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Mg2+, the most abundant divalent cation in living cells, plays a pivotal role in numerous enzymatic reactions and is of particular importance for organisms performing oxygenic photosynthesis. Its significance extends beyond serving as the central ion of the chlorophyll molecule, as it also acts as a counterion during the light reaction to balance the proton gradient across the thylakoid membranes. In this study, we investigated the effects of Mg2+ limitation on the physiology of the well-known model microorganism Synechocystis sp. PCC6803. Our findings reveal that Mg2+ deficiency triggers both morphological and functional changes. As seen in other oxygenic photosynthetic organisms, Mg2+ deficiency led to a decrease in cellular chlorophyll concentration. Moreover, the PSI-to-PSII ratio decreased, impacting the photosynthetic efficiency of the cell. In line with this, Mg2+ deficiency led to a change in the proton gradient built up across the thylakoid membrane upon illumination. [ABSTRACT FROM AUTHOR]

ABSTRACT Because of the stubborn resistance to antibiotics, treating clinically resistant bacteria is a tricky business. Although considerable attention has been devoted to preventing and treating infections from drug-resistant bacteria, few studies are available on combining metabolites and antibiotics to combat methicillin-resistant Staphylococcus aureus (MRSA). This study found that exogenous L-glutamine potentiated aminoglycoside (gentamicin)-mediated killing efficacy in a dose- and time-dependent manner in MRSA (USA300 cell line) and other Gram-positive-resistant bacteria including MRSA 252, Listeria monocytogenes, and Corynebacterium diphtheriae. L-glutamine promoted the uptake of gentamicin through increasing the membrane permeability and was correlated with disrupted pH gradient. Furthermore, L-glutamine decreased intracellular reactive oxygen species by glutathione, which also increased USA300 sensitivity to gentamicin. We also demonstrated that combined treatment with gentamicin and L-glutamine enhanced the survival of MRSA-infected mice. In conclusion, we developed a promising therapeutic strategy for treating Gram-positive-resistant bacterial infections. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) infection severely threatens human health due to high morbidity and mortality; it is urgent to develop novel strategies to tackle this problem. Metabolites belong to antibiotic adjuvants which improve the effect of antibiotics. Despite reports of L-glutamine being applied in antibiotic adjuvant for Gram-negative bacteria, how L-glutamine affects antibiotics against Gram-positive-resistant bacteria is still unclear. In this study, L-glutamine increases the antibacterial effect of gentamicin on MRSA, and it links to membrane permeability and pH gradient (ΔpH), resulting in uptake of more gentamicin. Of great interest, reduced reactive oxygen species (ROS) by glutathione was found under L-glutamine treatment; USA300 becomes sensitive again to gentamicin. This study not only offers deep understanding on ΔpH and ROS on bacterial resistance but also provides potential treatment solutions for targeting MRSA infection.

Hydrogen (H 2) metabolism in Escherichia coli occurs via reversible membrane-associated hydrogenase enzymes (Hyd). Hyd-3 and Hyd-4 with formate dehydrogenase H (FDH-H) form formate hydrogen lyase complexes. The changes of metabolic pathways and ΔpH (pH in -pH ex) regulation during fermentation of glucose, glycerol and formate in non H 2 -producing hypF (lack of all Hyds) and fdhF (lack of FDH-H) mutants at pH 7.5 were investigated. It was shown that specific growth rate was higher by ∼23% in hypF and fdhF , compared to wild type (wt), suggesting the negative effect of H 2 on bacterial growth. Moreover, it was shown that H 2 generation did not have a vital role in glucose and glycerol utilization rate at 0–72 h. The utilization of external formate was detected in wt (∼2.6 mM) and hypF (∼0.68 mM), but not in fdhF, due to the absence of enzyme responsible for formate metabolism. Nevertheless, the changes in ΔpH were not evident at 3 h. The ratio of generated end-products and regulation of ΔpH at late log (6 h) and exponential phase (24, 72 h) were various in hypF and fdhF due to formate disproportionation in hypF and proton generation, therewith absence of H 2 generation. Taken together it can be concluded that bacteria regulate generation of fermentation end-products via balancing the concentration of acids and ethanol to maintain ΔpH and redox potential values. The results obtained are important for development and regulation of H 2 production technology when applying mixed carbon sources. • Cell specific growth rate was higher by ∼23% in hypF and fdhF compared to wild type. • H 2 generation is not determinant in glucose and glycerol utilization rate at 0–72 h. • External formate was utilized in wild type and hypF but not in fdhF mutant. • Regulation of ΔpH at 6 h and 24 or 72 h was different in hypF and fdhF mutants. [ABSTRACT FROM AUTHOR]

The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin–Benson–Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants. [ABSTRACT FROM AUTHOR]

The pH of various chloroplast compartments, such as the thylakoid lumen and stroma, is light-dependent. Light illumination induces electron transfer in the photosynthetic apparatus, coupled with proton translocation across the thylakoid membranes, resulting in acidification and alkalization of the thylakoid lumen and stroma, respectively. Luminal acidification is crucial for inducing regulatory mechanisms that protect photosystems against photodamage caused by the overproduction of reactive oxygen species (ROS). Stromal alkalization activates enzymes involved in the Calvin–Benson–Bassham (CBB) cycle. Moreover, proton translocation across the thylakoid membranes generates a proton gradient (ΔpH) and an electric potential (ΔΨ), both of which comprise the proton motive force (pmf) that drives ATP synthase. Then, the synthesized ATP is consumed in the CBB cycle and other chloroplast metabolic pathways. In the dark, the pH of both the chloroplast stroma and thylakoid lumen becomes neutral. Despite extensive studies of the above-mentioned processes, the molecular mechanisms of how chloroplast pH can be maintained at proper levels during the light phase for efficient activation of photosynthesis and other metabolic pathways and return to neutral levels during the dark phase remain largely unclear, especially in terms of the precise control of stromal pH. The transient increase and decrease in chloroplast pH upon dark-to-light and light-to-dark transitions have been considered as signals for controlling other biological processes in plant cells. Forward and reverse genetic screening approaches recently identified new plastid proteins involved in controlling ΔpH and ΔΨ across the thylakoid membranes and chloroplast proton/ion homeostasis. These proteins have been conserved during the evolution of oxygenic phototrophs and include putative photosynthetic protein complexes, proton transporters, and/or their regulators. Herein, we summarize the recently identified protein players that control chloroplast pH and influence photosynthetic efficiency in plants.

Escherichia coli is able to ferment mixed carbon sources and produce various fermentation end-products. In this study, the function of FhlA protein in the specific growth rate (µ), metabolism, regulation of ΔpH and proton ATPase activity was investigated. Reduced µ in fhlA mutant of ∼25% was shown, suggesting the role of FhlA in the growth process. The utilization rate of glycerol is decreased in fhlA ∼ 2 fold, depending on the oxidation-reduction potential values. Bacteria regulate the activity of hydrogenase enzymes during growth depending on the external pH, which manifests as a lack of hydrogen gas generation during glycerol utilization at pH values below 5.9. It is suggested that cells maintain ΔpH during the fermentative growth via formate-lactate-succinate exchange. The decrement of the value of pHin, but not of pHex in mutant cells, is regulating ΔpH and consequently proton motive force generation. [ABSTRACT FROM AUTHOR]

The classical view of oxidative phosphorylation is that a proton motive force (PMF) generated by the respiratory chain complexes fuels ATP synthesis via ATP synthase. Yet, under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected these two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions. pH profiles of mitochondrial sub‐compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH sensor directly at ATP synthase's F1 and FO subunits, complex IV and in the matrix. Our results clearly show that ATP synthase activity substantially controls the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible ΔpH. In addition, we show how this changes lateral, transmembrane, and radial pH gradients in glycolytic and respiratory cells. SYNOPSIS: Mitochondrial F1FO ATP synthase synthesizes or hydrolyses ATP depending on the metabolic conditions and controlled by IF1. Analysis of pH values using local pH sensors at ATP synthase and ATP synthase subcomplexes discloses these activities, which essentially contribute to the establishment of radial, transmembrane, and lateral pH gradients in cristae. Complementing the recent report on intramitochondrial inhomogeneity of ΔΨm these findings provide a new perspective on the heterogeneity of protonic energy coupling. The ΔpH along and across the inner mitochondrial membrane is not homogeneous.Local pH at cristae rims is controlled by F1FO ATP synthase.Under OXPHOS conditions, the pH difference between FO and F1 of active ATP synthase is almost negligible (1.2 proton versus 1 proton equivalent).Consequently, IF1 is required to prevent the onset of ATP hydrolysis also under OXPHOS conditions. [ABSTRACT FROM AUTHOR]

Cyclic electron transport (CET) around photosystem I (PSI) was discovered more than 60 years ago but it was 2004 when two pathways of CET were proposed. The main pathway depends on the PROTON GRADIENT REGULATION 5 (PGR5) protein, whereas the minor pathway is mediated by the chloroplast NADH dehydrogenase-like (NDH) complex. Although it is still a topic of debate, as to whether PGR5 is really involved in CET, the Arabidopsis pgr5 mutant is severely defective in the ΔpH-dependent regulation of photosynthetic electron transport. PGR5 is necessary to induce a ΔpH-dependent component of nonphotochemical quenching (NPQ) of chlorophyll fluorescence at a high light intensity and this qE mechanism dissipates excessively absorbed light energy safely as heat from photosystem II antennae. The lumenal acidification also downregulates the electron transport at the cytochrome b6f complex. The latter is essential for protecting PSI and the pgr5 mutant cannot survive the fluctuating light intensity. Although the contribution of the NDH complex to the pmf formation is much smaller than that of the PGR5-dependent CET during steady-state photosynthesis, the NDH complex efficiently pumps protons probably in the absence of large pmf. In addition to the size of pmf, it is also necessary to regulate the partitioning pmf components, ΔpH, and membrane potential (ΔΨ) across the thylakoid membrane. Ion channels and transporters localized to the thylakoid membrane involve the regulation and form a regulatory network of photosynthesis with the CET machinery. [ABSTRACT FROM AUTHOR]

Upon exposure to environmental stress, the primary electron donor in photosystem I (PSI), P700, is oxidized to suppress the production of reactive oxygen species that could oxidatively inactivate the function of PSI. The illumination of rice leaves with actinic light induces intrinsic fluctuations in the opening and closing of stomata, causing the net CO2 assimilation rate to fluctuate. We examined the effects of these intrinsic fluctuations on electron transport reactions. Under atmospheric O2 conditions (21 kPa), the effective quantum yield of photosystem II (PSII) (Y(II)) remained relatively high while the net CO2 assimilation rate fluctuated, which indicates the function of alternative electron flow. By contrast, under low O2 conditions (2 kPa), Y(II) fluctuated. These results suggest that photorespiration primarily drove the alternative electron flow. Photorespiration maintained the oxidation level of ferredoxin (Fd) throughout the fluctuation of the net CO2 assimilation rate. Moreover, the relative activity of photorespiration was correlated with both the oxidation level of P700 and the magnitude of the proton gradient across the thylakoid membrane in 21 kPa O2 conditions. These results show that photorespiration oxidized P700 by stimulating the proton gradient formation when CO2 assimilation was suppressed by stomatal closure.

Chloroplastic ATP synthase plays a significant role in the regulation of proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes. However, the regulation of chloroplastic ATP synthase at chilling temperature and its role in photoprotection are little known. In our present study, we examined the chlorophyll fluorescence, P700 signal, and electrochromic shift signal at 25°C, and 6°C in tobacco (Nicotiana tabacum L. cv. Samsun). Although photosynthetic electron flow through both PSI and PSII were severely inhibited at 6°C, non-photochemical quenching and P700 oxidation ratio were largely increased. During the photosynthetic induction under high light, the formation of pmf at 6°C was similar to that at 25°C. However, the ΔpH was significantly higher at 6°C, owing to the decreased activity of chloroplastic ATP synthase (gH+). During illumination at 6°C and high light, a high ΔpH made PSI to be highly oxidized, preventing PSI from photoinhibition. These results indicate that the down-regulation of gH+ is critical to the buildup of ΔpH at low temperature, adjusting the redox state of PSI, and thus preventing photodamage to PSI. Our findings highlight the importance of chloroplastic ATP synthase in photoprotection at chilling temperature.

Importance: Methicillin-resistant Staphylococcus aureus (MRSA) infection severely threatens human health due to high morbidity and mortality; it is urgent to develop novel strategies to tackle this problem. Metabolites belong to antibiotic adjuvants which improve the effect of antibiotics. Despite reports of L-glutamine being applied in antibiotic adjuvant for Gram-negative bacteria, how L-glutamine affects antibiotics against Gram-positive-resistant bacteria is still unclear. In this study, L-glutamine increases the antibacterial effect of gentamicin on MRSA, and it links to membrane permeability and pH gradient (ΔpH), resulting in uptake of more gentamicin. Of great interest, reduced reactive oxygen species (ROS) by glutathione was found under L-glutamine treatment; USA300 becomes sensitive again to gentamicin. This study not only offers deep understanding on ΔpH and ROS on bacterial resistance but also provides potential treatment solutions for targeting MRSA infection., Competing Interests: The authors declare no conflict of interest.

Chloroplastic ATP synthase plays a significant role in the regulation of proton motive force (pmf) and proton gradient (ΔpH) across the thylakoid membranes. However, the regulation of chloroplastic ATP synthase at chilling temperature and its role in photoprotection are little known. In our present study, we examined the chlorophyll fluorescence, P700 signal, and electrochromic shift signal at 25°C, and 6°C in tobacco (Nicotiana tabacum L. cv. Samsun). Although photosynthetic electron flow through both PSI and PSII were severely inhibited at 6°C, non-photochemical quenching and P700 oxidation ratio were largely increased. During the photosynthetic induction under high light, the formation of pmf at 6°C was similar to that at 25°C. However, the ΔpH was significantly higher at 6°C, owing to the decreased activity of chloroplastic ATP synthase (g H+). During illumination at 6°C and high light, a high ΔpH made PSI to be highly oxidized, preventing PSI from photoinhibition. These results indicate that the down-regulation of g H+ is critical to the buildup of ΔpH at low temperature, adjusting the redox state of PSI, and thus preventing photodamage to PSI. Our findings highlight the importance of chloroplastic ATP synthase in photoprotection at chilling temperature. [ABSTRACT FROM AUTHOR]

Main conclusion: Respective contributions of PGR5- and NDH-dependent cyclic electron flows around photosystem I for generating the proton gradient across the thylakoid membrane are ~30 and ~5%. The proton concentration gradient across the thylakoid membrane (ΔpH) produced by photosynthetic electron transport is the driving force of ATP synthesis and non-photochemical quenching. Two types of electron transfer contribute to ΔpH formation: linear electron flow (LEF) and cyclic electron flow (CEF, divided into PGR5- and NDH-dependent pathways). However, the respective contributions of LEF and CEF to ΔpH formation are largely unknown. We employed fluorescence quenching analysis with the pH indicator 9-aminoacridine to directly monitor ΔpH formation in isolated chloroplasts of Arabidopsis mutants lacking PGR5- and/or NDH-dependent CEF. The results indicate that ΔpH formation is mostly due to LEF, with the contributions of PGR5- and NDH-dependent CEF estimated as only ~30 and ~5%, respectively. [ABSTRACT FROM AUTHOR]

Over-reduction of the photosynthetic electron transport ( PET) chain should be avoided, because the accumulation of reducing electron carriers produces reactive oxygen species ( ROS) within photosystem I ( PSI) in thylakoid membranes and causes oxidative damage to chloroplasts. To prevent production of ROS in thylakoid membranes the H+ gradient (ΔpH) needs to be built up across the thylakoid membranes to suppress the over-reduction state of the PET chain. In this study, we aimed to identify the critical component that stimulates ΔpH formation under illumination in higher plants. To do this, we screened ethyl methane sulfonate ( EMS)-treated Arabidopsis thaliana, in which the formation of ΔpH is impaired and the PET chain caused over-reduction under illumination. Subsequently, we isolated an allelic mutant that carries a missense mutation in the γ-subunit of chloroplastic CF0 CF1- ATP synthase, named hope2. We found that hope2 suppressed the formation of ΔpH during photosynthesis because of the high H+ efflux activity from the lumenal to stromal side of the thylakoid membranes via CF0 CF1- ATP synthase. Furthermore, PSI was in a more reduced state in hope2 than in wild-type ( WT) plants, and hope2 was more vulnerable to PSI photoinhibition than WT under illumination. These results suggested that chloroplastic CF0 CF1- ATP synthase adjusts the redox state of the PET chain, especially for PSI, by modulating H+ efflux activity across the thylakoid membranes. Our findings suggest the importance of the buildup of ΔpH depending on CF0 CF1- ATP synthase to adjust the redox state of the reaction center chlorophyll P700 in PSI and to suppress the production of ROS in PSI during photosynthesis. [ABSTRACT FROM AUTHOR]

A by-product of Wastewater Treatment Stations is sewage sludge. By treatment and processing, the sludge is made suitable for rational and environmentally safe use in agriculture. The aim of this study was to assess the influence of different doses of limed sewage sludge (50 %) on clay dispersion in soil samples with different textures (clayey and medium). The study was conducted with soil samples collected from native forest, on a Red Latosol (Brazilian classification: Latossolo Vermelho distroférrico) loamy soil in Londrina (PR) and a Red-Yellow Latosol (BC: Latossolo Vermelho-Amarelo distrófico) medium texture soil in Jaguapitã (PR). Pots were filled with 3 kg of air-dried fine earth and kept in greenhouse. The experiment was arranged in a randomized block design with six treatments: T1 control, and treatments with limed sewage sludge (50 %) as follows: T2 (3 t ha-1), T3 (6 t ha-1), T4 (12 t ha-1), T5 (24 t ha-1) and T6 (48 t ha-1) and five replications. The incubation time was 180 days. At the end of this period, the pots were opened and two sub-samples per treatment collected to determine pH-H2O, pH KCl (1 mol L-1), organic matter content, water-dispersible clay, ΔpH (pH KCl - pH-H2O) and estimated PZC (point of zero charge): PZC = 2 pH KCl - pH-H2O, as well as the mineralogy of the clay fraction, determined by X ray diffraction. The results showed no significant difference in the average values for water-dispersible clay between the control and the other treatments for the two soil samples studied and ΔpH was the variable that correlated best with water-dispersible clay in both soils.As Estações de Tratamento de Esgoto Sanitário (ETES) geram um subproduto (lodo de esgoto) que, quando tratado e processado, adquire características que permitem sua utilização agrícola de maneira racional e ambientalmente segura. Este trabalho objetivou avaliar a influência de diferentes doses de lodo de esgoto caleado (50 %) na dispersão de argilas em amostras de solos com diferentes texturas (muito argilosa e média). O estudo foi realizado com amostras de solos coletadas sob mata nativa, em Latossolo Vermelho distroférrico, textura muito argilosa, em Londrina (PR), e Latossolo Vermelho-Amarelo distrófico, textura media, em Jaguapitã (PR). Os vasos foram preenchidos com 3 kg de TFSA em casa de vegetação, em um delineamento de blocos ao acaso, com seis tratamentos - T1: controle; e os tratamentos com lodo de esgoto caleado (50%): T2 (3 t ha-1); T3 (6 t ha-1); T4 (12 t ha-1); T5 (24 t ha-1) e T6 (48 t ha-1), com cinco repetições. O tempo de incubação foi de 180 dias; após esse período, os vasos foram abertos e coletaram-se duas subamostras por tratamento, para determinar: pH-H2O, pH-KCl (1 mol L-1), teor de matéria orgânica, argila dispersa em água, ΔpH (pH-KCl - pH-H2O) e o PCZ estimado: PCZ = 2 pH-KCl - pH-H2O, além da mineralogia da fração argila por difração de raios X. Os resultados permitiram concluir que não houve diferença significativa quanto aos teores médios de argila dispersa em água entre o controle e os outros tratamentos estudados para as duas amostras de solo; ΔpH foi a variável que melhor se correlacionou com a argila dispersa em água nos dois solos.

• Through hydrogen‑deuterium exchange, reactions typically involving protonation can be studied. • The induction and recovery kinetics of NPQ and ΔpH are affected to different extents. • At certain light intensities, the magnitude of the ΔpH and ΔpD were equivalent, yet NPQ was slower to form and relax. [ABSTRACT FROM AUTHOR]

Non-photochemical quenching (NPQ) is one of the most important photoprotection mechanisms in photosynthetic organisms when they are exposed to excessive irradiation. The basic principle of NPQ, which is the safe dissipation of excessive absorbed light as heat, is identical in all photoautotrophs. However, crucial differences in its regulation and structural mechanisms exist in different phototrophs. Here, we investigated NPQ in the green alga Ulva linza coupled with inhibitors to alter the amplitude of the transthylakoid proton gradient (ΔpH) and/or de-expoxidation of xanthophyll cycle (XC) under high light conditions. The data demonstrates that NPQ started with a rapid initial rise within the first minute of illumination, followed by a decline before a further rise in quenching. During the whole phase, NPQ was triggered and completely controlled by ΔpH, then strengthened and modulated by zeaxanthin. NPQ relaxation was slower in U. linza when compared to plants and other green algae, and it may be mainly caused by the slow conversion of zeaxanthin to violaxanthin. NPQ in U. linza is controlled to a greater extent by XC, which show more similarities to Arabidopsis than to Chlamydomonas and may be an adaptive mechanism for its successful colonization of coastal ecosystems. [ABSTRACT FROM AUTHOR]

Here we show how the protein environment in terms of detergent concentration/protein aggregation state, affects the sensitivity to pH of isolated, native LHCII, in terms of chlorophyll fluorescence quenching. Three detergent concentrations (200, 20 and 6 μM n-dodecyl β-d-maltoside) have been tested. It was found that at the detergent concentration of 6 μM, low pH quenching of LHCII is close to the physiological response to lumen acidification possessing pK of 5.5. The analysis has been conducted both using arbitrary PAM fluorimetry measurements and chlorophyll fluorescence lifetime component analysis. The second led to the conclusion that the 3.5 ns component lifetime corresponds to an unnatural state of LHCII, induced by the detergent used for solubilising the protein, whilst the 2 ns component is rather the most representative lifetime component of the conformational state of LHCII in the natural thylakoid membrane environment when the non-photochemical quenching (NPQ) was absent. The 2 ns component is related to a pre-aggregated LHCII that makes it more sensitive to pH than the trimeric LHCII with the dominating 3.5 ns lifetime component. The pre-aggregated LHCII displayed both a faster response to protons and a shift in the pK for quenching to higher values, from 4.2 to 4.9. We concluded that environmental factors like lipids, zeaxanthin and PsbS protein that modulate NPQ in vivo could control the state of LHCII aggregation in the dark that makes it more or less sensitive to the lumen acidification. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy. [ABSTRACT FROM AUTHOR]

Light-induced and lumen acidity-dependent quenching (qE) of excited chlorophylls (Chl) in vivo has been amply documented in plants and algae, but not in cyanobacteria, using primarily the saturation pulse method of quenching analysis which is applied to continuously illuminated samples. This method is unsuitable for cyanobacteria because the background illumination elicits in them a very large Chl a fluorescence signal, due to a state 2 to state 1 transition, which masks fluorescence changes due to other causes. We investigated the qE problem in the cyanobacterium Synechococcus sp. PCC 7942 using a kinetic method (Chl a fluorescence induction) with which qE can be examined before the onset of the state 2 to state 1 transition and the attendant rise of Chl a fluorescence. Our results confirm the existence of a qE mechanism that operates on excited Chls a in Photosystem II core complexes of cyanobacteria. [ABSTRACT FROM AUTHOR]

Non-photochemical fluorescence quenching (NPQ) is mainly associated with the transthylakoid proton gradient (ΔpH) and xanthophyll cycle. However, the exact mechanism of NPQ is different in different oxygenic photosynthetic organisms. In this study, several inhibitors were used to study NPQ kinetics in the sea ice alga Chlamydomonas sp. ICE-L and to determine the functions of ΔpH and the xanthophyll cycle in the NPQ process. NHCl and nigericin, uncouplers of ΔpH, inhibited NPQ completely and zeaxanthin (Z) was not detected in 1 mM NHCl-treated samples. Moreover, Z and NPQ were increased in the samples containing N,N'-dicyclohexyl-carbodiimide (DCCD) under low light conditions. We conclude that ΔpH plays a major role in NPQ, and activation of the xanthophyll cycle is related to ΔpH. In dithiothreitol (DTT)-treated samples, no Z was observed and NPQ decreased. NPQ was completely inhibited when NHCl was added suggesting that part of the NPQ process is related to the xanthophyll cycle and the remainder depends on ΔpH. Moreover, lutein and β-carotene were also essential for NPQ. These results indicate that NPQ in the sea ice alga Chlamydomonas sp. ICE-L is mainly dependent on ΔpH which affects the protonation of PSII proteins and de-epoxidation of the xanthophyll cycle, and the transthylakoid proton gradient alone can induce NPQ. [ABSTRACT FROM AUTHOR]

A Typic Palexerult from northern Spain was subjected to acidity correction field tests over a period of 2 years. The experimental crop was a local rye variety and the amendments included gypsum, dolomite, limestone, and sugar foam waste, all at a 6000 kg ha–1 rate as calcium carbonate (CaCO3). General analyses were integrated with specific tests for soluble and easily exchangeable aluminum (Al) forms (Al-CaCl2) in addition to potassium chloride (KCl)–, barium chloride (BaCl2)–, and copper chloride (CuCl2)–extracted Al and adsorbed and amorphous Al. Two types of multiple linear regression models for production each year were established; some encompassed all studied variables and others the six Al forms only. As shown here, Al-KCl, Al-CaCl2, base saturation, and exchangeable Ca were the individual variables most strongly correlated with production, with R 2 = 0.865, within the topmost 12 cm. A principal component analysis exposed a substantial share of pH-dependent charge in organic matter on the cation exchange capacity of the soil. [ABSTRACT FROM PUBLISHER]

Moderately high temperature reduces photosynthetic capacities of leaves with large effects on thylakoid reactions of photosynthesis, including xanthophyll conversion in the lipid phase of the thylakoid membrane. In previous studies, we have found that leaf temperature of 40°C increased zeaxanthin accumulation in dark-adapted, intact tobacco leaves following a brief illumination, but did not change the amount of zeaxanthin in light-adatped leaves. To investigate heat effects on zeaxanthin accumulation and decay, zeaxanthin level was monitored optically in dark-adapted, intact tobacco and Arabidopsis thaliana leaves at either 23 or 40°C under 45-min illumination. Heated leaves had more zeaxanthin following 3-min light but had less or comparable amounts of zeaxanthin by the end of 45 min of illumination. Zeaxanthin accumulated faster at light initiation and decayed faster upon darkening in leaves at 40°C than leaves at 23°C, indicating that heat increased the activities of both violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). In addition, our optical measurement demonstrated in vivo that weak light enhances zeaxanthin decay relative to darkness in intact leaves of tobacco and Arabidopsis, confirming previous observations in isolated spinach chloroplasts. However, the maximum rate of decay is similar for weak light and darkness, and we used the maximum rate of decay following darkness as a measure of the rate of ZE during steady-state light. A simulation indicated that high temperature should cause a large shift in the pH dependence of the amount of zeaxanthin in leaves because of differential effects on VDE and ZE. This allows for the reduction in ΔpH caused by heat to be offset by increased VDE activity relative to ZE. [ABSTRACT FROM AUTHOR]

Phycobilisomes (PBSs) are the main accessory light-harvesting complexes in cyanobacteria and their movement between photosystems (PSs) affects cyclic and respiratory electron transport. However, it remains unclear whether the movement of PBSs between PSs also affects the transthylakoid proton gradient (ΔpH). We investigated the effect of PBS movement on ΔpH levels in a unicellular cyanobacterium Synechocystis sp. strain PCC 6803, using glycinebetaine to immobilize and couple PBSs to photosystem II (PSII) or photosystem I (PSI) by applying under far-red or green light, respectively. The immobilization of PBSs at PSII inhibited decreases in ΔpH, as reflected by the slow phase of millisecond-delayed light emission (ms-DLE) that occurs during the movement of PBSs from PSII to PSI. By contrast, the immobilization of PBSs at PSI inhibited the increase in ΔpH that occurs when PBSs move from PSI to PSII. Comparison of the changes in ΔpH and electron transport caused by the movement of PBSs between PSs indicated that the changes in ΔpH were most likely caused by respiratory electron transport. This will further improve our understanding of the physiological role of PBS movement in cyanobacteria. [ABSTRACT FROM AUTHOR]

Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) in the thylakoid membrane (TM) has been shown to play a role in protection and regulation of photosynthesis under moderate heat stress. However, the physiological significance of RCA bound to the TM (TM–RCA) without heat stress remains unknown. In this study, it is first shown, using experiments in vivo, that the TM–RCA varies in rice leaves at different development stages, under different environmental conditions, and in a rice mutant. Furthermore, it is shown that the amount of TM–RCA always increased when the Rubisco activation state and the pH gradient across the TM (ΔpH) decreased. It was then demonstrated in vitro that the RCA bound dynamically to TM and the amount of TM–RCA increased during Rubisco activation. A high level of ATP and a high pH value promoted the dissociation of RCA from the TM. Both the RCA association with and dissociation from the TM showed conformational changes related to the ATP level or pH as indicated by the changes in fluorescence intensity of 1-anilinonaphthalene-8-sulphonic acid (ANS) binding to RCA. These results suggest that the reversible association of RCA with the TM is ATP and pH (or ΔpH) dependent; it might be involved in the RCA activation of Rubisco, in addition to the previously discovered role in the protection and regulation of photosynthesis under heat stress. [ABSTRACT FROM PUBLISHER]

Differential absorbance measurements and fluorometry were applied to examine the impact of dicyclohexylcarbodiimide (DCCD, an inhibitor of H+ conductance in thylakoid membranes) and nigericin (a K+/H+ antiporter) on photoinduced redox state transients of chlorophyll P700 and the induction curves of chlorophyll fluorescence in pea ( Pisum sativum L., cv. Premium) leaves. The treatment of leaves with DCCD strongly modified the kinetics of P700+ absorbance changes (Δ A810) by promoting rapid photooxidation of P700. These characteristic changes in Δ A810 induction kinetics and P700+ accumulation did not appear when the leaves were treated with DCCD in the presence of nigericin. In addition to opposite modifications of Δ A810 kinetics evoked by permeability-modifying agents, the fluorescence induction curves differed conspicuously depending on leaf incubation in DCCD solutions with or without nigericin. The observed modifications of fluorescence induction curves and Δ A810 indicate that DCCD suppresses electron transport from photosystem II (PSII) to P700, whereas this inhibition is removed by nigericin. The results suggest that slowing down of the electron transport rate in the presence of DCCD was caused by elevation of ΔpH in thylakoids. The prevention of pH gradient formation in the presence of protonophore lowered also the steady-state P700+ level in far-red irradiated leaves and accelerated the subsequent dark reduction of P700. These findings indicate that PSI-driven cyclic electron flow is accelerated after the removal of the pH gradient. [ABSTRACT FROM AUTHOR]

Chilling leads to cytoplasmic acidification in chill-sensitive plants. A possible explanation for this observation is that a ΔpH-stat between the cytosol and vacuole (ΔpHvac-stat) is perturbed by chilling. To understand the nature of this ΔpHvac-stat, the effect of temperature, between 20 and 0 °C, on pyrophosphate (PPi)- or ATP-dependent acidification of vacuolar vesicles, isolated from mung bean hypocotyls, was determined. Over the temperature range investigated, the H+-influx mediated by PPase was balanced with the H+-efflux, which was PPi-dependently suppressed, and consequently a constant pH in vesicles (pHin) of ca. 5 was maintained against temperature changes. However, the ΔpHin driven by ATP decreased as the temperature dropped. Thus, the PPi-dependent H+-accumulation may function as an essential factor to form a ΔpHvac-stat against temperature changes. Next, to study the chilling sensitivity of PPi-dependent H+-accumulation, vacuolar vesicles were isolated from control seedlings or from seedlings chilled at 0 °C for 1 d. Chilling treatment resulted in a decrease in the H+-accumulation rate and in the steady-state ΔpHin formed by PPi, the causes of which were enhanced by PPi-dependent H+-efflux and reduced by H+-influx driven by PPase. Together, the results suggest that the decrease of PPi-dependent H+-accumulation associated with the ΔpHvac-stat could result in cytoplasmic acidification. [ABSTRACT FROM AUTHOR]

Abstract: To adapt to environmental changes, plant cells very likely possess a biochemical system, using vacuoles, for maintaining cytoplasmic pH homeostasis. A simple approach is to estimate the active H+ influx and H+ efflux of isolated vacuolar vesicles, although there is no good mathematical model to describe H+ flux. To establish a new quantitative model, vacuolar vesicles were isolated from hypocotyls of mung bean (Vigna radiata L.), and pyrophosphate (PPi)- or ATP-dependent acidification was monitored using acridine orange. The change of pH inside the vesicles (pHin) was calculated using a pH calibration curve relating fluorescence quenching with ΔpH. After formation of a steady state ΔpH, passive H+ efflux was monitored after terminating pumping with ethylenediaminetetraacetate, and the relative H+ permeability coefficient (p H+) was calculated. The H+ efflux simulated using the p H+ corresponded to the H+ efflux determined experimentally. H+ influx was then calculated by subtracting the predicted H+ efflux from the experimental net H+ influx. H+ influx into vesicles driven by H+-PPase or H+-ATPase decreased exponentially as the intravesicular pHin decreased, suggesting modulation of pumping by ΔpH, pHin, or both. Finally, the PPi- or ATP-dependent H+ accumulation determined experimentally was closely simulated by the predicted H+ influx and H+ efflux. The ability to predict H+ flux under different conditions provides a powerful tool for studying pH homeostasis. [Copyright &y& Elsevier]

The phenomenon of cyclic electron transport was first characterized in higher plant chloroplasts 50 years ago, yet there is still a debate about whether or not this is a physiological process. The recent isolation of mutants that appear to lack cyclic electron transport, as well as new data providing functional evidence for its occurrence, support the notion that this pathway plays an important role in plant responses to stress, providing a pH gradient across the thylakoid membrane to trigger non-photochemical quenching of chlorophyll fluorescence. At present, little is known about the regulation of cyclic electron transport, but it is possible that this is activated in response to a low redox potential in the chloroplast stroma. [ABSTRACT FROM PUBLISHER]

In a fructose-containing medium in which rye root-microsomal membrane vesicles had reached the equilibrium of uptake of fructose, the presence of both Mg2+ and ATP caused the efflux of fructose from the vesicles. Among nucleotides examined, ATP caused the largest efflux of fructose. The efflux of fructose dependent on Mg2+ and ATP was quite insensitive to a protonophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), which actually abolished MgATP-dependent proton accumulation in the vesicles, while it was largely inhibited by vanadate, which inhibits ATP-binding cassette transporters (ABCTs). The Michaelis-Menten constant (Km) of the efflux of fructose was 0.4 mM. It was observed that fructose stimulated the ATPase activity of the vesicles and that vanadate markedly decreased the fructose-stimulated ATPase activity. [ABSTRACT FROM AUTHOR]

The Tat pathway is a major route for protein export in prokaryotes and for protein targeting to thylakoids in chloroplasts. Based on in vitro studies, protein translocation through this pathway is thought to be strictly dependent on a transmembrane ΔpH. In this paper, we assess the ΔpH sensitivity of the Tat pathway in vivo. Using Chlamydomonas reinhardtii, we observed changes in the efficiency of thylakoid targeting in vivo by mutating the Tat signal of the Rieske protein. We then employed two endogenous pH probes located on the lumen side of the thylakoid membranes to estimate spectroscopically the ΔpH in vivo. Using experimental conditions in which the trans-thylakoid ΔpH was almost zero, we found no evidence for a ΔpH dependence of the Tat pathway in vivo. We confirmed this observation in higher plants using attached barley leaves. We conclude that the Tat pathway does not require a ΔpH under physiological conditions, but becomes ΔpH sensitive when probed in vitro/in organello because of the loss of some critical intracellular factors. [ABSTRACT FROM AUTHOR]

The interface between the c -subunit oligomer and the a subunit in the F0 sector of the ATP synthase is believed to form the core of the rotating motor powered by the protonic flow. Besides the essential cAsp61 and aArg210 residues (Escherichia coli numbering), a few other residues at this interface, although nonessential, show a high degree of conservation, among these aGlu219. The homologous residue aGlu210 in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus has been substituted by a lysine. Inner membranes prepared from the mutant strain showed approximately half of the ATP synthesis activity when driven both by light and by acid-base transitions. As estimated with the ACMA assay, proton pumping rates in the inner membranes were also reduced to a similar extent in the mutant. The most striking impairment of ATP synthesis in the mutant, a decrease as low as 12 times as compared to the wild-type, was observed in the absence of a transmembrane electrical membrane potential (Δφ) at low transmembrane pH difference (ΔpH). Therefore, the mutation seems to affect both the mechanism responsible for coupling F1 with proton translocation by F0 , and the mechanism determining the relative contribution of ΔpH and Δφ in driving ATP synthesis. [ABSTRACT FROM AUTHOR]

The effect of temperature on the rate of electron transfer through photosystems I and II (PSI and PSII) was investigated in leaves of barley (Hordeum vulgare L.). Measurements of PSI and PSII photochemistry were made in 21% O2 and in 2% O2, to limit electron transport to O2 in the Mehler reaction. Measurements were made in the presence of saturating CO2 concentrations to suppress photorespiration. It was observed that the O2 dependency of PSII electron transport is highly temperature dependent. At 10 °C, the quantum yield of PSII (ΦPSII) was insensitive to O2 concentration, indicating that there was no Mehler reaction operating. At high temperatures (>25 °C) a substantial reduction in ΦPSII was observed when the O2 concentration was reduced. However, under the same conditions, there was no effect of O2 concentration on the ΔpH-dependent process of non-photochemical quenching. The rate of electron transport through PSI was also found to be independent of O2 concentration across the temperature range. We conclude that the Mehler reaction is not important in maintaining a thylakoid proton gradient that is capable of controlling PSII activity, and present evidence that cyclic electron transport around PSI acts to maintain membrane energisation at low temperature. [ABSTRACT FROM AUTHOR]

The effects of the arginine modifying reagent phenylglyoxal (PGO) on solute transport was studied in two cellular systems: protoplasts isolated from the mesophyll of Vicia faba L. and XD cell suspension culture of Nicotiana tabacum L. cv. Xanthi. The solutes in the case of the protoplasts were the non-metabolizable glucose analog 3-O-methyl-D-glucose (MeG), and a non-metabolizable amino acid analog α-aminoisobutyric acid (AIB), whereas the solutes for the cell suspension were AIB and nitrate. Solute transport in both systems was rapidly inhibited by PGO. Exposure of the protoplasts to light enhanced the initial rate of MeG uptake. PGO rapidly inhibited MeG uptake in both the light and the dark, the half-time for inactivation being less than 3 min. Flux analysis of double-labeled MeG showed that initial MeG uptake was mediated mainly by the plasma membrane transport system and that it was inhibited by PGO. Maximal inhibition of initial MeG uptake rate was observed at PGO concentrations of 1 mM and above. PGO treatment altered rapidly the equilibrium distribution of the ΔpH probe dimethyloxazolidine (DMO) in both cellular systems, indicating dissipation of ΔpH between cell and medium. In the protoplasts, PGO inhibited both DMO and MeG uptake at pH 5.5; however, at pH 7.0, where ΔpH is minimal, only MeG uptake was inhibited. Our results suggest that PGO has two effects on glucose uptake: an indirect effect through ΔpH dissipation and a direct effect through interaction with essential arginyl residues in the glucose transporter. [ABSTRACT FROM AUTHOR]

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell-cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the ΔpH and the Δψ of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an α-helix/β-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus-cell fusion. [ABSTRACT FROM AUTHOR]

We investigated the temperature dependence and inhibitor sensitivity of the light-induced reversible changes in the circular dichroism (ΔCD) of chloroplast thylakoid membranes. Earlier, these changes, which originate from structural changes affecting the chiral macroorganization of the chromophores, were thought to be driven by photochemically generated proton and/or ion gradients in the thylakoids [Garab et al. (1988) Biochemistry 27: 2430]. However, more recently, these changes have been shown to be largely independent of the photochemical activity of thylakoids, and ΔCD has been observed in lamellar aggregates of the light harvesting chlorophyll a/b complex (LHC II) of Photosystem II [Barzda et al. (1996) Biochemistry 35: 8981]. Here, we show that in thylakoids (i) ΔCD is gradually and substantially decelerated upon gradually decreasing the temperature from 33 °C to 2 °C, and abruptly disappears above 35–37 °C; (ii) ΔCD is inhibited with nigericin with I50≈ 1 μM, which is about 10 times higher than the I50for the transmembrane ΔpH; (iii) ΔCD can be inhibited with dicyclohexylcarbodiimide that blocks proton binding at the lumenal side of LHC II; (iv) quinone antagonists, such as antimycin-A and myxothiazol, inhibit ΔCD without noticeably affecting the electron and proton transport, and the chiral macroorganization of the chromophores in the dark. We conclude that ΔCD is conditioned but not driven by the photochemical activity of the membranes, and the structural changes are given rise by a physical mechanism previously unrecognized in thylakoids, thermooptic effect described for liquid crystals. We discuss the possible link between the deactivation(s) of the excess excitation energy and ΔCD, the light-induced changes in the chiral macroorganization of the chromophores of the photophysical apparatus in thylakoids. [ABSTRACT FROM AUTHOR]

Photoinhibition of Photosystem II in unicellular algae in vivo is accompanied by thylakoid membrane energization and generation of a relatively high ΔpH as demonstrated by C-methylamine uptake in intact cells. Presence of ammonium ions in the medium causes extensive swelling of the thylakoid membranes in photoinhibited Chlamydomonas reinhardtii but not in Scenedesmus obliquus wild type and LF-1 mutant cells. The rise in ΔpH and the related thylakoid swelling do not occur at light intensities which do not induce photoinhibition. The rise in ΔpH and membrane energization are not induced by photoinhibitory light in C. reinhardtii mutant cells possessing an active Photosystem II but lacking cytochrome b/f, plastocyanin or Photosystem I activity and thus being unable to perform cyclic electron flow around Photosystem I. In these mutants the light-induced turnover of the D1 protein of Reaction Center II is considerably reduced. The high light-dependent rise in ΔpH is induced in the LF-1 mutant of Scenedesmus which can not oxidize water but otherwise possesses an active Reaction Center II indicating that PS II-linear electron flow activity and reduction of plastoquinone are not required for this process. Based on these results we conclude that photoinhibition of Photosystem II activates cyclic electron flow around Photosystem I which is responsible for the high membrane energization and ΔpH rise in cells exposed to excessive light intensities. [ABSTRACT FROM AUTHOR]

Formaldehyde accumulation ratios ([CHO]/[CHO]) as high as 12-fold were measured in anaerobic, CHOH-energized, whole cell suspensions of the ribulose monophosphate (RuMP)-type methylotrophic strain T15. Uptake kinetics were extremely rapid, enabling the attainment of equilibrium in only 10-30 s. Transport appears to be energy-dependent and associated with the protonmotive force (pmf). Anaerobic incubation with 5 μM carbonyl p-(trifluoromethoxy)-phenylhydrazone (FCCP) led to 70%-90% reduction of the accumulation ratio. Though not as pronounced, diminished uptake was also observed in the presence of 140 μM nigericin, 161 μM valinomycin and 90 mM KSCN, commensurate with their effects on pmf. Accumulation of CHO as a function of external pH followed a trend more similar to that of pmf than either δpH or δΨ. Preventing energization by incubation with 100 μM N,N′-dicyclohexylcarbodiimide (DCCD) led to nearly 80% inhibition of CHO transport. Over short time periods it was possible to 'chase' accumulated CHO from previously loaded cells by collapsing pmf; however, this technique also indicated that significant CHO incorporation began to occur within 3 min. [ABSTRACT FROM AUTHOR]

Upon exposure to environmental stress, the primary electron donor in photosystem I (PSI), P700, is oxidized to suppress the production of reactive oxygen species that could oxidatively inactivate the function of PSI. The illumination of rice leaves with actinic light induces intrinsic fluctuations in the opening and closing of stomata, causing the net CO2 assimilation rate to fluctuate. We examined the effects of these intrinsic fluctuations on electron transport reactions. Under atmospheric O2 conditions (21 kPa), the effective quantum yield of photosystem II (PSII) (Y(II)) remained relatively high while the net CO2 assimilation rate fluctuated, which indicates the function of alternative electron flow. By contrast, under low O2 conditions (2 kPa), Y(II) fluctuated. These results suggest that photorespiration primarily drove the alternative electron flow. Photorespiration maintained the oxidation level of ferredoxin (Fd) throughout the fluctuation of the net CO2 assimilation rate. Moreover, the relative activity of photorespiration was correlated with both the oxidation level of P700 and the magnitude of the proton gradient across the thylakoid membrane in 21 kPa O2 conditions. These results show that photorespiration oxidized P700 by stimulating the proton gradient formation when CO2 assimilation was suppressed by stomatal closure. [ABSTRACT FROM AUTHOR]

It is important to assess acid tolerance in lactic acid bacteria, particularly for probiotics, although it involves multiple mechanisms. Measuring the difference between intracellular and extracellular pH (ΔpH) using the fluorescent probe CFDA-SE is particularly effective for such assessments because it gives direct information on the level of tolerance in the extracellular acidic pH range from 7 to 2.5. It also enables acid adaptation to be induced and observed by slowly introducing HCl into the medium and decreasing the extracellular pH. The difference of acid tolerance between anaerobic and aerobic conditions in lactic acid bacteria can also be evaluated by measuring ΔpH.