69%. Although growth on nitrate alone might be expected to increase the demand for reductant, the cells responded by decreasing the assimilation of inorganic N. Cells were also grown in media containing different levels of inorganic phosphate (Pi). Comparison of the flux maps showed that decreasing Pi availability: (i) decreased flux through the oxPPP; (ii) increased the proportion of substrate fully oxidised by the TCA cycle; and (iii) decreased carbon conversion efficiency. These changes are consistent with redirection of metabolism away from biosynthesis towards cell maintenance as Pi is depleted. Although published genome-wide transcriptomic and metabolomic studies suggest that Pi starvation leads to the restructuring of carbon and nitrogen metabolism, the current analysis suggests that the impact on metabolic organisation is much less extreme.

Authors: Masakapalli, S

• Publication date: 2011
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: Oxford University, UK
• Language: English
• Keywords: radiorespirometric; steady state flux analysis; isotopic steady state; flux maps; 13C NMR; 14N NMR; metabolic flux analysis in plants; metabolic networks; Arabidopsis thaliana; pool correction; phosphate limitation; central carbon metabolism; nitrogen source; systems biology; phosphorus starvation; 13C MFA; mathematical modeling; nitrate; metabolic modeling; subcellular compartmentation; stable isotope labelling; suspension cultures; Pi levels; GC-MS; carbon conversion efficiency; carbohydrate oxidation
• Subjects: Systems Biology; Plant Sciences; Biochemistry; Plant metabolism; Computational modeling; Molecular Plant Physiology; Life Sciences