Reduction Of Extracellular Ferricyanide Research Articles

Role of respiratory terminal oxidases in the extracellular electron transfer ability of cyanobacteria.

Cyanobacteria are used as anode catalysts in photo-bioelectrochemical cells to generate electricity in a sustainable, economic, and environmental friendly manner using only water and sunlight. Though cyanobacteria (CB) possess unique advantage for solar energy conversion by virtue of its robust photosynthesis, they cannot efficiently perform extracellular electron transfer (EET). The reasons being, unlike dissimilatory metal reducing bacteria (that are usually exploited in microbial fuel cells to generate electricity), (1) CB do not possess any special features on their outer membrane to carry out EET and, (2) the electrons generated in photosynthetic electron transport chain are channeled into competing respiratory pathways rather than to the anode. CB, genetically engineered to express outer membrane cytochrome S (OmcS), was found to generate ∼nine-fold higher photocurrent compared to that of wild-type cyanobacterium in our previous work. In this study, each of the three respiratory terminal oxidases in Synechococcus elongatus PCC7942 namely bd-type quinol oxidase, aa3 -type cytochrome oxidase, and cbb3 -type cytochrome oxidase was knocked-out one at a time (cyd- , cox- , and cco- respectively) and its contribution for extracellular ferricyanide reduction and photocurrent generation was investigated. The knock-out mutant lacking functional bd-type quinol oxidase (cyd- ) exhibited greater EET by reducing more ferricyanide compared to other single knock-out mutants as well as the wild type. Further, cyd- omcs (the cyd- mutant expressing OmcS) was found to generate more photocurrent than the corresponding single knock out controls and the wild-type. This study clearly demonstrates that the bd-quinol oxidase diverted more electrons from the photosynthetic electron transport chain towards respiratory oxygen reduction and knocking it out had certainly enhanced the cyanobacterial EET.

How do erythrocytes contribute to the ABTS* scavenging capacity of blood?

It has been suggested lately that erythrocytes contribute significantly to the oxidant scavenging capacity (OSC) of blood and that surface adsorption of polyphenols enhances the antioxidant capacity of erythrocytes. The aim of this study was to examine the contribution of erythrocytes to the OSC of whole blood measured with a substrate not penetrating into the cells. Comparison of reduction of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical (ABTS*) by whole blood and blood plasma indicates that erythrocytes do contribute to ABTS* reduction but their contribution is lower with respect to plasma. ABTS* reduction by erythrocytes and its enhancement by polyphenols were inhibited by thiol reagents (N-ethylmaleimide and iodacetate). These reagents inhibited also the reduction of extracellular ferricyanide by erythrocytes and its enhancement by polyphenols. On this basis we postulate that the contribution of erythrocytes to the blood OSC estimated by ABTS* decolorization is at least partly due to the transmembrane-reducing system, which activity is routinely assayed by ferricyanide reduction.

Assessing the reductive capacity of cells by measuring the recycling of ascorbic and lipoic acids.

Most mammalian cells cannot synthesize vitamin C, or ascorbic acid, and thus must have efficient mechanisms for its intracellular recycling. Ascorbate can be recycled from both its oxidized forms using electrons from several intracellular reducing co-factors, including GSH and the reduced pyridine nucleotides. Methods have been developed to assess the ability of intact cells to recycle ascorbate, which include assay of extracellular ferricyanide reduction and measurement of the ability of the cells to reduce dehydroascorbic acid to ascorbate. Lipoic acid, a disulfide containing medium chain fatty acid, is also taken up by cells and reduced to dihydrolipoic acid, which can be measured upon efflux from the cells using Ellman's reagent. Together, these assays provide an estimate of the ability of different cell types to recycle ascorbate and to generate intracellular reducing equivalents required to maintain the redox status of the cells.

Reduction and uptake of methylene blue by human erythrocytes.

A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB(+)) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB(+). Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB(+) <5 microM selectively oxidized NAD(P)H, higher concentrations also oxidized both glutathione (GSH) and ascorbate, especially in the absence of d-glucose. MB(+)-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB(+)-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB(+), which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB(+), that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB(+) reduction may correspond to MB(+)-dependent NAD(P)H reductase activity in erythrocyte ghosts.

Generation of oxidant stress in cultured endothelial cells by methylene blue: protective effects of glucose and ascorbic acid

The thiazine dye methylene blue has long been used to stimulate cellular redox metabolism. To determine the extent to which it also generates oxidant stress in cells, its effects in cultured human-derived endothelial cells were studied. As expected, low concentrations of the dye (2–20 μM) activated the pentose phosphate pathway and oxidized both NADPH and NADH. Methylene blue enhanced extracellular ferricyanide reduction, indicating that the reduced form of the dye was present outside the cells. This reduction was greater when ferricyanide was added just before rather than 15 min after methylene blue, confirming that the dye is at least initially reduced at the cell surface. In the absence of glucose, methylene blue at concentrations above 5 μM increased intracellular oxidant stress, as manifest by oxidation of dihydrofluorescein and cellular GSH. Inclusion of glucose protected against these effects. In cells that had been loaded with ascorbate, the dye caused progressive oxidation of ascorbate, even in the presence of d-glucose. Loading cells with ascorbate also partially prevented oxidation of dihydrofluorescein by methylene blue. These results suggest that concentrations of the dye above 5 μM generated intracellular reactive oxygen species that were scavenged by ascorbate and GSH. Further, although d-glucose enhanced reduction of methylene blue, it ameliorated the oxidant stress generated by the dye.

Feasibility of Measuring Ferricyanide Reduction by Yeasts to Estimate Their Activity during Alcoholic Fermentation in Wine-Making Conditions

We assessed the feasibility of measuring the extracellular reduction of ferricyanide in the presence of an intermediate carrier (menadione) as a means of estimating the activity of yeasts during alcoholic fermentation. A spectrophotometric and a potentiometric approach were used. Comparison of specific reductase activity and gas production rate during the stationary phase indicated that measuring the menadione-catalyzed reduction of ferricyanide provides a good estimate of the total activity of the yeast cells in a fermenting must. The response observed following the addition of an electron acceptor (acetaldehyde) confirmed that the reductase activity of menadione is dependent on the availability of NADH. The stability of menadione in the fermentation medium, as assessed by the potentiometric method, suggested that electrochemical reoxidation of the ferrocyanide can act as a substitute for the addition of an electron acceptor when studying the redox regulation of fermenting yeasts.

Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes

Reduction of extracellular ferricyanide [Fe(CN) 6] −3 to ferrocyanide by intact cells reflects the activity of a trans-plasma membrane oxidoreductase that, in human red blood cells, utilizes ascorbic acid as an electron donor. We herein report that the flavonoids quercetin and myricetin, while inhibiting dehydroascorbic acid uptake—and thus the erythrocyte ascorbic acid content—effectively stimulate the extracellular reduction of ferricyanide. Other flavonoids such as rutin, acacetin, apigenin, and genistein do not show the same effect. The notion that quercetin or myricetin may serve as an intracellular donor for a trans-plasma membrane oxidoreductase is supported by the following lines of evidence: (i) they afford direct reduction of ferricyanide; (ii) extracellular reduction of ferricyanide was not mediated by direct effects of the flavonoids released by the cells and was abolished by the sulphydryl reagent parachloromercuribenzenesulfonic acid ( pCMBS); (iii) the intracellular concentrations of quercetin or myricetin well correlate with increases in ferricyanide reduction; (iv) the intracellular concentration of the flavonoids dramatically declines after ferricyanide exposure. Taken together, the results presented in this study demonstrate that myricetin and quercetin, which accumulate in large amounts in red blood cells, act as intracellular substrates of a pCMBS-sensitive trans-plasma membrane oxidoreductase. This may represent a novel mechanism whereby these flavonoids exert beneficial effects under oxidative stress conditions.

Mechanisms of ascorbic acid recycling in human erythrocytes

Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of d-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM d-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by d-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75–90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of d-glucose metabolism in the pentose cycle, but that in the absence of d-glucose DHA reduction can also utilize NADH.

Ferric reduction by iron-limited Chlamydomonas cells interacts with both photosynthesis and respiration.

Iron limitation led to a large increase in extracellular ferricyanide (Fe[III]) reductase activity in cells of the green alga Chlamydomonas reinhardtii Dangeard. Mass-spectrometric measurement of gas exchange indicated that ferricyanide reduction in the dark resulted in a stimulation of respiratory CO2 production without affecting the rate of respiratory O2 consumption, consistent with the previously postulated activation of the oxidative pentose phosphate pathway in support of Fe(III) reduction by iron-limited Chlamydomonas cells (X. Xue et al., 1998, J. Phycol. 34: 939-944). At saturating irradiance, the rate of ferricyanide reduction was stimulated almost 3-fold, and this stimulation was inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea. Ferricyanide reduction during photosynthesis resulted in approximately a 50% inhibition of photosynthetic CO2 fixation at saturating irradiance, and almost 100% inhibition of CO2 fixation at sub-saturating irradiance. Photosynthesis by iron-sufficient cells was not affected by ferricyanide addition. Addition of 250 microM ferricyanide to iron-limited cells in which photosynthesis was inhibited (either by the presence of glycolaldehyde, or by maintaining the cells at the CO2 compensation point) resulted in a stimulation in the rate of gross photosynthetic O2 evolution. Chlorophyll a fluorescence measurements indicated a large increase in non-photochemical quenching during ferricyanide reduction in the light; the increase in nonphotochemical quenching was abolished by the addition of nigericin. These results suggest that reduction of extracellular ferricyanide (mediated at the plasma membrane) interacts with both photosynthesis and respiration, and that both of these processes contribute NADPH in the light.

Ascorbate-dependent electron transfer across the human erythrocyte membrane

Reduction of extracellular ferricyanide by intact cells reflects the activity of an as yet unidentified trans-plasma membrane oxidoreductase. In human erythrocytes, this activity was found to be limited by the ability of the cells to recycle intracellular ascorbic acid, its primary trans-membrane electron donor. Ascorbate-dependent ferricyanide reduction by erythrocytes was partially inhibited by reaction of one or more cell-surface sulfhydryls with p-chloromercuribenzene sulfonic acid, an effect that persisted in resealed ghosts prepared from such treated cells. However, treatment of intact cells with the sulfhydryl reagent had no effect on NADH-dependent ferricyanide or ferricytochrome c reductase activities of open ghosts prepared from treated cells. When cytosol-free ghosts were resealed to contain trypsin or pronase, ascorbate-dependent reduction of extravesicular ferricyanide was doubled, whereas NADH-dependent ferricyanide and ferricytochrome c reduction were decreased by proteolytic digestion. The trans-membrane ascorbate-dependent activity was also found to be inhibited by reaction of sulfhydryls on its cytoplasmic face. These results show that the trans-membrane ferricyanide oxidoreductase is limited by the ability of erythrocytes to recycle intracellular ascorbate, that it does not involve the endofacial NADH-dependent cytochrome b 5 reductase system, and that it is a trans-membrane protein that contains sensitive sulfhydryl groups on both membrane faces.

Role of ascorbic acid in transferrin-independent reduction and uptake of iron by U-937 cells

The role of ascorbic acid in transferrin-independent ferric iron reduction and uptake was evaluated in cultured U-937 monocytic cells. Uptake of 55Fe by U-937 cells was doubled by 100 μM extracellular ascorbate, and by pre-incubation of cells with 100 μM dehydroascorbic acid, the two-electron-oxidized form of ascorbate. Reduction of extracellular ferric citrate also was enhanced by loading the cells with dehydroascorbic acid. Dehydroascorbic acid was taken up rapidly by the cells and reduced to ascorbate, such that the latter reached intracellular concentrations as high as 6 mM. However, some ascorbate did escape the cells and could be detected at concentrations of up to 1 μM in the incubation medium. Further, addition of ascorbate oxidase almost reversed the effects of dehydroascorbic acid on both 55Fe uptake and ferric citrate reduction. Thus, it is likely that extracellular ascorbate reduced ferric to ferrous iron, which was then taken up by the cells. This hypothesis also was supported by the finding that during loading with ferric citrate, only extracellular ascorbate increased the pool of intracellular ferrous iron that could be chelated with cell-penetrant ferrous iron chelators. In contrast to its inhibition of ascorbate-dependent ferric iron reduction, ascorbate oxidase was without effect on ascorbate-dependent reduction of extracellular ferricyanide. This indicates that the cells use different mechanisms for reduction of ferric iron and ferricyanide. Therefore, extracellular ascorbate derived from cells can enhance transferrin-independent iron uptake by reducing ferric to ferrous iron, but intracellular ascorbate neither contributes to this reduction nor modifies the redox status of intracellular free iron.

Ascorbate-mediated transplasma membrane electron transport in pulmonary arterial endothelial cells.

Pulmonary endothelial cells are capable of reducing certain electron acceptors at the luminal plasma membrane surface. Motivation for studying this phenomenon comes in part from the expectation that it may be important both as an endothelial antioxidant defense mechanism and in redox cycling of toxic free radicals. Pulmonary arterial endothelial cells in culture reduce the oxidized forms of thiazine compounds that have been used as electron acceptor probes for studying the mechanisms of transplasma membrane electron transport. However, they reduce another commonly studied electron acceptor, ferricyanide, only very slowly by comparison. In the present study, we examined the influence of ascorbate [ascorbic acid (AA)] and dehydroascorbate [dehydroascorbic acid (DHAA)] on the ferricyanide and thiazine reductase activities of the bovine pulmonary arterial endothelial cell surface. The endothelial cells were grown on microcarrier beads so that the reduction of ferricyanide and methylene blue could be studied colorimetrically in spectrophotometer cuvettes and in flow-through cell columns. The ferricyanide reductase activity could be increased 80-fold by adding DHAA to the medium, with virtually no effect on methylene blue reduction. The DHAA effect persisted after the DHAA was removed from the medium. AA also stimulated the ferricyanide reductase activity but was less potent, and the relative potencies of AA and DHAA correlated with their relative rates of uptake by the cells. The results are consistent with the hypothesis that AA is an intracellular electron donor for an endothelial plasma membrane ferricyanide reductase and that the stimulatory effect of DHAA is the result of increasing intracellular AA. Adding sufficient DHAA to markedly increase extracellular ferricyanide reduction had little effect on the plasma membrane methylene blue reductase activity, suggesting that pulmonary arterial endothelial cells have at least two separate transplasma membrane electron transport systems.

Ascorbate recycling in human erythrocytes: Role of GSH in reducing dehydroascorbate

Human erythrocytes regenerate ascorbate from its oxidized product, dehydroascorbate. The extent to which such ascorbate recycling occurs by a GSH-dependent mechanism was investigated. In the presence of glucose, erythrocytes took up over 90% of extracellular [ 14C]dehydroascorbate and rapidly converted it to [ 14C]ascorbate, which was trapped within the cells. Dehydroascorbate uptake and reduction was not associated with generation of a monoascorbyl free radical intermediate. Uptake and reduction of dehydroascorbate by glucosedepleted erythrocytes coordinately decreased GSH and raised GSSG concentrations in erythrocytes. This effect was reversed by D-glucose, but not by l-lactate. Conversely, depletion of cellular GSH decreased the ability of cells to recycle dehydroascorbate to ascorbate, as reflected in the extent to which cells were able to reduce extracellular ferricyanide. Monoascorbyl free radical was formed during the reduction of extracellular ferricyanide, indicating that one electron transfer steps were involved in this process. In GSH-depleted cells, addition of l-lactate as an energy source for glycolysis-dependent NADH regeneration did cause a partial recovery of the ability of cells to reduce ferricyanide. However, in resealed erythrocyte ghosts containing either 4 mM GSH or 400 μM NADH, only the GSH-containing ghosts supported regeneration of ascorbate from added dehydroascorbate. These results suggest that in human erythrocytes ascorbate regeneration from dehydroascorbate is largely GSH dependent, and that it occurs through either enzymatic or nonenzymatic reactions not involving the monoascorbyl free radical

Ascorbic acid recycling enhances the antioxidant reserve of human erythrocytes.

The role of ascorbate transport and metabolism in the response of human erythrocytes to an extracellular oxidant stress was investigated. Rates of entry and exit of [14C]dehydroascorbate from erythrocytes were more than 10-fold greater than those of [14C]ascorbate. Both the reduced and oxidized forms of the vitamin were transported largely by the glucose transporter. Inside erythrocytes, dehydroascorbate was converted to ascorbate, increasing intracellular ascorbate concentrations 2-3-fold over those in the medium. In such ascorbate-loaded cells, the membrane-impermeant oxidant ferricyanide induced a transmembrane oxidation of intracellular ascorbate to dehydroascorbate. The latter escaped the cells on the glucose transporter, which resulted in a halving of the net entry of [14C]dehydroascorbate in the presence of ferricyanide. Treatment of ascorbate-loaded cells with H2O2 and Cu2+ also oxidized ascorbate and induced efflux of [14C]dehydroascorbate. Ferricyanide-dependent intracellular oxidation of ascorbate resulted in a corresponding reduction of extracellular ferricyanide, which served as an integrated measure of ascorbate recycling. Ferricyanide reduction was proportional to the loading concentration of dehydroascorbate and was enhanced when loss of dehydroascorbate from cells was decreased, either by blockade of the glucose transporter or by concentrating the cells. Selective depletion of cellular ascorbate lowered rates of ferricyanide reduction by two-thirds, suggesting that ascorbate rather than NADH is the major donor for the transmembrane ferricyanide oxidoreductase activity. On the basis of the ascorbate-dependent rate of ferricyanide reduction, erythrocytes at a 45% hematocrit can regenerate the ascorbic acid present in whole blood every 3 min. Erythrocyte ascorbate recycling may thus contribute more to the antioxidant reserve of blood than is evident from plasma ascorbate concentrations alone.

Lipid peroxidation in Plasmodium falciparum-parasitized human erythrocytes

cis-Parinaric acid (PnA) was used as a fluorescent probe to study lipid peroxidation in nonparasitized and Plasmodium falciparum-parasitized erythrocytes, upon challenge by cumene hydroperoxide and tert-butyl hydroperoxide. Parasitized erythrocytes were less susceptible toward lipid peroxidation than nonparasitized erythrocytes with which they had been cultured. Furthermore, nonparasitized erythrocytes cultured together with parasitized cells, and thereafter isolated on a Percoll gradient, were less susceptible toward lipid peroxidation than erythrocytes kept under the same experimental conditions but in the absence of parasitized cells. We concluded, therefore, that the intracellular development of the parasite leads to an increase in the resistance against oxidative stress, not only of the host cell membrane of the parasitized erythrocyte, but also in the plasma membrane of the neighboring cells. The erythrocyte cytosol of parasitized cells and/or the intraerythrocytic parasite was required for the increased protection of the host cell membrane, since ghosts prepared from parasitized erythrocytes were more susceptible to lipid peroxidation than those prepared from nonparasitized ones. Vitamin E content of parasitized erythrocytes was lower than that of nonparasitized cells. However, parasitized erythrocytes promoted extracellular reduction of ferricyanide at higher rates, which might be indicative of a larger cytosolic reductive capacity. It is suggested that the improved response of intact erythrocytes is due to an increased reduction potential of the host-erythrocyte cytosol. The role of vitamin C as a mediator of this process is discussed.

Phytophthora elicitor inhibits ferricyanide reduction by cultured rose cells

Suspension cultured cells of Rosa damascena Mill. cv. Gloire de Guilan exhibited normal plasma membrane electron transport activities, including the reduction of extracellular ferricyanide to ferrocyanide, the reduction of Fe(III) chelate, and the oxidation of exogenous NADH. Elicitor preparations from autoclaved cell walls of Phytophthora sp. or from clarified fungal growth medium inhibited ferricyanide reduction (and stimulated ferrocyanide oxidation) by the rose cells. The effect occurred after a lag period of 10–15 min and lasted for at least 60 min. There was no inhibition of reduction of Fe(III) chelate; there was a stimulation of oxidation of exogenous NADH. The effect was observed after a treatment of the cells with salicylate, but not after treatment with ultraviolet radiation or cycloheximide, agents that otherwise mimic the effect of elicitor on rose cell plasma membrane activities. There was no effect of elicitor added in vivo or in vitro on the NADH-ferricyanide oxidoreductase activity of isolated plasma membrane. The inhibition of ferricyanide reduction and oxidation of ferrocyanide could not be attributed to an elicitor-dependent accumulation of O − 2 or H 2O 2, since added superoxide dismutase and catalase did not block the inhibition of ferricyanide reduction. It is suggested that the inhibition of ferricyanide reduction was related to a change in the redox state of cytoplasmic NADH/NAD +.

Extracellular ferricyanide reduction and nitrate reductase activity in the green alga Monoraphidium braunii

The unicellular green alga, Monoraphidium braunii, reduced extracellular ferricyanide at high rates. Impermeability of the membranes to ferri- and ferrocyanide was shown for untreated cells as for those permeabilized with 5% 1-propanol. Fe starvation stimulated reduction rates by 50%, light or glucose were ineffective. Extracellular acidification largely corresponded to formation of the new anionic valence of ferrocyanide. 1-Propanol used as permeabilizer for NR assays in the same samples apparently inhibited ferricyanide reduction, however addition of catalase showed that this was not due to interference with ferricyanide reduction but to partial reoxidation by H 2O 2. Between ferricyanide reduction and nitrate reduction no competitionwas found in an ‘in situ’ system with endogenous reductant in the presence of 1-propanol, neither with ‘induced’ algae showing high NR activity nor with ‘repressed’ algae grown in ammonium medium and showing only constitutive NR activity. According to this data the inducible high-activity NR and the inducible nitrate uptake system are not responsible for reduction of ferricyanide. The plasmalemma-bound, low activity, NR might have extracellular reducing activity, but also for this NR lack of competition in reduction of the two substrates indicated independent reactions in Monoraphidium.

Changes in intracellular redox and energy status during induced transplasma membrane electron transport in Cuscuta protoplasts

Extracellular reduction of ferricyanide was exhibited by isolated Cuscuta protoplasts. A larger decrease in NADH than NADPH levels of the ferricyanide-treated protoplasts pointed to the major involvement of the former as an electron donor. Glutathione levels were also found to be lowered in similarly treated tissue. The time-dependent variation in intracellular ATP levels in presence of ferricyanide supported the concept of plasma membrane ATPase activation during transplasma membrane electron transport in eukaryotes.

Interrelationships between trans-Plasma Membrane Electron/Proton Transfer Stoichiometry, Organic Acid Metabolism, and Nitrate Reduction in Dwarf Bean (Phaseolus vulgaris)

Iron deficiency in dwarf bean (Phaseolus vulgaris L.) induces an increased activity of a system in the rhizodermal cells, which reduces extracellular ferric salts, and an active proton efflux from the roots, which is coupled to accumulation of citrate and malate in the roots and subsequent export of these compounds in the xylem. During reduction of extracellular ferricyanide by Fe-deficient plants, the stoichiometry of electron transport to proton efflux is 2e(-)/1H(+), and citrate and malate levels in the roots are strongly decreased. Reduction of ferricyanide by Fe-sufficient plants has no influence on root and shoot levels of citrate and malate, but in such plants the process is characterized by a e(-)/H(+) efflux stoichiometry close to unity. Apparently, organic acid metabolism and transport are closely associated with the e(-)/H(+) efflux ratio. To assess the significance of organic acid metabolism as one of the direct intracellular components of the induced unbalanced e(-)/H(+) efflux by roots, we studied NO(3) (-) reduction in shoots and roots of Fe-deficient and Fe-sufficient plants. Nitrate reductase activity in the roots was positively correlated with the level of citrate and malate, whereas the enzyme activity in the leaves responded positively to the import of these organic acid anions.

Thansplasmalemma redox reactions and ion transport in photosynthetic and heterotrophic plant cells

Extracellular ferricyanide reduction, NADH and ferrocyanide oxidation were investigated by spectrophotometrical method on photosynthetic freshwater plants (Elodea canadensis Rich., Vallisneria spiralis L., Nitella flexilis L.) and heterotrophic tissues (roots of Triticum vulgare L., Hordeum vulgare L., Zea mays L., Pisum sativum L., Avena sativa L., Allium sativa L., Allium cepa L.). All species had ferricyanide reductase activity. The roots of land plants also carried out extracellular oxidation of NADH and ferrocyanide in contrast to leaves of the freshwater plants. External NADH stimulated ferricyanide reductase activity, but only with those objects that had external NADH oxidase activity. In all species ferricyanide decreased the membrane potential (MP), decreased the membrane resistance measured at a fixed current and inhibited K+ influx measured by flame photometry. The factors affecting ferricyanide reductase activity also influenced the inhibitory effect of ferricyanide on the MP and K+ transport. These results demonstrate a connection between transport, electrogenic and redox functions of the plasmalemma.