Mechanism of iron uptake by peanut plants : I. Fe reduction, chelate splitting, and release of phenolics

Iron deficiency in peanuts (Arachis hypogeae L.) caused an increase in release of caffeic acid, a higher rate of Fe^III reduction, and increased rates of both Fe^III chelate splitting and iron uptake.

Experiments on Fe^III reduction by phenolics (in vitro experiments) and by roots of Fe-deficient peanuts exclude the direct involvement of released phenolics in Fe^III reduction by roots: Fe^III reduction by phenolics had a pH optimum higher than 8.0 and was strongly dependent on the concentration and the stability of the supplied Fe^III chelates. In contrast, Fe^III reduction by roots of Fe-deficient peanuts had a pH optimum of about 5.0 and was less dependent on the stability of the supplied Fe^III chelates. Furthermore, the observed release of phenolics into nutrient solution would have to be at least 200 times higher to attain the reduction rates of roots of Fe-deficient peanuts. The results of these experiments support the idea of an enzymic reduction of Fe^III on the plasmalemma of cortical cells of roots.

     

Enhancement of ferric-mugineic acid uptake by iron deficient barley roots in the presence of excess free mugineic acid in the medium

To investigate the mechanism of mugineic acid-Fe^III uptake by barley roots, plasma membrane fractions were isolated from Fe-deficient barley roots using an aqueous two-phase partition method. Utilizing the plasma membrane vesicles, we developed an assay system for studying mugineic acid-⁵⁵Fe^III binding to the plasma membrane. However, no efficient active transport of mugineic acid-⁵⁵Fe^III into the plasma membrane vesicle was detected, because of large amount of non-specific adsorption of ⁵⁵Fe^III onto the vesicle. And the adsorption could be decreased by adding excess amount of free mugineic acid to the assay system. From the results it is speculated that an excess of free mugineic acids is necessary in the medium for effective uptake of mugineic acid-Fe^III by Fe-deficient barley roots. Support for this speculation came from a multi-compartment transport box experiment with excised roots of Fe-deficient barley.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - MA mugineic acid     

Depolarization of Cell Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L

Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe^IIIEDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (30-40 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts. Ferrocyanide has no effect. In the state of ferricyanide reduction, H⁺ (1H⁺/2 electrons) and K⁺ ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts.     

Iron-Stress Induced Redox Activity in Tomato (Lycopersicum esculentum Mill.) Is Localized on the Plasma Membrane

Tomato plants (Lycopersicum esculentum Mill.) were grown for 21-days in a complete hydroponic nutrient solution including Fe³⁺-ethylenediamine-di(o-hydroxyphenylacetate) and subsequently switched to nutrient solution withholding Fe for 8 days to induce Fe stress. The roots of Fe-stressed plants reduced chelated Fe at rates sevenfold higher than roots of plants grown under Fe-sufficient conditions. The response in intact Fe-deficient roots was localized to root hairs, which developed on secondary roots during the period of Fe stress. Plasma membranes (PM) isolated by aqueous two-phase partitioning from tomato roots grown under Fe stress exhibited a 94% increase in rates of NADH-dependent Fe³⁺-citrate reduction compared to PM isolated from roots of Fe-sufficient plants. Optimal detection of the reductase activity required the presence of detergent indicating structural latency. In contrast, NADPH-dependent Fe³⁺-citrate reduction was not significantly different in root PM isolated from Fe-deficient versus Fe-sufficient plants and proceeded at substantially lower rates than NADH-dependent reduction. Mg²⁺-ATPase activity was increased 22% in PM from roots of Fe-deficient plants compared to PM isolated from roots of Fe-sufficient plants. The results localized the increase in Fe reductase activity in roots grown under Fe stress to the PM.     

Voltammetric studies of Zn and Fe complexes of EDTA: Evidence for the push mechanism

The `push' hypothesis for the antioxidant action of Zn²⁺ is based on its displacement of iron from a low molecular weight pro-oxidant complex. In this study, the chemical plausibility of that proposed function is investigated by cyclic voltammetry. As a model for a pro-oxidative low molecular weight iron complex the Fe^II/IIIEDTA couple was examined. This complex was selected for its well-defined electrochemical, iron stability constants, and similarity to other low molecular weight chelates in physiological fluids in terms of logical binding sites, i.e. amino, and carboxylate groups. Also investigated were iron complexes of nitrilotriacetic acid and DL-glutamic acid. Results demonstrate that approximately 90% of the cyclic voltammetric peak current for Fe^IIIEDTA reduction and the EC′ current for the mediated reduction of H₂O₂ by Fe^II/IIIEDTA (Fenton Reaction) are lost when Zn²⁺ is introduced to a 1:1 molar ratio relative to iron. All experiments were conducted in HEPES buffered solutions at pH 7.4. Iron (II/III) complexes of nitrilotriacetic acid and DL-glutamic acid followed the same trends. Cyclic voltammetric experiments indicate that Zn²⁺ displaces Fe^III from EDTA despite the much larger stability constant for the iron complex (10^25.1) versus zinc (10^16.50). The hydrolysis aided displacement of Fe^III from EDTA by Zn²⁺ is considered by the equilibria modeling program, HySS. With Fe^III hydrolysis products included, Zn²⁺ is able to achieve 90% displacement of iron from EDTA, a result consistent with cyclic voltammetric observations. Published online December 2004     

Uptake of 5 9Fe from soluble 5 9Fe-humate complexes by cucumber and barley plants

The capability of cucumber (Cucumis sativus L., cv. Serpente cinese), a Strategy I plant and barley (Hordeum vulgaris L., cv. Europa), a Strategy II plant to use Fe complexed by a water-soluble humic fraction (WEHS) extracted from a peat, was studied. Uptake of ⁵⁹Fe from ⁵⁹Fe-WEHS by cucumber plants was higher at pH 6.0 than at pH 7.5. Roots of intact cucumber plants were able to reduce the Fe^III-WEHS complex either at pH 6.0 or 7.5, rates being higher in the assay medium buffered at pH 6.0. After supply of ⁵⁹Fe-WEHS, a large pool of root extraplasmatic ⁵⁹Fe was formed, which could be used to a large extent by Fe-deficient plants, particularly under acidic conditions. Uptake of ⁵⁹Fe from ⁵⁹Fe-WEHS by Fe-sufficient and Fe-deficient barley plants was examined during periods of high (morning) and low (evening) PS release. Uptake paralleled the diurnal rhythm of PS release. Furthermore, ⁵⁹Fe uptake was strongly enhanced by addition of PS to the uptake solution in both Fe-sufficient and Fe-deficient plants. High amount of root extraplasmatic ⁵⁹Fe was formed upon supply of Fe-WEHS, particularly in the evening experiment. Fe-deficient barley plants were able to utilize Fe from the root extraplasmatic pool, conceivably as a result of high rates of PS release. The results of the present work together with previous observations indicate that cucumber plants (Strategy I) utilize Fe complexed to WEHS, presumably via reduction of Fe^III-WEHS by the plasma membrane-bound reductase, while barley plants (Strategy II) use an indirect mechanism involving ligand exchange between WEHS and PS.     

Fe reduction in cell walls of soybean roots

Reduction of Fe^IIIEDTA by excised roots of soybean seedlings (Glycine max L.) is stimulated by l-malate in the bathing solution. Reduction occurs much more rapidly with roots of seedlings grown in the absence of iron than with roots of seedlings grown with iron. Cell-wall preparations from these roots catalyze reduction of Fe^IIIEDTA by NADH. They also contain NAD⁺-dependent l-malate dehydrogenase. Enzymic activity of the cell-wall preparations is not affected by previous iron nutrition of the plants, but the amount of l-malate in the roots is increased when seedlings have been deprived of iron. We propose that reduction of iron before absorption by soybean roots occurs in the cell-wall space, with l-malate secreted from the roots serving as the source of electrons. Part of the iron reductase activity of the cell walls can be solubilized by extraction with 1 molar NaCl. The enzyme has been partially purified.     

Evidence for a specific uptake system for iron phytosiderophores in roots of grasses

Roots of grasses in response to iron deficiency markedly increase the release of chelating substances (`phytosiderophores') which are highly effective in solubilization of sparingly soluble inorganic Fe<sup>III</sup> compounds by formation of Fe<sup>III</sup>phytosiderophores. In barley (Hordeum vulgare L.), the rate of iron uptake from Fe<sup>III</sup>phytosiderophores is 100 to 1000 times faster than the rate from synthetic Fe chelates (e.g. Fe ethylenediaminetetraacetate) or microbial Fe siderophores (e.g. ferrichrome). Reduction of Fe<sup>III</sup> is not involved in the preferential iron uptake from Fe<sup>III</sup>phytosiderophores by barley. This is indicated by experiments with varied pH, addition of bicarbonate or of a strong chelator for Fe<sup>II</sup> (e.g. batho-phenanthrolinedisulfonate). The results indicate the existence of a specific uptake system for Fe<sup>III</sup>phytosiderophores in roots of barley and all other graminaceous species. In contrast to grasses, cucumber plants (Cucumis sativus L.) take up iron from Fe<sup>III</sup>phytosiderophores at rates similar to those from synthetic Fe chelates. Furthermore, under Fe deficiency in cucumber, increased rates of uptake of Fe<sup>III</sup>phytosiderophores are based on the same mechanism as for synthetic Fe chelates, namely enhanced Fe<sup>III</sup> reduction and chelate splitting. Two strategies are evident from the experiments for the acquisition of iron by plants under iron deficiency. Strategy I (in most nongraminaceous species) is characterized by an inducible plasma membrane-bound reductase and enhancement of H<sup>+</sup> release. Strategy II (in grasses) is characterized by enhanced release of phytosiderophores and by a highly specific uptake system for Fe<sup>III</sup>phytosiderophores. Strategy II seems to have several ecological advantages over Strategy I such as solubilization of sparingly soluble inorganic Fe<sup>III</sup> compounds in the rhizosphere, and less inhibition by high pH. The principal differences in the two strategies have to be taken into account in screening methods for resistance to `lime chlorosis'.     

Cytosolic NADPH is the electron donor for extracellular fe reduction in iron-deficient bean roots

Pyridine nucleotides were determined in lateral roots of iron-deficient and iron-sufficient Phaseolus vulgaris L. cv Prelude. In iron-deficient plants, total NADP per gram fresh weight and the NADPH/NADP⁺ ratio were twice the values found in iron-sufficient plants. The NADPH/NADP⁺ ratio in iron-deficient plants was considerably lowered after a 2 minute incubation in 1 millimolar ferricyanide. Total NAD was not influenced by growth conditions and was mainly present in oxidized form.

These results indicate that NADPH is the electron donor for the high Fe^III reduction activity found in iron-deficient roots, a process that is part of the Fe-uptake mechanism.

     

The kinetics of the complex formation between iron(III)-ethylenediaminetetraacetate and hydrogen peroxide in aqueous solution

The kinetics of the formation of the purple complex [Fe^III(EDTA)O₂]³⁻, between Fe^III-EDTA and hydrogen peroxide was studied as a function of pH (8.22-11.44) and temperature (10-40 °C) in aqueous solutions using a stopped-flow method. The reaction was first-order with respect to both reactants. The observed second-order rate constants decrease with an increase in pH and appear to be related to deprotonation of Fe^III-EDTA ([Fe(EDTA)H₂O]⁻ ⇔ Fe(EDTA)OH]²⁻ + H⁺). The rate law for the formation of the complex was found to be d[Fe^IIIEDTAO₂]³⁻/dt=[(k₄[H⁺]/([H⁺] + K₁)][Fe^III-EDTA][H₂O₂], where k₄=8.15±0.05×10⁴ M⁻¹ s⁻¹ and pK₁=7.3. The steps involved in the formation of [Fe(EDTA)O₂]³⁻ are briefly discussed.     

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 NO₃⁻ 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.     

Physiological root responses of iron deficiency susceptible and tolerant tomato genotypes and their reciprocal F1 hybrids

By using two tomato genotypes line 227/1 (Fe chlorosis susceptible) and Roza (Fe chlorosis tolerant) and their reciprocal F₁hybrid, some root morphological changes, pH changes of nutrient solution, reduction capacity of Fe^III and uptake and root-to-shoot translocation of ⁵⁹Fe were studied under controlled environmental conditions in nutrient solution with 3 different Fe supplies as Fe EDDHA (i.e., 10^–7 M, severe Fe deficiency; 10^–6 M, intermediate Fe deficiency; 10^–4 M, adequate Fe supply). Tolerant parent `Roza' was less affected by low Fe supply than susceptible parent `line 227/1' as judged from the severity of leaf chlorosis. Under both Fe deficient conditions there were no differences between the reciprocal hybrids concerning the appearance of chlorosis. Under intermediate Fe deficiency, reciprocal F₁ hybrids (`line 227/1 × Roza' and `Roza × line' 227/1) showed an intermediate chlorosis between tolerant and susceptible parents. However, under severe Fe deficiency the reciprocal hybrids were more chlorotic than the tolerant parent irrespective of which parent was the cytoplasm contributor. A decreased Fe supply during preculture enhanced Fe^III reduction capacities of the parents and reciprocal hybrids. Differences in the tolerance to Fe deficiency always were better correlated with Fe^III reduction capacity of the genotypes than the Fe deficiency-induced release of H⁺ ions. Under both Fe deficient conditions the tolerant parent Roza had a much higher Fe^III reduction capacity than the susceptible parent line 227/1. The reduction capacity of the hybrids `Roza × line 227/1' was very similar to the capacity of the parent Roza, but higher than the capacity of the hybrids `line 227/1×Roza' at both Fe-deficient conditions. Under both Fe deficient conditions tolerant parent had higher number of lateral roots than the susceptible parent. Among the reciprocal hybrids `Roza × line 227/1' possessed more lateral roots than the `line 227/1 × Roza' under both Fe deficient conditions. Low Fe nutritional status resulted in marked increase in root uptake of ⁵⁹Fe. At adequate Fe supply, reciprocal hybrids and their parents did not differ in uptake and root-to-shoot translocation of Fe. However, under Fe-deficient conditions uptake and root-to-shoot translocation of ⁵⁹Fe were significantly higher in the Fe chlorosis tolerant than the susceptible parent. Based on the reduction capacity of Fe^III and uptake and root-to-shoot translocation of Fe, the F₁ hybrids obtained from the cross in which the maternal genotype was Roza appeared to be more tolerant than when the maternal genotype was the susceptible line 227/1. Uptake and translocation ratio of the F₁ hybrids obtained from `Roza × line 227/1' were similar to those of the parent Roza, but higher than the F<sub>1</sub> hybrids obtained from `line 227/1 × Roza', particularly under intermediate Fe deficiency. The results indicate that Fe^III reduction show a better relationship to Fe efficiency than Fe deficiency induced release of H⁺ ions. The inheritance of Fe deficiency tolerance of Roza seems not to be simple monogenic. It might be characterised by both, nuclear and extranuclear heredity. The intermediate responses of the reciprocal hybrids of the `line 227/1 × Roza' indicates that the Fe deficiency tolerance character of Roza is transferable by nuclear heredity. The better responses of the hybrids of `Roza × line 227/1' than the hybrids of `line 227/1 × Roza' may be due to maternal transmission from the parent Roza besides the nuclear transmission.     

Does Iron Deficiency in Pisum sativum Enhance the Activity of the Root Plasmalemma Iron Transport Protein?

Roots of Fe-sufficient and Fe-Deficient pea (Pisum sativum L.) were studied to determine the effect of Fe-deficiency on the activity of the root-cell plasmalemma Fe²⁺ transport protein. Rates of Fe(III) reduction and short-term Fe²⁺ influx were sequentially determined in excised primary lateral roots using Fe(III)-ethylene-diaminetetraacetic acid (Fe[III]-EDTA). Since the extracellular Fe²⁺ for membrane transport was generated by root Fe(III) reduction, rates of Fe²⁺ influx for each root system were normalized on the basis of Fe(III) reducing activity. Ratios of Fe²⁺ influx to Fe(III) reduction (micromole Fe²⁺ absorbed/micromole Fe[III] reduced) revealed no enhanced Fe²⁺ transport capacity in roots of Fe-deficient peas (from the parental genotype, Sparkle) or the functional Fe-deficiency pea mutant, E107 (derived from Sparkle), relative to roots of Fe-sufficient Sparkle plants. Data from studies using 30 to 100 micromolar Fe(III)-EDTA indicated a linear relationship between Fe²⁺ influx and Fe(III) reduction (Fe²⁺ generation), while Fe²⁺ influx saturated at higher concentrations of Fe(III)-EDTA. Estimations based on current data suggest the Fe²⁺ transport protein may saturate in the range of 10^−4.8 to 10⁻⁴ molar Fe²⁺. These results imply that for peas, the physiological rate limitation to Fe acquisition in most well-aerated soils would be the root system's ability to reduce soluble Fe(III)-compounds.     

Characterization of mugineic-acid-Fe transporter in Fe-deficient barley roots using the multi-compartment transport box method

Summary We have investigated the mugineicacid-Fe transport activity of Fe-deficient barley roots, using the multi-compartment transport box system. The roots maintained Fe transport activity for 20 h after excision. The following results were obtained. (1) In Fe-deficient roots, mugineic acid addition enhanced the transport of Fe by 32.2 times over that of the control (with FeC1₃ addition). (2) The mugineic-acid-⁵⁵Fe transport activity of Fe-deficient roots was 18.4-fold higher than that of the Fe-sufficient roots. (3) The mugineic-acid-⁵⁵Fe transport activity was decreased (7.13% based on the control) by treatment with 5 M carbonylcyanidem-chlorophenyl hydrazone (CCCP). Pretreatment with 0.1 mM dicyclohexyl carbodiimide (DCCD) lowered the transport activity (10.7% based on the control) and 1 mMN-ethylmaleimide (NEM) pretreatment reduced the transport activity to a value equivalent to 2.41% of that in the control. It is concluded that mugineicacid-Fe transporter is induced in its activity and/or amount by Fe-deficiency treatment and has an SH residue at its active site, and that the transporter needs the proton motive force produced by ATPase. We detected three polypeptides (14, 28 and 40 kDa) in the root plasma membrane that were induced under Fe-deficiency treatment.Abbreviations p-APMSF (p-amidinophenyl)methanesulfonyl fluoride hydrochloride - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD dicyclohexylcabodiimide - DMSO dimethyl sulfoxide - MA mugineic acid - NEM N-ethylmaleimide     

NADH-dependent Fe3+EDTA and oxygen reduction by plasma membrane vesicles from barley roots

Brüggemann, W. and Moog, P. R. 1989. NADH-dependent Fe³⁺EDTA and oxygen reduction by plasma membrane vesicles from barley roots. Biochemical properties of pyridine-dinucleotide-dependent Fe³⁺-EDTA reductase were analysed in purified plasma membranes (PM) from barley (Hordeum vulgare L. cv. Marinka) roots. The enzymatic activity preferred NADH over NADPH as electron donor and it was 3-fold increased in the presence of detergent. The reductase showed a pH optimum of 6.8 and saturable kinetics for NADH with K_m (NADH) of 125 μM and V_max of 143 nmol Fe (mg protein)^-1 min^-1 in the presence of 500 μM Fe³⁺EDTA. For the dependence of the reaction rate on the iron compound, K_m(Fe³⁺EDTA) of 120 μM and V_max of 184 nmol (mg protein)^-1 min^-1 were obtained. The activity was insensitive to superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6) and antimycin A, but stimulated by an oxygen-free reaction medium. It could be solubilized by 0.25% (w/v) Triton X-100. The solubilized enzyme revealed one band in native polyacrylamide gel electrophoresis (PAGE) and in isoelectric focussing (IEF) at pl 7.4 by enzyme staining. Major polypeptides with molecular weights of 94, 106, 120 and 205 kDa corresponded to the enzyme-stained band from native PAGE. Analysis of oxygen consumption by the membranes revealed the existence of NADH:CK oxidoreductase activity, which was stimulated by salicylhydroxamic acid (SHAM), chinhydron, Fe³⁺EDTA and Fe³⁺EDTA but not by K₃ [Fe(CN)₆] or K₄[Fe (CN)₆). The stimulating effect of the iron chelates on oxygen consumption was due to Fe²⁺ and could be suppressed by bathophenanthroline disulfonate (BPDS), SOD and p-chloromercurophenylsulfonic acid (PCMS). The results are discussed with respect to the nature of the stimulation.     

Response of lupins (Lupinus angustifolius L.) and peas (Pisum sativum L.) to Fe deficiency induced by low concentrations of Fe in solution or by addition of HCO3 -

Lupins appear to be more sensitive than peas to Fe deficiency. However, when grown in nutrient solutions between pH 5–6, little difference existed between them in their ability to acidify the solution or to release Fe^III reducing compounds. This experiment was aimed at determining whether differences between species which occurred when Fe deficiency was induced by withholding Fe from an acid solution, are maintained when Fe deficiency is induced by addition of HCO₃ ^-. Lupins and peas were grown in nutrient solutions at 0, 2 and 6 μM of Fe^III EDDHA and either with or without HCO₃ ^- (6 mM). Bicarbonate induced symptoms of Fe deficiency (chlorosis) in both lupins and peas, and markedly decreased the growth of shoots. Symptoms appeared sooner and were more severe in lupins than in peas. Growing plants without HCO₃ ^-, but at the lowest Fe level, decreased the growth and Fe concentration of shoots of lupins but did not induce chlorosis. Growing peas in this treatment, decreased Fe concentrations, but to a lesser extent than in lupins, and did not decrease growth. H⁺-ion extrusion and release of Fe^III reducing compounds was greater in lupins than in peas. Bicarbonate also decreased the growth of roots of lupins but increased the growth of roots of peas. Results indicate that when Fe deficiency is induced by HCO₃ ^-, then the response of lupins and peas are similar to their response in acid solution culture. Differences between species therefore could not be explained by their relative abilities to acidify or release Fe^III reducing compounds. Greater control of the distribution of Fe within the shoots, the presence of a pool of Fe within the roots, a lower threshold for Fe uptake, or a higher content of seed-Fe, may therefore be the reason for the lower sensitivity of peas than lupins to Fe deficiency.     

Disproportionation of hydroxylamine by water-soluble iron(III) porphyrinate compounds

The reactions of hydroxylamine (HA) with several water-soluble iron(III) porphyrinate compounds, namely iron(III) meso-tetrakis-(N-ethylpyridinium-2yl)-porphyrinate ([Fe^III(TEPyP)]⁵⁺), iron(III) meso-tetrakis-(4-sulphonatophenyl)-porphyrinate ([Fe^III(TPPS)]³⁻), and microperoxidase 11 ([Fe^III(MP11)]) were studied for different [Fe^III(Porph)]/[HA] ratios, under anaerobic conditions at neutral pH. Efficient catalytic processes leading to the disproportionation of HA by these iron(III) porphyrinates were evidenced for the first time. As a common feature, only N₂ and N₂O were found as gaseous, nitrogen-containing oxidation products, while NH₃ was the unique reduced species detected. Different N₂/N₂O ratios obtained with these three porphyrinates strongly suggest distinctive mechanistic scenarios: while [Fe^III(TEPyP)]⁵⁺ and [Fe^III(MP11)] formed unknown steady-state porphyrinic intermediates in the presence of HA, [Fe^III(TPPS)]³⁻ led to the well characterized soluble intermediate, [Fe^II(TPPS)NO]⁴⁻. Free-radical formation was only evidenced for [Fe^III(TEPyP)]⁵⁺, as a consequence of a metal centered reduction. We discuss the catalytic pathways of HA disproportionation on the basis of the distribution of gaseous products, free radicals formation, the nature of porphyrinic intermediates, the Fe^II/Fe^III redox potential, the coordinating capabilities of each complex, and the kinetic analysis. The absence of revealed either that no HAO-like activity was operative under our reaction conditions, or that , if formed, was consumed in the reaction milieu.     

Preparation, structures and properties of oxalate-bridged binuclear iron(III) complex: [(acac)2Fe(μ-ox)Fe(acac)2] and [(acac)2Fe(μ-ox)Fe(acac)2]·CH2ClCH2Cl

An oxalate-bridged binuclear iron(III) complex, [(acac)₂Fe(μ-ox)Fe(acac)₂], (acac⁻=acetylacetonate anion and ox²⁻=oxalate anion) was prepared. The complex crystallized as two types of crystals under different conditions: one had 1,2-dichloroethane as a solvent molecule of crystallization 2, the other did not 1. Both compounds have been characterized by X-ray crystallography, infrared spectroscopy, and thermogravimetric analysis. Compound 1 has also been characterized by UV-Vis and ¹H NMR spectroscopies, mass spectrometry, and electrochemistry. In both crystals, each iron(III) is coordinated in an octahedral arrangement by the oxygen atoms of an oxalate-bridging ligand and four oxygen atoms belonging to peripheral acac ligands in an octahedral arrangement. The intermetallic distance of Fe?Fe is 5.4368(9) Å in 1 and 5.438(2) Å in 2. Two iron(III) ions in each crystal are bridged by the oxalate and both lie in the oxalate-plane. The results of thermal analyses imply that the thermal stability of 2 is lower than that of 1. Cyclic voltammograms of 1 in acetonitrile and dichloromethane at low temperature showed two consecutive, quasi-Nernstian, one-electron reduction steps corresponding to the reduction of Fe^III-Fe^III to Fe^III-Fe^II followed by the reduction of Fe^III-Fe^II to Fe^II-Fe^II. The electrochemical comproportionation constants (K_c) of the equilibrium (Fe^III-Fe^III) + (Fe^II-Fe^II) ? 2(Fe^III-Fe^II) are 10^8.9 in acetonitrile medium and 10^8.5 in dichloromethane, respectively. The considerably large K_c values indicate that the main factor contributing to the stabilization of the Fe^III-Fe^II mixed-valence state is electronic delocalization through the oxalate-bridge.     

Ferric chelate reduction by suspension culture cells and roots of soybean: A kinetic comparison

The abilities of suspension cultures and intact roots of soybean (Glycine max L. cv. Hawkeye) to reduce ferric chelate were compared. Ferric chelate was supplied as ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) and reduction was measured spectrophotometrically using bathophenan-throlinedisulfonic acid (BPDS) as the ferrous scavenger. Ferric chelate reduction by cell suspension cultures showed typical saturation kinetics; however, no difference was observed between cells that had been continuously grown with Fe (+Fe) and those that had been grown for four days without added Fe (–Fe). Values for K_m and V_max, determined from a Lineweaver-Burk plot, were 57 M and nmoles mg^-1 dry weight for the +Fe cells and 50 M and 22 nmoles mg^-1 dry weight for the -Fe cells, respectively. Ferric chelate reduction by Fe-deficient roots also exhibited saturation kinetics, while roots grown with adequate Fe did not reduce ferric chelate. The K_m and V_max values for Fe-deficient roots were 45 M and 20 nmoles mg^-1 dry weight, respectively, and did not differ from values obtained for cells in culture. This study offers strong evidence that the mechanism responsible for the reduction of ferric chelate is the same for cultured cells and roots and that the process is controlled at the cellular level. We propose that suspension cultures can be used as an alternative to intact roots in the study of ferric chelate reduction.     

Iron availability in plant tissues-iron chlorosis on calcareous soils

The article describes factors and processes which lead to Fe chlorosis (lime chlorosis) in plants grown on calcareous soils. Such soils may contain high HCO₃ ^- concentrations in their soil solution, they are characterized by a high pH, and they rather tend to accumulate nitrate than ammonium because due to the high pH level ammonium nitrogen is rapidly nitrified and/or even may escape in form of volatile NH₃. Hence in these soils plant roots may be exposed to high nitrate and high bicarbonate concentrations. Both anion species are involved in the induction of Fe chlorosis.Physiological processes involved in Fe chlorosis occur in the roots and in the leaves. Even on calcareous soils and even in plants with chlorosis the Fe concentration in the roots is several times higher than the Fe concentration in the leaves. This shows that the Fe availability in the soil is not the critical process leading to chlorosis but rather the Fe uptake from the root apoplast into the cytosol of root cells. This situation applies to dicots as well as to monocots. Iron transport across the plasmamembrane is initiated by Fe^III reduction brought about by a plasmalemma located Fe^III reductase. Its activity is pH dependent and at alkaline pH supposed to be much depressed. Bicarbonate present in the root apoplast will neutralize the protons pumped out of the cytosol and together with nitrate which is taken up by a H⁺/nitrate cotransport high pH levels are provided which hamper or even block the Fe^III reduction.Frequently chlorotic leaves have higher Fe concentrations than green ones which phenomenon shows that chlorosis on calcareous soils is not only related to Fe uptake by roots and Fe translocation from the roots to the upper plant parts but also dependent on the efficiency of Fe in the leaves. It is hypothesized that also in the leaves Fe^III reduction and Fe uptake from the apoplast into the cytosol is affected by nitrate and bicarbonate in an analogous way as this is the case in the roots. This assumption was confirmed by the highly significant negative correlation between the leaf apoplast pH and the degree of iron chlorosis measured as leaf chlorophyll concentration. Depressing leaf apoplast pH by simply spraying chlorotic leaves with an acid led to a regreening of the leaves.