Abstract
Background 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 13C 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 13C labeled carbon. As a test case, we use uniformly labeled 13C-sucrose.ResultsWe 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 13C-labeled sub-pools and of 13C enrichment of metabolites relative to the enrichment of 13C-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.ConclusionThe 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.
Highlights
Conventional metabolomics approaches face the problem of hidden metabolic phenotypes where only fluxes are altered but pool sizes stay constant
The A. thaliana rosette might look homogeneous but, is a complex system that comprises leaves in different developmental stages. We investigated this system as a “proof of concept” study and demonstrate that metabolism can be quite different in single leaves across an A. thaliana rosette
We demonstrate that dissecting rosettes into single leaves or groups of leaves at similar developmental stages will generate more detailed physiological insights than the frequently used pools of whole rosettes
Summary
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, time consuming, may be cost intensive, and involve specialists for modeling. We present rapid stable isotope tracing assays and analysis strategies of 13C 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 13C labeled carbon. The most elegant method used in plant research on metabolic fluxes is the tracing of carbon isotopes using photosynthetic labeling. The perturbation of the plant is by far the lowest possible when using labeled C O2 as an entry point into the metabolism, e.g., [6]
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