-Action potential (AP) of excitable plant cells is an important signaling event that can differentially alter physicochemical and physiological processes in various parts of the same cell. In giant cells of characean algae, the AP propagation has minor effect on photosynthetic electron transport in areas with high activity of plasmalemmal H+-pump but inhibits linear electron flow in regions featuring high passive H+/OH- conductance of the plasma membrane (PM). Uneven spatial distributions of local periplasmic and cytoplasmic pH facilitate the operation of distinct (CO2-dependent and O2-mediated) pathways of photoinduced electron flow, which presumably accounts for differential influence of AP on photosynthesis. The excitation of Chara australis cell in the presence of methyl viologen (MV), a redox mediator with the prooxidant action, provides a convenient model system to clarify the influence of voltage-dependent ion fluxes across PM on photosynthetic activity of chloroplasts. This study shows that permeation of MV to their target sites in chloroplasts is restricted by PM in resting cells, but MV easily passes through ionic channels opened during the PM depolarization. This gated permeation of MV gives rise to strong non-photochemical quenching, decrease in the effective quantum yield of linear electron flow, apparent O2 uptake, and, finally, the enhanced ROS production, as detected by the fluorescent probe dichlorofluorescein. Taken together, the results indicate that the AP generation in the presence of MV acts as trigger for instant redirection of photosynthetic linear electron flow from CO2-dependent route to the path of O2 reduction with the eventual formation of H2O2 as a dominant and most stable ROS form.

Under nitrogen deprivation (-N), cyanobacterium Synechocystis sp. PCC 6803 exhibits growth arrest, reduced protein content, and remarkably increased glycogen accumulation. However, producing glycogen under this condition requires a two-step process with cell transfer from normal to -N medium. Metabolic engineering and chemical treatment for rapid glycogen accumulation can bypass the need for two-step cultivation. For example, recent studies indicate that individually disrupting hydrogen (H2) or poly(3-hydroxybutyrate) (PHB) synthesis, or treatment with methyl viologen (MV), effectively increases glycogen accumulation in Synechocystis. Here we explore the effects of disrupted H2 or poly(3-hydroxybutyrate) synthesis, together with MV treatment to on enhanced glycogen accumulation in Synechocystis grown in normal medium. Wild-type cells without MV treatment exhibited low glycogen content of less than 6% w/w dry weight (DW). Compared with wild type, disrupting PHB synthesis combined with MV treatment did not increase glycogen content. Disrupted H₂ production without MV treatment yielded up to 11% w/w DW glycogen content. Interestingly, when combined, disrupted H2 production with MV treatment synergistically enhanced glycogen accumulation to 51% and 59% w/w DW within 3 and 7 days, respectively. Metabolomic analysis suggests that MV treatment mediated the conversion of proteins into glycogen. Metabolomic and transcriptional-expression analysis suggests that disrupted H2 synthesis under MV treatment positively influenced glycogen synthesis. Disrupted H₂ synthesis under MV treatment significantly increased NADPH levels. This increased NADPH content potentially contributed to the observed enhancements in antioxidant activity against MV-induced oxidants, O2 evolution, and metabolite substrates levels for glycogen synthesis in normal medium, ultimately leading to enhanced glycogen accumulation in Synechocystis. KEY MESSAGE: Combining disrupted hydrogen-gas synthesis and the treatment by photosynthesis electron-transport inhibitor significantly enhance glycogen production in cyanobacteria.

The design and exploration of advanced materials as a durable multifunctional electrocatalyst toward sustainable energy generation and storage development is the most perdurable challenge in the domain of renewable energy research. Herein, a facile in situ solvothermal approach has been adopted to prepare a methylviologen-regulated crystalline metal phosphonate compound, [C12H14N2][Ni(C11H11N2)(H2hedp)2]2•6H2O (NIT1), (H4hedp = 1-hydroxyethane 1,1-diphosphonic acid) and well characterized by several techniques. The as-prepared NIT1 displays excellent bifunctional electrocatalytic activity with dynamic stability toward oxygen evolution reaction (η10 = 288 mV) and hydrogen evolution reaction (η10 = 228 mV) in alkaline (1.0 M KOH) and acidic mediums (0.5 M H2SO4), respectively. Such a low overpotential and Tafel slope (68 mV/dec for OER; 56 mV/dec for HER) along with long-term durability up to 20 h of NIT1 make it superior to benchmark the electrocatalyst and various nonprecious metal-based catalysts under similar experimental condition. Further, the electrochemical supercapacitor measurements (in three-electrode system) reveal that the NIT1 electrode possesses much higher specific capacity of 187.6 C g-1 at a current density of 2 A g-1 (272 C g-1 at 5 mV s-1) with capacitance retention of 75.2% over 10,000 cycles at 14 A g-1 (Coulombic efficiency > 99%) in 6 M KOH electrolyte medium. Finally for a practical application, an asymmetric supercapacitor device (coin cell) is assembled by NIT1 material. The as-fabricated device delivers the maximum energy density of 39.4 Wh kg-1 at a power density of 450 W kg-1 and achieves a wide voltage window of 1.80 V. Notably, the device endures a remarkable cycle performance with cyclic retention of 92% (Coulombic efficiency > 99%) even after 14,000 charge/discharge cycles at 10 A g-1. Nevertheless, the extraordinary electrochemical activities toward OER and HER as well as the high-performance device fabrication for LED illumination of such a noble metal-free lower-dimensional charge-transfer compound are truly path breaking and would be promising for the development of advanced multifunctional materials.

Bio-hydrogen production is an eco-friendly alternative to commercial H2 production, taking advantage of natural systems. Microbial hydrogenases play a main role in biological mechanisms, catalyzing proton reduction to molecular hydrogen (H2) formation under ambient conditions. Direct determination is an important approach to screen bacteria with active hydrogenase and accurately quantify the amount of H2 production. Here, we present a detailed protocol for determining hydrogenase activity based on H2 production using methyl viologen (MV2+) as an artificial reductant, directly monitored by gas chromatography. Recombinant Escherichia coli is used as a hydrogenase-enriched model in this study. Even so, this protocol can be applied to determine hydrogenase activity in all biological samples. Key features • This protocol is optimized for a wide variety of biological samples; both purified hydrogenase (in vitro) and intracellular hydrogenase (in vivo) systems. • Direct, quantitative, and accurate method to detect the amount of H2 by gas chromatography with reproducibility. • Requires only 2 h to complete and allows testing various conditions simultaneously. • Kinetic plot of H2 production allows to analyze kinetic parameters and estimate the efficiency of hydrogenase from different organisms.

The photosynthesis-induced accumulation of reactive oxygen species in chloroplasts can lead to oxidative stress, triggering changes in protein synthesis, degradation, and the assembly/disassembly of protein complexes. Using shot-gun proteomics, we identified methyl viologen-induced changes in protein abundance in wild-type Arabidopsis and oxidative stress-hypersensitive fsd1-1 and fsd1-2 knockout mutants, which are deficient in IRON SUPEROXIDE DISMUTASE 1 (FSD1). The levels of proteins that are localized in chloroplasts and the cytoplasm were modified in all lines treated with methyl viologen. Compared with the wild-type, fsd1 mutants showed significant changes in metabolic protein and chloroplast chaperone levels, together with increased ratio of cytoplasmic, peroxisomal, and mitochondrial proteins. Different responses in proteins involved in the disassembly of photosystem II-light harvesting chlorophyll a/b binding proteins were observed. Moreover, the abundance of PATELLIN 4, a phospholipid-binding protein enriched in stomatal lineage, was decreased in response to methyl viologen. Reverse genetic studies using patl4 knockout mutants and a PATELLIN 4 complemented line indicate that PATELLIN 4 affects plant responses to oxidative stress by effects on stomatal closure.

Time-dependent density functional theory (TDDFT) was applied to gain insights into the electronic and vibrational spectroscopic properties of an important electron transport mediator, methyl viologen (MV2+ ). An organic dication, MV2+ has numerous applications in electrochemistry that include energy conversion and storage, environmental remediation, and chemical sensing and electrosynthesis. MV2+ is easily reduced by a single electron transfer to form a radical cation species (MV•+ ), which has an intense UV-visible absorption near 600 nm. The redox properties of the MV2+ /MV•+ couple and light-sensitivity of MV•+ have made the system appealing for photo-electrochemical energy conversion (e.g., solar hydrogen generation from water) and the study of photo-induced charge transfer processes through electronic absorption and resonance Raman spectroscopic measurements. The reported work applies leading TDDFT approaches to investigate the electronic and vibrational spectroscopic properties of MV2+ and MV•+ . Using a conventional hybrid exchange functional (B3-LYP) and a long-range corrected hybrid exchange functional (ωB97X-D3), including with a conductor-like polarizable continuum model to account for solvation, the electronic absorption and resonance Raman spectra predicted are in good agreement with experiment. Also analyzed are the charge transfer character and natural transition orbitals derived from the TDDFT vertical excitations calculated. The findings and models developed further the understanding of the electronic properties of viologens and related organic redox mediators important in renewable energy applications and serve as a reference for guiding the interpretation of electronic absorption and Raman spectra of the ions.

Flavodiiron proteins Flv1/Flv3 accept electrons from photosystem (PS) I. In this work we investigated light adaptation mechanisms of Flv1-deficient mutant of Synechocystis PCC 6803, incapable to form the Flv1/Flv3 heterodimer. First seconds of dark-light transition were studied by parallel measurements of light-induced changes in chlorophyll fluorescence, P700 redox transformations, fluorescence emission at 77 K, and OCP-dependent fluorescence quenching. During the period of Calvin cycle activation upon dark-light transition, the linear electron transport (LET) in wild type is supported by the Flv1/Flv3 heterodimer, whereas in Δflv1 mutant activation of LET upon illumination is preceded by cyclic electron flow that maintains State 2. The State 2-State 1 transition and Orange Carotenoid Protein (OCP)-dependent non-photochemical quenching occur independently of each other, begin in about 10 s after the illumination of the cells and are accompanied by a short-term re-reduction of the PSI reaction center (P700+). ApcD is important for the State 2-State 1 transition in the Δflv1 mutant, but S-M rise in chlorophyll fluorescence was not completely inhibited in Δflv1/ΔapcD mutant. LET in Δflv1 mutant starts earlier than the S-M rise in chlorophyll fluorescence, and the oxidation of plastoquinol (PQH2) pool promotes the activation of PSII, transient re-reduction of P700+ and transition to State 1. An attempt to induce state transition in the wild type under high intensity light using methyl viologen, highly oxidizing P700 and PQH2, was unsuccessful, showing that oxidation of intersystem electron-transport carriers might be insufficient for the induction of State 2-State 1 transition in wild type of Synechocystis under high light.

Widely used in biomedical and bioanalytical applications, the detonation nanodiamonds (NDs) are generally considered to be biocompatible and non-toxic to a wide range of eukaryotic cells. Due to their high susceptibility to chemical modifications, surface functionalisation is often used to tune the biocompatibility and antioxidant activity of the NDs. The response of photosynthetic microorganisms to redox-active NDs is still poorly understood and is the focus of the present study. The green microalga Chlamydomonas reinhardtii was used to assess the potential phytotoxicity and antioxidant activity of NDs hosting hydroxyl functional groups at concentrations of 5-80 μg NDs/mL. The photosynthetic capacity of microalgae was assessed by measuring the maximum quantum yield of PSII photochemistry and the light-saturated oxygen evolution rate, while oxidative stress was assessed by lipid peroxidation and ferric-reducing antioxidant capacity. We demonstrated that hydroxylated NDs might reduce cellular levels of oxidative stress, protect PSII photochemistry and facilitate the PSII repair under methyl viologen and high light associated stress conditions. Factors involved in this protection may include the low phytotoxicity of hydroxylated NDs in microalgae and their ability to accumulate in cells and scavenge reactive oxygen species. Our findings could pave the way for using hydroxylated NDs as antioxidants to improve cellular stability in algae-based biotechnological applications or semi-artificial photosynthetic systems.

Butanol production by solventogenic Clostridia shows great potential to combat the energy crisis, but is still challenged by low butanol selectivity and high downstream cost. In this study, a novel cathodic electro-fermentation (CEF) system mediated by methyl viologen (MV) was proposed and sequentially optimized to obtain highly selective butanol production. Under the optimal conditions (-0.60 V cathode potential, 0.50 mM MV, 30 g/L glucose), 7.17 ± 0.55 g/L butanol production were achieved with the yield of 0.32 ± 0.02 g/g. With the supplement of 4 g/L butyric acid as co-substrate, butanol production further improved to 13.14 ± 1.14 g/L with butanol yield and selectivity as high as 0.43 ± 0.01 g/g and 90.44 ± 1.66%, respectively. The polarized electrode enabled the unbalanced fermentation towards butanol formation and MV further inhibited hydrogen production, both of which contributed to the high-level butanol production and selectivity. The MV-mediated CEF system is a promising approach for cost-effective bio-butanol production.

Gold nanoparticles-modified silver nanorod (AuNPs@AgNR) arrays were fabricated as surface-enhanced Raman spectroscopy (SERS) substrates. The coffee ring effect of the AuNPs@AgNR was explored as a preconcentration method for enriching the target analytes and increasing the \"hot spots.\" Furthermore, methyl viologen (MV) as a toxic herbicide used in agricultural production was successfully determined to investigate the application of the coffee ring effect on AuNPs@AgNR arrays and density functional theory (DFT) was employed to calculate its vibrational modes of corresponding characteristic peaks. Good linearity was obtained in the range 0.10-100 mg/L, and the limit of detection (LOD) of MV was estimated to be 0.01 mg/L, which was lower than the US maximum residue limits (MRLs). This method was also applied to practical detection of MV in river water and apple peel with LODs of 0.10 mg/L and 0.05 mg/L, respectively. SERS results suggest that the coffee ring on AuNPs@AgNR arrays provides a promising way for monitoring environmental pollution and food safety caused by pesticides.