A model for NADH-tetrazolium reductase

Summary In the presence of light, reduced nicotinamide adenine dinucleotide (NADH) and riboflavin formed a complex which was able to reduce certain tetrazolium salts. Neither NADH (10^–3 M) nor riboflavin (10^–4 M) alone was able to induce tetrazolium reduction in the presence of oxygen, but in a nitrogen atmosphere photoreduction of riboflavin induced reduction of tetrazolium salts. Only electrophilic nitro and thiazolyl substituted tetrazolium salts with more positive redox potentials were reduced by the NADH-riboflavin complex, and only monoformazans were produced from the ditetrazolium salts. The reduction kinetics of these tetrazolium salts are given, and the spectral area capable for induction of electron transfer in the NADH-riboflavin complex is screened. It is concluded that the electron transfer in flavin nucleotide dependent dehydrogenase systems will probably proceed without direct interference with the apoenzyme. This may have practical implications for the histochemistry of tetrazolium reductases especially as regards fixation. The catalytic action of light on tetrazolium reduction should also be taken into consideration when tetrazolium salts are used as electron acceptors in a histochemical reaction.     

The link between the electrogenic and redox functions of the plasmalemma and the energy metabolism of the cell

A dependence of the plasmalemma redox activity, determined by the reduction of external electron acceptors (ferricyanide, nitro-blue tetrazolium), on the energy state of the cell, which was modified by light conditions or introduction of glucose into the media, was shown on leaves of Elodea canadensis Rich. Glucose (10 m M ) and light (40 W m^-2) caused hyperpolarization of the membrane potential and stimulated the redox activity of the plasmalemma. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) completely inhibited the light activation of electrogenic and redox functions of the plasmalemma. The light saturation intensity for membrane potential and ferricyanide reductase activity was 10–30% of the light saturation of photosynthesis. Membrane potential, K⁺ transport and plasmalemma redox activity changed in parallel in response to light and darkness and when DCMU was added. Ferricyanide reductase activity is suggested to be a simple parameter for characterizing the energy state of the cell. The functional significance of the light-induced hyperpolarization of the membrane potential is discussed.     

Blue light effects on stomata are mediated by the guard cell plasma membrane redox system distinct from the proton translocating ATPase

Abstract. The effects of blue light on stomata are critically analysed. Blue-light-induced increase in stomatal conductance is preceded by membrane hyperpolarization, proton efflux, potassium uptake and malate synthesis in guard cells. Hypothetically, a flavin containing plasma membrane redox system can pump protons out of guard cells on illumination with blue light. It is proposed that this electrogenic proton pump requires NAD(P)H but does not involve ATP/ATPase.     

The role of plasma membrane redox activity in light effects in plants

Stimulations by light of electron transport at the plasma membrane make it possible that redox activity is involved in light-induced signal transduction chains. This is especially true in cases where component(s) of the chain are also located at the plasma membrane. Photosynthetic reactions stimulate transplasma membrane redox activity of mesophyll cells. Activity is measured as a reduction of the nonpermeating redox probe, ferricyanide. The stimulation is due to production of a cytosolic electron donor from a substance(s) transported from the chloroplast. It is unknown whether the stimulation of redox activity is a requirement for other photosynthetically stimulated processes at the plasma membrane, but a reduced intermediate may regulate proton excretion by guard cells. Blue light induces an absorbance change (LIAC) at the plasma membrane whose difference spectrum resembles certainb-type cytochromes. This transport of electrons may be due to absorption of light by a flavoprotein. The LIAC has been implicated as an early step in certain blue light-mediated morphogenic events. Unrelated to photosynthesis, blue light also stimulates electron transport at the plasma membrane to ferricyanide. The relationship between LIAC and transmembrane electron flow has not yet been determined, but blue light-regulated proton excretion and/or growth may depend on this electron flow. No conclusions can be drawn regarding any role for phytochrome because of a paucity of information concerning the effects of red light on redox activity at the plasma membrane.     

Effect of sink size on photosynthesis and carbohydrate content of leaves of three spring wheat varieties

Effects of CO₂ on stomatal movements of Commelina communis L. were studied with plants, epidermal strips and guard cell protoplasts. With plants, the stomatal response induced by a blue light pulse was studied for different ambient CO₂ concentration ranging from CO₂-deprived air to 100 Pa in darkness or under red light. It was observed that the blue light response could be obtained not only under a red light background but also in darkness and CO₂-free air, the two responses being quite similar.
With epidermal strips, the effect of CO₂ on ferricyanide reductase activity at the guard cell plasmalemma was studied by transmission electron microscopy. In the presence of ferric ions, reduced ferricyanide gives an electron dense precipitate of Prussian Blue. In darkness and air, no precipitate was observed. In darkness and CO₂-free air as well as under light and normal air, a precipitate was found along the plasmalemma of the guard cells, indicating a ferricyanide reductase activity. With guard cell protoplasts suspended in a medium either in equilibrium with air or in a CO₂-free medium the H⁺ extrusion induced by a blue light pulse added to a red light background was measured. A low CO₂ content was obtained by adding photosynthetic algae to the suspension of guard cell protoplasts. In a CO₂-free medium the rate of H⁺ extrusion was enhanced.
The results are discussed on the basis of a possible competition for reducing power between CO₂ fixation and a putative blue light dependent redox chain located on the plasma membrane.     

Light perception in guard cells

Abstract. Guard cells perceive light via two photoreceptor systems: a blue-light-dependent photosystem and the guard cell chloroplast. Chloroplasts stimulate stomatal opening by transducing photosynthetic active radiation into proton pumping at the guard cell plasma membrane. In addition, guard cell chloroplasts fix CO₂ photosynthetically. Sugar from guard cell photosynthesis can contribute to the osmotic build-up required for opening. The blue-light-dependent photosystem activates proton pumping at the guard cell plasma membrane and stimulates starch hydrolysis. Available information on the photobiological properties of guard cells makes it possible to describe stomatal function in terms of the cellular components regulating stomatal movements. The blue light response is involved in stomatal opening in the early morning and stomatal responses to sunflecks. The guard cell chloroplast is likely to be involved in stomatal adaptations to sun, shade and to temperature. Interactions between these photosystems, a third photoreceptor in guard cells, phytochrome, and other mechanisms transducing stomatal responses such as VPD and carbon dioxide, provide the cellular basis for stomatal regulation.     

NADH dehydrogenase from bovine neutrophil membranes. Purification and properties

A membrane-associated NADH dehydrogenase from beef neutrophils was purified to homogeneity, using detergent (cholate plus Triton X-100) extraction and chromatography on DEAE-Sepharose CL-6B, agarose-hexane-NAD, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 17,500, but the enzyme was highly aggregated (Mr greater than 450,000) in nondenaturing gels containing 0.1% Triton X-100. The protein band in nondenaturing gels was also stained for activity using NADH and nitro blue tetrazolium. The enzyme showed greatest electron acceptor activity with ferricyanide (100%), followed by cytochrome c (3.5%), dichloroindophenol (2.7%), and cytochrome b5 (0.34%). No activity was seen with oxygen. The Km values for NADH and ferricyanide were 18 and 9.5 microM, respectively, and NAD+ was a weak competitive inhibitor (Ki = 118 microM). No activity was seen with NADPH. No effects were seen with mitochondrial respiratory inhibitors such as azide, cyanide, or rotenone, but p-chloromercuribenzoate was strongly inhibitory and N-ethylmaleimide was weakly inhibitory. No free flavin was detectable in enzyme preparations. Based upon kinetic, physical, and inhibition properties, this NADH dehydrogenase differs from those previously described in microsomes and erythrocyte plasma membrane.     

Abaxial and Adaxial Stomatal Responses to Light of Different Wavelengths and to Phenylacetic Acid on Isolated Epidermes of Commelina communis L.

Abaxial and adaxial stomatal responses to light of differentwavelengths and to phenylacetic acid (PAA), a molecule knownto form complexes with irradiated flavins, were examined onisolated epidermes of Commelina communis L. Blue light was superiorto red and green in promoting opening. Potassium accumulationand malate production were common to both abaxial and adaxialstomatal cells, but the photosensitivity was markedly higherin the former than in the latter. PAA suppressed opening andpotassium accumulation in guard cells, but hardly affected thelevel of epidermal malate; CO₂-free air failed to reverse thesesuppressions. The PAA-effect was more substantial in blue lightthan in red, green or darkness; thus, a flavin photoreceptoris indicated. Because of the overall effect of PAA under allconditions it is suggested that, in addition to its interactionwith blue light reception, PAA also has a more general effecton guard cells.     

Direct imaging of dehydrogenase activity within living cells using enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP)

Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q(10) of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD(+)) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.     

Demonstration of two 3,3'-diaminobenzidine oxidation reactions associated with photosynthetic membranes in anaerobic light-grown Rhodospirillum rubrum.

Photosynthetic membranes of anaerobic light-grown Rhodospirillum rubrum oxidized 3,3'-diaminobenzidine. When glutaraldehyde-treated cells were exposed to 3,3'-diaminobenzidine in the light aerobically, the oxidation appeared to occur by two systems. One reaction was stimulated by white light and the second required molecular oxygen. The O2-dependent activity was inhibited by KCN.     

Reduction of exogenous cytochrome c by Neurospora crassa conidia: effects of superoxide dismutase and blue light.

The reduction of externally added cytochrome c by Neurospora crassa conidia was observed. The reduction was stimulated by antimycin A and suppressed partially by superoxide dismutase. When conidia were treated with diethyldithiocarbamate, which inactivated endogenous superoxide dismutase, the cytochrome c reduction was stimulated. Blue light also stimulated the cytochrome c reduction. Azide, which inhibits photochemical reactions mediated by flavins, suppressed the blue light effect. Superoxide dismutase partially suppressed the cytochrome c reduction in the light. The results suggest that O2(-) participates in the cytochrome c reduction by conidia and the flavins or flavoproteins are candidates for the receptor pigment of blue light to stimulate the cytochrome c reduction. It was also suggested that the redox component(s), which could directly transfer its reducing equivalents to exogenous cytochrome c, was present at the surface of conidia.     

Photoactivation of NADP-Malate Dehydrogenase by a Heterogeneous Photochemical System in Zea mays

A heterogeneous photochemical electron relay system was constructed, mimicking the chloroplast electron transport reaction in order to activate the NADP-malate dehydrogenase in light. The photocatalyst acridine orange or proflavin sensitized EDTA-dependent reduction of ferredoxin. In a complete system, consisting of a dye donor couple, ferredoxin, thioredoxin and ferredoxin-thioredoxin reductase, light activation of purified NADP-MDH was observed in vitro. The chloroplast mediated redox activation of enzyme essentially required ferredoxin, while heterogeneous photochemical mediated activation of enzyme need not require ferredoxin. The heterogeneus photochemical system activated NADP-MDH by eight fold similar to chloroplasts mediated ferredoxin dependent redox activation but was not affected by the presence of disalicylinden propanediamine-1, 2-disulphonic acid while there was complete inhibition of chloroplasts mediated activation of NADP-MDH in presence of this inhibitor. These observations suggest that a thiol mediator is essential for reductive activation of NADP-MDH and ferredoxin is not required for photochemical activation.     

Bioenergetic processes in guard cells related to stomatal function

The energy required for ion uptake in guard cells is provided by two important bioenergetic processes, namely respiration and photosynthesis. The blue light-sensitive plasma membrane redox system is considered as the third bioenergetic phenomenon, since it uses blue light to create a proton gradient across the membrane. The unique features of respiration and photosynthesis in guard cells and their role in stomatal function are emphasized. Evidence for and against the blue light-sensitive components on plasma membrane (ATPase/distinct redox chain) and the photoreceptors (flavins, carotenoids, pterins) in guard cells are presented. The information on ion channels and their response to various kinds of secondary messengers including G-proteins, phosphoinositides, diacylglycerol, calcium, cAMP and protein kinases are reviewed. A model is presented indicating the possible mechanism of perception and transduction by guard cells of external signals and their interaction with different bioenergetic components.     

Effect of riboflavie and cyanide on blue light induction of betacyanin formation in Amamnthus caudatus seedlings

The role of phyto chrome and flavins in blue light induction of betacyanin formation was studied in etiolated, three-day-old Amaranthus caudatus L. seedlings, using the criterion of far-red reversibility and exogenously applied riboflavin and KCN. The effect of riboflavin was studied using high fluence rate blue light (42.7 :nmol m⁻²s⁻¹nm⁻¹ at 450 nm). When present in the incubation medium during illumination, riboflavin promoted the far-red reversibility with short light treatments and suppressed the inductive action of continuous illumiaation. If added after light treatments, it promoted betacyanin formation. The filtration of blue light through the riboflavin solution caused profound changes in light quality without affecting the far-red reversibility after 30 mm illumination. The effect of 1 mM KCN was tested with 70'% lower fluence of blue light. Cyanide caused the suppression of the inductive effect with 5 min blue light, which was accompanied by an enhancement of betacyanin induction by the terminal far-red light pulse. With 30 min blue light, however, it caused the appearance of far-red reversibility. The inductive effect of continuous blue illumination was slightly promoted by this Inhibitor. These results demonstrate that the effect of blue light on the pbyto chrome system is complex, whereas the physiological (inductive) action of the flavin triplet state is limited to low fluence, short blue light treatments.     

Reactive oxygen species in programmed death of pea guard cells

Hydrogen peroxide potentiates CN(-)-induced apoptosis of guard cells recorded as destruction of cell nuclei in the epidermis from pea leaves. A still stronger effect was exerted by the addition of H2O2 and NADH, which are the substrates of the plant cell wall peroxidase producing O2*- coupled to the oxidation of NADH. The CN(-)-or (CN(-) + H2O2)-induced destruction of guard cell nuclei was completely removed by nitroblue tetrazolium (NBT) oxidizing O2*- and preventing there-by the subsequent generation of H2O2. The reduced NBT was deposited in the cells as formazan crystals. Cyanide-induced apoptosis was diminished by mannitol and ethanol, which are OH* traps. The dyes Rose Bengal (RB) and tetramethylrhodamine ethyl ester (TMRE) photosensitizing singlet oxygen production suppressed the CN(-)-induced destruction of the cell nuclei in the light. This suppression was removed by exogenous NADH, which reacts with 1O2 yielding O2*-. Incubation of leaf slices with RB in the light lowered the photosynthetic O2 evolution rate and induced the permeability of guard cells for propidium iodide, which cannot pass across intact membranes. Inhibition of photosynthetic O2 evolution by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea or bromoxynil prevented CN(-)-induced apoptosis of guard cells in the light but not in the dark. RB in combination with exogenous NADH caused H2O2 production that was sensitive to NBT and estimated from dichlorofluorescein (DCF) fluorescence. Data on NBT reduction and DCF and TMRE fluorescence obtained using a confocal microscope and data on the NADH-dependent H2O2 production are indicative of generation of reactive oxygen species in the chloroplasts, mitochondria, and nuclear region of guard cells as well as with participation of apoplastic peroxidase. Cyanide inhibited generation of reactive oxygen species in mitochondria and induced their generation in chloroplasts. The results show that H2O2, OH*, and O2*- resources utilized for H2O2 production are involved in apoptosis of guard cells. It is likely that singlet oxygen generated by RB in the light, judging from the permeability of the plasmatic membrane for propidium iodide, makes Photosystem II of chloroplasts inoperative and induces necrosis of the guard cells.     

Effects of Cations and Abscisic Acid on Chlorophyll a Fluorescence in Guard Cells of Vicia faba

The effects of cations and abscisic acid on chloroplast activity in guard cells of Vicia faba were investigated by analysis of the transient of chlorophyll a fluorescence. When epidermal strips containing guard cells as the only living cells were incubated in water and illuminated with strong light, chlorophyll a fluorescence rose rapidly to a high intensity and then declined slowly to a stationary level. The rate of this decline was enhanced by K⁺ or Na⁺, and the effect of these cations was greater when added with phosphate than with chloride as the anion. Ca²⁺ suppressed the enhancement by Na⁺ and, to a lesser extent, that by K⁺. Abscisic acid also suppressed the enhancement by K⁺ and Na⁺. Since the fluorescence decline reflects the increase of intrathylakoid H⁺ concentration necessary for photophosphorylation, the acceleration of the decline by K⁺ (or Na⁺ in the absence of Ca²⁺) implicates chloroplast activity in ion accumulation by guard cells in the light. The differential effects of phosphate and chloride suggest that chloroplast activity may be involved in malate formation in guard cells in the light.     

Blue light-modulation of chlorophyll a fluorescence transients in guard cell chloroplasts

Chlorophyll a fluorescence transients from mesophyll and single guard cell pairs of Vicia faba were measured by microspectrofluorometry. In both chloroplast types, fluorescence induction (O to P) was similar under actinic blue and green light. In slow transients from mesophyll cell chloroplasts, blue and green light induced identical, typical rapid quenching from P to S, and the M peak. In contrast, the P to S transient from guard cell (GC) chloroplasts irradiated with blue light showed a much slower quenching rate, and the P to T transition showed no M peak. Actinic green light induced mesophyll-like transients in GC chloroplasts, including rapid quenching from P to S and the M peak. Detection of these transients in single pairs of GC and isolated protoplasts ruled out mesophyll contamination as a signal source. Green light induced a rapid quenching and the M peak in GC chloroplasts from several species. The effect of CO₂ concentration on the fluorescence transients was investigated in the presence of HCO₃⁻ at pH 6.8 and 10.0. In transients induced by green light in both chloroplast types, a pH increase concomitant with a reduction in CO₂ concentration caused an increase in the initial rate of quenching and the elimination of the M peak. Actinic blue light induced mesophyll-like transients from GC chloroplasts in the presence of 10 micromolar KCN, a concentration at which the blue light-induced stomatal opening is inhibited. Addition of 100 to 200 micromolar phosphate also caused large increases in fluorescence quenching rates and a M peak. These results indicate that blue light modulates photosynthetic activity in GC chloroplasts. This blue light effect is not observed in the absence of transduction events connected with the blue light response and in the presence of high phosphate concentrations.     

Partial purification of a plasma membrane flavoprotein and NAD(P)H-oxidoreductase activity

Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H-dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two-phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg^?1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non-covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H-oxidoreductase activity and suggests the presence of plasma membrane bound pterins.     

Relationship of Plasmalemma Redox Activity to K+ Uptake by Dunaliella salina Cells

The plasmalemma-bond redox system localized within the plasmalemma of unicellular green alga Dunaliella salina was studied. This system oxidized exogenous NADH, increased O2 consumption to 165 % and increased the pH of the external medium, while K+ influx was inhibited. With no NADH added, ferricyanide stimulated K+ uptake about 3 folds. In the presence of exogenous NADH, ferricyanide was rapidly reduced and the external medium was acidified, generating a greater electrochemical proton gradient across the plasmalemma, thus resulting an 6-fold increase of K+ influx. Typical inhibitors of plasmalemma H+-ATPase and redox system inhibited K+ uptake to different extent. That the inhibition of K+ uptake by vanadate could be resumed partly by addition of NADH and ferricyanide indicated that plasmalemma redox system operated in association with the H+-ATPase to exert an influence on K+ transportation. A model was presented in which the implication of two possible redox chains and H+-ATPase in generating an electrochemical potential gradient for protons (△uH+) was discussed.     

Purification and characterization of the molybdoenzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini

The enzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase from Bacillus niacini could be purified to homogeneity by means of anion exchange chromatography, hydrophobic interaction chromatography, gel filtration, and chromatography on hydroxylapatite. During enrichment procedures both enzymes showed a significant loss in specific activity. The molecular weight of nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase was determined to be about 300,000 and 120,000, respectively. They were highly substrate specific and transferred electrons only to artificial acceptors of high redox potential. The K _m for their specific substrates was about 1.0 mM for both enzymes, and their pH optimum was determined to be 7.5. For nicotinate dehydrogenase a content of 8.3 mol iron, 1.5 mol acid-labile sulfur, 2.0 mol flavin, and 1.5 mol molybdenum per mol of enzyme was determined. Both enzymes contained FAD and Fe/S center. After inhibition by KCN, thiocyanate was detected, and subsequently the initial nicotinate dehydrogenase activity was restored by the addition of Na₂S indicating the presence of cyanolyzable sulfur. 6-Hydroxynicotinate dehydrogenase seemed to contain the same type of constituents as determined for nicotinate dehydrogenase. A partial immunological identity of the enzymes could be shown by antibodies raised against nicotinate dehydrogenase.Abbreviations DCPIP 2,6-dichlorophenol-indophenol - EEO electroendosmosis - FTTC fluorescein isothiocyanate - HAP hydroxylapatite - 6-HDH 6-hydroxynicotinate dehydrogenase - NBT nitroblue tetrazolium chloride - NDH nicotinate dehydrogenase - MTT thiazolyl blue - PES phenazine ethosulfate - PMSF phenylmethyl sulfonyl fluoride - TEMED N,N,N',N'-tetramethyl-ethylenediamine