Establishment of a GC‐MS‐based 13C‐positional isotopomer approach suitable for investigating metabolic fluxes in plant primary metabolism. (23rd September 2021)

• Record Type: Journal Article
• 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.; dos Anjos, Leticia; Kopka, Joachim; Daloso, Danilo M.
• Is Part Of: Plant journal. Volume 108:Number 4(2021)
• Page Start: 1213
• Page End: 1233
• Publication Date: 2021-09-23
• Subjects: 13C‐metabolic flux analysis -- gluconeogenesis -- guard cells -- metabolic regulation -- phosphoenolpyruvate cfrearboxylase -- tricarboxylic acid cycle -- isotopomer analysis; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.15484
• Languages: English
• ISSNs: 0960-7412

Abstract

SUMMARY: 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 phospho enol pyruvate 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. Significance Statement: We established a GC‐MS‐based 13 C‐labelling approach that provides 13 C‐derived positional information for glucose, malate and glutamate, thereby overcoming the main limitation of MS‐based 13 C‐metabolic flux analysis, namely the lack of positional 13 C‐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.