Utilization of microbial siderophores in iron acquisition by oat

Iron uptake by oat (Avena sativa cv Victory) was examined under hydroponic chemical conditions that required direct utilization of microbial siderophores for iron transport. Measurements of iron uptake rates by excised roots from the hydroxamate siderophores, ferrichrome, ferrichrome A, coprogen, ferrioxamine B (FOB), and rhodotorulic acid (RA) showed all five of the siderophores supplied iron, but that FOB and RA were preferentially utilized. FOB-mediated iron uptake increased four-fold when roots were preconditioned to iron stress and involved an active, iron-stress induced transport system that was inhibited by 5 millimolar sodium azide or 0.5 millimolar dinitrophenol. Kinetic studies indicated partial saturation with an apparent K_m of 5 micromolar when FOB was supplied at 0.1 to 50 micromolar concentrations. Whole plant experiments confirmed that 5 micromolar FOB was sufficient for plant growth. Siderophore-mediated iron transport was inhibited by Cr-ferrichrome, an analog of ferrated siderophore. Our results confirm the existence of a microbial siderophore iron transport system in oat which functions within the physiological concentrations produced and used by soil microorganisms.     

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.     

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.     

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.     

Iron acquisition by plants: the reduction of ferrisiderophores by higher plant NADH: Nitrate reductase

Siderophores are avid Fe³⁺-chelators of microbial origin. Plant roots are colonized by fungi and bacteria which synthesize siderophores, and plants have been shown to metabolize these substances to obtain iron. We have previously shown that nitrate reductase from squash catalyzed the reduction of the ferrisiderophore ferrioxamine B with the subsequent loss of Fe²⁺. Using a spectrophotometric assay which traps Fe²⁺ in a ferrozine complex, we have noted that the substrate diversity of nitrate reductase as a ferrisiderophore reductase includes ferrichrome A, ferrichrome, ferrirhodotorulic acid, ferrischizokinen, and the novel siderophore ferri-‘AAHS’. These reductions were inhibited by polyclonal antibodies against nitrate reductase, but ferrisiderophore reductase activity, as evidenced with ferrirhodotorulic acid, was unaffected by low concentrations of azide. In addition, maximal activity occurred between pH 4 and 5, and appaarent K_m values were approx. 100 μmolar. Thus, we suggest that plant nitrate reductases might be involved in iron assimilation as well as nitrate reduction.     

The growth of wheat plants in humic acid solutions under axenic conditions

Summary A technique is described for growing wheat plants in nutrient solutions containing C¹⁴-labelled humic acid under axenic conditions. The general appearance of axenic plants was indistinguishable from plants grown in association with microbes. C¹⁴-labelled humic acid enhanced the growth of both roots and shoots showing that by-products of microbial degradation of humic acid are unnecessary for this enhanced plant growth. Thus humic acid had a direct effect on the growth processes. The C¹⁴-labelled humic acid was taken up by the roots and virtually none was transported to the shoot. Only some 30 to 40 per cent of the incorporated radioactivity was associated with the root cell walls and thus more than 60 per cent was in the cytoplasm and may have influenced the biochemical processes involved in the regulation of plant growth.     

Nitrogen Nutrition and Cytokinin Activity in Solanum tuberosum

In water culture experiments with potato plants (Solanum tuberosum L. cv. Ostara), the influence of continuous nitrogen nutrition (constant supply of NO₃^?) and discontinuous nitrogen nutrition (interruption of NO₃^? supply, i. e., nitrogen withdrawal for 6 days) on the endogeneous cytokinin level in the roots, shoots and exudate of decapitated plants was studied. Harvests took place at intervals of 3 days. The chlorophyll formation test (cucumber cotyledons) and soya callus test were used to determine the cytokinin activity. With continuous nitrogen, the cytokinin activity decreased slightly with time in both roots and shoots but rose in the exudate. With discontinuous nitrogen, the nitrogen withdrawal led to a temporary, pronounced increase in cytokinin activity in the roots; at the same time, the cytokinin activity in the exudate decreased sharply. It is assumed that this temporary increase in cytokinin activity in the roots is a reflection of increased meristem activity in the roots. In the shoots, the cytokinin activity decreased during the nitrogen withdrawal period. These nitrogen-induced changes in cytokinin activity in the roots and shoots of potato should presumably have an important influence on the physiological age of the shoot, with all its consequences in the further development of the plant. Zeatin riboside was likely the main cytokinin component involved.     

A Method for Detection of Pseudobactin, the Siderophore Produced by a Plant-Growth-Promoting Pseudomonas Strain, in the Barley Rhizosphere

Detection in the rhizosphere of the siderophore produced by an inoculated microorganism is critical to determining the role of microbial iron chelators on plant growth promotion. We previously reported the development of monoclonal antibodies (MAb) to ferric pseudobactin, the siderophore of plant-growth-promoting Pseudomonas strain B10. One of these MAb reacted less strongly to pseudobactin than to ferric pseudobactin. The MAb reacted to Al(III), Cr(III), Cu(II), and Mn(II) complexes of pseudobactin at a level similar to the level at which it reacted to ferric pseudobactin and reacted less to the Zn(II) complex, but these metals would make up only a small fraction of chelated pseudobactin in soil on the basis of relative abundance of metals and relative binding constants. Fourteen-day-old barley plants grown in limed and autoclaved soil were inoculated with 10⁹ CFU of Pseudomonas strain Sm1-3, a strain of Pseudomonas B10 Rif^r Nal^r selected for enhanced colonization, and sampled 3 days later. Extraction and analysis of the roots and surrounding soil using the MAb in an immunoassay indicated a concentration of 3.5 × 10^-10 mol of ferric pseudobacting g^-1 (wet weight). This is the first direct measurement of a pseudobactin siderophore in soil or rhizosphere samples.     

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.     

Iron uptake by plants from microbial siderophores : a study with 7-nitrobenz-2 oxa-1,3-diazole-desferrioxamine as fluorescent ferrioxamine B analog

The synthetically produced fluorescent siderophore NBD-desferrioxamine B (NBD-DFO), an analog of the natural siderophore ferrioxamine B, was used to study iron uptake by plants. Short-term (10-hour) ⁵⁵Fe uptake rates by cotton (Gossypium spp.) and maize (Zea mays L.) plants from the modified siderophore were similar to those of the natural one. In longer-term uptake experiments (3 weeks), both siderophore treatments resulted in similar leaf chlorophyll concentration and dry matter yield. These results suggest that the synthetic derivative acts similarly to the natural siderophore. The NBD-DFO is fluorescent only when unferrated and can thus be used as a probe to follow iron removal from the siderophore. Monitoring of the fluorescence increase in a nutrient solution containing Fe³⁺-NBD-DFO showed that iron uptake by plants occurs at the cell membrane. The rate of iron uptake was significantly lower in both plant species in the presence of antibiotic agent, thus providing evidence for iron uptake by rhizosphere microbes that otherwise could have been attributed to plant uptake. Confocal fluorescence microscopy revealed that iron was taken up from the complex by cotton plants, and to a much lesser extent by maize plants. The active cotton root sites were located at the main and lateral root tips. Significant variations in the location and the intensity of the uptake were noticed under nonaxenic conditions, which suggested that rhizosphere microorganisms play an important role in NBD-DFO-mediated iron uptake.     

Mobilisation of iron and manganese by plant-borne and synthetic metal chelators

Phytosiderophores released by roots of iron-deficient grasses mobilise Fe, Zn, Mn and Cu in calcareous soils. Mobilisation of Fe, Zn and Cu can be explained as the chelation of these metal cations by phytosiderophores. Mobilisation of Mn could not be so explained because phytosiderophores have a much smaller affinity for Mn than for Fe, Cu and Zn. Model experiments have been made with freshly precipitated Fe(OH)₃ and different soils to study the mobilisation of iron and manganese by plant-borne chelating phytosiderophores, the synthetic metal chelators DTPA and the microbial metal chelator sulphonated ferrioxamine B (FOB). Compared with the synthetic chelator DTPA, the plant-borne chelating phytosiderophores mobilised Fe very efficiently, but no change was observed in the Mn mobilisation by phytosiderophores.Different phytosiderophores, as well as the microbial metal chelator FOB, were used to compare the mobilisation of iron and manganese in a calcareous soil.     

Characterization of ‘Schizokinen’; a dihydroxamate-type siderophore produced by Rhizobium leguminosarum IARI 917

The Rhizobia comprise one of the most important groups of beneficial bacteria, which form nodules on the roots (rarely on the stems) of leguminous plants. They live within the nodules and reduce atmospheric nitrogen to ammonia, which is further assimilated by plants into required nitrogenous compounds. The Rhizobia in return obtain nutrition from the plant. Rhizobia are free-living soil bacteria and have to compete with other microorganisms for the limited available iron in the rhizosphere. In order to acquire iron Rhizobia have been shown to express siderophore-mediated iron transport systems. Rhizobium leguminosarum IARI 917 was investigated for its ability to produce siderophore. It was found to produce a dihydroxamate type siderophore under iron restricted conditions. The siderophore was purified and chemically characterized. The ESMS, MS/MS and NMR analysis indicate the dihydroxamate siderophore to be ‘schizokinen’, a siderophore reported to be produced by Bacillus megaterium that shares a similar structure to ‘rhizobactin 1021’ produced by Sinorhizobium meliloti 1021. This is the first report of production of schizokinen by a strain of R. leguminosarum, therefore it was carefully investigated to confirm that it is indeed ‘schizokinen’ and not a degradation product of ‘rhizobactin 1021’. Since ferric–siderophore complexes are transported across the outer membrane (OM) into the periplasm via an OM receptor protein, R. leguminosarum IARI 917 was investigated for the presence of an OM receptor for ‘ferric–schizokinen’. SDS PAGE analysis of whole cell pellet and extracted OM fractions indicate the presence of a possible iron-repressible OM receptor protein with the molecular weight (MW) of approximately 74 kDa.     

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.     

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.     

Effect of Nitrate and Ammonium Nutrition of Nonnodulated Phaseolus vulgaris L. on Phosphoenolpyruvate Carboxylase and Pyruvate Kinase Activity

Young bean plants (Phaseolus vulgaris L. var Saxa) were fed with 3.5 or 10 millimolar N in either the form of NO₃⁻ or NH₄⁺, after being grown on N-free nutrient solution for 8 days. The pH of the nutrient solutions was either 6 or 4. The cell sap pH and the extractable activities of phosphoenolpyruvate carboxylase and of pyruvate kinase from roots and primary leaves were measured over several days.

The extractable activity of phosphoenolpyruvate carboxylase (based on soluble protein) from primary leaves increased with NO₃⁻ nutrition, whereas with NH₄⁺ nutrition and on N-free nutrient solution the activity remained at a low level. Phosphoenopyruvate carboxylase activity from the roots of NH₄⁺-fed plants at pH 4 was finally somewhat higher than from the roots of plants grown on NO₃⁻ at the same pH. There was no difference in activity from the root between the N treatments when pH in the nutrient solutions was 6. The extractable activity of pyruvate kinase from roots and primary leaves seemed not to be influenced by the N nutrition of the plants.

The results are discussed in relation to the physiological function of both enzymes with special regard to the postulated functions of phosphoenolpyruvate carboxylase in C3 plants as an anaplerotic enzyme and as part of a cellular pH stat.

     

AhDMT1, a Fe<Superscript>2+</Superscript> transporter,is involved in improving iron nutrition and N<Subscript>2</Subscript> fixation in nodules of peanut intercropped with maize in calcareous soils

Peanut (Arachis hypogaea L.) is an important legume providing edible proteins and N₂ fixation. However, iron deficiency severely reduces peanut growth in calcareous soils. The maize/peanut intercropping effectively improves iron nutrition and N₂ fixation of peanut under pot and field conditions on calcareous soils. However, little was known of how intercropping regulates iron transporters in peanut. We identified AhDMT1 as a Fe²⁺ transporter which was highly expressed in mature nodules with stronger N₂ fixation capacity. Promoter expression analysis indicated that AhDMT1 was localized in the vascular tissues of both roots and nodules in peanut. Short-term Fe-deficiency temporarily induced an AhDmt1 expression in mature nodules in contrast to roots. However, analysis of the correlation between the complex regulation pattern of AhDmt1 expression and iron nutrition status indicated that sufficient iron supply for long term was a prerequisite for keeping AhDmt1 at a high expression level in both, peanut roots and mature nodules. The AhDmt1 expression in peanut intercropped with maize under 3 years greenhouse experiments was similar to that of peanut supplied with sufficient iron in laboratory experiments. Thus, the positive interspecific effect of intercropping may supply sufficient iron to enhance the expression of AhDmt1 in peanut roots and mature nodules to improve the iron nutrition and N₂ fixation in nodules. This study may also serve as a paradigm in which functionally important genes and their ecological significance in intercropping were characterized using a candidate gene approach.     

Effect of cadmium on iron uptake in cucumber roots: A Mössbauer-spectroscopic study

The uptake and accumulation of iron in cucumber roots exposed to cadmium were investigated with Fe sufficient and deficient cucumber plants using Mössbauer spectroscopy, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and ferric chelate reductase activity measurements. Both Fe sufficient and Fe deficient plants were applied. In the case of Fe sufficient cucumber roots grown in nutrient solution with 10 μM Cd no changes were found in the occurrence of Fe species (mostly hydrous ferric oxides and ferric-carboxylate complexes) compared to the control where no Cd was added. In the Fe deficient roots pretreated with 0, 0.1, 1, 10 and 100 μM Cd for 3 h then supplied also with 0.5 mM ⁵⁷Fe-citrate for 30 min, Fe^II was identified in a hexaaqua complex form. The relative amount of Fe^II was decreasing simultaneously with increasing Cd concentration, while the relative occurrence of Fe^III species and total Fe concentration were increasing. The results support the inhibitory effect of Cd on Fe-chelate reduction. Although the reductase activity at 10 and 100 μM Cd treatment was lower than in the iron sufficient control plants, Fe^II could be identified by Mössbauer spectroscopy whereas in the Fe sufficient control, this form was below detection limit. These data demonstrate that the influx and the reoxidation of Fe^II was decreased by Cd, consequently, they refer to the competition of Cd²⁺ and Fe²⁺ during the membrane transport and the inhibition of the reoxidation process.     

Microbial Processes Associated with Roots of Bulbous Rush Coated with Iron Plaques

Bulbous rush (Juncus bulbosus) is a pioneer species in acidic, iron-rich, coal mining lakes in the eastern part of Germany. Juncus roots are coated with iron plaques, and it has been suggested that microbial processes under the iron plaques might be supportive for Juncus plant growth. The objectives of this work were to enumerate the microbes involved in the turnover of iron and organic root exudates in the rhizoplane, to investigate the effect of oxygen and pH on the utilization of these exudates by the rhizobacteria, and to study the ability of the root-colonizing microbiota to reduce sulfate. Enumeration studies done at pH 3 demonstrated that 10⁶ Fe(III) reducers and 10⁷ Fe(II) oxidizers g (fresh wt root)^–1 were associated with Juncus roots. When roots were incubated in goethite-containing medium without and with supplemental glucose, Fe(II) was formed at rates approximating 1.1 mmol g (fresh wt root) ^–1 d^–1 and 3.6 mmol g (fresh wt root)^–1 d^–1 under anoxic conditions, respectively. These results suggest that a rapid microbially mediated cycling of iron occurs in the rhizosphere of Juncus roots under changing redox conditions. Most-probable-number estimates of aerobes and anaerobes capable of consuming root exudates at pH 3 were similar in the rhizosphere sediment and in Juncus roots, but numbers of aerobes were significantly higher than those of anaerobes. At pH 3, supplemental organic exudates were primarily subject to aerobic oxidation to CO₂ and not subject to fermentation. However, at pH 4.5, root exudates were also rapidly utilized under anoxic conditions. Root-associated sulfate reduction was not observed at pH 3 to 4.5 but was observed at pH 4.9. The pH increased during all root-incubation studies both under oxic and anoxic conditions. Thus, as result of the microbial turnover of organic root exudates, pH and CO₂ levels might be elevated at the root surface and favor Juncus plants to colonize acidic habitats.