Mobilization of iron and other micronutrient cations from a calcareous soil by plant-borne,microbial, and synthetic metal chelators

Mobilization of Fe, Zn, Cu, and Mn by various chelators from a calcareous soil was measured using a simple dialysis tube/complexing resin system. Root exudates from Fe-deficient barley increased the concentrations of all four metals in solution by, on average, a factor of 20, and the addition of complexing resin as a sink for heavy metal cations forced steady state solution concentrations to be reached sooner. Root exudates mobilized increasing amounts of the various micronutrients in the following order: Cu<Fe<Zn<Mn. Phytosiderophores isolated from root exudates of Fe-deficient barley mobilized similar amounts of Cu and Zn but somewhat more Fe and considerably more Mn than crude exudate. The synthetic chelators EDDHA and DTPA showed low specificity in micronutrient mobilization, but the microbial siderophore Desferal was relatively more specific, preferentially mobilizing Fe and Mn. The data indicates that phytosiderophores are capable of increasing the amount of complexed cations in solution. Despite their lack of specificity, phytosiderophores were just as effective as Desferal increasing the availability of Fe. Thus, phytosiderophores, as plant-borne chelators, are certainly of significance for the Fe nutrition of cereals grown in calcareous soils.     

The determination of ferric iron in plants by HPLC using the microbial iron chelator desferrioxamine E

Common methods for plant iron determination are based on atomic absorption spectroscopy, radioactive measurements or extraction with subsequent spectrophotometry. However, accuracy is often a problem due to background, contamination and interfering compounds. We here describe a novel method for the easy determination of ferric iron in plants by chelation with a highly effective microbial siderophore and separation by high performance liquid chromatography (HPLC). After addition of colourless desferrioxamine E (DFE) to plant fluids, the soluble iron is trapped as a brown-red ferrioxamine E (FoxE) complex which is subsequently separated by HPLC on a reversed phase column. The formed FoxE complex can be identified due to its ligand-to-metal charge transfer band at 435 nm. Alternatively, elution of both, DFE and FoxE can be followed as separate peaks at 220 nm wavelength with characteristic retention times. The extraordinarily high stability constant of DFE with ferric iron of K=10³² enables extraction of iron from a variety of ferrous and ferric iron compounds and allows quantitation after separation by HPLC without interference by coloured by-products. Thus, iron bound to protein, amino acids, citrate and other organic acid ligands and even insoluble ferric hydroxides and phosphates can be solubilized in the presence desferrioxamine E. The “Ferrioxamine E method” can be applied to all kinds of plant fluids (apoplasmic, xylem, phloem, intracellular) either at physiological pH or even at acid pH values. The FoxE complex is stable down to pH 1 allowing protein removal by perchloric acid treatment and HPLC separation in the presence of trifluoroacetic acid containing eluents. Published online December 2004     

Reduction of iron by extracellular iron reductases: implications for microbial iron acquisition

The extracellular enzymatic reduction of iron by microorganisms has not been appropriately considered. In this study the reduction and release of iron from ferrioxamine were examined using extracellular microbial iron reductases and compared to iron mobilization by chemical reductants, and to chelation by EDTA and desferrioxamine. A flavin semiquinone was formed during the enzymatic reduction of ferrioxamine, which was consistent with the 1 e(-) reduction of iron by an enzyme. The rates for the enzymatic reactions were substantially faster than both the 2 e(-) chemical reductions and the chelation reactions. The rapid rates of the enzymatic reduction reactions demonstrated that these enzymes are capable of accomplishing the extracellular mobilization of iron required by microorganisms. The data suggest that mechanistically there are two phases for the mobilization and transport of iron by those microorganisms that produce both extracellular iron reductases and siderophores, with reduction being the principle pathway.     

The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe²⁺) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of ⁵⁵Fe³⁺ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.     

Root-microbial effects on plant iron uptake from siderophores and phytosiderophores

Collaborative experiments were conducted to determine whether microbial populations associated with plant roots may artifactually affect the rates of Fe uptake and translocation from microbial siderophores and phytosiderophores. Results showed nonaxenic maize to have 2 to 34-fold higher Fe-uptake rates than axenically grown plants when supplied with 1 μM Fe as either the microbial siderophore, ferrioxamine B (FOB), or the barley phytosiderophore, epi-hydroxymugineic acid (HMA). In experiments with nonsterile plants, inoculation of maize or oat seedlings with soil microorganisms and amendment of the hydroponic nutrient solutions with sucrose resulted in an 8-fold increase in FOB-mediated Fe-uptake rates by Fe-stressed maize and a 150-fold increase in FOB iron uptake rates by Fe-stressed oat, but had no effect on iron uptake by Fe-sufficient plants. Conversely, Fe-stressed maize and oat plants supplied with HMA showed decreased uptake and translocation in response to microbial inoculation and sucrose amendment. The ability of root-associated microorganisms to affect Fe-uptake rates from siderophores and phytosiderophores, even in short-term uptake experiments, indicates that microorganisms can be an unpredictable confounding factor in experiments examining mechanisms for utilization of microbial siderophores or phytosiderophores under nonsterile conditions.     

Siderophores sorbed on Ca-montmorillonite as an iron source for plants

Previous investigations have shown significant sorption of siderophores to the solid phase in soils, and clay surfaces in particular. The ability of plants to utilize Fe from this reservoir is therefore of great interest. This research focused on the ability of the hydroxamate siderophore ferrioxamine B (FOB) sorbed to Ca-montmorillonite – prevailing in soils – to supply Fe to peanuts (Arachis hypogeae L.). Remediation of Fe deficiency by the sorbed siderophore was found to be similar to that by the free (unsorbed) form. The concentration needed to achieve complete remediation of chlorosis was one order of magnitude higher than that of the optimal FeEDDHA [Fe-ethylenediamine-di(o-hydroxyphenylacetic acid)]. Using dialysis tubes, it was shown that Fe uptake from the sorbed siderophore is executed mainly via long-range pathways and does not require close proximity to the plant roots. It was hypothesized that the process involves chelating agents in solution, which transport the Fe from the immobilized siderophore and enable its uptake by the plant. Under calcareous conditions, the ability of the sorbed FOB to supply Fe was significantly impaired, probably as a result of inactivation of the bridging mechanism. Various possible shuttle compounds were examined. EDDHA was found to be a very efficient shuttle compound, which caused complete remediation of Fe deficiency, even under very harsh calcareous conditions. The findings support our hypothesis and imply the effectiveness of a ligand-exchange mechanism to strategy I plants (commonly attributed to strategy II plants). We suggest that the secretion of substances with chelating abilities, which is usually considered a less effective means of Fe acquisition mechanism, takes on more importance in this context.     

Water-extractable humic substances enhance iron deficiency responses by Fe-deficient cucumber plants

The ability of Fe-deficient cucumber plants to use iron complexed to a water-extractable humic substances fraction (WEHS), was investigated. Seven-day-old Fe-deficient plants were transferred to a nutrient solution supplemented daily for 5 days with 0.2 μM Fe as Fe-WEHS (5 μg org. C mL^-1), Fe-EDTA, Fe-citrate or FeCl₃. These treatments all allowed re-greening of the leaf tissue, and partial recovery of dry matter accumulation, chlorophyll and iron contents. However, the recovery was faster in plants supplied with Fe-WEHS and was already evident 48 h after Fe supply. The addition of 0.2 μM Fe to the nutrient solution caused also a partial recovery of the dry matter and iron accumulation in roots of Fe-deficient cucumber plants, particularly in those supplied with Fe-WEHS. The addition of WEHS alone (5 μg org. C mL^-1, 0.04 μM Fe) to the nutrient solution slightly but significantly increased iron and chlorophyll contents in leaves of Fe-deficient plants; in these plants, dry matter accumulation in leaves and roots was comparable or even higher than that measured in plants treated with Fe-citrate or FeCl₃. After addition of the different iron sources for 5 days to Fe-deficient roots, morphological modifications (proliferation of lateral roots, increase in the diameter of the sub-apical zones and amplified root-hair formation) and physiological responses (enhanced Fe(III)-chelate reductase and acidification of the nutrient solution) induced by Fe deficiency, were still evident, particularly in plants treated with the humic molecules. The presence of WEHS caused also a further acidification of the nutrient medium by Fe-deficient plants. The Fe-WEHS complex (1 μM Fe) could be reduced by intact cucumber roots, at rates of reduction higher than those measured for Fe-EDTA at equimolar iron concentration. Plasma membrane vesicles, purified by two-phase partition from root microsomes of Fe-deficient plants, were also able to reduce Fe-WEHS. Results show that Fe-deficient cucumber plants can use iron complexed to water soluble humic substances, at least in part via reduction of complexed Fe(III) by the plasma membrane Fe(III)-chelate reductase of root cells. In addition, the stimulating effect of humic substances on H⁺ release might be of relevance for the overall response of the plants to iron shortage. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

微生物铁载体运输系统

铁是大多数生物生长所必需的元素,在生命活动中占有极其重要的地位.尽管自然界中铁元素含量丰富,但是大多敬好氧生物包括微生物都面临着铁缺乏的问题,铁载体运输系统是微生物获得铁元素的重要途径.本文就铁载体运输系统的组成、调控机制及当前微生物中的铁载体运输系统的进展作以综述.     

Root exudates as mediators of mineral acquisition in low-nutrient environments

Plant developmental processes are controlled by internal signals that depend on the adequate supply of mineral nutrients by soil to roots. Thus, the availability of nutrient elements can be a major constraint to plant growth in many environments of the world, especially the tropics where soils are extremely low in nutrients. Plants take up most mineral nutrients through the rhizosphere where micro-organisms interact with plant products in root exudates. Plant root exudates consist of a complex mixture of organic acid anions, phytosiderophores, sugars, vitamins, amino acids, purines, nucleosides, inorganic ions (e.g. HCO₃ ^–, OH^–, H⁺), gaseous molecules (CO₂, H₂), enzymes and root border cells which have major direct or indirect effects on the acquisition of mineral nutrients required for plant growth. Phenolics and aldonic acids exuded directly by roots of N₂-fixing legumes serve as major signals to Rhizobiaceae bacteria which form root nodules where N₂ is reduced to ammonia. Some of the same compounds affect development of mycorrhizal fungi that are crucial for phosphate uptake. Plants growing in low-nutrient environments also employ root exudates in ways other than as symbiotic signals to soil microbes involved in nutrient procurement. Extracellular enzymes release P from organic compounds, and several types of molecules increase iron availability through chelation. Organic acids from root exudates can solubilize unavailable soil Ca, Fe and Al phosphates. Plants growing on nitrate generally maintain electronic neutrality by releasing an excess of anions, including hydroxyl ions. Legumes, which can grow well without nitrate through the benefits of N₂ reduction in the root nodules, must release a net excess of protons. These protons can markedly lower rhizosphere pH and decrease the availability of some mineral nutrients as well as the effective functioning of some soil bacteria, such as the rhizobial bacteria themselves. Thus, environments which are naturally very acidic can pose a challenge to nutrient acquisition by plant roots, and threaten the survival of many beneficial microbes including the roots themselves. A few plants such as Rooibos tea (Aspalathus linearis L.) actively modify their rhizosphere pH by extruding OH^– and HCO₃ ^– to facilitate growth in low pH soils (pH 3 – 5). Our current understanding of how plants use root exudates to modify rhizosphere pH and the potential benefits associated with such processes are assessed in this review.     

The use of microbial siderophores for foliar iron application studies

Experiments were conducted to assess the distribution of foliar applied Fe-containing compounds using microbial siderophores. Fe was measured in leaf fluid obtained by centrifugation according to a determination method based on Fe chelation by desferrioxamine E and HPLC separation on a reversed phase column. To avoid sample Fe contamination, treatments were only applied to a part of the leaf following a systematic and reproducible procedure and iron concentration was exclusively determined in fluid obtained from non-treated leaf surfaces. The increase in leaf fluid Fe concentration associated with the distribution of leaf applied Fe-siderophores, Fe–EDTA and FeSO₄ × 7H₂O was evaluated using Vicia faba L., Nicotiana tabacum L. and Citrus madurensis Lour. plants. The method proved useful to investigate the process of leaf Fe penetration and its distribution within the plant. Evidence of the penetration and distribution of leaf applied Fe-rhizoferrin, Fe-coprogen hydrolysis products and Fe-dimerum acid is presented in this study.     

Mechanisms of iron acquisition from siderophores by microorganisms and plants

Most bacteria, fungi, and some plants respond to Fe stress by the induction of high-affinity Fe transport systems that utilize biosyrthetic chelates called siderophores. To competitively acquire Fe, some microbes have transport systems that enable them to use other siderophore types in addition to their own. Bacteria such as Escherichia coli achieve this ability by using a combination of separate siderophore receptors and transporters, whereas other microbial species, such as Streptomyces pilosus, use a low specificity, high-affinity transport system that recognizes more than one siderophore type. By either strategy, such versatility may provide an advantage under Fe-limiting conditions; allowing use of siderophores produced at another organism's expense, or Fe acquisition from siderophores that could otherwise sequester Fe in an unavailable form.Plants that use microbial siderophores may also be more Fe efficient by virtue of their ability to use a variety of Fe sources under different soil conditions. Results of our research examining Fe transport by oat indicate parity in plant and microbial requirements for Fe and suggest that siderophores produced by root-colonizing microbes may provide Fe to plants that can use the predominant siderophore types. In conjunction with transport mechanisms, ecological and soil chemical factors can influence the efficacy of siderophores and phytosiderophores. A model presented here attempts to incorporate these factors to predict conditions that may govern competition for Fe in the plant rhizosphere. Possibly such competition has been a factor in the evolution of broad transport capabilities for different siderophores by microorganisms and plants.     

The role of phytosiderophores in acquisition of iron and other micronutrients in graminaceous species: An ecological approach

Phytosiderophores (PS) are released in graminaceous species (Gramineae) under iron (Fe) and zinc (Zn) deficiency stress and are of great ecological significance for acquisition of Fe and presumably also of Zn. The potential for release of PS is much higher than reported up to now. Rapid microbial degradation during PS collection from nutrient solution-grown plants is the main cause of this underestimation. Due to spatial separation of PS release and microbial activity in the rhizosphere a much slower degradation of PS can be assumed in soil-grown plants. Concentrations of PS up to molar levels have been calculated under non-sterile conditions in the rhizosphere of Fe-deficient barley plants.Besides Fe, PS mobilize also Zn, Mn and Cu. Despite this unspecific mobilization, PS mobilize appreciable amounts of Fe in calcareous soils and are of significance for chlorosis resistance of graminaceous species. In most species the rate of PS release is high enough to satisfy the Fe demand for optimal growth on calcareous soils.In contrast to the chelates ZnPS and MnPS, FePS are preferentially taken up in comparison with other soluble Fe compounds. In addition, the specific uptake system for FePS (translocator) is regulated exclusively by the Fe nutritional status. Therefore, it seems appropriate to retain the term phytosiderophore instead of phytochelate.     

Molecular evidence for phytosiderophore‐induced improvement of iron nutrition of peanut intercropped with maize in calcareous soil

Peanut/maize intercropping is a sustainable and effective agroecosystem that evidently enhances the Fe nutrition of peanuts in calcareous soils. So far, the mechanism involved in this process has not been elucidated. In this study, we unravel the effects of phytosiderophores in improving Fe nutrition of intercropped peanuts in peanut/maize intercropping. The maize ys3 mutant, which cannot release phytosiderophores, did not improve Fe nutrition of peanut, whereas the maize ys1 mutant, which can release phytosiderophores, prevented Fe deficiency, indicating an important role of phytosiderophores in improving the Fe nutrition of intercropped peanut. Hydroponic experiments were performed to simplify the intercropping system, which revealed that phytosiderophores released by Fe‐deficient wheat promoted Fe acquisition in nearby peanuts and thus improved their Fe nutrition. Moreover, the phytosiderophore deoxymugineic acid (DMA) was detected in the roots of intercropped peanuts. The yellow stripe1‐like (YSL) family of genes, which are homologous to maize yellow stripe 1 (ZmYS1), were identified in peanut roots. Further characterization indicated that among five AhYSL genes, AhYSL1, which was localized in the epidermis of peanut roots, transported Fe(III)–DMA. These results imply that in alkaline soil, Fe(III)–DMA dissolved by maize might be absorbed directly by neighbouring peanuts in the peanut/maize intercropping system.     

Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5m and was completely inhibited at an iron concentration of 10m. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.     

Mechanism and stoichiometry of the redox reaction between iron(III) and caffeic acid

The stoichiometry of the redox reaction of caffeic acid with iron(III) was determined at pH 2.5. A linear increase in the yield of iron(II) was found with increasing iron(III) concentration until reached constant values when iron(III)/caffeic acid molar ratios were higher than 9. The reaction proceeds through two steps each having different rates, and involving intermediates with different redox activities. A mechanism of the redox reaction consistent with our results is proposed.