Fe Reductase Research Articles

Extracellular Fe(III) reductase structure reveals a modular organization enabling S-layer insertion and electron transfer to insoluble substrates

The thermophilic anaerobic Gram-positive bacterium Carboxydothermus ferrireducens utilizes insoluble Fe(III) oxides as electron acceptors in respiratory processes using an extracellular 11-heme cytochrome cOmhA as a terminal reductase. OmhA is able to transfer electrons to soluble and insoluble Fe(III) compounds, substrates of multiheme oxidoreductases, and soluble electron shuttles. The crystal structure of OmhA at 2.5 Å resolution shows that it consists of two functionally distinct parts: the cytochrome с electron transfer and the S-layer binding domains. Nonaheme C-terminal subdomain of the cytochrome с domain isstructurally similar to the extracellular multiheme cytochrome OcwA from the metal-reducing Gram-positive bacterium "Thermincola potens." S-layer binding domain of OmhA is responsible for interaction with the S-layer that surrounds the Carboxydothermus ferrireducens cell envelope. The structural foundations enabling the embedding of extracellular multiheme cytochromes to the S-layer of a Gram-positive-type cell wall and putative electron transfer pathways to insoluble minerals are discussed.

The Geomagnetic Field Is a Contributing Factor for an Efficient Iron Uptake in Arabidopsis thaliana.

The Earth’s magnetic field, defined as the geomagnetic field (GMF), is an unavoidable environmental factor for all living organisms. Variation in the GMF intensity was found to affect the content of some nutrients and their associated channels and transporters in Arabidopsis thaliana. In this work, we observed that reduction of the GMF to near null magnetic field (NNMF) affects the accumulation of metals in plant tissues, mainly iron (Fe) and zinc (Zn) content, while the content of others metals such as copper (Cu) and manganese (Mn) is not affected. Accordingly, Fe uptake genes were induced in the roots of NNMF-exposed plants and the root Fe reductase activity was affected by transferring GMF-exposed plant to NNMF condition. Under Fe deficiency, NNMF-exposed plants displayed a limitation in the activation of Fe-deficiency induced genes. Such an effect was associated with the strong accumulation of Zn and Cu observed under NNMF conditions. Overall, our results provide evidence on the important role of the GMF on the iron uptake efficiency of plants.

Enhancement of Iron Acquisition in Rice by the Mugineic Acid Synthase Gene With Ferric Iron Reductase Gene and OsIRO2 Confers Tolerance in Submerged and Nonsubmerged Calcareous Soils.

Iron (Fe) is an essential micronutrient for plants. Plants encounter Fe deficiency when grown in calcareous soil with low Fe availability, leading to reduced crop yield and agricultural problem. Rice acquires Fe from the soil via Strategy I–related system (ferrous ion uptake by OsIRT1) and Strategy II system (ferric ion uptake by chelation). However, rice plants have a weak ability in Fe(III) reduction and phytosiderophore secretion. We previously produced an Fe deficiency–tolerant rice harboring OsIRT1 promoter-refre1/372 (for higher Fe(III) reductase ability) and a 35S promoter-OsIRO2 (for higher phytosiderophore secretion). In this study, we produced a new Fe deficiency–tolerant rice by the additional introduction of a barley IDS3 genome fragment with refre1/372 and OsIRO2 (named as IRI lines) for further enhancement in Strategy II phytosiderophore productivity and better growth performance in various environments. Our results show that an enhanced tolerance was observed in OsIRO2 introduced line at the early growth stage, refre1/372 introduced line in the late stage, and RI line in all stages among five types of cultivation method. Moreover, we demonstrated that new IRI rice lines exhibited enhanced tolerance to Fe deficiency compared to nontransgenic (NT) rice and rice lines harboring the overexpressing OsIRO2 or the IDS3 fragment under submerged calcareous soil. The yields of IRI lines were ninefold higher than the NT line. Furthermore, under Fe-limited nonsubmerged calcareous soil condition (a new cultivation condition), IRI lines also conferred enhanced tolerance than NT, lines introducing only the OsIRT1 promoter-refre1/372 or overexpressing OsIRO2, and lines harboring both. Our results demonstrate that further enhancement of the Strategy II Fe uptake system by the mugineic acid synthase gene in addition to Fe uptake by enhanced ferric Fe reduction and phytosiderophore production in rice contributes Fe deficiency tolerance and broaden its utility in calcareous soil cultivation under paddy or nonpaddy field conditions.

Cadmium tolerance is associated with the root-driven coordination of cadmium sequestration, iron regulation, and ROS scavenging in rice

Excess cadmium (Cd) is a serious threat to agriculture and the environment. High Cd availability showed no significant decline in growth, chlorophyll synthesis, soluble protein, cell and membrane stability in Sonarbangla (Cd-tolerant), while these were severely affected in BRRI 72 (Cd-sensitive). Atomic absorption spectroscopy analysis demonstrated a huge increment of Cd and Fe in root and shoot of BRRI 72; however, Sonarbangla only exhibited a significant increase of Cd in roots. It suggests that excess Cd in Sonarbangla possibly retained in roots through vacuolar sequestration without interfering cell functions. This was further confirmed by the increased accumulation of cysteine, glutathione, and phytochelatin along with OsPCS1 and OsHMA3 upregulation, possibly facilitated by nitric oxide in roots of Sonarbangla. Further, Fe chelate reductase activity in conjunction with the genes (OsFRO1, OsNRAMP1, OsIRT1, and OsYSL15) associated with Fe availability significantly upregulated in BRRI 72 but not in Sonarbangla in response to Cd. It advises that Fe acquisition and transport were tightly regulated in Cd-tolerant Sonarbangla. Furthermore, elevated CAT, APX, GR, NO in root along with shoot sugar helps rice plants to withstand Cd-induced oxidative damage. Finally, reciprocal grafting combining Sonarbangla rootstock with either BRRI 72 or Sonarbangla scion showed Sonarbangla type tolerance along with no changes of H2O2 and Fe reductase activity in roots under high Cd. It indicates that the signal inducing the responses to adjust Cd stress is originated in the root system. These messages deliver essential background for further breeding program to produce Cd-free rice.

Natural Variation in Physiological Responses of Tunisian Hedysarum carnosum Under Iron Deficiency.

Iron (Fe) is an essential element for plant growth and development. The cultivation of leguminous plants has generated strong interest because of their growth even on poor soils. Calcareous and saline soils with poor mineral availability are wide-spread in Tunisia. In an attempt to select better forage crops adapted to Tunisian soils, we characterized Fe deficiency responses of three different isolates of Hedysarum carnosum, an endemic Tunisian extremophile species growing in native stands in salt and calcareous soil conditions. H. carnosum is a non-model crop. The three isolates, named according to their habitats Karkar, Thelja, and Douiret, differed in the expression of Fe deficiency symptoms like morphology, leaf chlorosis with compromised leaf chlorophyll content and photosynthetic capacity and leaf metal contents. Across these parameters Thelja was found to be tolerant, while Karkar and Douiret were susceptible to Fe deficiency stress. The three physiological and molecular indicators of the iron deficiency response in roots, Fe reductase activity, growth medium acidification and induction of the IRON-REGULATED TRANSPORTER1 homolog, indicated that all lines responded to -Fe, however, varied in the strength of the different responses. We conclude that the individual lines have distinct adaptation capacities to react to iron deficiency, presumably involving mechanisms of whole-plant iron homeostasis and internal metal distribution. The Fe deficiency tolerance of Thelja might be linked with adaptation to its natural habitat on calcareous soil.

Ayçiçeği Genotiplerinin Demir Noksanlığına Karşı Tolerans Düzeylerinin Belirlenmesi

Demir (Fe) noksanlığı dünyada bitkilerde ve insanlarda görülen önemli bir beslenme problemidir. Bu sorun aynı zamanda Türkiye‘de de yaygın bir beslenme problemi olup ayçiçeği, önemli derecede verim ve kalite kayıplarına yol açan Fe noksanlığına duyarlı bir tür olarak bilinmektedir. Bu çalışmada farklı ayçiçeği genotiplerinin Fe noksanlığına karşı duyarlılığı test edilmiştir. Çalışmada, bitki materyali olarak, TR-6149-SA, TR-3080 ve 6480 genotipleri kullanılmıştır. Bitkiler, Fe‘siz (0 µmol Fe) ve Fe (100 µmol Fe) içeren su kültürü yetiştirme ortamında test edilmiştir. Deneme sonunda bitkilerde simptom derecesi, SPAD değeri, klorofil konsantrasyonu, yeşil aksam kuru madde verimi, Fe-redüktaz enzim aktivitesi, yeşil aksam Fe konsantrasyonu ve büyüme ortamının pH değeri ölçülmüştür. Sonuçlar ayçiçeği bitkisinin Fe noksanlığını etkileyen en önemli faktörlerin Fe alımı ve Fe-redüktaz aktivitesi olduğunu göstermiştir. Çeşitlerin Fe noksanlığına karşı toleransta önemli rolü olan köklerin Fe redüktaz enzim aktivitesinin TR-3080 nolu genotipde Fe noksanlığı koşullarında diğer genotiplerden daha yüksek olduğu bulunmuştur. Sonuç olarak, uygulanan değişik Fe konsantrasyonlarına karşı genotiplerin tepkilerinin önemli derecede farklılık gösterdiği ve Fe noksanlığına karşı tolerant genotiplerin belirlenmesinde Fe redüktaz enzim aktivitesinin önemli rol oynayabileceği ortaya çıkmıştır.

Bioenergy grass [Erianthus ravennae (L.) Beauv.] secretes two members of mugineic acid family phytosiderophores which involved in their tolerance to Fe deficiency

ABSTRACTRavenna grass, Erianthus ravennae (L.) Beauv. (E. ravennae) is a potential high biomass-energy crop with low input requirements. Iron (Fe) deficiency in calcareous soils is a widespread agronomic problem which reduces crop yields. Fe is sparingly soluble under aerobic conditions at high soil pH, such as in calcareous soils; therefore, plants cannot take up enough Fe. Increasing crop productivity of giant grasses, such as Ravenna grass in calcareous soil, has a positive effect by alleviating environmental problems. However, the growth character in calcareous soil and Fe homeostatic trait of Ravenna grass are largely unknown. In this study, we analyzed characteristics of Ravenna grass. The growth of E. ravennae plants were impaired in calcareous soil compared to those in the normal soil. In calcareous soil, the growth of E. ravennae plants differ among the water and fertilizer conditions; E. ravennae plants were grown better in the submerged condition adding micronutrient among conditions. These results suggested that impaired growth of E. ravennae in calcareous soil might be micronutrient shortage. We found that E. ravennae roots possess Fe reductase activities which were upregulated under Fe-deficient conditions. E. ravennae produced and secreted mugineic acid (MA) and deoxymugineic acid (DMA) to acquire Fe from the soil. The amount of MA was higher than that of DMA. Thus, E. ravennae might have both partial Strategy-I and Strategy-II Fe uptake systems. E. ravennae intercropped with transgenic rice plants producing and secreting MA through the introduction of the barley MA synthase gene showed improved growth compared to monocropped E. ravennae plants, suggesting that the increased amounts of MA enhanced their tolerance to Fe deficiency. Our results suggest that there is a considerable potential to improve the growth of E. ravennae plants in calcareous soils by enhancement of their Fe uptake systems through increase of MA production.

A new transgenic rice line exhibiting enhanced ferric iron reduction and phytosiderophore production confers tolerance to low iron availability in calcareous soil.

Iron (Fe) deficiency is a critical agricultural problem, especially in calcareous soil, which is distributed worldwide. Rice plants take up Fe(II) from soil through a OsIRT1 transporter (Strategy I-related system) and also take up Fe(III) via a phytosiderophore-based system (Strategy II system). However, rice plants are susceptible to low-Fe conditions because they have low Fe(III) reduction activity and low-level phytosiderophore secretion. Previously, we produced transgenic rice plants expressing a mutationally reconstructed yeast ferric chelate reductase, refre1/372, under the control of the OsIRT1 promoter. This transgenic rice line exhibited higher Fe(III) chelate reductase activity and tolerance to Fe deficiency. In addition, we produced transgenic rice overexpressing the Fe deficiency-inducible transcription factor, OsIRO2, which regulates the expression of various genes involved in the strategy II Fe(III) uptake system, including OsNAS1, OsNAAT1, OsDMAS1, OsYSL15, and TOM1. This transgenic rice exhibited improved phytosiderophore secretion ability and tolerance to Fe deficiency. In the present research, transgenic rice plants that possess both the OsIRT1 promoter-refre1/372 and the 35S promoter-OsIRO2 (RI lines) were produced to enhance both Strategy I Fe(II) reductase ability and Strategy II phytosiderophore productivity. RI lines exhibited enhanced tolerance to Fe-deficient conditions at the early and middle-late stages of growth in calcareous soil, compared to both the non-transgenic line and lines harboring either OsIRT1 promoter-refre1/372 or 35S promoter-OsIRO2 alone. RI lines also exhibited a 9-fold higher yield than the non-transgenic line. Moreover, we successfully produced Fe-deficiency-tolerant Tachisugata rice, which is a high-biomass variety used as fodder. Collectively, our results demonstrate that combined enhancement of two Fe uptake systems in rice is highly effective in conferring tolerance to low Fe availability in calcareous soil.

Effects of Fe-deficient conditions on Fe uptake and utilization in P-efficient soybean

Phosphorus (P)-efficient soybean (Glycine max) plants absorb and utilize P with high efficiency. To investigate the effects of iron (Fe)-deficient conditions on the absorption and utilization of Fe in P-efficient soybean plants, two soybean cultivars with different P efficiency, the 03-3 (P-efficient variety) and Bd-2 (P-inefficient variety), were used in this study. The two soybean cultivars were grown in nutrient solution containing Fe concentrations of 0 (Fe0), 20 (Fe20), 40 (Fe40), or 80 (Fe80) μM for 7 days. The Fe reductase activity of roots was higher in 03-3 plants grown under the Fe0, Fe20, and Fe40 treatments than in Bd-2 plants and the total Fe uptake was greater in 03-3 plants under the Fe40 treatment. GmFRD3a was much more highly expressed in the stem of 03-3 than in that of Bd-2, and significantly more iron was transported to 03-3 plant shoots during Fe0 treatment. Chlorosis in young leaves caused by Fe deficiency under the Fe0 and Fe20 treatments was alleviated by increased Fe concentration in shoots. Increased levels of active Fe in young 03-3 leaves under Fe-deprivation conditions (Fe0) and maintenance of stable Fe concentrations in 03-3 shoots subjected to Fe20, Fe40, and Fe80 treatments suggested that the P-efficient 03-3 cultivar is also Fe-efficient. It is suggested that 03-3 soybean cultivar should be a good resource for application to farm field.

Role of Silicon Counteracting Cadmium Toxicity in Alfalfa (Medicago sativa L.).

Cadmium (Cd) is one of the most phytotoxic elements causing an agricultural problem and human health hazards. This work investigates whether and how silicon (Si) ameliorates Cd toxicity in Alfalfa. The addition of Si in Cd-stressed plants caused significant improvement in morpho-physiological features as well as total protein and membrane stability, indicating that Si does have critical roles in Cd detoxification in Alfalfa. Furthermore, Si supplementation in Cd-stressed plants showed a significant decrease in Cd and Fe concentrations in both roots and shoots compared with Cd-stressed plants, revealing that Si-mediated tolerance to Cd stress is associated with Cd inhibition in Alfalfa. Results also showed no significant changes in the expression of two metal chelators [MsPCS1 (phytochelatin synthase) and MsMT2 (metallothionein)] and PC (phytochelatin) accumulation, indicating that there may be no metal sequestration or change in metal sequestration following Si application under Cd stress in Alfalfa. We further performed a targeted study on the effect of Si on Fe uptake mechanisms. We observed the consistent reduction in Fe reductase activity, expression of Fe-related genes [MsIRT1 (Fe transporter), MsNramp1 (metal transporter) and OsFRO1 (ferric chelate reductase] and Fe chelators (citrate and malate) by Si application to Cd stress in roots of Alfalfa. These results support that limiting Fe uptake through the down-regulation of Fe acquisition mechanisms confers Si-mediated alleviation of Cd toxicity in Alfalfa. Finally, an increase of catalase, ascorbate peroxidase, and superoxide dismutase activities along with elevated methionine and proline subjected to Si application might play roles, at least in part, to reduce H2O2 and to provide antioxidant defense against Cd stress in Alfalfa. The study shows evidence of the effect of Si on alleviating Cd toxicity in Alfalfa and can be further extended for phytoremediation of Cd toxicity in plants.

Biochemical and molecular changes in rice seedlings (Oryza sativa L.) to cope with chromium stress.

Chromium (Cr) is very toxic to both humans and plants. This investigation aimed to understand the physiological and molecular responses of rice seedlings to Cr stress. Cr toxicity did not significantly affect morphological features and Cr accumulation in roots and shoots in Pokkali but not in BRRI 51, although there was a reduction in chlorophyll concentration in leaves of both genotypes. These results imply that Pokkali has mechanisms to cope with Cr supplementation. We therefore performed quantitative real-time PCR on the expression pattern of two chelator genes, OsPCS1 and OsMT1, but there were no significant changes in expression in roots and shoots of Pokkali and BRRI 51 following Cr stress. This suggests that there was no metal sequestration following heavy metal stress in roots of these genotypes. Moreover, no expression of two heavy metal transporter genes, OsHMA3 and OsNRAMP1, was induced after Cr stress in roots and shoots, suggesting that these transporter genes are not induced by Cr stress or might not be involved in Cr uptake in rice. We also performed a targeted study on the effect of Cr on Fe uptake mechanisms. Our studies showed a consistent reduction in Fe uptake, Fe reductase activity and expression of Fe-related genes (OsFRO1 and OsIRT1) under Cr stress in both roots and leaves of Pokkali. In contrast, these parameters and genes were significantly increased in Cr-sensitive BRRI 51 under Cr stress. The results confirm that limiting Fe uptake through the down-regulation of Fe reductase and Fe transporter genes is the main strategy of Cr-tolerant Pokkali to cope with Cr stress. Finally, increased CAT, POD and GR activity and elevated glutathione and proline synthesis might provide strong antioxidant defence against Cr stress in Pokkali. Taken together, our findings reveal that Cr stress tolerance in rice (Pokkali) is not related to metal sequestration but is associated with reduced Fe transport and increased antioxidant defence.

The Effect of Nitrate Levels and Harvest Times on Fe, Zn, Cu, and K, Concentrations and Nitrate Reductase Activity in Lettuce and Spinach

The Effect of Nitrate Levels and Harvest Times on Fe, Zn, Cu, and K, Concentrations and Nitrate Reductase Activity in Lettuce and Spinach

Iron partitioning at an early growth stage impacts iron deficiency responses in soybean plants (Glycine max L.).

Iron (Fe) deficiency chlorosis (IDC) leads to leaf yellowing, stunted growth and drastic yield losses. Plants have been differentiated into ‘Fe-efficient’ (EF) if they resist to IDC and ‘Fe-inefficient’ (IN) if they do not, but the reasons for this contrasting efficiency remain elusive. We grew EF and IN soybean plants under Fe deficient and Fe sufficient conditions and evaluated if gene expression and the ability to partition Fe could be related to IDC efficiency. At an early growth stage, Fe-efficiency was associated with higher chlorophyll content, but Fe reductase activity was low under Fe-deficiency for EF and IN plants. The removal of the unifoliate leaves alleviated IDC symptoms, increased shoot:root ratio, and trifoliate leaf area. EF plants were able to translocate Fe to the aboveground plant organs, whereas the IN plants accumulated more Fe in the roots. FRO2-like gene expression was low in the roots; IRT1-like expression was higher in the shoots; and ferritin was highly expressed in the roots of the IN plants. The efficiency trait is linked to Fe partitioning and the up-regulation of Fe-storage related genes could interfere with this key process. This work provides new insights into the importance of mineral partitioning among different plant organs at an early growth stage.

Mechanisms associated with differential tolerance to Fe deficiency in okra (Abelmoschus esculentus Moench)

The study aimed at characterizing the physiological, biochemical and molecular mechanisms conferring differential tolerance to Fe deficiency in two contrasting Okra genotypes (BARI-1 and Orca Onamica). Fe deficiency caused greater decline in morpho-physiological traits in Orca Onamica compared to BARI-1, suggesting greater Fe-efficiency in BARI-1. Fe reductase, proton extrusion and phenol content in roots increased greatly in BARI-1 suggesting these responses contribute immensely to Fe-efficiency in this genotype. Semi-quantitative RT-PCR revealed the upregulation of ZIP1 (metal transporter) and IRT1 (iron-regulated transporter) genes in Fe-deficient roots of BARI-1 and Orca Onamica, albeit to a lesser extent. Also, BARI-1 was less affected by oxidative stress but showed enhanced scavenging activity that may stabilize antioxidant capacities and protect against oxidative damage under Fe deficiency. In addition, increased SOD and GR activities in BARI-1 suggest better capacity to detoxify ROS and to facilitate generating antioxidant metabolites, respectively. Furthermore, HPLC (high-performance liquid chromatography) data showed increased glutathione, cysteine, citrate and ascorbic acid in roots of BARI-1 and to a lesser extent in Orca Onamica under Fe deficiency. Elevated glutathione, ascorbic acid and cysteine suggest better protection of Okra from Fe-deficiency induced oxidative stress. In addition, a significant increase in citrate and Fe-citrate was observed only in xylem sap of BARI-1 under Fe deficiency, indicating a role for citrate in long-distance transport of Fe. These findings provide first evidence on the mechanisms of differential Fe-efficiency in Okra. This will allow the improvement of Fe biofortification in Okra and other plants.

Enzymatic exploration of catalase from a nanoparticle producing and biodecolorizing algae Shewanella xiamenensis BC01

Shewanella xiamenensis (SXM) was found to produce nanoparticles (NPs) under aerobic condition. The oxidoreductase enzymatic activities including of catalase, manganese peroxidase, laccase, NADH dehydrogenase, flavin reductase, azoreductase and Fe reductase are first investigated. Catalase showed the greatest enzymatic activity among all oxidoreductases in SXM, which with strong activities in multiple substrates of ABTS, guaiacol and 2,6-DMP. The optimum temperature, pH, concentrations of H2O2 and 2,6-DMP for this enzyme were found to be 65°C, pH 4.0, 128.7mM and 10mM, respectively. Finally, from the kinetic parameters and structure simulation of catalase, implied that SXM would potentially apply in bioremediation, microbe fuel cells (MFCs) and nano-biotechnology based on its distinguished enzymatic system.

Cultivar variability of iron uptake mechanisms in rice (Oryza sativa L.)

Rice (Oryza sativa L.) is the most important staple food in the world. It is rich in genetic diversity and can grow in a wide range of environments. Iron (Fe) deficiency is a major abiotic stress in crop production and in aerobic soils, where Fe forms insoluble complexes, and is not readily available for uptake. To cope with Fe deficiency, plants developed mechanisms for Fe uptake, and although rice was described as a Strategy II plant, recent evidence suggests that it is capable of utilizing mechanisms from both Strategies. The main objective of this work was to compare two cultivars, Bico Branco (japonica) and Nipponbare (tropical japonica), to understand if the regulation of Fe uptake mechanisms could be cultivar (cv.) dependent. Plants of both cultivars were grown under Fe-deficient and -sufficient conditions and physiological and molecular responses to Fe deficiency were evaluated. Bico Branco cv. developed more leaf chlorosis and was more susceptible to Fe deficiency, retaining more nutrients in roots, than Nipponbare cv., which translocated more nutrients to shoots. Nipponbare cv. presented higher levels of Fe reductase activity, which was significantly up-regulated by Fe deficiency, and had higher expression levels of the Strategy I-OsFRO2 gene in roots, while Bico Branco cv. induced more genes involved in Strategy II.These new findings show that rice cultivars have different responses to Fe deficiency and that the induction of Strategy I or II may be rice cultivar-dependent, although the utilization of the reduction mechanisms seems to be an ubiquitous advantage.

Direct uptake of copper from plasma ceruloplasmin by cells that do and do not express CTR1 (996.2)

The main Cu‐binding protein of blood plasma, ceruloplasmin (Cp), has mainly been studied for its role in mediating cellular iron efflux but also has other functions, and as shown by i.v. infusion of 67Cu‐Cp, its Cu enters most major organs. To determine whether it delivers Cu directly to cells and the mechanisms involved, we incubated purified radio‐copper‐labeled and unlabeled Cp with cells and measured rates of Cu uptake and accumulation. Uptake was demonstrated for human mammary epithelial cell monolayers (PMC42 cells) with tight junctions, and with mouse embryonic fibroblasts expressing/not expressing Ctr1. Cp‐Cu was internalized. It could not be washed from the cell surface even with acidic buffers, and was found in the cytosol. Inhibitors of endocytosis did not impair uptake. Cu deficient cells exposed to Cp increased their cellular Cu contents; and holo‐Cp was converted to apo‐Cp during cell treatment, with no loss of total Cp protein. Uptake was almost the same in cells not expressing Ctr1. Excess ionic Cu(II) and Cu(I) virtually eliminated Cp‐Cu uptake. The presence of Cu(II) and Fe(III) reductase activities on the cell surface was verified. We conclude that Cp‐Cu delivers Cu to cells by a process involving cell surface reduction of Cp‐Cu(II), release of apo‐Cp, and uptake of the resulting Cu(I) by Ctr1 and an additional as yet unidentified Cu(I) transporter.Grant Funding Source: Supported by PHS Grant R15 GM100464

Identification of Multiple Soluble Fe(III) Reductases in Gram-Positive Thermophilic Bacterium Thermoanaerobacter indiensis BSB-33.

Thermoanaerobacter indiensis BSB-33 has been earlier shown to reduce Fe(III) and Cr(VI) anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI) reduction activity in both the membrane and cytoplasm. The soluble fraction prepared from T. indiensis cells grown at 60°C was found to contain the majority of Fe(III) reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III) reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS) with the help of T. indiensis proteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs), thioredoxin reductase (Trx), NADP(H)-nitrite reductase (Ntr), and thioredoxin disulfide reductase (Tdr) were determined to be responsible for Fe(III) reductase activity. Amino acid sequence and three-dimensional (3D) structural similarity analyses of the T. indiensis Fe(III) reductases were carried out with Cr(VI) reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI) reducing dihydrolipoamide dehydrogenase from Thermus scotoductus and thioredoxin disulfide reductase from Desulfovibrio desulfuricans. It appears that in addition to their iron reducing activity T. indiensis LPDs and Tdr are possibly involved in Cr(VI) reduction as well.

Exploring metal effects and synergistic interactions of ferric stimulation on azo-dye decolorization by new indigenous Acinetobacter guillouiae Ax-9 and Rahnella aquatilis DX2b

The first-attempt study deciphered metal-interacting effects on dye-decolorizing capabilities of indigenous bioelectricity-generating strains, Acinetobacter guillouiae Ax-9 and Rahnella aquatilis DX2b. Most of the metallic ions were inhibitory to color removal capabilities of these strains. However, with supplementation of 5 mM ferric chloride, specific decolorization rate (SDR) of Ax-9 increased by 55.48% compared to Fe(3+)-free conditions. In contrast, SDR of DX2b decreased 75.35% due to the inhibition of ferric chloride. On the other hand, ferric citrate could stimulate SDR of DX2b for 21.5% at same dosage. Enzymatic assay indicated that Fe reductase activity was consistent with synergistic effects of ferric chloride on Ax-9, and ferric citrate on DX2b. Protein analysis via SDS-PAGE and identification of Tandem MS/MS afterwards showed that outer membrane protein (Omp) primarily deals with decolorization as a channeling regulation. Moreover, molecular modeling and bioinformatics data also provided detailed evidences to confirm the biological significance of Omp.

The Effects of Iron Deficiency on Lead Accumulation in Ailanthus altissima (Mill.) Swingle Seedlings

Understanding the effects of root Fe deficiency on the uptake and translocation of toxic metals can be important for improving the phytoremediation strategies of polluted soils. The present study investigated how Fe nutritional status affects the uptake and root-to-shoot-translocation of Pb in hydroponically grown seedlings of (Mill.) Swingle. The interactions of Fe deficiency and Pb were assessed by measuring the root Fe(III) reductase activity, carboxylic acids concentration in root exudates, root and shoot biomass, and accumulation of Pb and other metals (Fe, Zn, Mn, and Cu) in roots and leaves of Fe-sufficient (+Fe) and Fe-deficient (-Fe) plants. The results indicate that Fe deficiency induced 18-fold higher Fe(III) reductase activity in roots compared with +Fe plants, which was followed by increased root exudation of citric acid (28.2 ± 1.39 in +Fe and 498 ± 256.4 μmol g DW 2 h in -Fe plants). Iron deficiency also induces a significant decrease of root and shoot dry weight compared with the control +Fe plants, whereas 2-wk Pb (20 μM) treatment did not influence root and shoot growth. Iron-sufficient plants accumulated more Pb (56.8 ± 17.29 μg g) in leaves than -Fe plants (21.5 ± 8.10 μg g). Two weeks of exposure to Pb significantly decreased Fe(III) reductase activity and accumulation of Fe, Zn, and Mn in the roots of -Fe plants. It is hypothesized that 2 wk of root exposure to Pb blocks functioning of a specific Fe transport system activated under Fe deficiency.