Root-mediated ferric reduction-responses to iron deficiency, exogenously induced changes in hormonal balance and inhibition of protein synthesis

The effects of the auxin analogue 2,4-dichloro-phenoxyaceticacid (2,4-D), the translation inhibitor cycloheximide (CHM)and iron deficiency on electron transport activities in rootshave been investigated. Deprivation of an external Fe supplycaused an increase in capacity to reduce both FeEDTA and theartificial electron acceptor ferricyanide. With respect to theiron stress-induced redox activity, bean (Phaseolus vulgarisL.) roots reduced both substrates at equal rates, while rootsof Plantago lanceolata L. exhibited clearly higher rates withFeEDTA. The iron stress induced FeEDTA and ferricyanide reductionactivities were unequally affected by CHM in both species, reflectingthe differences in substrate specificity. Application of 2,4-D via the nutrient solution induced an increasein ferricyanide reduction rates in iron-sufficient roots ofP. lanceolata; the reduction of FeEDTA was only slightly affected.The presence of 2,4-D caused no further enhancement in reductionactivity of plants grown without Fe. However, iron-deficientplants exhibited a distinct developmental pattern of ferricreduction activities when treated with 2,4-D, resembling thedevelopment of 2,4-D-induced ferricyanide reduction by iron-sufficientplants. This information is taken as support that the reduction of ferricyanideand FeEDTA can in part be separately regulated. The data alsoseem to indicate that the amount of Fe²⁺ is the main cause forinduction or repression of ferric chelate reduction by rootsof iron-efficient plants. Key words: Phaseolus vulgaris, Plantago, ferric iron reduction, cycloheximide, 2,4-dichlorophenoxyacetic acid     

Effects of various inhibitors on in vivo reduction by Plantago lanceolata L. roots

Roots of Plantago lanceolata L. showed an iron stress-induced increase in the rates of electron transport to the extracytoplasmatic acceptors FeEDTA and ferricyanide. No significant changes in the reduction of hexachloroiridate were observed with respect to the iron-nutritional status of the plants. The reduction activity of iron-deficient roots was inhibited by the translation inhibitor cycloheximide (CHM) and the amino acid analog p-fluorophenylalanine (FPA). In both cases, the reduction of FeEDTA and ferricyanide was affected to a different extent, providing evidence for enzyme heterogeneity. Resupply of FeEDTA to iron-deficient plants resulted in a qualitatively similar pattern of decrease in FeEDTA and ferricyanide reduction rates, although a longer time period was required for the decrease of the redox activity by iron resupply compared to the effect of inhibitors of protein synthesis.Inhibitors of the plasma membrane (PM)-bound H⁺-ATPase decreased the FeEDTA reduction activity of iron-deficient plants. In contrast, the reduction of ferricyanide and hexachloroiridate was not inhibited. Oxidation of ferrocyanide occurs in both iron-deficient and iron-sufficient plants at comparable rates. The reaction was decreased by the H⁺-ATPase inhibitor orthovanadate.The results are interpreted in terms of a simultaneous action of distinct redox systems in iron-deficient roots. The role of proton extrusion in the regulation of iron stress-induced electron transport is discussed.     

Reduction of extracytoplasmic acceptors by roots of Plantago lanceolata L. Evidence for enzyme heterogeneity

The capacity of iron-stressed (-Fe plants) and non-stressed (+Fe plants) roots of Plantago lanceolata L. to reduce acceptors differing in their midpoint potentials has been characterized. Highest reduction activity in iron sufficient roots was observed with the artificial acceptors hexachloroiridate (HCI) and ferricyanide. FeEDTA and ferric citrate (FeCitr) were reduced at equal rates with respect to maximal velocity; marked differences between the two latter reactions have been observed in the affinity for the substrate. Iron starvation increased the reduction of FeEDTA and ferricyanide, the rates of HCI and FeCitr reduction were not significantly affected. With the exception of HCI, the reduction rates of all acceptors were diminished by inhibitors of protein synthesis. The inhibition was more pronounced in iron stressed roots compared to +Fe grown plants. Protoplasts isolated from Plantago roots were capable of reducting FeCitr, but failed to reduce FeEDTA. The kinetics (K_m) of FeCitr reduction by root protoplasts resembled the characteristics of intact plant roots. The existence of distinct redox systems in root cells and their physiological significance are discussed with reference to results obtained with material reduced in biological complexity.     

Pyridine nucleotide pool size changes in iron-deficient Plantago lanceolata roots during reduction of external oxidants

The involvement of pyridine nucleotides in the reduction of extracytoplasmatic electron acceptors by iron-deficient Plantago lanceolata L. roots has been examined by measuring the changes in NAD(P)H and NAD(P)⁻ induced by various external acceptors. Exposure of the plants to FeEDTA, ferricyanide, ferric citrate or hexachloroiri-date resulted in a transient decrease in NADPH and an increase in NAD⁻. No major differences in this pattern were observed between acceptors which were assumed to be reduced by different enzymes. The application of the membrane-permeable oxidant nitro blue tetrazolium led to similar changes in reduced and oxidized pyridine nucleotides and decreased the reduction of external acceptors. The amino acid analog p -fluorophenylalanine caused a transient decline in both NADPH level and NADPH/ NADP⁻ ratio and a decrease in the ratio of NADH to NAD⁻ without affecting the level of NADH. Exposure of the plants to the translation inhibitor cycloheximide increased both NADH and NADPH concentrations. A comparison of the redox activities and pyridine nucleotide fractions after inhibitor treatment revealed that the constitutive, but not iron stress-induced redox activity correlates with NADPH levels. These results are interpreted as confirming that the redox systems on the root plasma membrane are separately regulated. Possible metabolic reactions during the reduction processes are discussed.     

Ferric chelate reduction by sunflower (Helianthus annuus L.) leaves: influence of light, oxygen, iron-deficiency and leaf age

The presence of ferric chelate reducing activity in sunflower[Helianthus annuus L.) leaves has been studied by submergingleaf discs in a solution with Fe(III)-ethylenediaminetetra-acetate(FeEDTA), batho-phenanthroline disulphonate (BPDS) and vacuuminfiltration. The effect of different factors on the Fe(III)reduction rate was studied. Ferric reduction rate was about10-fold higher in the light than in darkness. The light effectwas greatly inhibited by 3-(3,4-dichloro-phenyl)-1,1-dimethylurea(DCMU), a photosystem II inhibitor. Several inhibitors of redoxsystems [cis-platinum (II) diamine dichloride (cis-platin),p-nitro-phenylacetate (p-NPA) and p-hydroxymercuribenzoic acid(pHMB)] decreased the FeEDTA reduction rate. The greatest inhibitionwas produced by the - SH group reagent pHMB (17% of control,in light). The FeEDTA reduction rate was much higher in theabsence of O₂ than with air or 100% O₂. Superoxide dismutase(SOD) decreased FeEDTA reduction with air in the light. Youngleaves reduced Fe(III)-chelate at a higher rate than did olderleaves. In iron-deficient plants, leaves did not exhibit enhancedferric chelate-reducing activity as was observed in roots. Itis suggested that at least two different redox systems or twostates of the same redox system work in the light and in darkness. Key words: Iron, leaves, plasma membrane-redox, light, oxygen level     

The induction of the “turbo reductase” is inhibited by cycloheximide, cordycepin and ethylene inhibitors in Fe-deficient cucumber (Cucumis sativus L.) plants

Summary Dicotyledonous plants respond to Fe deficiency by enhancing the capacity of their roots to reduce Fe(III) to Fe(II). It has been suggested that there are two different ferric redox systems in the roots: the standard reductase, active with ferricyanide and not inducible by Fe deficiency, and the turbo reductase, active with both ferricyanide and ferric chelates and inducible by Fe deficiency. We have used different experimental approaches to test whether or not the Fe(III)-reducing capacity of cucumber (Cucumis sativus L. cv. Ashley) roots can be explained by considering the standard and the turbo reductase as the same enzyme. For this, we used both Fe-sufficient and Fe-deficient plants, which were treated with ethylene inhibitors (cobalt or silver thiosulfate; found to inhibit the turbo reductase in a previous work), a protein synthesis inhibitor (cycloheximide), or an mRNA polyadenylation inhibitor (cordycepin). At different times after application of these inhibitors, reduction of both ferricyanide and Fe(III)-EDTA were determined. In addition, we studied the effects of pH and temperature on the reduction of ferricyanide and Fe(III)-EDTA by both Fe-sufficient and Fe-deficient plants. Results suggest that there are, at least, two different ferric redox systems in the roots. Enhancement of Fe(III)-reducing capacity (turbo reductase) by Fe-deficient plants probably requires the de novo synthesis of a (or several) protein(s), which has a high turnover rate and whose expression is presumably regulated by ethylene.Abbreviations Ch-R ferric chelate reductase - CHM cycloheximide - CN-R ferricyanide reductase - EDDHA N,N-ethylene bis[2-(2-hydroxyphenyl)-glycine] - EDTA ethylenediamine-tetraacetic acid - Ferrozine 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine - HEDTA N-hydroxyethylethylene-diaminetriacetic acid - STS silver thiosulfate     

Evidence for electron transport across the plasma membrane of Zea mays root cells

Exogenous ferricyanide is reduced by roots of Z. mays. In contrast to oxidation of exogenous electron donors, ferricyanide reduction occurs mostly at the apical 5 mm of the root. Using just this portion of the root, it is shown that the activity is neither a consequence of uptake of ferricyanide followed by excretion of its reduced form, nor of leakage of a reductant. Addition of ferricyanide for 40 s or 5 min results in an apparent oxidation of NADPH but not of NADH; rates of ferricyanide reduction vary together with levels of NADPH but not of NADH in the presence or absence of oxygen. It is concluded that an enzyme which can oxidize cytoplasmic NADPH and transfer the electrons to an external acceptor exists at the cell surface of maize roots. This finding extends the results of others who showed similar redox activity at the surface of Fe-depleted dicotyledonous roots, and indicates that an energy source other than ATP exists at the cell surface of a variety of plants under unstressed conditions.     

Reduction of root iron in Plantago lanceolata during recovery from Fe deficiency

Changes in root-associated Fe(III) reductase activity and Fe concentration during recovery from temporary iron starvation were investigated in hydroponically grown Plantago lanceolata L. Within two days, interruption of the Fe supply resulted in enhanced rates of reduction by intact plant roots. Transfer of iron deficient plants to a solution containing various amounts of chelated Fe caused a transient increase in reduction activity before the rates declined to a level determined by the amount of Fe added. Repression of root-associated redox activity was independent of the Fe concentration in the preculture. When iron deficient plants were submitted to a supply of Fe localized to a part of the root system (split-root plants), the decrease in reduction rates was much more pronounced in the Fe-deprived portion of the roots than in the Fe-supplied one. No correlation was observed between root Fe concentration and Fe(III) reductase activity. Continued growth of split-root plants in the +Fe/-Fe regime increased the reduction rates of the +Fe-grown portion of the root system over the rates in iron sufficient plants with non-divided roots. The results are discussed in relation to putative factors mediating intra- and interorgan regulation of iron nutrition.     

Reduction of root iron in Plantago lanceolata during recovery from Fe deficiency

Changes in root-associated Fe(III) reductase activity and Fe concentration during recovery from temporary iron starvation were investigated in hydroponically grown Plantago lanceolata L. Within two days, interruption of the Fe supply resulted in enhanced rates of reduction by intact plant roots. Transfer of iron deficient plants to a solution containing various amounts of chelated Fe caused a transient increase in reduction activity before the rates declined to a level determined by the amount of Fe added. Repression of root-associated redox activity was independent of the Fe concentration in the preculture. When iron deficient plants were submitted to a supply of Fe localized to a part of the root system (split-root plants), the decrease in reduction rates was much more pronounced in the Fe-deprived portion of the roots than in the Fe-supplied one. No correlation was observed between root Fe concentration and Fe(III) reductase activity. Continued growth of split-root plants in the +Fe/-Fe regime increased the reduction rates of the +Fe-grown portion of the root system over the rates in iron sufficient plants with non-divided roots. The results are discussed in relation to putative factors mediating intra- and interorgan regulation of iron nutrition.     

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.     

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.     

The proton-pumps at the plasmalemma of Catharanthus roseus cells

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide (FIC) reduction which is associated with a proton translocation and a decrease in the ATP content of the cells. The H+ efflux and the ATP consumption may be counteracted by vanadate, a specific inhibitor of the ATPase activity, and by Na2WO4 which prevents FIC reduction. From these data it is concluded that the redox chain could be coupled with ATP hydrolysis for electrogenic proton extrusion which may involve a redox control mechanism for the plasmamembrane ATPase.     

Inhibition of iron deficiency stress response in cucumber by rare earth elements.

We investigated the influence of the trivalent scandium (Sc), chromium (Cr), gallium (Ga), yttrium (Y) and lanthanum (La) on both the function and activity of ferric chelate reductase (FCR) in cucumber (Cucumis sativus L.) roots. Cucumber seedlings were grown for 1week in a nutrient solution without Fe or in some experiments with 10microM FeEDTA. Intact root systems were assayed for FCR activity in a medium at pH 5.0 containing 100microM FeEDTA with the ferrous chelating agent Ferrozine. Addition of 100microM concentrations of the EDTA complexes of Sc, Cr, Ga, Y and La did not inhibit FCR in Fe-deficient roots. When Fe-deficient roots were grown with 10microM LaCl(3), ScCl(3), or YCl(3) for 3days, FCR activity decreased to 23%, 15% and 1%, respectively, of the activity of Fe-deficient plants grown without trivalent metal addition. Additionally, these trivalent metals suppressed proton secretion. Growth of Fe-deficient plants with 80microM Ga(2)(SO(4))(3) decreased FCR activity to 35% of the control activity while 80microM CrEDTA did not affect FCR activity. With the addition of either FeEDTA or YCl(3), FCR activity decreased to less than 5% of the activity of the Fe-deficient control roots in 3days. Addition of FeEDTA, but not Y, resulted in recovery from Fe deficiency as indicated by increasing chlorophyll content of leaves.     

PHYTOPLANKTON PLASMA MEMBRANE REDOX ACTIVITY: EFFECT OF IRON LIMITATION AND INTERACTION WITH PHOTOSYNTHESIS1

Phytoplankton plasma membrane electron transport activity was determined by monitoring the reduction of the impermeant artificial electron acceptor ferricyanide in a range of diatoms. The results revealed that constitutive plasma membrane electron transport activity of marine diatoms is high compared with chlorophytes and higher plant cells. Diatom plasma membrane electron transport activity was not significantly increased by iron limitation. This lack of induction on iron limitation indicates that diatoms have an iron acquisition strategy that is distinct from chlorophytes and the dicotyledon higher plants that exhibit marked increases in plasma membrane ferricyanide reductase activity on iron limitation. The interaction of the constitutive plasma membrane electron transport with photosynthesis was also investigated. We found that 1) ferricyanide reduction at the plasma membrane was progressively inhibited in response to increasing irradiances; 2) the presence of extracellular ferricyanide, but not the reduced couple ferrocyanide, caused a marked inhibition of carbon fixation at high irradiance; and 3) extracellular electron acceptors ferricyanide and hexachloroiridate (but not ferrocyanide) induced an immediate and reversible decrease in fluorescence yields (F_o and F_m). The extent to which extracellular electron acceptors affected CO₂ fixation, F_o, and F_m was related to the level of constitutive ferricyanide reductase activity, the species with highest ferricyanide reduction rates being most sensitive. The data suggest that consumption of electrons and/or reductant at the plasma membrane by external acceptors may compete directly with CO₂ fixation for electrons, alter cytosolic‐chloroplast redox poise, and/or induce a redox‐signaling cascade that alters photosynthetic metabolism.     

THE ENERGETICS OF EXTRACELLULAR FE(III) REDUCTION BY IRON-LIMITED CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

Fe-limited cells of the green alga Chlamydomonas reinhardtii (Fe-limited growth rate = 0.3 d⁻¹) reduced extracellular Fe(III) to Fe(II) when Fe(III) was supplied as ferricyanide or Fe(III)-EDTA; Fe(III) reduction was stimulated by light. In both darkness and during photosynthesis, ferricyanide reduction was accompanied by a decrease in cellular NADPH levels, with a concomitant increase in NADP⁺. NADH and NAD⁺ levels were not measurably altered during ferricyanide reduction. Furthermore, cellular hexose monophosphate levels declined and 6-phosphogluconate levels increased during ferricyanide reduction. Levels of most glycolytic and tricarboxylic acid cycle intermediates were mostly unaltered. Ferricyanide reduction was also associated with a decrease in cellular ATP levels, a concomitant increase in ADP and AMP, and increased extracellular acidification. The acidification was sensitive to inhibition by the H⁺-ATPase inhibitor N,N' -dicyclohexylcarbodiimide (DCCD). We conclude that the oxidative pentose phosphate pathway provides reducing equivalents for Fe(III) reduction in darkness and also contributes reducing equivalents to Fe(III) reduction during photosynthesis. The decline in ATP was likely due to activation of the plasma membrane H⁺-ATPase during ferricyanide reduction and was not directly associated with provision of reducing equivalents.     

Effect of primary leaves on 59Fe uptake by roots and 59Fe distribution in the shoot of iron sufficient and iron deficient bean (Phaseolus vulgaris L.) plants

A study has been made on the effect of primary leaves on iron (Fe) distribution in the shoot. Bean (Phaseolus vulgaris L.) seedlings were precultured in nutrient solution with 8×10^-5 M FeEDTA for 4 days, and then grown further with either 8×10^-5 M FeEDTA (+Fe) or without Fe supply (-Fe) for another 5 days. Thereafter, both +Fe and -Fe plants were treated in three different ways: undisturbed; one primary leaf removed; or one primary leaf shaded, starting two hours before supply ⁵⁹FeEDTA to the roots. The +Fe plants were supplied with 8×10^-5 M ⁵⁹FeEDTA, and the -Fe plants with only 1×10^-6 M ⁵⁹FeEDTA. After 1 to 8 hour uptake periods, plants were harvested and ⁵⁹Fe in different organs was determined. Removal or shading of one primary leaf did not affect ⁵⁹Fe uptake by roots and ⁵⁹Fe translocation to the shoot in +Fe plants. In the -Fe plants, however, removal of one primary leaf decreased ⁵⁹Fe uptake by roots, whereas shading of one primary leaf had no effect on ⁵⁹Fe uptake but slightly enhanced ⁵⁹Fe translocation from roots to the shoot. The quantity of ⁵⁹Fe in primary leaves was positively correlated with quantity of ⁵⁹Fe in the stem in the -Fepplants, but not in the +Fe plants. In both, the +Fe and -Fe plants, the quantity of ⁵⁹Fe in the shoot apex was positively correlated with ⁵⁹Fe in primary leaves. The results suggest that irrespective of the Fe nutritional status of plants, the source of Fe for the shoot apex is Fe retranslocated from primary leaves.     

Regulation of transplasmalemma electron transport in oat mesophyll cells by sphingoid bases and blue light

Long-chain sphingoid bases inhibit transplasmalemma electron transport in certain animal cells in part by inhibiting protein phosphorylation. As a first step in determining whether similar regulatory processes exist for cell surface redox activity in plants, peeled leaf segments of Avena sativa L. cv Garry were exposed to sphingoid bases and other long chain lipids. Sphingoid bases which are the most active inhibitors of protein kinase C in animal cells inhibit transplasmalemma electron transport by mesophyll cells in the dark as measured by reduction of exogenous ferricyanide. In white light, however, the same compounds markedly stimulate redox activity. The stimulation by sphingoid bases in the light is not eliminated by the inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). Redox activity remaining in the presence of DCMU and sphingoid bases can be observed in blue but not red light. A tentative hypothesis considering the involvement of two separate redox systems is presented in an attempt of explain the disparate action of sphingoid bases on electron transport across the plasmalemma.     

NO removal in continuous BioDeNOx reactors: Fe(II)EDTA2- regeneration, biomass growth, and EDTA degradation

BioDeNOx is a novel technique for NOx removal from industrial flue gases. In principle, BioDeNOx is based on NO absorption into an aqueous Fe(II)EDTA2- solution combined with biological regeneration of that scrubber liquor in a bioreactor. The technical and economical feasibility of the BioDeNOx concept is strongly determined by high rate biological regeneration of the aqueous Fe(II)EDTA2- scrubber liquor and by EDTA degradation. This investigation deals with the Fe(II)EDTA2- regeneration capacity and EDTA degradation in a lab-scale BioDeNOx reactor (10-20 mM Fe(II)EDTA2-, pH 7.2 +/- 0.2, 55 degrees C), treating an artificial flue gas (1.5 m3/h) containing 60-155 ppm NO and 3.5-3.9% O2. The results obtained show a contradiction between the optimal redox state of the aqueous FeEDTA solution for NO absorption and the biological regeneration. A low redox potential (below -150 mV vs. Ag/AgCl) is needed to obtain a maximal NO removal efficiency from the gas phase via Fe(II)EDTA2- absorption. Fe(III)EDTA- reduction was found to be too slow to keep all FeEDTA in the reduced state. Stimulation of Fe(III)EDTA- reduction via periodical sulfide additions (2 mM spikes twice a week for the conditions applied in this study) was found to be necessary to regenerate the Fe(II)EDTA2- scrubber liquor and to achieve stable operation at redox potentials below -150 mV (pH 7.2 +/- 0.2). However, redox potentials of below -200 mV should be avoided since sulfide accumulation is unwanted because it is toxic for NO reduction. Very low values for biomass growth rate and yield, respectively, 0.043/d and 0.009 mg protein per mg ethanol, were observed. This might be due to substrate limitations, that is the electron acceptors NO and presumably polysulfide, or to physiological stress conditions induced by the EDTA rich medium or by radicals formed in the scrubber upon the oxidation of Fe(II)EDTA2- by oxygen present in the flue gas. Radicals possibly also induce EDTA degradation, which occurs at a substantial rate: 2.1 (+/-0.1) mM/d under the conditions investigated.     

Inhibition of trans-membrane hexacyanoferrate III reductase activity and proton secretion of maize (Zea mays L.) roots by thenoyltrifluoroacetone

Summary Intact plants can reduce external oxidants by an appearingly trans-membrane electron transport. In vivo an increase in net medium acidification accompanies the reduction of the apoplastic substrate. Up to now, several NAD(P)H dehydrogenases,b-type cytochromes, and a phylloquinone have been identified and partially purified from plant plasma membranes. The occurrence of a quinone in the plasma membrane of maize roots supports the hypothetical model of a proton-transferring redox system, i.e., an electron transport chain with a quinone as mobile electron and proton carrier. In the present study the trans-membrane electron transport system of intact maize (Zea mays L.) roots was investigated. Flow-through and ionostat systems have been used to estimate the electron and proton transport activity of this material. Application of 4,4,4-trifluoro-1-(2-thienyl)-butane-1,3-dione (thenoyltrifluoroacetone) inhibited the reduction of ferricyanide in the incubation solution of intact maize roots up to 70%. This inhibition could not be washed off by rinsing the roots with fresh incubation medium. The acidification of the medium induced after ferricyanide application was inhibited to about 62%. The effects of thenoyltrifluoroacetone on proton fluxes in the absence of ferricyanide have been characterized in a pH-stat system. The net medium acidification by maize roots was inhibited up to 75% by thenoyltrifluoroacetone in the absence of ferricyanide, while dicumarol inhibited net acidification completely. The inhibition of H⁺-ATPase activity was estimated with plasma membrane vesicles isolated by phase partitioning and treated with 0.05% (w/v) Brij 58. ATP-dependent proton gradients and P_i release were measured after preincubation with the effectors. The proton pumping activity by those plasma membrane vesicles was inhibited by dicumarol (53.6%) and thenoyltrifluoroacetone (77.8%), while the release of P_i was unaffected by both inhibitors.Abbreviations Brij 58 polyoxyethylene 20-cetyl ether - duroquinone tetramethyl-p-benzoquinone - HCF III hexacyanoferrate III - TTFA thenoyltrifluoroacetone - vitamin K₁ 2-methyl-3-phytyl-1,4-naphthoquinone - vitamin K₃ 2-methyl-1,4-naphthoquinone     

Source of electrons for extracellular Fe(III) reduction in iron-deficient bean roots

Iron-deficient Phaseolus vulgaris L. cv. Prelude developed a high reducing capacity for extracellular Fe(III) at the root surface. This reduction was competitively inhibited by Nitro-Blue Tetrazolium salt (Nitro-BT) which was deposited as a blue precipitate within the epidermis cells of the youngest root parts. Root respiration was not influenced by Nitro-BT. The intracellular reduction of Nitro-BT could largely be prevented by excess extracellular Fe(III)EDTA. Iron-sufficient control plants reduced both extracellular Fe(III)EDTA and intracellular Nitro-BT at a much slower rate. A role of cytosolic NADH or NADPH as direct electron donors for the enhanced Fe(III) reduction at the plasmalemma is indicated. NAD⁺-3-phosphate dehydrogenase activity was higher in preparations from iron-deficient root parts than in preparations from control root parts. Ferricyanide, dichlorophenolindophenol and phenazine methosulfate were also reduced at an increased rate by iron-deficient roots. We conclude that a trans-plasma membrane electron transfer, mediated by a membrane-bound reductase, is responsible for the reduction of extracellular Fe(III).