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.

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.

Changes in intracellular NAD status affect stomatal development in an abscisic acid-dependent manner.

Nicotinamide adenine dinucleotide (NAD) plays a central role in redox metabolism in all domains of life. Additional roles in regulating posttranslational protein modifications and cell signaling implicate NAD as a potential integrator of central metabolism and programs regulating stress responses and development. Here we found that NAD negatively impacts stomatal development in cotyledons of Arabidopsis thaliana. Plants with reduced capacity for NAD+ transport from the cytosol into the mitochondria or the peroxisomes exhibited reduced numbers of stomatal lineage cells and reduced stomatal density. Cotyledons of plants with reduced NAD+ breakdown capacity and NAD+ -treated cotyledons also presented reduced stomatal number. Expression of stomatal lineage-related genes was repressed in plants with reduced expression of NAD+ transporters as well as in plants treated with NAD+ . Impaired NAD+ transport was further associated with an induction of abscisic acid (ABA)-responsive genes. Inhibition of ABA synthesis rescued the stomatal phenotype in mutants deficient in intracellular NAD+ transport, whereas exogenous NAD+ feeding of aba-2 and ost1 seedlings, impaired in ABA synthesis and ABA signaling, respectively, did not impact stomatal number, placing NAD upstream of ABA. Additionally, in vivo measurement of ABA dynamics in seedlings of an ABA-specific optogenetic reporter - ABAleon2.1 - treated with NAD+ showed increases in ABA content suggesting that NAD+ impacts on stomatal development through ABA synthesis and signaling. Our results demonstrate that intracellular NAD+ homeostasis as set by synthesis, breakdown and transport is essential for normal stomatal development, and provide a link between central metabolism, hormone signaling and developmental plasticity.

Hydrogen Sulfide Increases Production of NADPH Oxidase-Dependent Hydrogen Peroxide and Phospholipase D-Derived Phosphatidic Acid in Guard Cell Signaling.

Hydrogen sulfide (H2S) is an important gaseous signaling molecule in plants that participates in stress responses and development. l-Cys desulfhydrase 1, one of the enzymatic sources of H2S in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of H2S in stomatal closure and the interplay between H2S and other second messengers of the guard cell signaling network, such as hydrogen peroxide (H2O2) and phospholipase D (PLD)-derived phosphatidic acid in Arabidopsis (Arabidopsis thaliana). Both NADPH oxidase isoforms, respiratory burst oxidase homolog (RBOH)D and RBOHF, were required for H2S-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that H2S stimulates H2O2 production in Arabidopsis guard cells. Additionally, we observed an interplay between H2S and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for H2S-induced stomatal closure, and most of the H2S-dependent H2O2 production required the activity of PLDα1. Finally, we showed that H2S induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of H2S in the signaling network that controls stomatal closure, and suggest that H2S regulates NADPH oxidase and PLD activity in guard cells.