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 within the specialized guard cells of plants.

Contents 1018 I. 1018 II. 1019 III. 1022 IV. 1025 V. 1026 VI. 1029 1030 References 1030 SUMMARY: Stomata are leaf epidermal structures consisting of two guard cells surrounding a pore. Changes in the aperture of this pore regulate plant water-use efficiency, defined as gain of C by photosynthesis per leaf water transpired. Stomatal aperture is actively regulated by reversible changes in guard cell osmolyte content. Despite the fact that guard cells can photosynthesize on their own, the accumulation of mesophyll-derived metabolites can seemingly act as signals which contribute to the regulation of stomatal movement. It has been shown that malate can act as a signalling molecule and a counter-ion of potassium, a well-established osmolyte that accumulates in the vacuole of guard cells during stomatal opening. By contrast, their efflux from guard cells is an important mechanism during stomatal closure. It has been hypothesized that the breakdown of starch, sucrose and lipids is an important mechanism during stomatal opening, which may be related to ATP production through glycolysis and mitochondrial metabolism, and/or accumulation of osmolytes such as sugars and malate. However, experimental evidence supporting this theory is lacking. Here we highlight the particularities of guard cell metabolism and discuss this in the context of the guard cells themselves and their interaction with the mesophyll cells.

Roles of sucrose in guard cell regulation.

The control of stomatal aperture involves reversible changes in the concentration of osmolytes in guard cells. Sucrose has long been proposed to have an osmolytic role in guard cells. However, direct evidence for such a role is lacking. Furthermore, recent evidence suggests that sucrose may perform additional roles in guard cells. Here, we provide an update covering the multiple roles of sucrose in guard cell regulation, highlighting the knowledge accumulated regarding spatiotemporal differences in the synthesis, accumulation, and degradation of sucrose as well as reviewing the role of sucrose as a metabolic connector between mesophyll and guard cells. Analysis of transcriptomic data from previous studies reveals that several genes encoding sucrose and hexose transporters and genes involved in gluconeogenesis, sucrose and trehalose metabolism are highly expressed in guard cells compared with mesophyll cells. Interestingly, this analysis also showed that guard cells have considerably higher expression of C4 -marker genes than mesophyll cells. We discuss the possible roles of these genes in guard cell function and the role of sucrose in stomatal opening and closure. Finally, we provide a perspective for future experiments which are required to fill gaps in our understanding of both guard cell metabolism and stomatal regulation.

Klotho increases antioxidant defenses in astrocytes and ubiquitin-proteasome activity in neurons.

Klotho is an antiaging protein, and its levels decline with age and chronic stress. The exogenous administration of Klotho can enhance cognitive performance in mice and negatively modulate the Insulin/IGF1/PI3K/AKT pathway in terms of metabolism. In humans, insulin sensitivity is a hallmark of healthy longevity. Therefore, this study aimed to determine if exogenous Klotho, when added to neuronal and astrocytic cell cultures, could reduce the phosphorylation levels of certain insulin signaling effectors and enhance antioxidant strategies in these cells. Primary cell cultures of cortical astrocytes and neurons from mice were exposed to 1 nM Klotho for 24 h, with or without glucose. Klotho decreased pAKT and mTOR levels. However, in astrocytes, Klotho increased FOXO-3a activity and catalase levels, shielding them from intermediate oxidative stress. In neurons, Klotho did not alter FOXO-3 phosphorylation levels but increased proteasome activity, maintaining lower levels of PFKFB3. This study offers new insights into the roles of Klotho in regulating energy metabolism and the redox state in the brain.

Feedback regulation by trehalose 6-phosphate slows down starch mobilization below the rate that would exhaust starch reserves at dawn in Arabidopsis leaves.

Trehalose 6-phosphate (Tre6P), a sucrose signaling metabolite, inhibits transitory starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves and potentially links starch turnover to leaf sucrose status and demand from sink organs (Plant Physiology, 163, 2013, 1142). To investigate this relationship further, we compared diel patterns of starch turnover in ethanol-inducible Tre6P synthase (iTPS) lines, which have high Tre6P and low sucrose after induction, with those in sweet11;12 sucrose export mutants, which accumulate sucrose in their leaves and were predicted to have high Tre6P. Short-term changes in irradiance were used to investigate whether the strength of inhibition by Tre6P depends on starch levels. sweet11;12 mutants had twofold higher levels of Tre6P and restricted starch mobilization. The relationship between Tre6P and starch mobilization was recapitulated in iTPS lines, pointing to a dominant role for Tre6P in feedback regulation of starch mobilization. Tre6P restricted mobilization across a wide range of conditions. However, there was no correlation between the level of Tre6P and the absolute rate of starch mobilization. Rather, Tre6P depressed the rate of mobilization below that required to exhaust starch at dawn, leading to incomplete use of starch. It is discussed how Tre6P interacts with the clock to set the rate of starch mobilization.

Stainless steel and polyethylene surfaces functionalized with silver nanoparticles.

The antimicrobial effects of a stainless steel surface and a polyethylene surface functionalized with silver nanoparticles on the adhesion of different bacteria and the changes in physical and chemical characteristics of these surfaces that influence biofilm formation were evaluated. The functionalized surfaces of polyethylene and stainless steel were more hydrophobic than the control ones. The bacterial surfaces were hydrophilic. The adhesion of all bacteria was thermodynamically favorable (ΔGadhesion<0) on all surfaces functionalized and control. The numbers of adhered cells of Staphylococcus aureus, Escherichia coli, and Pseudomonas fluorescens were not significantly different (p > 0.05) between the control and functionalized surfaces, reaching values compatible with biofilm formation. Analysis of atomic absorption spectrometry using water and reconstituted skim milk as simulants showed no release of Ag from the functionalized surfaces. In conclusion, the surfaces that were functionalized with silver nanoparticles were modified in hydrophobicity, roughness, and did not avoid bacterial adhesion. Additional studies of surfaces functionalized with silver nanoparticles should be conducted addressing the adsorption technique of silver nanoparticles on the stainless steel surface as well as in the preparation of the polyethylene surface to allow the contact of microorganism with the antimicrobial agent.