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

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. RESULTS: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 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. CONCLUSION: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.

SUBMITTER: Dethloff F

PROVIDER: S-EPMC5648436 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Rapid in situ <sup>13</sup>C tracing of sucrose utilization in Arabidopsis sink and source leaves.

Dethloff Frederik F Orf Isabel I Kopka Joachim J

Plant methods 20171018

<h4>Background</h4>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 <sup>13</ ...[more]

PMID: 29075313

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Project description:BACKGROUND AND AIMS: In crops other than sugarcane there is good evidence that the size and activity of carbon sinks influence source activity via sugar-related regulation of the enzymes of photosynthesis, an effect that is partly mediated through coarse regulation of gene expression. METHODS: In the current study, leaf shading treatments were used to perturb the source-sink balance in 12-month-old Saccharum spp. hybrid 'N19' (N19) by restricting source activity to a single mature leaf. Changes in leaf photosynthetic gas exchange variables and leaf and culm sugar concentrations were subsequently measured over a 14 d period. In addition, the changes in leaf gene response to the source-sink perturbation were measured by reverse northern hybridization analysis of an array of 128 expressed sequence tags (ESTs) related to photosynthetic and carbohydrate metabolism. KEY RESULTS: Sucrose concentrations in immature culm tissue declined significantly over the duration of the shading treatment, while a 57 and 88% increase in the assimilation rate (A) and electron transport rate (ETR), respectively, was observed in the source leaf. Several genes (27) in the leaf displayed a >2-fold change in expression level, including the upregulation of several genes associated with C(4) photosynthesis, mitochondrial metabolism and sugar transport. Changes in gene expression levels of several genes, including Rubisco (EC 4.1.1.39) and hexokinase (HXK; EC 2.7.1.1), correlated with changes in photosynthesis and tissue sugar concentrations that occurred subsequent to the source-sink perturbation. CONCLUSIONS: These results are consistent with the notion that sink demand may limit source activity through a kinase-mediated sugar signalling mechanism that correlates to a decrease in source hexose concentrations, which, in turn, correlate with increased expression of genes involved in photosynthesis and metabolite transport. The signal feedback system reporting sink sufficiency and regulating source activity may be a potentially valuable target for future genetic manipulation to increase sugarcane sucrose yield.

Dataset Information

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

Publications

Rapid in situ <sup>13</sup>C tracing of sucrose utilization in Arabidopsis sink and source leaves.

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