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

• Published in: The Plant journal : for cell and molecular biology 2021-11, Vol.108 (4), p.1213-1233
• Main Authors: Lima, Valéria F., Erban, Alexander, Daubermann, André G., Freire, Francisco Bruno S., Porto, Nicole P., Cândido‐Sobrinho, Silvio A., Medeiros, David B., Schwarzländer, Markus, Fernie, Alisdair R., Anjos, Leticia, Kopka, Joachim, Daloso, Danilo M.
• Format: Article
• Language: English
• Subjects: 13C‐metabolic flux analysis; Carbon dioxide; Electron impact; Fluctuations; Fluxes; Gas chromatography; Gluconeogenesis; Glucose; Glycolysis; Guard cells; In vivo methods and tests; Ionization; isotopomer analysis; Labeling; Leaves; Malate; Mass spectrometry; Mass spectroscopy; Metabolic flux; metabolic regulation; Metabolism; Mitochondria; NMR; Nuclear magnetic resonance; Phosphoenolpyruvate carboxylase; phosphoenolpyruvate cfrearboxylase; Photosynthesis; Thioredoxin; tricarboxylic acid cycle
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.15484

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 (Glu). A map of electron impact (EI)‐mediated MS fragmentation was created and validated by 13C‐positionally labelled references via GC‐EI‐MS and GC‐atmospheric pressure chemical ionization‐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 Glu. 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 Glu are restricted by the mitochondrial thioredoxin system in illuminated leaves. Significance Statement We established a GC‐MS‐based 13C‐labelling approach that provides 13C‐derived positional information for glucose, malate and glutamate, thereby overcoming the main limitation of MS‐based 13C‐metabolic flux analysis, namely the lack of positional 13C‐labelling information. We provide proof‐of‐concept that the approach is applicable to investigate both gluconeogenic metabolic fluxes and the contribution of the PEPc‐derived CO2 assimilation to the synthesis of malate and glutamate.