¹³ C-Metabolic flux analysis (¹³ C-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 ¹³ C-positional labelling information in glucose, malate and glutamate (Glu). A map of electron impact (EI)-mediated MS fragmentation was created and validated by ¹³ C-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 ¹³ C-MFA data from leaves and guard cells, and ¹³ C-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 ¹³ C-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 ¹³ C-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.

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