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

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 intensiv...

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Autores principales: Dethloff, Frederik, Orf, Isabel, Kopka, Joachim
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648436/
https://www.ncbi.nlm.nih.gov/pubmed/29075313
http://dx.doi.org/10.1186/s13007-017-0239-6
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author Dethloff, Frederik
Orf, Isabel
Kopka, Joachim
author_facet Dethloff, Frederik
Orf, Isabel
Kopka, Joachim
author_sort Dethloff, Frederik
collection PubMed
description 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 (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. 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 (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. 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-017-0239-6) contains supplementary material, which is available to authorized users.
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spelling pubmed-56484362017-10-26 Rapid in situ (13)C tracing of sucrose utilization in Arabidopsis sink and source leaves Dethloff, Frederik Orf, Isabel Kopka, Joachim Plant Methods Research 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 (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. 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 (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. 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-017-0239-6) contains supplementary material, which is available to authorized users. BioMed Central 2017-10-18 /pmc/articles/PMC5648436/ /pubmed/29075313 http://dx.doi.org/10.1186/s13007-017-0239-6 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Dethloff, Frederik
Orf, Isabel
Kopka, Joachim
Rapid in situ (13)C tracing of sucrose utilization in Arabidopsis sink and source leaves
title Rapid in situ (13)C tracing of sucrose utilization in Arabidopsis sink and source leaves
title_full Rapid in situ (13)C tracing of sucrose utilization in Arabidopsis sink and source leaves
title_fullStr Rapid in situ (13)C tracing of sucrose utilization in Arabidopsis sink and source leaves
title_full_unstemmed Rapid in situ (13)C tracing of sucrose utilization in Arabidopsis sink and source leaves
title_short Rapid in situ (13)C tracing of sucrose utilization in Arabidopsis sink and source leaves
title_sort rapid in situ (13)c tracing of sucrose utilization in arabidopsis sink and source leaves
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648436/
https://www.ncbi.nlm.nih.gov/pubmed/29075313
http://dx.doi.org/10.1186/s13007-017-0239-6
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