Oxidative stress,antioxidant activity and Fe(III)-chelate reductase activity of five <Emphasis Type=Italic>Prunus</Emphasis> rootstocks explants in response to Fe deficiency

The aim of this work was to investigate whether Fe reduction and antioxidant mechanisms were expressed differently in five Prunus rootstocks (‘Peach seedling,’ ‘Barrier,’ ‘Cadaman,’ ‘Saint Julien 655/2’ and ‘GF-677’). These rootstocks differ in their tolerance to Fe deficiency when grown in the absence of Fe (−Fe) or in presence of bicarbonate (supplied as 5 or 10 mM NaHCO₃). Fe deficiency conditions, especially bicarbonate, were shown to decrease Fe and total chlorophyll (CHL) concentration. In the (−Fe)-treated roots of all rootstocks and in the 5 mM NaHCO₃-treated ones of the tolerant ‘GF-677’ the Fe(III)-chelate reductase (FCR) activity was stimulated. On the contrary, apart from the ‘GF-677,’ FCR activity was greatly inhibited by the 10 mM NaHCO₃. From the results obtained with decapitated rootstocks, it is not entirely clear whether or not the presence of shoot apex was a prerequisite to induce FCR function in all rootstocks tested. In the leaves of rootstocks exposed to the (−Fe) treatment, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities were enhanced whereas the levels of the non-enzymatic antioxidants (FRAP values) were increased in the Fe-deprived leaves, irrespective of the rootstock. Except for ‘Peach seedling,’ foliar SOD activity was stimulated by the presence of NaHCO₃. Furthermore, POD activity was increased in the ‘Saint Julien 655/2’ and ‘GF-677,’ but was depressed in the ‘Barrier’ rootstocks exposed to 10 mM NaHCO₃. As a result of 10 mM NaHCO₃, the expression of a Cu/Zn-SOD and a POD isoform was diminished in the leaves of ‘Peach seedling’ and ‘Barrier,’ respectively. By contrast, an additional isoform of both POD and Mn–SOD were expressed in the leaves of ‘GF-677’ exposed to 10 mM NaHCO₃ suggesting that the tolerance of rootstocks to Fe deficiency is associated with induction of an antioxidant defense mechanism. Although CAT activity was increased in the 5 mM NaHCO₃-treated leaves of ‘GF-677,’ specifically the 10 mM NaHCO₃ treatment resulted in a decrease of CAT activity and an accumulation of H₂O₂, indicating that bicarbonate-induced Fe deficiency may cause more severe oxidative stress in the rootstocks, than the absence of Fe. A general link between Fe deficiency-induced oxidative stress and Fe reduction-sensing mechanism is also discussed.     

Light-regulated, tissue-specific, and cell differentiation-specific expression of the Arabidopsis Fe(III)-chelate reductase gene AtFRO6

Iron is an essential element for almost all living organisms, actively involved in a variety of cellular activities. To acquire iron from soil, strategy I plants such as Arabidopsis (Arabidopsis thaliana) must first reduce ferric to ferrous iron by Fe(III)-chelate reductases (FROs). FRO genes display distinctive expression patterns in several plant species. However, regulation of FRO genes is not well understood. Here, we report a systematic characterization of the AtFRO6 expression during plant growth and development. AtFRO6, encoding a putative FRO, is specifically expressed in green-aerial tissues in a light-dependent manner. Analysis of mutant promoter-beta-glucuronidase reporter genes in transgenic Arabidopsis plants revealed the presence of multiple light-responsive elements in the AtFRO6 promoter. These light-responsive elements may act synergistically to confer light responsiveness to the AtFRO6 promoter. Moreover, no AtFRO6 expression was detected in dedifferentiated green calli of the korrigan1-2 (kor1-2) mutant or undifferentiated calli derived from wild-type explants. Conversely, AtFRO6 is expressed in redifferentiated kor1-2 shoot-like structures and differentiating calli of wild-type explants. In addition, AtFRO7, but not AtFRO5 and AtFRO8, also shows a reduced expression level in kor1-2 green calli. These results suggest that whereas photosynthesis is necessary but not sufficient, both light and cell differentiation are necessary for AtFRO6 expression. We propose that AtFRO6 expression is light regulated in a tissue- or cell differentiation-specific manner to facilitate the acquisition of iron in response to distinctive developmental cues.     

Changes in sugar beet leaf plasma membrane Fe(III)-chelate reductase activities mediated by Fe-deficiency, assay buffer composition, anaerobiosis and the presence of flavins

Summary Different assay conditions induce changes in the ferric chelate reductase activities of leaf plasma membrane preparations from Fe-deficient and Fe-sufficient sugar beet. With an apoplasttype assay medium the ferric chelate reductase activities did not change significantly when Fe(III)-EDTA was the substrate. However, with ferric citrate as substrate, the effect depended on the citrateto-Fe ratio. When the citrate-to-Fe ratio was 20 1, the effects were practically unappreciable. However, with a lower citrate-to-Fe ratio of 5 1 the activities were significantly lower with the apoplast-type medium than with the standard assay medium. Our data also indicate that anaerobiosis during the assay facilitates the reduction of ferric malate and Fe(III)-EDTA by plasma membrane preparations. Anaerobiosis increased by approximately 50% the plasma membrane ferric chelate reductase activities when Fe(III)-EDTA was the substrate. With ferric malate anaerobiosis increased activities by 70–90% over the values obtained in aerobic conditions. However, with ferric citrate the increase in activity by anaerobiosis was not significant. We have also tested the effect of riboflavin, flavin adenine dinucleotide, and flavin mononucleotide on the plasma membrane ferric chelate reductase activities. The presence of flavins generally increased activities in plasma membrane preparations from control and Fe-deficient plants. Increases in activity were generally moderate (lower than twofold). These increases occurred with Fe(III)-EDTA and Fe(III)-citrate as substrates.Abbreviations BPDS bathophenantroline disulfonate - FC ferric chelate - FC-R ferric chelate reductase - PM plasma membrane     

Fe(III) Reducing Microorganisms from Iron Ore Caves Demonstrate Fermentative Fe(III) Reduction and Promote Cave Formation

The banded iron formations (BIF) of Brazil are composed of silica and Fe(III) oxide lamina, and are largely covered by a rock cap of BIF fragments in a goethite matrix (canga). Despite both BIF and canga being highly resistant to erosion and poorly soluble, >3,000 iron ore caves (IOCs) have formed at their interface. Fe(III) reducing microorganisms (FeRM) can reduce the Fe(III) oxides present in the BIF and canga, which could account for the observed speleogenesis. Here, we show that IOCs contain a variety of microbial taxa with member species capable of dissimilatory Fe(III) reduction, including the Chloroflexi, Acidobacteria and the Alpha- Beta- and Gammaproteobacteria; however, Fe(III) reducing enrichment cultures from IOCs indicate the predominance of Firmicutes and Enterobacteriaceae, despite varying the carbon/electron donor, Fe(III) type, and pH. We used model-based inference to evaluate multiple candidate hypotheses that accounted for the variation in medium chemistry and culture composition. Model selection indicated that none of the tested variables account for the dominance of the Firmicutes in these cultures. The addition of H₂ to the headspace of the enrichment cultures enhanced Fe(III) reduction, while addition of N₂ resulted in diminished Fe(III) reduction, indicating that these Enterobacteriaceae and Firmicutes were reducing Fe(III) during fermentative growth. These results suggest that fermentative reduction of Fe(III) may play a larger role in iron-rich environments than expected. Our findings also demonstrate that FeRM are present within the IOCs, and that their reductive dissolution of Fe(III) oxides, combined with mass transport of solubilized Fe(II) by groundwater, could contribute to IOC formation.     

Iron requirement for and effects of promoters and inhibitors of ethylene action on stimulation of Fe(III)-chelate reductase in roots of strategy I species

Stimulation of root Fe(III) reductase activity by iron additions to iron-deficient growth media may be the result of iron activation of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase required for ethylene biosynthesis. Two different ethylene inhibitors, aminooxyacetic acid (AOA) (20 m; ACC synthase inhibitor) and cobalt (3 m CoCl₂; ACC oxidase inhibitor), were used to study the effects of iron supply and cobalt inhibition on ethylene action in controlling the activity of Fe(III)-chelate reductase in pea (Pisum sativum L.) roots. Supplying 20 gm m Fe(III)-N,N-ethylenebis[2-(2-hydroxypheyl)-glycine [Fe(III)-EDDHA] to either cobalt-treated, iron-deficient Sparkle (normal parent) or E107 (brz mutant genotype) pea seedlings reversed the negative effects of cobalt on root Fe(III)-reductase activity. Re-supplying 20 m Fe(III)-EDDHA to iron-deficient, AOA-treated seedlings did not enhance root Fe(III)-reductase. Apparently, cobalt competes with iron for the active site in ACC oxidase during ethylene synthesis. Inhibition of root reductase activity by cobalt treatment lowered manganese, zinc, magnesium and potassium content of mutant E107 pea seedlings. In contrast, iron enhancement of root reductase activity in iron-deficient, cobalt-treated E107 seedlings resulted in higher seedling accumulations of manganese, zinc, magnesium and potassium. These results support the hypothesis that root cell plasma membrane reductase activity plays a role in cation uptake by root cells.     

Technical advance: reduction of Fe(III)-chelates by mesophyll leaf disks of sugar beet. Multi-component origin and effects of Fe deficiency

The characteristics of the Fe(III)-chelate reductase activity have been investigated in mesophyll disks of Fe-sufficient and Fe-deficient sugar beet leaves. The Fe(III)-chelate reductase activity of mesophyll disks was light dependent and increased markedly when the epidermis was removed. Iron(III)-citrate was photo-reduced directly by light in the absence of plant tissue. Total reductase activity was the sum of enzymatic mesophyll reduction, enzymatic reduction carried out by organelles exposed at the disk edge and reduction caused by the release of substances both by exposed mesophyll cells and at the disk edge. Compounds excreted were shown by HPLC to include organic anions, mainly oxalate, citrate and malate. When expressed on a leaf surface basis, Fe deficiency decreased the total mesophyll Fe(III)-chelate reductase activity. However, Fe-sufficient disks reduced less Fe than the Fe-deficient ones when expressed on a chlorophyll basis. The optimal pH values for Fe(III) reduction were always in the range 6.0-6.7. In control leaves Fe(III)-citrate and Fe(III)-malate were the substrates that led to the highest Fe reduction rates. In Fe-deficient leaves Fe(III)-malate led to the highest Fe reduction rates, followed by Fe(III)-EDTA and then Fe(III)-citrate. K:(m) values for the total reductase activity, enzymatic mesophyll reduction and enzymatic reduction carried out by organelles at the disk edge were obtained.     

Adhesion of Dissimilatory Fe(III)-Reducing Bacteria to Fe(III) Minerals

The metabolism of dissimilatory iron-reducing bacteria (DIRB) may provide a means of remediating contaminated subsurface soils. The factors controlling the rate and extent of bacterial F(III) mineral reduction are poorly understood. Recent research suggests that molecular-scale interactions between DIRB cells and Fe(III) mineral particles play an important role in this process. One of these interactions, cell adhesion to Fe(III) mineral particles, appears to be a complex process that is, at least in part, mediated by a variety of surface proteins. This study examined the hypothesis that the flagellum serves as an adhesin to different Fe(III) minerals that range in their surface area and degree of crystallinity. Deflagellated cells of the DIRB Shewanella algae BrY showed a reduced ability to adhere to hydrous ferric oxide (HFO) relative to flagellated cells. Flagellated cells were also more hydrophobic than deflagellated cells. This was significant because hydrophobic interactions have been previously shown to dominate S. algae cell adhesion to Fe(III) minerals. Pre-incubating HFO, goethite, or hematite with purified flagella inhibited the adhesion of S. algae BrY cells to these minerals. Transposon mutagenesis was used to generate a flagellum-deficient mutant designated S. algae strain NF. There was a significant difference in the rate and extent of S. algae NF adhesion to HFO, goethite, and hematite relative to that of S. algae BrY. Amiloride, a specific inhibitor of Na + -driven flagellar motors, inhibited S. algae BrY motility but did not affect the adhesion of S. algae BrY to HFO. S.algae NF reduced HFO at the same rate as S. algae BrY. Collectively, the results of this study support the hypothesis that the flagellum of S. algae functions as a specific Fe(III) mineral adhesin. However, these results suggest that flagellum-mediated adhesion is not requisite for Fe(III) mineral reduction.     

Electrochemical Regeneration of Fe(III) To Support Growth on Anaerobic Iron Respiration

Here we describe artificial help for the respiratory electron flow supporting anaerobic growth of Thiobacillus ferrooxidans through exogenous electrolysis. Flux between H₂ and a anode through cells was accomplished with electrochemical regeneration of iron. The electrochemical help resulted in a 12-fold increase in yield compared with the yield observed in its absence.     

Iron(III)-adriamycin and Iron(III)-daunorubicin complexes: physicochemical characteristics, interaction with DNA, and antitumor activity

Fe(III) complexes of two anthracyclines, adriamycin and daunorubicin, have been studied. Using potentiometric and spectroscopic measurements, we have shown that adriamycin and daunorubicin form two well-defined species with Fe(III), which can be formulated as respectively Fe(HAd)3 and Fe(HDr)3. In these formulas, HAd and HDr stand for adriamycin and daunorubicin in which the 1,4-dihydroxy-anthraquinone moiety is half-deprotonated. Both complexes are six-membered chelates. The stability constant is beta = (2.5 +/- 0.5) X 10(28) for both complexes. Interaction with DNA has been studied showing that, despite strong coordination to Fe(III), anthracyclines are able to intercalate between DNA bases pairs, releasing the metal. These complexes display antitumor activity against P 388 leukemia that compares with that of the free drug. Fe(HAd)3, unlike adriamycin, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. Moreover, it is shown that the triferric adriamycin compound so called "quelamycin" is in fact a mixture of Fe(HAd)3 and polymeric ferric hydroxide.     

Adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to crystalline Fe(III) oxides

Shewanella alga BrY adhesion to hydrous ferric oxide, goethite, and hematite was examined. Adhesion to each oxide followed the Langmuir adsorption model. No correlation between adhesion and Fe(III) oxide surface area or crystallinity was observed. Zeta potential measurements suggested that electrostatic interactions do not influence S. alga BrY adhesion to these minerals. Cell adhesion does not appear to explain the recalcitrance of crystalline Fe(III) oxides to bacterial reduction. Received: 12 May 2000 / Accepted: 19 June 2000     

Biocatalytic generation of Mn(III)-chelate as a chemical oxidant of different environmental contaminants

The objective of this study was to investigate the enzymatic generation of the Mn(3+) -malonate complex and its application to the process of oxidizing several organic compounds. The experimental set-up consisted of an enzymatic reactor coupled to an ultrafiltration membrane, providing continuous generation of Mn(3+) -malonate from a reaction medium containing versatile peroxidase (an enzyme produced by Bjerkandera adusta strain BOS55), H(2) O(2) , MnSO(4,) and malonate. The effluent of the enzymatic reactor was introduced into a batch-stirred reactor to oxidize three different classes of compounds: an azo dye (Orange II), three natural and synthetic estrogens, and a polycyclic aromatic hydrocarbon (anthracene). The enzymatic reactor provided the Mn(3+) complex under steady-state conditions, and this oxidative species was able to transform the three classes of xenobiotics considerably (90-99%) with negligible loss of activity.     

Role of Humic-Bound Iron as an Electron Transfer Agent in Dissimilatory Fe(III) Reduction

The dissimilatory Fe(III) reducer Geobacter metallireducens reduced Fe(III) bound in humic substances, but the concentrations of Fe(III) in a wide range of highly purified humic substances were too low to account for a significant portion of the electron-accepting capacities of the humic substances. Furthermore, once reduced, the iron in humic substances could not transfer electrons to Fe(III) oxide. These results suggest that other electron-accepting moieties in humic substances, such as quinones, are the important electron-accepting and shuttling agents under Fe(III)-reducing conditions.     

Facilitation of Fe(II) antoxidation by Fe(III) complexing agents

The rate of oxidation of Fe(II) by atmospheric oxygen at pH 7.0 is significantly enhanced by low molecular weight Fe(III)-complexing agents in the order EDTA ≈ nitrilotriacetate > citrate > phosphate > oxalate. This simple effect of Fe(III) binding probably accounts for the “ferroxidase” activity exhibited by transferrin and ferritin.     

Selective adsorption of apoferritin on immobilized Fe(III): demonstration of Fe(III) binding sites

Immobilized metal ion affinity chromatography has been used to demonstrate and partially characterize Fe(III) binding sites on apoferritin. Binding of Fe(III) to these sites is influenced by pH, but not affected by high ionic strength. These results suggest that both ionic and coordinate covalent interactions are important in the formation of the Fe(III): apoferritin complex. This is, to our knowledge, the first demonstration of direct Fe(III) binding to apoferritin. Other immobilized metal ions, including Zn(II), Ni(II), Cu(II), Cr(III), Co(II), and Tb(III), displayed little or no adsorption of apoferritin. The analytical technique of immobilized metal ion affinity chromatography also shows great promise in the purification of apoferritin, ferritin, and other iron-binding proteins.     

Plant regeneration from leaf protoplasts of evening primrose (Oenothera hookeri)

A protocol is presented for regenerating plants from leaf protoplasts of Oenothera. The method uses (1) embedding of isolated protoplasts at high cell densities in thin alginate layers, (2) initial culture in B5 medium containing 3 mg l^–1 α-naphthaleneacetic acid (NAA) and 1 mg l^-1 6-benzylaminopurine (BAP), (3) reduction of the osmotic pressure of the culture medium at early stages of culture and (4) plating of microcolonies recovered from the alginate onto solid B5 medium with 3 mg l^–1 NAA and 1 mg l^–1 BAP. The shortest time required from protoplast isolation to the appearance of shoot initials was 7 weeks. The efficiency of the procedure for protoplast to cell line formation is high (about 80%). Received: 17 February 1997 / Revision received: 6 November 1997 / Accepted: 15 November 1997     

Fe(III) and Fe(II) ions different effects on Enterococcus hirae cell growth and membrane-associated ATPase activity

Enterococcus hirae is able to grow under anaerobic conditions during glucose fermentation (pH 8.0) which is accompanied by acidification of the medium and drop in its oxidation-reduction potential (E(h)) from positive values to negative ones (down to ～-200 mV). In this study, iron (III) ions (Fe(3+)) have been shown to affect bacterial growth in a concentration-dependent manner (within the range of 0.05-2 mM) by decreasing lag phase duration and increasing specific growth rate. While iron(II) ions (Fe(2+)) had opposite effects which were reflected by suppressing bacterial growth. These ions also affected the changes in E(h) values during bacterial growth. It was revealed that ATPase activity with and without N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F(0)F(1)-ATPase, increased in the presence of even low Fe(3+) concentration (0.05 mM) but decreased in the presence of Fe(2+). It was established that Fe(3+) and Fe(2+) both significantly inhibited the proton-potassium exchange of bacteria, but stronger effects were in the case of Fe(2+) with DCCD. Such results were observed with both wild-type ATCC9790 and atpD mutant (with defective F(0)F(1)) MS116 strains but they were different with Fe(3+) and Fe(2+). It is suggested that the effects of Fe(3+) might be due to interaction of these ions with F(0)F(1) or there might be a Fe(3+)-dependent ATPase different from F(0)F(1) in these bacteria that is active even in the presence of DCCD. Fe(2+) inhibits E. hirae cell growth probably by strong effect on E(h) leading to changes in F(0)F(1) and decreasing its activity.     

Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor

The mechanisms for Fe(III) oxide reduction in Geobacter species are of interest because Fe(III) oxides are the most abundant form of Fe(III) in many soils and sediments and Geobacter species are prevalent Fe(III)-reducing microorganisms in many of these environments. Protein abundance in G. sulfurreducens grown on poorly crystalline Fe(III) oxide or on soluble Fe(III) citrate was compared with a global accurate mass and time tag proteomic approach in order to identify proteins that might be specifically associated with Fe(III) oxide reduction. A total of 2991 proteins were detected in G. sulfurreducens grown with acetate as the electron donor and either Fe(III) oxide or soluble Fe(III) citrate as the electron acceptor, resulting in 86% recovery of the genes predicted to encode proteins. Of the total expressed proteins 76% were less abundant in Fe(III) oxide cultures than in Fe(III) citrate cultures, which is consistent with the overall slower rate of metabolism during growth with an insoluble electron acceptor. A total of 269 proteins were more abundant in Fe(III) oxide-grown cells than in cells grown on Fe(III) citrate. Most of these proteins were in the energy metabolism category: primarily electron transport proteins, including 13 c-type cytochromes and PilA, the structural protein for electrically conductive pili. Several of the cytochromes that were more abundant in Fe(III) oxide-grown cells were previously shown with genetic approaches to be essential for optimal Fe(III) oxide reduction. Other proteins that were more abundant during growth on Fe(III) oxide included transport and binding proteins, proteins involved in regulation and signal transduction, cell envelope proteins, and enzymes for amino acid and protein biosynthesis, among others. There were also a substantial number of proteins of unknown function that were more abundant during growth on Fe(III) oxide. These results indicate that electron transport to Fe(III) oxide requires additional and/or different proteins than electron transfer to soluble, chelated Fe(III) and suggest proteins whose functions should be further investigated in order to better understand the mechanisms of electron transfer to Fe(III) oxide in G. sulfurreducens.     

Iron (III) reduction: A novel activity of the human NAD(P)H:oxidoreductase

NAD(P)H:quinone oxidoreductase (NQO1; EC 1.6.99.2) catalyzes a two-electron transfer involved in the protection of cells from reactive oxygen species. These reactive oxygen species are often generated by the one-electron reduction of quinones or quinone analogs. We report here on the previously unreported Fe(III) reduction activity of human NQO1. Under steady state conditions with Fe(III) citrate, the apparent Michaelis-Menten constant (Km(app)) was approximately 0.3 nM and the apparent maximum velocity (Vmax(app)) was 16 U mg(-1). Substrate inhibition was observed above 5 nM. NADH was the electron donor, Km(app)= 340 microM and Vmax(app) = 46 Umg(-1). FAD was also a cofactor with a Km(app) of 3.1 microM and Vmax(app) of 89 U mg(-1). The turnover number for NADH oxidation was 25 s(-1). Possible physiological roles of the Fe(III) reduction by this enzyme are discussed.     

Mechanisms for accessing insoluble Fe(III) oxide during dissimilatory Fe(III) reduction by Geothrix fermentans

Mechanisms for Fe(III) oxide reduction were investigated in Geothrix fermentans, a dissimilatory Fe(III)-reducing microorganism found within the Fe(III) reduction zone of subsurface environments. Culture filtrates of G. fermentans stimulated the reduction of poorly crystalline Fe(III) oxide by washed cell suspensions, suggesting that G. fermentans released one or more extracellular compounds that promoted Fe(III) oxide reduction. In order to determine if G. fermentans released electron-shuttling compounds, poorly crystalline Fe(III) oxide was incorporated into microporous alginate beads, which prevented contact between G. fermentans and the Fe(III) oxide. G. fermentans reduced the Fe(III) within the beads, suggesting that one of the compounds that G. fermentans releases is an electron-shuttling compound that can transfer electrons from the cell to Fe(III) oxide that is not in contact with the organism. Analysis of culture filtrates by thin-layer chromatography suggested that the electron shuttle has characteristics similar to those of a water-soluble quinone. Analysis of filtrates by ion chromatography demonstrated that there was as much as 250 microM dissolved Fe(III) in cultures of G. fermentans growing with Fe(III) oxide as the electron acceptor, suggesting that G. fermentans released one or more compounds capable of chelating and solubilizing Fe(III). Solubilizing Fe(III) is another strategy for alleviating the need for contact between cells and Fe(III) oxide for Fe(III) reduction. This is the first demonstration of a microorganism that, in defined medium without added electron shuttles or chelators, can reduce Fe(III) derived from Fe(III) oxide without directly contacting the Fe(III) oxide. These results are in marked contrast to those with Geobacter metallireducens, which does not produce electron shuttles or Fe(III) chelators. These results demonstrate that phylogenetically distinct Fe(III)-reducing microorganisms may use significantly different strategies for Fe(III) reduction. Thus, it is important to know which Fe(III)-reducing microorganisms predominate in a given environment in order to understand the mechanisms for Fe(III) reduction in the environment of interest.