Genotypical differences among graminaceous species in release of phytosiderophores and uptake of iron phytosiderophores

Graminaceous species can enhance iron (Fe) acquisition from sparingly soluble inorganic Fe(III) compounds by release of phytosiderophores (PS) which mobilize Fe(III) by chelation. In most graminaceous species Fe deficiency increases the rate of PS release from roots by a factor of 10–20, but in some species, for example sorghum, this increase is much less. The chemical nature of PS can differ between species and even cultivars.The various PS are similarly effective as the microbial siderophore Desferal (ferrioxamine B methane sulfonate) in mobilizing Fe(III) from a calcareous soil. Under the same conditions the synthetic chelator DTPA (diaethylenetriamine pentaacetic acid) is ineffective.The rate of Fe(III)PS uptake by roots of graminaceous species increases by a factor of about 5 under Fe deficiency. In contrast, uptake of Fe from both synthetic and microbial Fe(III) chelates is much lower and not affected by the Fe nutritional status of the plants. This indicates that in graminaceous species under Fe deficiency a specific uptake system for FePS is activated. In contrast, the specific uptake system for FePS is absent in dicots. In a given graminaceous species the uptake rates of the various FePS are similar, but vary between species by a factor of upto 3. In sorghum, despite the low rate of PS release, the rate of FePS uptake is particularly high.The results indicate that release of PS and subsequent uptake of FePS are under different genetic control. The high susceptibility of sorghum to Fe deficiency (lime-chlorosis) is most probably caused by low rates of PS release in the early seedling stage. Therefore in sorghum, and presumably other graminaceous species also, an increase in resistance to lime chlorosis could be best achieved by breeding for cultivars with high rates of PS release. In corresponding screening procedures attention should be paid to the effects of iron nutritional status and daytime on PS release as well as on rapid microbial degradation of PS.     

Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii

The basic question addressed in this study is how energy metabolism is adjusted to cope with iron deficiency in Chlamydomonas reinhardtii. To investigate the impact of iron deficiency on bioenergetic pathways, comparative proteomics was combined with spectroscopic as well as voltametric oxygen measurements to assess protein dynamics linked to functional properties of respiratory and photosynthetic machineries. Although photosynthetic electron transfer is largely compromised under iron deficiency, our quantitative and spectroscopic data revealed that the functional antenna size of photosystem II (PSII) significantly increased. Concomitantly, stress-related chloroplast polypeptides, like 2-cys peroxiredoxin and a stress-inducible light-harvesting protein, LhcSR3, as well as a novel light-harvesting protein and several proteins of unknown function were induced under iron-deprivation. Respiratory oxygen consumption did not decrease and accordingly, polypeptides of respiratory complexes, harboring numerous iron-sulfur clusters, were only slightly diminished or even increased under low iron. Consequently, iron-deprivation induces a transition from photoheterotrophic to primarily heterotrophic metabolism, indicating that a hierarchy for iron allocations within organelles of a single cell exists that is closely linked with the metabolic state of the cell.     

A new role for heme, facilitating release of iron from the bacterioferritin iron biomineral

Bacterioferritin (BFR) from Escherichia coli is a member of the ferritin family of iron storage proteins and has the capacity to store very large amounts of iron as an Fe(3+) mineral inside its central cavity. The ability of organisms to tap into their cellular stores in times of iron deprivation requires that iron must be released from ferritin mineral stores. Currently, relatively little is known about the mechanisms by which this occurs, particularly in prokaryotic ferritins. Here we show that the bis-Met-coordinated heme groups of E. coli BFR, which are not found in other members of the ferritin family, play an important role in iron release from the BFR iron biomineral: kinetic iron release experiments revealed that the transfer of electrons into the internal cavity is the rate-limiting step of the release reaction and that the rate and extent of iron release were significantly increased in the presence of heme. Despite previous reports that a high affinity Fe(2+) chelator is required for iron release, we show that a large proportion of BFR core iron is released in the absence of such a chelator and further that chelators are not passive participants in iron release reactions. Finally, we show that the catalytic ferroxidase center, which is central to the mechanism of mineralization, is not involved in iron release; thus, core mineralization and release processes utilize distinct pathways.     

Light-mediated release of phytosiderophores in wheat and barley under iron or zinc deficiency

The effect of varied light intensity (50 – 600 mol m-2 s-1) on the rate of phytosiderophore release was studied under zinc (Zn) deficiency using a bread (Triticum aestivum L. cv. Aroona) and a durum wheat cultivar (Triticum durum Desf. cv. Durati) differing in zinc (Zn) efficiency and under iron (Fe) deficiency using a barley cultivar (Hordeum vulgare L. Europe). Plants were grown under controlled environmental conditions in nutrient solution for 15 days (wheat plants) or 11 days (barley plants). Phytosiderophore release was determined by measuring capacity of root exudates to mobilize copper (Cu) from a Cu-loaded resin.With increasing light intensity visual Zn deficiency symptoms such as whitish-brown lesions on leaf blade developed rapidly and severely in wheat, particularly in the durum cultivar Durati. In wheat plants supplied well with Zn, increases in light intensity from 100 to 600 mol m-2 s-1 did not clearly affect the rate of phytosiderophore release. However, under Zn deficiency increases in light intensity markedly enhanced release of phytosiderophores in Zn-deficient Aroona, but not in Zn-inefficient Durati. When Fe-deficient barley cultivar Europe was grown first at 220 mol m-2 s-1 and then exposed to 600 mol m-2 s-1 for 24 and 48 h, the rate of release of phytosiderophores was enhanced about 4-fold and 7-fold, respectively. Transfer of Fe-deficient plants from 600 to 50 mol m-2 s-1 for 48 h reduced the rate of release of phytosiderophores by a factor of 7. The effect of light on phytosiderophore release was similar regardless of whether the rate of phytosiderophore release was expressed per plant or per unit dry weight of roots.The results demonstrate a particular role of light intensity in phytosiderophore release from roots under both Zn and Fe deficiency. It is suggested that in the studies concerning the role of phytosiderophore release in expression of Zn or Fe efficiency among and within cereals, a special attention should be given to the light conditions.     

Effect of nicotianamine on iron re-mobilization in de-rooted tomato seedlings

Summary An exogenous supply of nicotianamine is essential for the redistribution of⁵⁹iron via the symplast and the phloem to newly developing organs in de-rooted seedlings of the nicotianamine-less tomato mutantchloronerva. This observation supports the idea that nicotianamine could function as a translocator of iron within the symplast and the phloem.Abbreviations EDDHA ethylenediamine-N,N-bis(o-hydroxy-phenylacetic acid) - NA nicotianamine=(2S, 3S,3S)-N-[N-(3-amino-3-carboxypropyl)-3-amino-3-carboxypropyl]-azetidine-2-carboxylic acid This paper is part 36 in the seriesThe normalizing factor for the tomato mutant chloronerva. For part 35 see Stephan and Grün (1989)     

The effect of pH on the kinetics of iron release from human transferrin

The rate of iron release from the N-terminal and C-terminal monoferrictransferrins (Fe_N-transferrin and Fe_C-transferrin, respectively) has been studied at 37°C over the pH range 3.5–10.6 using EDTA as the accepting chelate. Fe_N-transferrin is the more facile except above pH 8.2. Plots of log₁₀k_obs against pH showed a deviation for both monoferrictransferrins between pH 5.6 and 6.0 and studies above and below this transition point indicated that iron release occures by different mechanisms. At low pH (< 5.6) the rate of release from Fe_N-transferrin is independent of the presence of EDTA or NaClO₄, whereas Fe_c-transferrin shows a small but significant increase with increasing EDTA concentration. Rapid protonation of both monoferrictransferrins is followed by relatively slow release of Fe³⁺ which is subsequently chelated by EDTA. The slower release from Fe_c-transferrin is probably due to its greater binding strength for iron and the greater conformational stability of the C-terminal domain. Above pH 6.0 iron release from both monoferrictransferrins increases as the concentration of EDTA is increased. Direct attacks of EDTA probably occurs giving Fe-transferrin (HCO₃). EDTA as a transition state or intermediate. The factors which may lead to the observed pH dependence of the rate include (i) protonation of groups directly bound to the iron, (ii) conformers which differ in degree of protonation and (iii) the degree of protonation of the attacking chelating agent. It is suggested that an increase in conformational fluctuations as the pH is lowered may play a very important role. Studies with differrictransferrin at pH 4.53 and 7.40 showed that when iron is released to EDTA the rate is independent of the occupancy of the other site; that is, the two sites are exhibiting non-co-operativity.     

Synthesis and structure of iron (III) and iron (II) complexes in S4P2 environment created by diethyldithiocarbamate and 1,2-bis(diphenylphosphino)ethane chelation: Investigation of the electronic structure of the complexes by Mössbauer and magnetic studies

Iron (II) and iron (III) complexes, [Fe^II(DEDTC)₂(dppe)] · CH₂Cl₂ (1), [Fe^II(ETXANT)₂(dppe)] (2) (DEDTC = diethyldithiocarbamate, ETXANT = ethyl xanthate, dppe = 1,2-bis (diphenylphosphino) ethane), and [Fe^III(DEDTC)₂(dppe)] [Fe^IIICl₄] (3) have been synthesized and characterized. Since 3 contains two magnetic centers, an anion metathesis reaction has been conducted to replace the tetrahedral FeCl₄⁻ by a non-magnetic BPh₄⁻ ion producing [Fe^III(DEDTC)₂(dppe)]BPh₄ (4) for the sake of unequivocal understanding of the magnetic behavior of the cation of 3. With the similar end in view, the well-known FeCl₄⁻ ion, the counter anion of 3, is trapped as PPh₄[Fe^IIICl₄] (5) and its magnetic property from 298 to 2 K has been studied. Besides the spectroscopic (IR, UV-Vis, NMR, EPR, Mass and XPS) characterization of the appropriate compounds, especially 2, others viz. 1, 3 and 4 have been structurally characterized by X-ray crystallography. While Fe^II complexes, 1 and 2, are diamagnetic, the Fe^III systems, namely the cations of 3, and 4 behave as low-spin (S = 1/2) paramagnetic species from 298 to 50 K. Below 50 K 3 shows gradual increase of χ_MT up to 2 K suggesting ferromagnetic behavior while 4 exhibits gradual decrease of magnetic moment from 60 to 2 K, indicating the occurrence of weak antiferromagnetic interaction. These conclusions are supported by the Mössbauer studies of 3 and 4. The Mössbauer pattern of 1 exhibits a doublet site for diamagnetic (2-400 K) Fe^II. The compounds 1, 2 and 4 encompass interesting cyclic voltammetric responses involving Fe^II, Fe^III and Fe^IV.     

Characterization of the DNA-binding site in the ferric uptake regulator protein from Escherichia coli by UV crosslinking and mass spectrometry

Ferric uptake regulator protein (Fur) is activated by its cofactor iron to a state that binds to a specific DNA sequence called 'Fur box'. Using mass spectrometry-based methods, we showed that Tyr 55 of Escherichia coli Fur, as well as the two thymines in positions 18 and 19 of the consensus Fur Box, are involved with binding. A conformational model of the Fur-DNA complex is proposed, in which DNA is in contact with each H4 [A52-A64] Fur helix. We propose that this interaction is a common feature for the Fur-like proteins, such as Zur and PerR, and their respective DNA boxes.     

Comparison of ferric iron generation by different species of acidophilic bacteria immobilized in packed-bed reactors

Flooded packed-bed bioreactors, prepared by immobilizing four different species of acidophilic iron-oxidizing bacteria on porous glass beads, were compared for their ferric iron-generating capacities when operated in batch and continuous flow modes over a period of up to 9 months, using a ferrous iron-rich synthetic liquor and acid mine drainage (AMD) water. The bacteria used were strains of Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, a Ferrimicrobium-like isolate (TSTR) and a novel Betaproteobacterium (isolate PSTR), which were all isolated from relatively low-temperature mine waters. Three of the bacteria used were chemoautotrophs, while the Ferrimicrobium isolate was an obligate heterotroph. Greater biomass yields achievable with the Ferrimicrobium isolate resulted in greater iron oxidation efficiency in the newly commissioned bioreactor containing this bacterium, though long-term batch testing with organic carbon-free solution resulted in similar maximum iron oxidation rates in all four bioreactors. Two of the bioreactors (those containing immobilized L. ferrooxidans and Ferrimicrobium TSTR) were able to generate significantly lower concentrations of ferrous iron than the others when operated in batch mode. In contrast, when operated as continuous flow systems, the bioreactor containing immobilized PSTR was superior to the other three when challenged with either synthetic or actual AMD at high flow rates. The least effective bacterium overall was At. ferrooxidans, which has previously been the only iron-oxidizer used in the majority of reports describing ferric iron-generating bioreactors. The results of these experiments showed that different species of iron-oxidizing acidophiles have varying capacities to oxidize ferrous iron when immobilized in packed-bed bioreactors, and that novel isolates may be superior to well-known species.     

Isolation and characterisation of EfeM, a periplasmic component of the putative EfeUOBM iron transporter of Pseudomonas syringae pv. syringae

The EfeM protein is a component of the putative EfeUOBM iron-transporter of Pseudomonas syringae pathovar syringae and is thought to act as a periplasmic, ferrous-iron binding protein. It contains a signal peptide of 34 amino acid residues and a C-terminal ‘Peptidase_M75’ domain of 251 residues. The C-terminal domain contains a highly conserved ‘HXXE’ motif thought to act as part of a divalent cation-binding site. In this work, the gene (efeM or ‘Psyr_3370’) encoding EfeM was cloned and over-expressed in Escherichia coli, and the mature protein was purified from the periplasm. Mass spectrometry confirmed the identity of the protein (M_W 27,772 Da). Circular dichroism spectroscopy of EfeM indicated a mainly α-helical structure, consistent with bioinformatic predictions. Purified EfeM was crystallised by hanging-drop vapor diffusion to give needle-shaped crystals that diffracted to a resolution of 1.6 Å. This is the first molecular study of a peptidase M75 domain with a presumed iron transport role.     

Rapid mobilization of ferritin iron by ascorbate in the presence of oxygen

L-(—)-ascorbate mobilizes iron from horse-spleen ferritin in the presence of oxygen at pH 8.0. The reaction is strongly stimulated by Cu²⁺. Dehydroascorbate and other stable oxidation products of ascorbate are ineffective. We present evidence that monodehydroascorbate mobilizes ferritin iron by reduction.     

Biosynthetic pathway of phytosiderophores in iron-deficient graminaceous plants: A new assay system for the detection of nicotianamine amino-transferase activity

A new assay system for the detection of nicotianamine amino-transferase activity was developed. The activity of nicotianamine amino-transferase which participated in biosynthetic pathway of MAs from methionine in graminaceous plants was induced by the iron deficiency treatment.     

Investigation of the release of iron from ferritin by naturally occurring antioxidants

Ferritin is the main intracellular iron storage protein. The release of iron from ferritin in the presence of a number of phenolic based compounds of nutritional significance was studied at physiological pH. The release of iron was measured by monitoring the formation of the iron(II)-ferrozine complex. The kinetics of this process were studied in Hepes buffer (pH 7.00), at 37 degrees C. The order of ability to remove iron from ferritin is epigallocatechin>gallic acid methyl ester approximately equal to sinapic acid>ferulic acid. The presence of the oxyradical scavenger urea resulted in a slight inhibition in the release of iron from ferritin by both gallic acid methyl ester and epigallocatechin. The ability of each reagent to release iron is interpreted on the basis of their ability to (a) reduce the bound iron and (b) complex the iron with the oxidised form of the phenol, thus mobilising it from the protein. These studies indicate that some phenolic based compounds that have been epidemiologically associated with a negative effect on iron absorption in man, can individually mobilise and release iron from ferritin under suitable conditions.     

Effects of chelators on iron uptake and release by the brain in the rat

The iron chelators desferrioxamine (DFO), pyridoxal isonicotinoyl hydrazone (PIH), 2,2-bipyridine, diethylenetriamine penta-acetic acid (DTPA) and 1,2 dimethyl-3-hydroxy pyrid-4-one (CP20) were analysed for their ability to change⁵⁹Fe uptake and release from the brain of 15- and 63-day rats either during or after intravenous injection of⁵⁹Fe-¹²⁵I-transferrin. DTPA was the only chelator unable to significantly reduce iron uptake into the brain of 15-day rats. This indicates that iron is not released from transferrin at the luminal surface of brain capillary endothelial cells. CP20 was able to reduce iron uptake in the brain by 85% compared to 28% with DFO. Only CP20 was able to significantly reduce brain iron uptake in 63 day rats. Once⁵⁹Fe had entered the brain no chelator used was able to mediate its release. All of the chelators except CP20 had similar effects on femur iron uptake as they did on brain uptake, suggesting similar iron uptake mechanisms. It is concluded that during the passage of transferrin-bound iron into the brain the iron is released from transferrin within endothelial cells after endocytosis of transferrin.     

Critical examination for the presence of a low molecular weight fraction in serum iron

Native human pool serum and individual sera were ultrafiltered by Pellicon ultrafilters (Millipore) and the ultrafiltrates were extracted by an ammonium pyrrolidinedithicarbamate/methylisobutylketone system after addition of different internal iron standards to three of four identical ultrafiltrates. The extracts were examined for iron content by atomic absorption spectrometry. During ultrafiltration pH 7.4 was miantained by a constant atmosphere of a CO₂/air mixture.Low molecular weigth iron in native human sera from normal, normal orally iron substituted and siderotic individuals was found to be less than 0.05 μg/100 ml. Elevating serum citrate to 3-fold normal had no effect on this result.More iron became ultrafiltrable if the serum pH were lowered by citric acid as compared with hydrochloric acid.     

Relationship between hypolimnetic phosphorus and iron release from eleven lakes in Maine, USA

We studied five eutrophic (high phosphorus) and six mesotrophic/oligotrophic (low phosphorus) lakes in Maine, USA, all of which are dimictic and develop anoxic hypolimnia during stratification. The lakes were sampled during the stratified period from May to September 1999. Late summer hypolimnetic total phosphorus (P) concentrations in the high-P lakes ranged from 185 to 460ppb; epilimnetic total P increased up to 30ppb from the spring to the fall overturn. During the same period, the low-P lakes had hypolimnetic total P concentrations in the range of 6–19ppb.Individual high-P lakes demonstrated strong temporal correlations between aqueous hypolimnetic dissolved Fe and total P concentrations (R ²0.88) with an average molar Fe:P ratio of 11.9±4.2. For the combined data, the high-P lakes exhibited strong correlation between hypolimnetic Fe and P concentrations (R ²=0.82). The low-P lakes, however, did not show a good correlation between the hypolimnetic Fe and P concentrations. Among the low-P lakes two lakes had hypolimnetic Fe fluxes comparable to the Fe fluxes of the high-P lakes. These two lakes had considerably higher hypolimnetic Fe:P ratios than all other lakes studied here. There were no significant differences in surface sediment Fe(III) or P fractions that correlated with the differences in the relationship between aqueous concentrations of Fe and P in these two outlier low-P lakes. A model for the generation of hypolimnetic acid neutralization capacity (ANC) was developed based on microbially-catalyzed reduction of Fe(III) hydroxide, Mn(IV) oxide and sulfate. Reduction of Fe(III) hydroxide was the most important contributor to the increase in the hypolimnetic ANC in all high-P and the two outlier lakes. Assuming that all hypolimnetic P was due to the reduction of Fe(III) hydroxide by bacteria and sulfide, average summer hypolimnetic P flux for each lake was predicted using the sediment reducible Fe(III):P ratio. The observed and predicted average P fluxes in the high-P lakes corresponded reasonably, suggesting that in these lakes internal P release is closely related to the reduction of Fe(III) hydroxide. Other release or sequestration mechanisms may operate for the release and availability of P in the low-P lakes.     

Relationships of nicotianamine and other amino acids with nickel, zinc and iron in Thlaspi hyperaccumulators

Experimental evidence suggests that nicotianamine (NA) is involved in the complexation of metal ions in some metal-hyperaccumulating plants. Closely-related nickel (Ni)- and zinc (Zn)-hyperaccumulating species were studied to determine whether a correlation exists between the Ni and Zn concentrations and NA in foliar tissues. A liquid chromatography-mass spectrometry (LC-MS) procedure was developed to quantify the NA and amino acid contents using the derivatizing agent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. A strong correlation emerged between Ni and NA, but not between Zn and NA. Concentrations of NA and L-histidine (His) also increased in response to higher Ni concentrations in the hydroponic solution supplied to a serpentine population of Thlaspi caerulescens. An inversely proportional correlation was found between the iron (Fe) and Ni concentrations in the leaves. Correlations were also found between Zn and asparagine. The results obtained in this study suggest that NA is involved in hyperaccumulation of Ni but not Zn. The inverse proportionality between the Ni and Fe concentrations in the leaf may suggest that Ni and Fe compete for complexation to NA.     

LFR1 ferric iron reductase of Leishmania amazonensis is essential for the generation of infective parasite forms

The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. The parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages, where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly, indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections. Here, we identify and functionally characterize a second member of the Leishmania iron acquisition pathway, the ferric iron reductase LFR1. The LFR1 gene is up-regulated under iron deprivation and accounts for all the detectable ferric reductase activity exposed on the surface of Leishmania amazonensis. LFR1 null mutants grow normally as promastigote insect stages but are defective in differentiation into the vertebrate infective forms, metacyclic promastigotes and amastigotes. LFR1 overexpression partially restores the abnormal morphology of infective stages but markedly reduces parasite viability, precluding its ability to rescue LFR1 null replication in macrophages. However, LFR1 overexpression is not toxic for amastigotes lacking the ferrous iron transporter LIT1 and rescues their growth defect. In addition, the intracellular growth of both LFR1 and LIT1 null parasites is rescued in macrophages loaded with exogenous iron. This indicates that the Fe(3+) reductase LFR1 functions upstream of LIT1 and suggests that LFR1 overexpression results in excessive Fe(2+) production, which impairs parasite viability after intracellular transport by LIT1.     

The effect of various chelating agents on the mobilization of iron from reticulocytes in the presence and absence of pyridoxal isonicotinoyl hydrazone

The chelating agent pyridoxal isonicotinoyl hydrazone (PIH) has recently been shown to mobilize ⁵⁹Fe from reticulocytes loaded with non-heme ⁵⁹Fe. In this study, various chelating agents were tested for their ability to effect the mobilization of iron from reticulocytes by PIH. They fall into several groups. The largest group includes chelators such as citrate, ethylenediaminetetracetic acid and desferrioxamine, which fail to affect PIH-induced iron mobilization and do not mobilize iron per se. Either these chelators do not enter reticulocytes or they do not take up iron from PIH-Fe complexes. The second group includes chelators such as 2,2′-bipyridine, 1,10-phenanthroline, bathophenanthroline sulfonate and N,N′-ethylenebis(o-hydroxyphenylglycine) which inhibit PIH-induced iron mobilization from reticulocytes and, when added together with PIH, induce radioiron accumulation in an alcohol-soluble fraction of reticulocytes. It appears that these chelators enter the cell and compete with PIH for ⁵⁹Fe(II), but having bound iron are unable to cross the cell membrane. Spectral analysis suggests that Fe(II) chelators such as 2,2′-bipyridine and 1,10-phenanthroline remove iron from Fe(II)PIH but are not able to do so from Fe(III)PIH. Then there are compounds such as 2,3-dihydroxybenzoic acid and catechol which potentiate PIH-induced iron mobilization although they are unable to mobilize iron from reticulocytes by themselves. Lastly, there is a group of miscellaneous compounds which include chelators that either potentiate the iron-mobilizing effect of PIH as well as mobilizing iron from reticulocytes by themselves (tropolone), or that reduce PIH-induced iron mobilization while themselves having an iron-mobilizing effect (N,N′-bis(2,3-dihydroxybenzoyl)-1,6-diaminohexane). In further experiments, heme was found to stimulate globin synthesis in reticulocytes, the heme synthesis of which was inhibited by PIH, suggesting that PIH is probably not toxic to the cells.