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  Establishment of a GC-MS-based 13C-positional isotopomer approach suitable for investigating metabolic fluxes in plant primary metabolism

Lima, V. F., Erban, A., Daubermann, A. G., Freire, F. B. S., Porto, N. P., Cândido-Sobrinho, S. A., et al. (2021). Establishment of a GC-MS-based 13C-positional isotopomer approach suitable for investigating metabolic fluxes in plant primary metabolism. The Plant Journal. doi:10.1111/tpj.15484.

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Genre: Journal Article
Alternative Title : The Plant Journal

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 Creators:
Lima, Valéria F.1, Author
Erban, A.2, Author              
Daubermann, André G.1, Author
Freire, Francisco Bruno S.1, Author
Porto, Nicole P.1, Author
Cândido-Sobrinho, Silvio A.1, Author
Medeiros, D.B.3, Author              
Schwarzländer, Markus1, Author
Fernie, A. R.3, Author              
dos Anjos, Leticia1, Author
Kopka, J.2, Author              
Daloso, Danilo M.1, Author
Affiliations:
1external, ou_persistent22              
2Applied Metabolome Analysis, Department Willmitzer, Max Planck Institute of Molecular Plant Physiology, Max Planck Society, ou_1753338              
3Central Metabolism, Department Willmitzer, Max Planck Institute of Molecular Plant Physiology, Max Planck Society, ou_1753339              

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Free keywords: 13C-metabolic flux analysis, gluconeogenesis, guard cells, metabolic regulation, PEPc, TCA cycle, isotopomer analysis
 Abstract: Summary 13C-Metabolic flux analysis (13C-MFA) has greatly contributed to our understanding of plant metabolic regulation. However, the generation of detailed in vivo flux maps remains a major challenge. Flux investigations based on nuclear magnetic resonance have resolved small networks with high accuracy. Mass spectrometry (MS) approaches have broader potential but have hitherto been limited in their power to deduce flux information due to lack of atomic level position information. Herein we established a gas chromatography (GC) coupled to MS-based approach that provides 13C-positional labelling information in glucose, malate and glutamate. A map of electron impact (EI)-mediated mass spectral fragmentation was created and validated by 13C-positionally labelled references via GC-EI-MS and GC-atmospheric pressure chemical ionization (APCI)-MS technologies. The power of the approach was revealed by analysing previous 13C-MFA data from leaves and guard cells and 13C-HCO3 labelling of guard cells harvested in the dark and after the dark-to-light transition. We demonstrated that the approach is applicable to established GC-EI-MS-based 13C-MFA without the need for experimental adjustment but will benefit in the future from paired analyses by the two GC-MS platforms. We identified specific glucose carbon atoms that are preferentially labelled by photosynthesis and gluconeogenesis and provide an approach to investigate the phosphoenolpyruvate carboxylase (PEPc)-derived 13C-incorporation into malate and glutamate. Our results suggest that gluconeogenesis and the PEPc-mediated CO2 assimilation into malate are activated in a light-independent manner in guard cells. We further highlight that the fluxes from glycolysis and PEPc toward glutamate are restricted by the mitochondrial thioredoxin system in illuminated leaves.

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Language(s): eng - English
 Dates: 2021-09-06
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Identifiers: DOI: 10.1111/tpj.15484
 Degree: -

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Title: The Plant Journal
  Other : Plant J.
Source Genre: Journal
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Publ. Info: Oxford : Blackwell Science
Pages: - Volume / Issue: - Sequence Number: - Start / End Page: - Identifier: ISSN: 0960-7412
CoNE: https://pure.mpg.de/cone/journals/resource/954925579095_1