Dethloff Frederik, Orf Isabel, Kopka Joachim
Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Kraepelinstr. 2-10, 80804 Munich, Germany.
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
Plant Methods. 2017 Oct 18;13:87. doi: 10.1186/s13007-017-0239-6. eCollection 2017.
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 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 C labeled carbon. As a test case, we use uniformly labeled C-sucrose.
We present petiole and hypocotyl feeding assays for the rapid in situ feeding (≤ 4 h) of isotopically labeled metabolic precursor to whole rosettes. The assays are assessed by conventional gas chromatography-mass spectrometry based metabolite profiling that was extended by joined differential analysis of C-labeled sub-pools and of C enrichment of metabolites relative to the enrichment of C-sucrose within each sample. We apply these analyses to the sink to source transition continuum of leaves from single 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.
传统代谢组学方法面临隐藏代谢表型的问题，即仅通量发生改变而代谢物池大小保持不变。代谢通量实验用于检测此类隐藏的通量表型。然而，这些实验耗时、成本可能很高，并且需要专家进行建模。我们填补了传统代谢组学与通量建模之间的空白。我们提出了快速稳定同位素示踪测定法和碳标记数据的分析策略。为此，我们将检测代谢物池大小显著相对变化的传统代谢组学方法与对碳标记碳的差异利用分析相结合。作为测试案例，我们使用均匀标记的碳 - 蔗糖。
我们提出了叶柄和下胚轴饲喂测定法，用于将同位素标记的代谢前体快速原位饲喂（≤4小时）到整个莲座叶丛中。通过基于传统气相色谱 - 质谱的代谢物谱分析对这些测定法进行评估，该分析通过对碳标记子池以及每个样品中代谢物相对于碳 - 蔗糖富集的碳富集进行联合差异分析得到扩展。我们将这些分析应用于单个莲座叶丛叶片从库到源转变的连续过程，并表征代谢物池的相关相对变化以及蔗糖衍生碳分配的先前隐藏变化。我们比较了蔗糖作为碳源在主要为库叶到主要为源叶中的贡献，并确定了一组在库到源转变期间具有不同碳利用的初级代谢物。
所提出的饲喂测定法和数据评估策略代表了一个快速且易于使用的工具包，用于增强代谢组学研究，该研究将差异池大小分析与从定义的稳定同位素标记代谢前体筛选差异碳利用相结合。
