Rapid in situ 13C tracing of sucrose utilization in Arabidopsis sink and source leaves

Frederik Dethloff, Isabel Orf, Joachim Kopka

Conventional metabolomics approaches face the problem of hidden metabolic phenotypes where only fluxes are altered but pool sizes stay constant. Metabolic flux experiments are used to detect such hidden flux phenotypes. These experiments are, however, time consuming, may be cost intensive, and involve specialists for modeling. We fill the gap between conventional metabolomics and flux modeling. We present rapid stable isotope tracing assays and analysis strategies of (13)C labeling data. For this purpose, we combine the conventional metabolomics approach that detects significant relative changes of metabolite pool sizes with analyses of differential utilization of (13)C labeled carbon. As a test case, we use uniformly labeled (13)C-sucrose. We present petiole and hypocotyl feeding assays for the rapid in situ feeding (≤ 4 h) of isotopically labeled metabolic precursor to whole Arabidopsis thaliana rosettes. The assays are assessed by conventional gas chromatography-mass spectrometry based metabolite profiling that was extended by joined differential analysis of (13)C-labeled sub-pools and of (13)C enrichment of metabolites relative to the enrichment of (13)C-sucrose within each sample. We apply these analyses to the sink to source transition continuum of leaves from single A. thaliana rosettes and characterize the associated relative changes of metabolite pools, as well as previously hidden changes of sucrose-derived carbon partitioning. We compared the contribution of sucrose as a carbon source in predominantly sink to predominantly source leaves and identified a set of primary metabolites with differential carbon utilization during sink to source transition. The presented feeding assays and data evaluation strategies represent a rapid and easy-to-use tool box for enhanced metabolomics studies that combine differential pool size analysis with screening for differential carbon utilization from defined stable isotope labeled metabolic precursors.