Concurrent isotope-assisted metabolic flux analysis and transcriptome profiling reveal responses of poplar cells to altered nitrogen and carbon supply

Reduced nitrogen is indispensable to plants. However, its limited availability in soil combined with the energetic and environmental impacts of nitrogen fertilizers motivates research into molecular mechanisms toward improving plant nitrogen use efficiency (NUE). We performed a systems-level investigation of this problem by employing multiple 'omics methodologies on cell suspensions of hybrid poplar (Populus tremula x Populus alba). Acclimation and growth of the cell suspensions in four nutrient regimes ranging from abundant to deficient supplies of carbon and nitrogen revealed that cell growth under low-nitrogen levels was associated with substantially higher NUE. To investigate the underlying metabolic and molecular mechanisms, we concurrently performed steady-state C-13 metabolic flux analysis with multiple isotope labels and transcriptomic profiling with cDNA microarrays. The C-13 flux analysis revealed that the absolute flux through the oxidative pentose phosphate pathway (oxPPP) was substantially lower (similar to threefold) under low-nitrogen conditions. Additionally, the flux partitioning ratio between the tricarboxylic acid cycle and anaplerotic pathways varied from 84%:16% under abundant carbon and nitrogen to 55%:45% under deficient carbon and nitrogen. Gene expression data, together with the flux results, suggested a plastidic localization of the oxPPP as well as transcriptional regulation of certain metabolic branchpoints, including those between glycolysis and the oxPPP. The transcriptome data also indicated that NUE-improving mechanisms may involve a redirection of excess carbon to aromatic metabolic pathways and extensive downregulation of potentially redundant genes (in these heterotrophic cells) that encode photosynthetic and light-harvesting proteins, suggesting the recruitment of these proteins as nitrogen sinks in nitrogen-abundant conditions.