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

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.

Toward the apoplast metabolome: Establishing a leaf apoplast collection approach suitable for metabolomics analysis.

The leaf apoplast contains several compounds that play important roles in the regulation of different physiological processes in plants. However, this compartment has been neglected in several experimental and modelling studies, which is mostly associated to the difficulty to collect apoplast washing fluid (AWF) in sufficient amount for metabolomics analysis and as free as possible from symplastic contamination. Here, we established an approach based in an infiltration-centrifugation technique that use little leaf material but allows sufficient AWF collection for gas chromatography mass spectrometry (GC-MS)-based metabolomics analysis in both tobacco and Arabidopsis. Up to 54 metabolites were annotated in leaf and apoplast samples from both species using either 20% (v/v) methanol (20% MeOH) or distilled deionized water (ddH2O) as infiltration fluids. The use of 20% MeOH increased the yield of the AWF collected but also the level of symplastic contamination, especially in Arabidopsis. We propose a correction factor and recommend the use of multiple markers such as MDH activity, protein content and conductivity measurements to verify the level of symplastic contamination in MeOH-based protocols. Neither the concentration of sugars nor the level of primary metabolites differed between apoplast samples extracted with ddH2O or 20% MeOH. This indicates that ddH2O can be preferentially used, given that it is a non-toxic and highly accessible infiltration fluid. The infiltration-centrifugation-based approach established here uses little leaf material and ddH2O as infiltration fluid, being suitable for GC-MS-based metabolomics analysis in tobacco and Arabidopsis, with great possibility to be extended for other plant species and tissues.

Unveiling the dark side of guard cell metabolism.

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a13C-HCO3 labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells.