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

Abstract

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 Cpositionally 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-HCO 3 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 PEPcmediated CO 2 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.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening

“…That said, cytosolic MDH4 and especially PEPc were highly abundant in Arabidopsis GCs and present in larger amounts compared to MCs (Fig. 5h ), in agreement with the overall idea that anaplerotic CO 2 fixation is the main pathway of carbon assimilation in GCs 17 , 50 , 52 , 57 , 66 . For example, early 14 C-labelling experiments showed that CO 2 was fixed into Mal 2– at high rates both in the dark and the light in GCs, whereas in MCs, 14 C was enriched in 3-PGA and sucrose in the light and Mal 2– only in the dark 50 .…”

“…For example, early 14 C-labelling experiments showed that CO 2 was fixed into Mal 2– at high rates both in the dark and the light in GCs, whereas in MCs, 14 C was enriched in 3-PGA and sucrose in the light and Mal 2– only in the dark 50 . Furthermore, recent 13 C-feeding experiments demonstrated a faster 13 C enrichment in Mal 2– in isolated epidermal fragments compared with the whole rosette of Arabidopsis 17 , 52 , 67 . Fixation of CO 2 via PEP carboxylation ultimately leads to the formation of Mal 2– , further highlighting the central role of this metabolite for GCs.…”

“…Besides energy, starch biosynthesis requires the supply of carbohydrate precursors. There are three possible carbon sources for GC starch synthesis: photoassimilates from GC photosynthesis, sugars imported from the mesophyll, or organic acids previously accumulated within the GCs, which can be converted back to starch via gluconeogenesis 33 , 34 , 49 – 52 . While it is well established that the coordinated actions of the photosystems and CBB cycle deliver the precursors required for starch biosynthesis in MCCs 53 , it is unclear to what extent the CBB cycle is active in GCCs and what its relative contribution is to the pool of accumulated starch 33 , 34 .…”

Simultaneous adjustments of major mitochondrial pathways through redox regulation of dihydrolipoamide dehydrogenase (mtLPD1)

“…Such discrepancies are, however, best explained by the different subcellular localization of both TRXs and the specific enzyme targets of TRX o1 such as SDH and fumarase (Daloso et al, 2015; Hou et al, 2021). The direct impact on both enzymes was discussed to support the regulation of the different flux modes of the TCA-cycle and its inactivation in the light in addition to PDH regulation (Sweetlove et al, 2010; Araújo et al, 2011; Daloso et al, 2015; Lima et al, 2021). Interestingly, the latter statement is further supported by our metabolite analysis since pyruvate significantly accumulates in both TRX single mutants on a short-term (Fig.…”

Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments

“…Since their inception, isotopic labeling experiments (ILEs) have been applied for various purposes, such as pathway elucidation, model validation, and determination of intracellular flux rates in a host of organisms from bacteria [ 1 ] to eukaryotes [ 2 , 3 ], plants [ 4 – 6 ] and mammalian cell lines [ 7 , 8 ]. Thus, they have contributed significantly to strain characterization, screening and rational strain development.…”