(210g) Understanding Nitrogen Metabolism in the Biofuel Crop Poplar by Isotope-Assisted Metabolic Flux Analysis

Nitrogen crucially determines the growth and productivity of photosynthetic organisms. However, it is often supplied by means of artificial fertilizers, whose production is energetically intensive and significantly relies on non-renewable fossil fuels. Therefore, the investigation of nitrogen use efficiency (NUE), particularly in plants that are potential biofuel crops, will have wide-ranging consequences for biofuel economy and sustainability. Poplar, a biofuel crop, uses an array of sophisticated metabolic and regulatory mechanisms to deposit, remobilize and thereby conserve nitrogen. The transport of nitrogen from senescing leaves to the bark storage protein (BSP) during winter is one such mechanism. This recycling of nitrogen is closely related to the status of carbon availability in plants as evidenced by previous studies which show that photoperiod perception, growth, presence of glutamine or calcium and protein phosphorylation affect accumulation of BSP (Zhu. B and Coleman. G.D., Plant Physiology 126:342-351, 2001). The involvement of multiple factors in regulation of BSP production suggests that significant metabolic rewiring occurs during nitrogen recycling.

To study this phenomenon we employed metabolic engineering tools such as steady state isotope-assisted metabolic flux analysis (MFA) to quantify metabolic fluxes in cells of the tree poplar (Populus trichocarpa). Metabolic fluxes are the rates of carbon flow through metabolic pathways (measured in mol time^-1 g biomass^-1) and are important indicators of cellular functions (Stephanopoulos, G. & Stafford, D.E., Chemical Engineering Science 57: 2595-2602, 2002). MFA involves conducting isotope labeling experiments (ILEs) wherein isotopically labeled (e.g. ¹³C, ¹⁵N, ¹⁷O) substrates are fed to the plant cell culture or tissue and the labeling patterns of biomass components such as proteins, lipids, sugars and nucleotides, are measured using nuclear magnetic resonance (NMR) or mass spectrometry (MS). Fluxes are estimated by fitting the labeling patterns to a mathematical model of the metabolic network.

Poplar suspension cells, subjected to environmental or genetic perturbations that affect BSP metabolism, will be grown under 100% 1-¹³C and 30% U-¹³C glucose in separate ILEs. Comparative metabolic flux maps for poplar cells under the various treatments will be constructed to evaluate the changes in metabolism during BSP accumulation and N recycling. The differences in fluxes between conditions and their implications on cell physiology will be discussed. The results of our MFA experiments are anticipated to reveal the metabolic rewiring that occurs in poplar cells when they deposit and remobilize nitrogen, and eventually provide insights toward engineering photosynthetic organisms with high NUE.