SIDEROPHORE‐INDEPENDENT IRON UPTAKE BY IRON‐LIMITED CELLS OF THE CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition by iron‐limited cyanobacteria is typically considered to be mediated mainly by siderophores, iron‐chelating molecules released by iron‐limited cyanobacteria into the environment. In this set of experiments, iron uptake by iron‐limited cells of the cyanobacterium Anabaena flos‐aquae (L.) Bory was investigated in cells resuspended in siderophore‐free medium. Removal of siderophores decreased iron‐uptake rates by ～60% compared to siderophore‐replete conditions; however, substantial rates of iron uptake remained. In the absence of siderophores, Fe(III) uptake was much more rapid from a weaker synthetic chelator [N‐(2‐hydroxyethyl)ethylenediamine‐N,N′,N′‐triacetic acid (HEDTA); log K_cond = 28.64 for Fe(III)HEDTA(OH)^?] than from a very strong chelator [N,N′‐bis(2‐hydroxybenzyl)‐ethylenediamine‐N,N′‐diacetic acid (HBED); log K_cond = 31.40 for Fe(III)HBED^?], and increasing chelator:Fe(III) ratios decreased the Fe(III)‐uptake rate; these results were evident in both short‐term (4 h; absence of siderophores) and long‐term (116 h; presence of siderophores) experiments. However, free (nonchelated) Fe(III) provided the most rapid iron uptake in siderophore‐free conditions. The results of the short‐term experiments are consistent with an Fe(III)‐binding/uptake mechanism associated with the cyanobacterial outer membrane that operates independently of extracellular siderophores. Iron uptake was inhibited by temperature‐shock treatments of the cells and by metabolically compromising the cells with diphenyleneiodonium; this finding indicates that the process is dependent on active metabolism to operate and is not simply a passive Fe(III)‐binding mechanism. Overall, these results point to an important, siderophore‐independent iron‐acquisition mechanism by iron‐limited cyanobacterial cells.     

Fe chelates from compost microorganisms improve Fe nutrition of soybean and oat

Iron availability to plants is often limited when soil pH is 7 or higher. In C rich, but Fe limiting environments, microorganisms may produce organic chelators that complex Fe and increase its availability to plants. Seedlings of soybean (Glycine max L.) and oat (Avena sativa L.) plants, with Fe-efficient or inefficient uptake mechanisms, were grown in an Fe free nutrient solution at pH 7.5. Experiments (using a complete factorial design) were conducted in which these seedlings were transferred to a fresh nutrient solution and treated with Fe sources (FeCl₃, FeEDDHA, and Fe complexed with chelators produced by compost microorganisms (CCMs) after their enrichment in an Fe free, glucose medium), Fe concentrations (0 and 6.7 M) and antibiotic (0 and 100 mg streptomycin L^-1). Dry weight of soybean plants and Fe uptake were significantly (P 0.05) higher when Fe was supplied as ⁵⁹FeCCM than as⁵⁹ FeCl₃ and similar to when Fe was supplied as⁵⁹ FeEDDHA. Dry weight of the Fe-inefficient Tam 0-312 oat cultivar was also significantly higher when Fe was supplied as FeCCM. Fe uptake by oat, when supplied as ⁵⁹FeCCM, was twice that for⁵⁹ FeEDDHA and ⁵⁹FeCl₃. Chlorophyll concentration in plants supplied with FeCCM and FeEDDHA was significantly greater (P 0.05) than in minus Fe control plants and in FeCl₃ supplied plants. Activities of catalase and peroxidase, measured as indicators of Fe nutrition in soybean and oats, were generally increased when Fe was supplied with FeCCM as compared to the other Fe sources. The experimental conditions in which the CCMs were produced are similar to those in soil after amendment with manures or other readily available organic materials. These CCMs can bind with Fe, even under slightly alkaline conditions, and effectively improve Fe nutrition of soybean and oat.     

Short-term effects of rhizosphere microorganisms on fe uptake from microbial siderophores by maize and oat

Effects of rhizosphere microorganisms on Fe uptake by oat (Avena sativa) and maize (Zea mays) were studied in short-term (10 h) nutrient solution experiments. Fe was supplied either as microbial siderophores (pseudobactin [PSB] or ferrioxamine B [FOB]) or as phytosiderophores obtained as root exudates from barley (epi-3-hydroxy-mugineic acid [HMA]) under varied population densities of rhizosphere microorganisms (axenic, uninoculated, or inoculated with different microorganism cultures). When maize was grown under axenic conditions and supplied with FeHMA, Fe uptake rates were 100 to 300 times higher compared to those in plants supplied with Fe siderophores. Fe from both sources was taken up without the involvement of an extracellular reduction process. The supply of FeHMA enhanced both uptake rate and translocation rate to the shoot (more than 60% of the total uptake). However, increased density of microorganisms resulted in a decrease in Fe uptake rate (up to 65%), presumably due to microbial degradation of the FeHMA. In contrast, when FeFOB or FePSB was used as the Fe source, increased population density of microorganisms enhanced Fe uptake. The enhancement of Fe uptake resulted from the uptake of FeFOB and FePSB by microorganisms adhering to the rhizoplane or living in the free space of cortical cells. The microbial apoplastic Fe pool was not available for root to shoot transport or, thus, for utilization by the plants. These results, in addition to the low uptake rate under axenic conditions, are in contrast to earlier hypotheses suggesting the existence of a specific uptake system for Fe siderophores in higher plants. The bacterial siderophores PSB and FOB were inefficient as Fe sources for plants even when supplied by stem injection. It was concluded that microorganisms are involved in degradation processes of microbial siderophores, as well as in competition for Fe with higher plants.     

Fusarinines and dimerum acid, mono- and dihydroxamate siderophores from Penicillium chrysogenum, improve iron utilization by strategy I and strategy II plants

Cucumber, as a strategy I plant, and Maize as a strategy II plant, were cultivated in hydroponic culture in the presence of a ferrated siderophore mixture (1 M) from a culture of Penicillium chrysogenumisolated from soil. The siderophore mixture significantly improved the iron status of these plants as measured by chlorophyll concentration to the same degree as a 100-fold higher FeEDTA supply. Analysis of the siderophore mixture from P. chrysogenum by HPLC and electrospray mass spectrometry revealed that besides the trihydroxamates, coprogen and ferricrocin, large amounts of dimerum acid and fusarinines were present which represent precursor siderophores or breakdown products of coprogen. In order to prove the iron donor properties of dimerum acid and fusarinines for plants, purified coprogen was hydrolyzed with ammonia and the hydrolysis products consisting of dimerum acid and fusarinine were used for iron uptake by cucumber and maize. In short term experiments radioactive iron uptake and translocation rates were determined using ferrioxamine B, coprogen and hydrolysis products of coprogen. While the trihydroxamates revealed negligible or intermediate iron uptake rates by both plant species, the fungal siderophore mixture and the ammoniacal hydrolysis products of coprogen showed high iron uptake, suggesting that dimerum acid and fusarinines are very efficient iron sources for plants. Iron reduction assays using cucumber roots or ascorbic acid also showed that iron bound to hydrolysis products of coprogen was more easily reduced compared to iron bound to trihydroxamates. Ligand exchange studies with epi-hydroxymugineic acid and EDTA showed that iron was easily exchanged between coprogen hydrolysis products and phytosiderophores or EDTA. The results indicate that coprogen hydrolysis products are an excellent source for Fe nutrition of plants.     

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

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

Iron availability in plant tissues-iron chlorosis on calcareous soils

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

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

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

Iron and cadmium uptake by duodenum of hypotransferrinaemic mice

Absorption from food is an important route for entry of the toxic metal, cadmium, into the body. Both cadmium and iron are believed to be taken up by duodenal enterocytes via the iron regulated, proton-coupled transporter, DMT1. This means that cadmium uptake could be enhanced in conditions where iron absorption is increased. We measured pH dependent uptake of ¹⁰⁹Cd and ⁵⁹Fe by duodenum from mice with an in vitro method. Mice with experimental (hypoxia, iron deficiency) or hereditary (hypotransferrinaemia) increased iron absorption were studied. All three groups of mice showed increased ⁵⁹Fe uptake (p<0.05) compared to their respective controls. Hypotransferrinaemic and iron deficient mice exhibited an increase in ¹⁰⁹Cd uptake (p<0.05). Cadmium uptake was not, however, increased by lowering the medium pH from 7.4 to 6. In contrast, ⁵⁹Fe uptake (from ⁵⁹FeNTA₂) and ferric reductase activity was increased by lowering medium pH in control and iron deficient mice (p<0.05). The data show that duodenal cadmium uptake can be increased by hereditary iron overload conditions. The uptake is not, however, altered by lowering medium pH suggesting that DMT1-independent uptake pathways may operate.     

Effect of primary leaves on 59Fe uptake by roots and 59Fe distribution in the shoot of iron sufficient and iron deficient bean (Phaseolus vulgaris L.) plants

A study has been made on the effect of primary leaves on iron (Fe) distribution in the shoot. Bean (Phaseolus vulgaris L.) seedlings were precultured in nutrient solution with 8×10^-5 M FeEDTA for 4 days, and then grown further with either 8×10^-5 M FeEDTA (+Fe) or without Fe supply (-Fe) for another 5 days. Thereafter, both +Fe and -Fe plants were treated in three different ways: undisturbed; one primary leaf removed; or one primary leaf shaded, starting two hours before supply ⁵⁹FeEDTA to the roots. The +Fe plants were supplied with 8×10^-5 M ⁵⁹FeEDTA, and the -Fe plants with only 1×10^-6 M ⁵⁹FeEDTA. After 1 to 8 hour uptake periods, plants were harvested and ⁵⁹Fe in different organs was determined. Removal or shading of one primary leaf did not affect ⁵⁹Fe uptake by roots and ⁵⁹Fe translocation to the shoot in +Fe plants. In the -Fe plants, however, removal of one primary leaf decreased ⁵⁹Fe uptake by roots, whereas shading of one primary leaf had no effect on ⁵⁹Fe uptake but slightly enhanced ⁵⁹Fe translocation from roots to the shoot. The quantity of ⁵⁹Fe in primary leaves was positively correlated with quantity of ⁵⁹Fe in the stem in the -Fepplants, but not in the +Fe plants. In both, the +Fe and -Fe plants, the quantity of ⁵⁹Fe in the shoot apex was positively correlated with ⁵⁹Fe in primary leaves. The results suggest that irrespective of the Fe nutritional status of plants, the source of Fe for the shoot apex is Fe retranslocated from primary leaves.     

Evaluation of 59Fe-lignosulfonates complexes as Fe-sources for plants

Iron chlorosis is a wide-spread limiting factor of production in agriculture. To cope with this problem, synthetic chelates (like EDTA or EDDHA) of Fe are used in foliar-spray or in soil treatments; however, these products are very expensive. Therefore paper-production byproducts, like Lignosulfonates (LS), with varying content of carboxylate and sulfonate groups, were tested with respect to their ability to maintain Fe in the solution of soils and to feed plants grown in hydroponics with Fe through foliar sprays or application to the nutrient solution. Results show that LS had a low capability to solubilize ⁵⁹Fe-hydroxide and that preformed ⁵⁹Fe(III)-LS complexes had poor mobility through a soil column (pH 7.5) and scarce stability when interacting with soils compared to ⁵⁹Fe(III)-EDDHA. However when ⁵⁹Fe(III)-LS were supplied to roots in a hydroponic system, they demonstrated an even higher capability to fed Fe-deficient tomato plants than ⁵⁹Fe(III)-EDDHA. Hence, data here presented indicate that the low Fe use efficiency from Fe-LS observed in soil-applications is due to interactions of these Fe-sources with soil colloids rather than to the low capability of roots to use them. Foliar application experiments of ⁵⁹Fe(III)-LS or ⁵⁹Fe(III)-EDTA to Fe-deficient cucumber plants show that uptake and reduction rates of Fe were similar between all these complexes; on the other hand, when ⁵⁹Fe(III)-LS were sprayed on Fe-deficient tomato leaves, they showed a lower uptake rate, but a similar reduction rate, than ⁵⁹Fe(III)-EDTA did. In conclusion, Fe-LS may be a valid, eco-compatible and cheap alternative to synthetic chelates in dealing with Fe chlorosis when applied foliarly or in the nutrient solution of hydroponically grown plants.     

Kinetics of iron passage through subcellular compartments of rabbit reticulocytes

Summary The kinetics of the separate processes of Fe₂(III)-transferrin binding to the transferrin receptor, transferrin-receptor internalization, iron dissociation from transferrin, iron passage through the membrane, and iron mobilization into the cytoplasm were studied by pulse-chase experiments using rabbit reticulocytes and⁵⁹Fe,¹²⁵I-labeled rabbit transferrin. The binding of⁵⁹Fe-transferrin to transferrin receptors was rapid with an apparent rate constant of 2×10⁵ m ^–1 sec^–1. The rate of internalization of⁵⁹Fe-transferrin was directly measured at 520±100 molecules of Fe₂(III)-transferrin internalized/sec/cell with 250±43 sec needed to internalize the entire complement of reticulocyte transferrin receptors. Subsequent to Fe₂(III)-transferrin internalization the flux of⁵⁹Fe was followed through three compartments: internalized transferrin, membrane, and cytosol.A process preceding iron dissociation from transferrin and a reaction involving membrane-associated iron required 17±2 sec and 34±5 sec, respectively. Apparent rate constants of 0.0075±0.002 sec^–1 and 0.0343±0.0118 sec^–1 were obtained for iron dissociation from transferrin and iron mobilization into the cytosol, respectively. Iron dissociation from transferrin is the rate-limiting step. An apparent rate constant of 0.0112±0.0025 sec^–1 was obtained for processes involving iron transport through the membrane although at least two reactions are likely to be involved. Based on mechanistic considerations, iron transport through the membrane may be attributed to an iron reduction step followed by a translocation step. These data indicate that the uptake of iron in reticulocytes is a sequential process, with steps after the internalization of Fe₂(III)-transferrin that are distinct from the handling of transferrin.     

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.     

Iron uptake and translocation by tomato plants as influenced by root temperature and manganese nutrition

The uptake of iron (Fe) by VF-36 tomato plants as influenced by root temperature and manganese (Mn) nutrition was studied. Following a 1-week pretreatment period of various levels of Fe and Mn in the nutrient solution in a controlled temperature room, the uptake of ⁵⁹Fe from ferric ethylenediamine di(O-hydroxyphenylacetate) (FeEDDHA) at 1 μmole per liter was studied for periods of 1 to 5 days.     

Impact of nitrogen form on iron uptake and distribution in maize seedlings in solution culture

Comparative studies on the effect of nitrogen (N) form on iron (Fe) uptake and distribution in maize (Zea mays L. cv Yellow 417) were carried out through three related experiments with different pretreatments. Experiment 1: plants were precultured in nutrient solution with 1.0×10^–4 M FeEDTA for 6 d and then exposed to NO₃–N or NH₄–N solution with 1.0×10^–4 M FeEDTA or without for 7 d. Experiment 2: plants were precultured with ⁵⁹FeEDTA for 6 d and were then transferred to the solution with different N forms, and 0 and 1.0×10^–4 M FeEDTA for 8 d. Experiment 3: half of roots were supplied with ⁵⁹FeEDTA for 5 d and then cut off, with further culturing in treatment concentrations for 7 d. In comparison to the NH₄-fed plants, young leaves of the NO₃-fed plants showed severe chlorosis under Fe deficiency. Nitrate supply caused Fe accumulation in roots, while NH₄–N supply resulted in a higher Fe concentration in young leaves and a lower Fe concentration in roots. HCl-extractable (active) Fe was a good indicator reflecting Fe nutrition status in maize plants. Compared with NO₃-fed plants, a higher proportion of ⁵⁹Fe was observed in young leaves of the Fe-deficient plants fed with NH₄–N. Ammonium supply greatly improved ⁵⁹Fe retranslocation from primary leaves and stem to young leaves. Under Fe deficiency, about 25% of Fe in primary leaves of the NH₄-fed plants was mobilized and retranslocated to young leaves. Exogenous Fe supply decreased the efficiency of such ⁵⁹Fe retranslocation. The results suggest that Fe can be remobilized from old to young tissues in maize plants but the remobilization depends on the form of N supply as well as supply of exogenous Fe.     

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.     

Effects of phosphorus and iron fertilizers on the growth of two soybean varieties at two soil temperatures

The influence of FeEDDHA (0, 0.2 and 2 μg Fe g⁻¹ soil) and NaH₂PO₄·H₂O (0 and 120 μg Pg⁻¹ soil) on the growth of two Fe-ineffective soybean (Glycine max L. Merr.) varieties (anoka and T203) on a calcareous soil at two soil temperatures (16 and 24°C) was compared under greenhouse conditions. The two soybean varieties differed in the following respects: (a) T203 accumulated smaller concentrations of Fe in washed tops than Anoka under comparable conditions; (b) T203 was more susceptible to Fe deficiency and its accentuation by high levels of fertilizer P than Anoka; (c) T203 accumulated lower quantities of Mn in tops than Anoka under comparable conditions; (d) T203, but not Anoka, developed Mn deficiency symptoms when treated with P and 2 μg Fe g⁻¹ at 16°C. Fe deficiency was more severe in both varieties at the higher soil temperature due apparently to: (a) greater plant concentration of P in tops at 24°C; and/or (b) an increased rate of plant growth and greater dilution of Fe in young tissue at 24°C. Foliar P concentration was increased much more than foliar Fe concentration by an increase in soil temperature. Severely Fe deficient T203 plants grown without FeEDDHA at 24°C accumulated less foliar Mn than their FeEDDHA counterparts. Comparisons of Fe effectiveness of various soybean cultivars based on relative responses to FeEDDHA can be influenced by differential effects on Mn nutrition.     

Reduction and transport of Fe from siderophores

Soils contain siderophores produced by bacteria and fungi; however, the role of siderophores in Fe nutrition of plants is uncertain. The Strategy I plant cucumber (Cucumis sativus L.) was used in an investigation of ferric chelate reduction activity and uptake and transport of Fe from ferric hydroxyethylethylenetriacetic acid (FeHEDTA) and ferric N,N–di–(2–hydroxybenzoyl)–ethylenediamine– N,N-diacetic acid (FeHBED) and the hydroxamate siderophores, ferric rhodotorulic acid (FeRA) and ferric ferrioxime B (FeFOB). Cucumber seedlings were grown in a hydroponic medium without Fe or supplied with 10 M FeHEDTA. Iron-deficient cucumber roots readily reduced FeHEDTA, while Fe-sufficient roots had low levels of ferric chelate reduction activity. The siderophore FeRA was reduced by Fe-deficient roots at 8% of the rate of FeHEDTA, while FeFOB was not reduced. The highly stable synthetic chelate FeHBED was reduced at 16% the rate of FeHEDTA. Fe transport to shoots by Fe-deficient seedlings from the slowly reducible complexes ⁵⁹FeRA and ⁵⁹FeHBED was, respectively, 74% and 73% of that transported from ⁵⁹FeHEDTA. The ferrous complexing agent, bathophenanthrolinedisulfonic acid (BPDS), had a strong inhibitory effect on uptake and transport of Fe from ⁵⁹FeHEDTA or ⁵⁹FeRA into shoots. An average of 11% as much Fe was transported to shoots of Fe-deficient seedlings from ⁵⁹FeFOB as from ⁵⁹FeHEDTA. Neither the Fe nutritional status of the seedlings nor the presence of BPDS influenced the uptake and transport of Fe from ⁵⁹FeFOB. It is concluded that cucumber roots may take up substantial amounts of Fe from FeRA and FeHBED following reduction, while small amounts of Fe may be taken up from FeFOB by a mechanism not involving reduction of the ferric siderophore at the root surface.     

The Role of Ligand Exchange in the Uptake of Iron from Microbial Siderophores by Gramineous Plants

The siderophore rhizoferrin, produced by the fungus Rhizopus arrhizus, was previously found to be as an efficient Fe source as Fe-ethylenediamine-di(o-hydroxphenylacetic acid) to strategy I plants. The role of this microbial siderophore in Fe uptake by strategy II plants is the focus of this research. Fe-rhizoferrin was found to be an efficient Fe source for barley (Hordeum vulgare L.) and corn (Zea mays L.). The mechanisms by which these Gramineae utilize Fe from Fe-rhizoferrin and from other chelators were studied. Fe uptake from 59Fe-rhizoferrin, 59Fe-ferrioxamine B, 59Fe-ethylenediaminetetraacetic acid, and 59Fe-2[prime]-deoxymugineic acid by barley plants grown in nutrient solution at pH 6.0 was examined during periods of high (morning) and low (evening) phytosiderophore release. Uptake and translocation rates from Fe chelates paralleled the diurnal rhythm of phytosiderophore release. In corn, however, similar uptake and translocation rates were observed both in the morning and in the evening. A constant rate of the phytosiderophore's release during 14 h of light was found in the corn cv Alice. The results presented support the hypothesis that Fe from Fe-rhizoferrin is taken up by strategy II plants via an indirect mechanism that involves ligand exchange between the ferrated microbial siderophore and phytosiderophores, which are then taken up by the plant. This hypothesis was verified by in vitro ligand-exchange experiments.     

Identification of the ferrioxamine B receptor,FoxB, inEscherichia coli K12

The photoreactivep-azidobenzoyl analog of ferrioxamine B was used to show that ferrioxamine-B-mediated iron transport is separate and distinct from coprogen-mediated iron transport inEscherichia coli. Photolysis of this analog inhibited uptake of [⁵⁹Fe]ferrioxamine B but not [⁵⁹Fe]coprogen or [⁵⁹Fe]ferrichrome. Conversely, photolysis of thep-azidobenzoyl analog of coprogen B inhibited uptake of [⁵⁹Fe]coprogen but not [⁵⁹Fe]ferrioxamine B or [⁵⁹Fe]ferrichrome. Photolabeling of outer membranes withp-azidobenzoyl-[⁵⁹Fe]ferrioxamine B resulted in the labeling of two iron-regulated peptides with molecular masses of about 66 and 26 kDa. Expression of these peptides was increased when ferrioxamine B was the sole iron source. Both peptides were present in outer membrane preparations of thefhuF mutant H1717, but the 66 kDa peptide was not inducible. These results are evidence for an outer membrane receptor inE. coli unique for linear ferrioxamines.     

Characterization of Pyoverdinpss, the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin_pss, in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin_pss of approximately 50 μg/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of ≥ 10 μM. Pyoverdin_pss was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin_pss and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin_pss was structurally unique since amino acid analysis of reductive hydrolysates yielded β-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin_pss exhibited a relatively high affinity constant for Fe(III), with values of 10²⁵ at pH 7.0 and 10³² at pH 10.0. Iron uptake assays with [⁵⁵Fe]pyoverdin_pss demonstrated rapid active uptake of ⁵⁵Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin_pss. The chemical and biological properties of pyoverdin_pss are discussed in relation to virulence and iron uptake during plant pathogenesis.