Ferric Reductase Activity Research Articles

Transferrin as a source of iron for Campylobacter rectus

Background and Objective:Campylobacter rectus is considered as one of the bacterial species of etiological importance in periodontitis. Iron-containing proteins such as transferrin are found in periodontal sites and may serve as a source of iron for periodontopathogens. The aim of this study was to investigate the capacity of C. rectus to assimilate transferrin-bound iron to support its growth.Design: Growth studies were performed in broth media pretreated with an iron-chelating resin and supplemented with various iron sources. The uptake of iron by C. rectus was monitored using 55Fe-transferrin. Transferrin-binding activity was assessed using a microplate assay while the degradation of transferrin and iron removal was evaluated by polyacrylamide gel electrophoresis. A colorimetric assay was used to determine ferric reductase activity.Results: Holotransferrin (iron-saturated form) but not apotransferrin (iron-free form) was found to support growth of C. rectus in an iron-restricted culture medium. Incubation of holotransferrin with cells of C. rectus resulted in removal of iron from the protein. A time dependent intracellular uptake of iron by C. rectus cells from 55Fe-transferrin was demonstrated. This uptake was significantly increased when bacteria were grown under an iron-limiting condition. Cells of C. rectus did not show transferrin-binding activity or proteolytic activity toward transferrin. However, a surface-associated ferric reductase activity was demonstrated.Conclusion: To survive and multiply in periodontal sites, periodontopathogens must possess efficient iron-scavenging mechanisms. In this study, we showed the capacity of C. rectus to assimilate iron from transferrin to support its growth. The uptake of iron appears to be dependent on a ferric reductive pathway.

Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis

The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.

Fur and the Novel Regulator YqjI Control Transcription of the Ferric Reductase Gene yqjH in Escherichia coli

Iron acquisition in aerobic habitats is complicated by the low solubility of ferric hydroxides. Siderophores that bind ferric iron with high affinity are used to mobilize iron. The reduction of ferric iron to the ferrous form can be coupled to the release of iron from siderophores. Iron is also stored intracellularly as a ferric mineral in proteins, such as ferritin, and must be reduced during release. In Escherichia coli, the yqjH gene encodes a putative ferric siderophore reductase that is also part of the Fur regulon. Here we show that YqjH has ferric reductase activity and is required for iron homeostasis in E. coli. Divergently transcribed from yqjH is the yqjI gene, which encodes a novel member of the winged-helix family of transcriptional regulators and also contains an N-terminal extension similar to the Ni(2+)-binding C-terminal tail of SlyD. Deletion of yqjI leads to constitutive high-level activity of the yqjH and yqjI promoters. Purified YqjI binds inverted repeat target sequences within the yqjH and yqjI promoters. We also observed that YqjI-dependent transcriptional repression is reduced when cells are exposed to elevated nickel levels, resulting in increased expression of yqjH and yqjI. YqjI binding to nickel or iron reduces YqjI DNA-binding activity in vitro. Furthermore, we found that elevated nickel stress levels disrupt iron homeostasis in E. coli and that deletion of yqjH increases nickel toxicity. Our results suggest that the YqjI protein controls expression of yqjH to help maintain iron homeostasis under conditions (such as elevated cellular nickel levels) that disrupt iron metabolism.

A cytochrome b561 with ferric reductase activity from the parasitic blood fluke, Schistosoma japonicum.

BackgroundIron has an integral role in numerous cellular reactions and is required by virtually all organisms. In physiological conditions, iron is abundant in a largely insoluble ferric state. Ferric reductases are an essential component of iron uptake by cells, reducing iron to the soluble ferrous form. Cytochromes b561 (cyts-b561) are a family of ascorbate reducing transmembrane proteins found in most eukaryotic cells. The identification of the ferric reductase duodenal cytochrome b (dcytb) and recent observations that other cyts-b561 may be involved in iron metabolism have opened novel perspectives for elucidating their physiological function.Methodology/Principal FindingsHere we have identified a new member of the cytochrome b561 (Sjcytb561) family in the pathogenic blood fluke Schistosoma japonicum that localises to the outer surface of this parasitic trematode. Heterologous expression of recombinant Sjcyt-b561 in a Saccharomyces cerevisiae mutant strain that lacks plasma membrane ferrireductase activity demonstrated that the molecule could rescue ferric reductase activity in the yeast.Significance/ConclusionsThis finding of a new member of the cytochrome b561 family further supports the notion that a ferric reductase function is likely for other members of this protein family. Additionally, the localisation of Sjcytb561 in the surface epithelium of these blood-dwelling schistosomes contributes further to our knowledge concerning nutrient acquisition in these parasites and may provide novel targets for therapeutic intervention.

Chromate reductase activity of the Paracoccus denitrificans ferric reductase B (FerB) protein and its physiological relevance

The homodimeric flavoprotein FerB of Paracoccus denitrificans catalyzed the reduction of chromate with NADH as electron donor. When present, oxygen was reduced concomitantly with chromate. The recombinant enzyme had a maximum activity at pH 5.0. The stoichiometric ratio of NADH oxidized to chromate reduced was found to be 1.53±0.09 (O(2) absent) or>2 (O(2) present), the apparent K (M) value for chromate amounted to 70±10μM with the maximum rate of 2.9±0.3μmol NADH s(-1) (mg protein)(-1). Diode-array spectrophotometry and experiments with one-electron acceptors provided evidence for oxygen consumption being due to a flavin semiquinone, formed transiently during the interaction of FerB with chromate. At the whole-cell level, a ferB mutant strain displayed only slightly diminished rate of chromate reduction when compared to the wild-type parental strain. Anaerobically grown cells were more active than cells grown aerobically. The activity could be partly inhibited by antimycin, suggesting an involvement of the respiratory chain. Chromate concentrations above ten micromolars transiently slowed or halted culture growth, with the effect being more pronounced for the mutant strain. It appears, therefore, that, rather than directly reducing chromate, FerB confers a protection of cells against the oxidative stress accompanying chromate reduction. With a strain carrying the chromosomally integrated ferB promoter-lacZ fusion, it was shown that the ferB gene is not inducible by chromate.

Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.

Role of Candida albicans Aft2p transcription factor in ferric reductase activity, morphogenesis and virulence

The ability of Candida albicans to act as an opportunistic fungal pathogen is linked to its ability to switch among different morphological forms. This conversion is an important feature of C. albicans and is correlated with its pathogenesis. Many conserved positive and negative transcription factors regulate morphogenetic transition of C. albicans. Here, we show the results of functional analysis of CaAFT2, which is an orthologue of the Saccharomyces cerevisiae AFT2 gene. We have cloned CaAFT2 which has an ability to complement the S. cerevisiae aft1Δ mutant strain growth defect. Interestingly, although disruption of the AFT2 gene did not affect cell growth in solid and liquid iron-limited conditions, the cell surface ferric reductase activity was significantly decreased. Importantly, deletion of AFT2 in C. albicans led to growth of a smooth colony with no peripheral hyphae. Moreover, virulence of an aft2Δ/aft2Δ mutant was markedly attenuated in a mouse model. Our results suggest that CaAft2p represents a novel activator and that it functions in ferric reductase activity, morphogenesis and virulence in C. albicans.

Plasma membrane ferric reductase activity of iron-limited algal cells is inhibited by ferric chelators

Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.

Chlorella vulgarisAldehyde Reductase Is Capable of Functioning as Ferric Reductase and of Driving the Fenton Reaction in the Presence of Free Flavin

The free flavin-dependent Fenton reaction was detected in cell-free extracts of Chlorella. The corresponding enzyme was purified to homogeneity, and its N-terminal sequence was highly homologous to those of aldo-keto reductase family enzymes. The purified enzyme displayed aldehyde reductase activity in the presence of NADPH. Additionally, it showed ferric reductase activity and drove the Fenton reaction in the presence of free FAD and NADH.

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP(+) reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k(cat)/K(m) value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k(cat)/K(m) value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.

Effects of carboxylic acids on the uptake of non-transferrin-bound iron by astrocytes

The concentrations of non-transferrin-bound iron are elevated in the brain during pathological conditions such as stroke and Alzheimer's disease. Astrocytes are specialised for sequestering this iron, however little is known about the mechanisms involved. Carboxylates, such as citrate, have been reported to facilitate iron uptake by intestinal cells. Citrate binds iron and limits its redox activity. The presence of high citrate concentrations in the interstitial fluid of the brain suggests that citrate may be an important ligand for iron transport by astrocytes. This study investigates whether iron accumulation by cultured rat astrocytes is facilitated by citrate or other carboxylates. Contrary to expectations, citrate, tartrate and malate were found to block iron accumulation in a concentration-dependent manner; α-ketoglutarate had limited effects, while fumarate, succinate and glutarate had no effect. This blockade was not due to an inhibition of ferric reductase activity. Instead, it appeared to be related to the capacity of these carboxylates to bind iron, since phosphate, which also binds iron, diminished the capacity of citrate, tartrate and malate to block the cellular accumulation of iron. These findings raise the possibility that citrate may have therapeutic potential in the management of neurodegenerative conditions that involve cellular iron overload.

Aspergillus oryzae flavohemoglobins promote oxidative damage by hydrogen peroxide

Apart from their well-established role in nitric oxide detoxification, flavohemoglobins (FHbs) are also believed to be involved in protection against oxidative stress in some yeast and bacteria. However, different studies have reported contradictory results in this regard. Here, we investigate the relationship between two FHbs in Aspergillus oryzae (cytosolic FHb1 and mitochondrial FHb2) and oxidative stress. The strains deficient in the two FHbs exhibited higher resistance to hydrogen peroxide than the wild-type. In addition, the FHb2 overexpression strain showed hypersensitivity to hydrogen peroxide. Flavin reductase accompanied by the ferric reductase activities of the two FHbs were observed in correspondence with this expression. The reductase activities of the FHbs were attributed to their C-terminal flavin reductase domains. The reduced intracellular free iron can subsequently promote oxidative damage by accelerating the Fenton reaction in the cytosol and mitochondria (corresponding to the subcellular localizations of the two FHbs). This study is the first to show that fungal FHbs have a deleterious effect on oxidative protection, and suggests that the accelerated Fenton reaction induced by FHbs might be responsible for this effect.

Effects of Glyphosate Application on Seed Iron and Root Ferric (III) Reductase in Soybean Cultivars

Previous research demonstrated that plant nutrient assimilation was reduced by glyphosate (Gly). A 2 year field experiment investigated the effects of Gly at drift rate (12.5% of commercial use rate) on Fe concentrations in leaves and seeds of Gly-sensitive (GS) soybean, and a greenhouse experiment evaluated Gly effects on Fe assimilation using root in vivo ferric reductase activity (FRA) in two GS and one Gly-resistant (GR) soybean cultivars. Field studies showed that Gly drift rates resulted in a significant decrease in the Fe concentration in seeds and leaves compared to the nontreated plants. In greenhouse studies, leaf Fe and FRA were inhibited in GS cultivars Hutcheson and DP 5110 and the GR cultivar AG 4604RR and leaf Fe was positively correlated with root FRA (p < 0.0001). These results indicate that Gly can interfere with Fe assimilation in both GS and GR soybean. Understanding the implication of Gly on Fe nutrition in soybean seed would help soybean agronomists and breeders seeking to improve seed mineral nutrition qualities.

High stability ferric chelates result in decreased iron uptake by the green alga Chlorella kessleri owing to decreased ferric reductase activity and chelation of ferrous iron

Cells of the green alga Chlorella kessleri Fott et Nováková use a reductive mechanism for iron acquisition. Iron-limited cells acquired iron more rapidly from a chelator with a lower stability constant for Fe3+ (hydroxyethylethylenediaminetriacetic acid (HEDTA)) than from a chelator with a higher stability constant (N,N′-di[2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED)). Furthermore, iron uptake rates decreased with increasing chelator concentrations at constant iron concentration. The negative effects of elevated HBED levels on iron uptake could be partly alleviated by the addition of Ga3+, which suggests that iron-free chelator has a negative effect on iron acquisition by competing for Fe2+ with the ferrous transport system. Furthermore, ferric reductase activity progressively decreased with increasing concentrations of both chelators (in the iron-free form). This effect was not alleviated by Ga3+ addition and was apparently caused by the direct inhibition of the reductase. Overall, we conclude that chelators with high stability constants for Fe3+ decrease iron acquisition rates by Strategy I organisms via three separate mechanisms.

Structure analysis of the flavoredoxin from Desulfovibrio vulgaris Miyazaki F reveals key residues that discriminate the functions and properties of the flavin reductase family

The crystal structure of flavoredoxin from Desulfovibrio vulgaris Miyazaki F was determined at 1.05 A resolution and its ferric reductase activity was examined. The aim was to elucidate whether flavoredoxin has structural similarity to ferric reductase and ferric reductase activity, based on the sequence similarity to ferric reductase from Archaeoglobus fulgidus. As expected, flavoredoxin shared a common overall structure with A. fulgidus ferric reductase and displayed weak ferric reductase and flavin reductase activities; however, flavoredoxin contains two FMN molecules per dimer, unlike A. fulgidus ferric reductase, which has only one FMN molecule per dimer. Compared with A. fulgidus ferric reductase, flavoredoxin forms three additional hydrogen bonds and has a significantly smaller solvent-accessible surface area. These observations explain the higher affinity of flavoredoxin for FMN. Unexpectedly, an electron-density map indicated the presence of a Mes molecule on the re-side of the isoalloxazine ring of FMN, and that two zinc ions are bound to the two cysteine residues, Cys39 and Cys40, adjacent to FMN. These two cysteine residues are close to one of the putative ferric ion binding sites of ferric reductase. Based on their structural similarities, we conclude that the corresponding site of ferric reductase is the most plausible site for ferric ion binding. Comparing the structures with related flavin proteins revealed key structural features regarding the discrimination of function (ferric ion or flavin reduction) and a unique electron transport system.

Ferrous Iron Production Mediated by Tetrathionate Hydrolase in Tetrathionate-, Sulfur-, and Iron-GrownAcidithiobacillus ferrooxidansATCC 23270 Cells

When tetrathionate-grown Acidithiobacillus ferrooxidans ATCC 23270 cells were incubated with ferric ions and tetrathionate at pH 3.0, ferrous ions were produced enzymatically. Fe(3+)-reductase, which catalyzes Fe(3+) reduction with tetrathionate, was purified to homogeneity not only from tetrathionate-grown, but also from sulfur- and iron-grown A. ferrooxidans ATCC 23270 cells. The results for apparent molecular weight measured by SDS-PAGE (52.3 kD) and the N-terminal amino acid sequences of the purified enzymes from iron-, sulfur, and tetrathionate-grown cells (AVAVPMDSTG) indicate that Fe(3+)-reductase corresponds to tetrathionate hydrolase. The evidence that tetrathionate-grown A. ferrooxidans ATCC 23270 cells have high iron-oxidizing activity at the early log phase, comparable to that of iron-grown ATCC 23270 cells, is supported by our finding that tetrathionate hydrolase produces Fe(2+) from tetrathionate during growth on tetrathionate. This is the first report on ferric reductase activity associated with tetrathionate hydrolase.

Iron Deficiency‐induced Increase of Root Branching Contributes to the Enhanced Root Ferric Chelate Reductase Activity

In various plant species, Fe deficiency increases lateral root branching. However, whether this morphological alteration contributes to the Fe deficiency-induced physiological responses still remains to be demonstrated. In the present research, we demonstrated that the lateral root development of red clover (Trifolium pretense L.) was significantly enhanced by Fe deficient treatment, and the total lateral root number correlated well with the Fe deficiency-induced ferric chelate reductase (FCR) activity. By analyzing the results from Dasgan et al. (2002), we also found that although the two tomato genotypes line227/1 (P1) and Roza (P2) and their reciprocal F1 hybrid lines ("P1 x P2" and "P2 x P1") were cultured under two different lower Fe conditions (10(-6) and 10(-7) M FeEDDHA), their FCR activities are significantly correlated with the lateral root number. More interestingly, the -Fe chlorosis tolerant ability of these four tomato lines displays similar trends with the lateral root density. Taking these results together, it was proposed that the Fe deficiency-induced increases of the lateral root should play an important role in resistance to Fe deficiency, which may act as harnesses of a useful trait for the selection and breeding of more Fe-efficient crops among the genotypes that have evolved a Fe deficiency-induced Fe uptake system.

The role of Dcytb in iron metabolism: an update

Dcytb (duodenal cytochrome b) is an iron-regulated ferric reductase highly expressed in duodenal enterocytes. Its location and strong regulation by iron has indicated it plays an important role in iron absorption. Expression of Dcytb in cells (Caco-2 and MDCK) was found to increase both ferric reductase activity and stimulate uptake of (59)Fe. An additional increase in cupric reductase activity was found in MDCK (Madin-Darby canine kidney) cells expressing Dcytb. Expression and purification of Dcytb in insect cells reveals that Dcytb is a di-haem protein and that the haems are reducible by ascorbate, indicating that ascorbate is the likely intracelluar electron donor. Studies underway in Dcytb-knockout mice reveal that Dcytb is the only iron-regulated ferric reductase in the duodenal mucosa and that loss of Dcytb affects iron absorption.

Histoplasma capsulatum secreted γ‐glutamyltransferase reduces iron by generating an efficient ferric reductant

The intracellular fungal pathogen Histoplasma capsulatum (Hc) resides in mammalian macrophages and causes respiratory and systemic disease. Iron limitation is an important host antimicrobial defence, and iron acquisition is critical for microbial pathogenesis. Hc displays several iron acquisition mechanisms, including secreted glutathione-dependent ferric reductase activity (GSH-FeR). We purified this enzyme from culture supernatant and identified a novel extracellular iron reduction strategy involving gamma-glutamyltransferase (Ggt1) activity. The 320 kDa complex was composed of glycosylated protein subunits of about 50 and 37 kDa. The purified enzyme exhibited gamma-glutamyl transfer activity as well as iron reduction activity in the presence of glutathione. We cloned and manipulated expression of the encoding gene. Overexpression or RNAi silencing affected both GGT and GSH-FeR activities concurrently. Enzyme inhibition experiments showed that the activity is complex and involves two reactions. First, Ggt1 initiates enzymatic breakdown of GSH by cleavage of the gamma-glutamyl bond and release of cysteinylglycine. Second, the thiol group of the released dipeptide reduces ferric to ferrous iron. A combination of kinetic properties of both reactions resulted in efficient iron reduction over a broad pH range. Our findings provide novel insight into Hc iron acquisition strategies and reveal a unique aspect of Ggt1 function in this dimorphic mycopathogen.

The FRO2 ferric reductase is required for glycine betaine’s effect on chilling tolerance in Arabidopsis roots

FRO2 (At1g01580) codes for an NADPH-dependent ferric reductase in plasma membranes of root epidermal cells with a demonstrated role in iron uptake by plants. Ferric reductase activity has been shown to be the rate-limiting step for iron uptake in strategy I plants like Arabidopsis and in rice, but it has been unclear whether FRO genes have other physiological functions. We hypothesized that FRO2 was involved in chilling stress tolerance because its expression was upregulated by treatment of plants with glycine betaine (GB), a chemical that prevents reactive oxygen species (ROS) signaling in chilling stress. This idea was confirmed by showing that the FRO2 null mutant frd1-1 failed to respond to GB in chilling assays either in relation to root growth recovery or inhibition of ROS accumulation. Measurements of ferric reductase activity in wild-type plants treated with GB before chilling showed no significant GB effect compared with controls. In addition, 35S-FRO2 transgenics with elevated mRNA levels did not have improved chilling tolerance. However, ferric reductase activity in wild-type plants or 35S-FRO2 transgenics pretreated with GB was several-fold higher after chilling compared with non-pretreated controls. These experiments identify a new physiological function for FRO2, i.e. blocking ROS accumulation during chilling. They also suggest that GB has a major effect on FRO2 activity posttranscriptionally in the cold.