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.

Metabolism-mediated mechanisms underpin the differential stomatal speediness regulation among ferns and angiosperms.

Recent results suggest that metabolism-mediated stomatal closure mechanisms are important to regulate differentially the stomatal speediness between ferns and angiosperms. However, evidence directly linking mesophyll metabolism and the slower stomatal conductance (gs ) in ferns is missing. Here, we investigated the effect of exogenous application of abscisic acid (ABA), sucrose and mannitol on stomatal kinetics and carried out a metabolic fingerprinting analysis of ferns and angiosperms leaves harvested throughout a diel course. Fern stomata did not respond to ABA in the time period analysed. No differences in the relative decrease in gs was observed between ferns and the angiosperm following provision of sucrose or mannitol. However, ferns have slower gs responses to these compounds than angiosperms. Metabolomics analysis highlights that ferns have a higher accumulation of secondary rather than primary metabolites throughout the diel course, with the opposite being observed in angiosperms. Our results indicate that metabolism-mediated stomatal closure mechanisms underpin the differential stomatal speediness regulation among ferns and angiosperms, in which the slower stomatal closure in ferns is associated with the lack of ABA-responsiveness, to a reduced capacity to respond to mesophyll-derived sucrose and to a higher carbon allocation toward secondary metabolism, which likely modulates both photosynthesis-gs and growth-stress tolerance trade-offs.

The sucrose-to-malate ratio correlates with the faster CO2 and light stomatal responses of angiosperms compared to ferns.

Stomatal responses to environmental signals differ substantially between ferns and angiosperms. However, the mechanisms that lead to such different responses remain unclear. Here we investigated the extent to which leaf metabolism contributes to coordinate the differential stomatal behaviour among ferns and angiosperms. Stomata from all species were responsive to light and CO2 transitions. However, fern stomatal responses were slower and minor in both absolute and relative terms. Angiosperms have higher stomatal density, but this is not correlated with speed of stomatal closure. The metabolic responses throughout the diel course and under different CO2 conditions differ substantially among ferns and angiosperms. Higher sucrose content and an increased sucrose-to-malate ratio during high CO2 -induced stomatal closure was observed in angiosperms compared to ferns. Furthermore, the speed of stomatal closure was positively and negatively correlated with sugars and organic acids, respectively, suggesting that the balance between sugars and organic acids aids in explaining the faster stomatal responses of angiosperms. Our results suggest that mesophyll-derived metabolic signals, especially those associated with sucrose and malate, may also be important to modulate the differential stomatal behaviour between ferns and angiosperms, providing important new information that helps in understanding the metabolism-mediated mechanisms regulating stomatal movements across land plant evolution.

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.

Thioredoxins regulate the metabolic fluxes throughout the tricarboxylic acid cycle and associated pathways in a light-independent manner.

The metabolic fluxes throughout the tricarboxylic acid cycle (TCAC) are inhibited in the light by the mitochondrial thioredoxin (TRX) system. However, it is unclear how this system orchestrates the fluxes throughout the TCAC and associated pathways in the dark. Here we carried out a13C-HCO3 labelling experiment in Arabidopsis leaves from wild type (WT) and mutants lacking TRX o1 (trxo1), TRX h2 (trxh2), or both NADPH-dependent TRX reductase A and B (ntra ntrb) exposed to 0, 30 and 60 min of dark or light conditions. No 13C-enrichment in TCAC metabolites in illuminated WT leaves was observed. However, increased succinate content was found in parallel to reductions in Ala in the light, suggesting the latter operates as an alternative carbon source for succinate synthesis. By contrast to WT, all mutants showed substantial changes in the content and 13C-enrichment in TCAC metabolites under both dark and light conditions. Increased 13C-enrichment in glutamine in illuminated trxo1 leaves was also observed, strengthening the idea that TRX o1 restricts in vivo carbon fluxes from glycolysis and the TCAC to glutamine. We further demonstrated that both photosynthetic and gluconeogenic fluxes toward glucose are increased in trxo1 and that the phosphoenolpyruvate carboxylase (PEPc)-mediated 13C-incorporation into malate is higher in trxh2 mutants, as compared to WT. Our results collectively provide evidence that TRX h2 and the mitochondrial NTR/TRX system regulate the metabolic fluxes throughout the TCAC and associated pathways, including glycolysis, gluconeogenesis and the synthesis of glutamine in a light-independent manner.