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Soil Science Society of America Journal 63:1681-1687 (1999)
© 1999 Soil Science Society of America

DIVISION S-3-SOIL BIOLOGY & BIOCHEMISTRY

Kinetics of Iron Complexing and Metal Exchange in Solutions by Rhizoferrin, A Fungal Siderophore

M. Shenkera, Y. Hadarb and Y. Chena

a Dep. of Soil and Water Sci., Faculty of Agricultural, Food, and Environmental Quality Sci., The Hebrew Univ. of Jerusalem, P.O. Box 12, Rehovot, Israel
b Dep. of Microbiol. and Plant Pathol., Faculty of Agricultural, Food and Environmental Quality Sci., The Hebrew Univ. of Jerusalem, P.O. Box 12, Rehovot, Israel

shenker{at}agri.huji.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Material and methods
 Results
 Discussion
 REFERENCES
 
Rhizoferrin, a siderophore produced by Rhizopus arrhizus, has been shown in previous studies to be an outstanding Fe carrier to plants. Yet, calculations based on stability constants and thermodynamic equilibrium lead to contradicting conclusions. In this study a kinetic approach was employed to elucidate apparent contradictions and to determine the behavior of rhizoferrin under conditions representing soil and nutrient solutions. Stability of Fe3+ complexes in nutrient solution, rate of metal exchange with Ca, and rate of Fe extraction by the free ligand were monitored for rhizoferrin and other chelating agents by 55Fe labeling. Ferric complexes of rhizoferrin, desferri–ferrioxamine–B (DFOB) and ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) were found to be stable in nutrient solution at pH 7.5 for 31 d, while ferric complexes of ethylenediaminetetraacetic acid (EDTA) and mugineic acid (MA) lost 50% of the chelated Fe within 2 d. Iron–calcium exchange in Ca solutions at pH 8.7 revealed rhizoferrin to hold Fe at nonequilibrium state for 3 to 4 wk at 3.3 mM Ca and for longer periods at lower Ca concentrations. Ethylenediaminetetraacetic acid lost the ferric ion at a faster rate under the same conditions. Iron extraction from freshly prepared Fe hydroxide at pH 8.7 and with 3.2 mM Ca was slow and followed the order: DFOB > EDDHA > MA >= rhizoferrin > EDTA. Based on these results we suggest that a kinetic rather than equilibrium approach should be the basis for predictions of Fe chelates' efficiency. We conclude that the nonequilibrium state of rhizoferrin is of crucial importance for its behavior as an Fe carrier to plants.

Abbreviations: DFOB, desferri–ferrioxamine–B • EDDHA, ethylenediamine-di(o-hydroxyphenylacetic acid) • EDTA, ethylenediaminetetraacetic acid • MA, mugineic acid • NS, nurtient solution


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Material and methods
 Results
 Discussion
 REFERENCES
 
THE RHIZOSPHERE OF LIVING PLANTS is highly populated with microorganisms. Under rhizosphere Fe-limiting conditions that prevail in calcareous soils, siderophores are produced and released to the soil solution, and hence, rhizosphere solutions contains various chelating agents at any given time. Iron utilization by plants involves processes like Fe extraction and mobilization from the solid phase by various Fe chelating agents (including siderophores), ligand exchange of the bound Fe, metal exchange between chelating agents, and competition between all potential Fe consumers for the chelated Fe.

Siderophores are highly specific chelators for Fe3+ (Matzanke et al., 1989; Albrecht-Gary and Crumbliss, 1998); however, competition with other metal ions, such as Ca2+, Mg2+, and others, which are often present in soil solution at much higher concentrations than Fe3+, may result in the binding of these metals to the siderophore. Once all stability constants and all solution components are known, a computer program may be used to calculate each metal–ligand species concentration at equilibrium. Such calculations may explain the efficiency or inefficiency of various chelating agents as Fe carriers in nutrient or soil solutions at various pH levels (Lindsay, 1979; Chaney, 1988). Yet, as pointed out by Buyer and Sikora (1990), this information is not enough to predict the actual distribution of metals between competing chelates. Thus, both kinetic and equilibrium approaches should be followed. Siderophores of various sources are excreted in the rhizosphere as free ligands. These free siderophores will complex and exchange metals according to kinetic and concentration considerations, as well as according to their stability constants. Hence, in case of a slow exchange process, nonequilibrium might be considered the rule rather than the exception in the soil. This approach explains results such as low Fe-mobilization specificity by the highly specific and well documented chelate EDDHA, as was found by Treeby et al. (1989).

Rhizoferrin (Fig. 1) is a siderophore produced by Rhizopus arrhizus, a common rhizosphere fungus. Its ferric complex was found to be as effective as FeEDDHA in supplying Fe to plants, whereas other microbial siderophores were found to be rather ineffective. Both dicots and monocots in nutrient culture positively reacted to Fe–rhizoferrin supply as a sole source for Fe (Shenker et al., 1992; Shenker et al., 1995 a; Yehuda et al., 1996). To the best of our knowledge the stability constants of rhizoferrin were studied by two groups (Shenker et al., 1996; Carrano et al., 1996) and two sets of data are available for Fe3+–rhizoferrin. The respective stability constants are numerically different: KML according to Shenker et al. (1996) is 13.7 at 0.1 M ionic strength, while according to Carrano et al. (1996) it is 25.3 at 2.0 M. However, since the first group had referred to the ligand as L-4 while the second had designated it as L-6, the comparison between the two sets of data is better presented by either the pFe or the apparent stability constant (Kapp), which are both indifferent to the definition of the free ligand. The pFe value is defined as the -log of the free Fe3+ concentration in equilibrium when total ligand and total Fe3+ concentrations are 10-5 and 10-6 M, respectively. The Kapp of a ligand (L), metal (M) and their complex (ML) is defined by: , where Ltotal and MLtotal represent the sum of all protonated species of the ligand or its metal complex, respectively, Mn+ is the free metal, and the brackets represent concentration (at any given ionic strength). The obtained values for pFe at pH 7.4, 6.0, and 4.0 are 19.7, 17.0, and 13.2, respectively, according to Carrano et al. (1996) at an ionic strength of 2.0 M, and 20.9, 18.0, and 13.1, respectively, according to Shenker et al. (1996) at an ionic strength of 0.1 M. The calculated log(Kapp) values at pH 7.0, 6.0, and 4.0 are 18.0, 16.0, and 12.2, respectively, according to Carrano et al. (1996) at an ionic strength of 2.0 M, and 19.1, 17.0, and 12.1, respectively, according to Shenker et al. (1996) at an ionic strength of 0.1 M. It is of interest to compare these values with the pFe values of 35.5 for enterobactin, 25.2 for ferrichrome, and 26.5 for ferrioxamine B (all values are at pH 7.4, Carrano et al., 1996), and with Kapp values of 21.8 for EDTA, 23.9 for EDDHA, and 24.2 for ferrioxamine B (values are at pH 7.0 and ionic strength of 0.1 M, Shenker et al., 1996). Thus, it is shown that both sets of data indicate rhizoferrin to have a relatively low stability constant for Fe3+, as compared with many siderophores and synthetic chelates. The stability constants of rhizoferrin used in this paper (Table 1) are all from Shenker et al. (1996), since Carrano et al. (1996) did not determine the stability constants of the siderophore with other metal ions, such as Ca. Zn, Cu, and Fe2+, and their data do not allow any prediction of metal exchange by rhizoferrin.



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  		Fig. 1 Rhizoferrin: chemical structure of the siderophore

 

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  		Table 1 Overall stability constants as log ß of rhizoferrin (L) with protons (H) and with the metals (M) Fe3+, Fe2+, Cu2+, Zn2+ and Ca2+ at 25°C and infinite dilution (converted by Davies' equation from Shenker et al., 1996)

 
As an outcome of the relatively low stability constant and specificity for Fe3+ of rhizoferrin at the studied pH levels, we did not expect this chelator to be as effective an Fe source for plants as it was found to be. We, therefore, hypothesized that ferric rhizoferrin acts for a long period in its nonequilibrium state. This seems to be the case for other siderophores and phytosiderophores that are all excreted as free ligands rather than as ferric complexes, although further evidence is required. Computer programs that are used to date, such as GEOCHEM-PC (Parker et al. 1995), are capable of computing solution composition at equilibrium, but they can not be employed for the prediction of actual composition in nonequilibrium states. Consequently, experimental nonequilibrium data are essential to the understanding of siderophore function in the rhizosphere.

The objectives of this research were to study the empirical Fe3+ binding capacity of natural and synthetic chelating agents with an emphasis on rhizoferrin, making a comparison to their equilibrium-predicted Fe3+ binding capacity, and to investigate rates of metal exchange and of Fe extraction from Fe hydroxide under conditions relevant for soil or plant nutrient solutions.


   Material and methods
TOP
 ABSTRACT
 INTRODUCTION
 Material and methods
Results
 Discussion
 REFERENCES
 
Rhizoferrin Production and Purification
Rhizoferrin was produced and purified as described by Shenker et al. (1995a). Briefly, rhizoferrin was produced from an Fe-deficient surface culture of Rhizopus arrhizus and purified by a sequence of three columns: coarse molecular sieving (Sephadex G-25, Pharmacia, Uppsala, Sweden); anion exchange (Whatman, DE-52, Whatman Biosystem, Maidstone, UK); and desalting (Sephadex G-10). Rhizoferrin was lyophilized to yield the pure tetra-sodium salt.

Calculation of Stability of Iron Complexes in Nutrient Solution
The following composition of plant nutrient solution was used to calculate the stability of Fe–rhizoferrin and other Fe complexes at 5 x 10-6 M with GEOCHEM-PC (Parker et al., 1995) (M): 7 x 10-4 K2SO4; 10-4 KCl; 2 x 10-3 Ca(NO3)2; 5 x 10-4 MgSO4; 10-4 KH2PO4; 10-5 H3BO3; 5 x 10-7 MnSO4; 5 x 10-7 ZnSO4; 2 x 10-7 CuSO4; 10-8 (NH4)6Mo7O24. This nutrient solution is referred to hereafter as NS. Stability of the Fe-complex was calculated for a pH range of 4.0 to 8.0, assuming that the calcium phosphates hydroxyapatite and ß-Ca3(PO4)2(c) do not precipitate. Stability constants for rhizoferrin at 0.1 M ionic strength that were previously determined (Shenker et al., 1996) were converted to thermodynamic stability constants according to the charge of each species using the Davies' equation (Davies, 1962) and were incorporated at infinite dilution (see Table 1) into the GEOCHEM-PC data base. Sources of stability constants for other chelating agents were as follows: EDTA and EDDHA [Martell and Smith (1974)]; MA [Murakami et al. (1989)]; and desferri–ferrioxamine–B (DFOB) [data base of GEOCHEM-PC ver. 2.0 (Parker et al., 1995)]. The log ß at zero ionic strength for the following L–M–H; (ligand–metal–proton) species, where L = DFOB, were: Fe3+, 32.6 for 1–1–0, 33.5 for 1–1–1; Fe2+, 18.2 for 1–1–0 and 24.0 for 1–1–1; protonation constants, 10.3 for LH, 19.8 for LH2, and 28.4 for LH3. Stability constants used for the solid phases, FePO4(c), FePO4 · 2H2O(strengite), and amorphous Fe(OH)3, were taken from Lindsay (1979). The amorphous Fe3+ hydroxide stability constant was used because this form is the most soluble of Fe3+ hydroxides; hence, it controls the Fe3+ concentration.

Stability in Nutrient Solution (Experimental)
Sterile NS was prepared by filtration through a 0.2-µm sterile, vacuum filter unit (Tamar, Israel). Iron-55 was added to 50 mL sterile NS under sterile conditions as a complex of one of the following chelates: rhizoferrin, EDDHA (Sigma, St. Louis), EDTA (Baker Analyzed, St. Louis), DFOB (Desferal, Ciba-Geigy, Basel, Switzerland), and MA (kindly provided by Dr. Satoshi Mori, Lab. of Plant Nutrition and Fertilizer, Univ. of Tokyo, Japan). All chelate solutions for this experiment and for the following experiments were quantified by the Cu–CAS method (Shenker et al., 1995b) to ensure accurate ligand:metal ratios. Final concentrations were 5 µM Fe and 5.5 µM chelate. As a control, 55Fe was added without any chelate. Unchelated Fe was assumed to precipitate as Fe hydroxide at the solution pH (7.5), which was maintained by 2 mM HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2- ethanesulfonic acid], Sigma). The solutions were sampled periodically, filtered through 0.2-µm syringe filter (Schleicher and Schuell, Dassel, Germany) in order to remove Fe hydroxide, and counted by liquid scintillation analyzer (1600 TR, Packard Instruments, Downers Grove, IL). The experiment was conducted under sterile conditions in order to eliminate microbial degradation of the chelates and to ensure that no new siderophore production occurred during the course of the experiment.

Preparation of Labeled Iron-55 Chelates
A solution containing unferrated chelate was stirred for 1 h with a trace amount of 55FeCl3 (0.08 MBq, Amersham Pharmacia, Amersham, UK) and with 80 µM FeCl3 in 160 µM HCl (yielding a specific activity of 59 MBq mmol-1 Fe). Chelate concentration was 10% higher than that of Fe. The pH was then raised to 7.5 by addition of HEPES buffer to a final concentration of 25 mM and by NaOH. The solution was stirred for 3 h at this pH and then filtered through a sterile 0.2-µm syringe filter to exclude Fe hydroxide precipitates and to sterilize the solution.

Calcium–Iron Exchange
The kinetics of Fe to Ca and Ca to Fe exchange at various Ca concentrations were monitored in solutions of the following composition (M): 1.1 x 10-4 rhizoferrin or EDTA; 10-4 55FeCl3 at specific activity of 10 MBq mmol-1 Fe; 0, 1.3 x 10-5, 4 x 10-5, 1.2 x 10-4, 3.7 x 10-4, 1.1 x 10-3, or 3.3 x 10-3 CaCl2. A typical Ca concentration of soil solutions is represented by 3.3 mM. The high chelate concentration was chosen to represent expected siderophore concentration in the rhizosphere. All solutions were buffered to pH 8.7 by 100 mM HEPES to ensure that no adsorption of Fe–rhizoferrin to Fe hydroxide would occur. A metal exchange experiment in the direction of metal A (Fe or Ca) to metal B (Ca or Fe, respectively) was performed according to the following sequence: the tested chelate was added to the salt solution of metal A, equilibrated for 90 min under gentle shaking, buffered to final pH using a HEPES solution and equilibrated for another 30 min under gentle shaking. A salt solution of metal B was then added and the solutions were stored in the dark at 25 C. The solutions were periodically sampled, filtered through a 0.2-µm syringe filter in order to remove Fe hydroxide, and counted using a liquid scintillation analyzer.

Iron Extraction from Iron Hydroxide
Iron-55 hydroxide was prepared by raising the pH of 55FeCl3 (at a specific activity of 59 MBq mmole-1 Fe) to 8.7 using KOH and HEPES additions. After an incubation period of 1 h, CaCl2 was added, and after an additional 10 min the tested chelate was added. No chelate was added to the control solution. The final concentrations were (M): 3.2 x 10-3 Ca; 5 x 10-5 tested chelate; and 10-4 HEPES. The total amount of Fe introduced as 55Fe hydroxide was twice that of the chelate. Preparation and handling were done under sterile conditions to prevent microbial degradation of the chelates and to eliminate possible siderophore production during incubation. The solutions were periodically sampled, filtered through a 0.2-µm syringe filter, and counted using a liquid scintillation analyzer. The aging of the Fe hydroxide during incubation was not monitored. Rather, it was assumed following Schwertmann (1988) that its transformation into more crystallized hydroxides of lower solubility is very slow and may last for several years. Each of the experiments was repeated three times and showed similar tendencies.


   Results
TOP
 ABSTRACT
 INTRODUCTION
 Material and methods
 Results
Discussion
 REFERENCES
 
Calculated Stability in Nutrient Solution at Equilibrium vs. Experimental Results
The stability of Fe complexed by rhizoferrin, DFOB, EDDHA, EDTA, and MA in NS for a pH range of 4.0 to 8.0, as calculated by GEOCHEM-PC, is shown in Fig. 2 . Based on the assumption that ß-Ca3(PO4)2(c) or less soluble calcium phosphates do not precipitate within the measurement period due to kinetic considerations, the Fe3+ in the Fe3+–rhizoferrin complex is exchanged for Ca2+ as the pH is raised. Therefore, in equilibrium at pH 7.0 to 7.5, Ca–rhizoferrin rather than Fe–rhizoferrin is the principal form of the siderophore. FeEDTA is a little more stable than Fe–rhizoferrin, but similarly, as pH increases, Fe is exchanged by competing metals in the following order of priority: Ca2+ >> Zn2+ > Mn2+ > Cu2+. Mugineic acid is shown to be a more efficient Fe3+ chelator at high pH values, and it loses only a small fraction of the iron, which is replaced by Zn2+ and Cu2+ (calculated values other than those of Fe3+–complexes are not shown). EDDHA, which is much more specific to Fe3+, complexes Fe efficiently up to pH 8, and only a small fraction of it is exchanged as the pH increases, mainly for Cu2+. The only chelator that prevails in the NS throughout the pH range of 4 to 8 as a Fe3+ complex is FeFOB. The unchelated Fe precipitates as FePO4 (strengite) up to pH 6.2 and thereafter as amorphous Fe(OH)3.



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  		Fig. 2 Concentration of chelated Fe as % of total Fe in equilibrium of various chelates (L) in a nutrient solution (NS) with 5 µM Fe3+ and L, vs. solution pH, as calculated by GEOCHEM-PC. For NS composition see text

 
The results shown here for rhizoferrin predict that this siderophore will not be useful as a Fe source for plants at pH levels above 7.0 to 7.5. Nevertheless, Fe–rhizoferrin has been shown previously to be very efficient as a Fe supplier for both strategy I and strategy II plants at this pH level (Shenker et al., 1992; Shenker et al., 1995a, Yehuda et al., 1996). To elucidate this contradiction, an experiment was designed to measure the actual capability of Fe–rhizoferrin, Fe-DFOB, FeEDDHA, FeEDTA, and Fe-MA to maintain Fe in NS at pH 7.5. The results as, indicated by residual soluble 55Fe for a period of 31 d, are shown in Fig. 3 . While EDTA and MA lost 50% of their 55Fe within 2 d, rhizoferrin, FOB, and EDDDHA retained almost all of their Fe for as long as 31 d.



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  		Fig. 3 Experimental stability of chelated Fe3+ in nutrient solution (NS) with 5 µM Fe and 5.5 µM of various chelates at pH 7.5. For NS composition see text

 
A comparison of equilibrium-predicted to actual, measured Fe levels in the solution indicates that nonequilibrium status governs concentrations of the complexed metals for a period of at least 31 d.

Calcium–Iron Exchange
According to the stability constants of rhizoferrin and EDTA with Ca2+ and Fe3+ and the solution composition of the above experiment or that of soil solution, Fe3+ chelated by both chelates is expected to be exchanged for Ca2+ as the pH increases. Since rhizoferrin was shown to stabilize Fe3+ in the solution for a long period in a nonequilibrium state, the kinetics of Fe–Ca exchange in a solution containing various Ca2+ concentrations was studied. Both directions of the metal exchange reaction were examined: the exchange of complexed Fe by free Ca2+ ions and the exchange of complexed Ca2+ by free Fe3+ ions. The first reaction represents a situation in which a nutrient solution is amended with a Fe–L complex, whereas the second reaction, namely Ca for Fe exchange, represents exchange reactions expected in calcareous soils. In the latter case the free siderophore is excreted into the rhizosphere and as a result of the Ca2+ concentration in soil solutions that exceeds that of Fe3+ by several orders of magnitude, a Ca2+–L rather than Fe3+–L complex is expected to form initially. EDTA was used as a control because it has a similar preference to the exchange of Fe3+ for Ca2+ at equilibrium in high pH solutions.

A time course change of Fe3+ remaining in the solutions, as a percentage of total Fe, measured in solutions containing 110 µM of the tested chelate (rhizoferrin or EDTA), 100 µM Fe3+, and various Ca2+ concentrations at pH 8.7, is shown in Fig. 4 . Calculated equilibrium concentrations for all cases, assuming 30.4 Pa (0.0003 atm.) partial pressure of CO2, predict that almost all the Fe (>99.5% for EDTA, and >99.9% for rhizoferrin) would appear in a solid phase as an Fe hydroxide.



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  		Fig. 4 Ca to Fe and Fe to Ca exchange of EDTA and rhizoferrin at pH 8.7, chelate concentration -110 µM, Fe -100 µM, and Ca concentrations (mM): 3.3 ({blacksquare}, {square}), 1.1 ({blacktriangleup}, {triangleup}), and 0.37 (•, {circ}). Closed symbols: Ca added first; Open symbols: Fe added first. Expressed as the percentage soluble (chelated) Fe of total Fe

 
As predicted by our calculations, CaEDTA did not exchange Fe from the added 55FeCl3 in solutions containing 3.3, 1.1, or 0.37 mM Ca2+ (Fig. 4). FeEDTA exchanged its Fe3+ for Ca2+ and approached equilibrium within 2 to 3 wk at 3.3 mM Ca, whereas at lower Ca concentrations the rate of metal exchange was slower.

Unlike EDTA, rhizoferrin exhibited an unexpected response in the first 7 to 10 d (Fig. 4). During the first 10 d, Ca2+ did not eliminate Fe3+ from the Fe–rhizoferrin complex by metal exchange, as expected by equilibrium calculations. Only later was the Fe3+ exchanged for Ca2+ and equilibrium was approached after another 20 d at 3.3 mM Ca and later at lower Ca2+ concentrations. In the case of 55Fe3+ added to Ca–rhizoferrin solution, 55Fe was initially complexed in the opposite direction to that predicted by equilibrium calculations. This reaction reached a maximum of 70 to 80% complexed Fe3+ after 6 to 7 d. At this point the reaction changed direction. A few days were needed to release Fe3+ from the complex and equilibrium was approached after another 10 to 20 d at 3.3 mM Ca and after longer periods at lower Ca levels.

Iron Extraction from Iron Hydroxide
Since complexation of Fe by siderophores in soil depends on Fe extraction from the solid phase, especially from Fe hydroxides, an experiment was designed to monitor the kinetics of 55Fe extraction from freshly prepared 55Fe hydroxide at a Ca concentration typical for calcareous soils. The tested chelates were rhizoferrin, EDDHA, EDTA, DFOB, and MA.

The level of soluble Fe at pH 8.7, without the presence of a chelate, was extremely low (Fig. 5) . EDTA, as expected at this high pH value and in the presence of 3.2 mM Ca, did not extract any Fe from the hydroxide. On the other hand, DFOB, EDDHA, MA, and rhizoferrin did extract Fe from the hydroxide at a high rate during the first several hours, and at a much slower rate thereafter. After 15 d of incubation only 9, 7.5, 5, and 5 µM Fe were extracted by 50 µM of DFOB, EDDHA, MA, and rhizoferrin, respectively.



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  		Fig. 5 Iron-55 extraction from freshly prepared Fe hydroxide by 50 µM DFOB, EDDHA, MA, rhizoferrin or EDTA, in presence of 3.2 mM Ca, at pH 8.7 buffered by 100 mM HEPES

 

   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 Material and methods
 Results
 Discussion
REFERENCES
 
The overall stability constants of rhizoferrin complexes with various metals of importance to plants and soils have been reported previously (Shenker et al., 1996). The apparent stability constants (Kapp,) of rhizoferrin with these metals at pH 7.0 and 0.1 M ionic strength are given in Table 2 along with those of EDTA, EDDHA, DFOB, and MA. It is evident from these data that rhizoferrin is a specific ferric chelate, but at high pH values that prevail in calcareous soils and in the presence of Ca at concentrations typical for soil solutions, most of the siderophore is predicted to complex Ca2+ rather than Fe3+ at equilibrium (Fig. 2). Based on equilibrium calculations it is expected that rhizoferrin will not be an efficient Fe mediator for plants at high pH levels in nutrient solutions and in soils. Yet, the results of the present study indicate that although Fe–rhizoferrin is not a stable complex (in the thermodynamic sense) at high pH values, its empirical stability at pH 7.5 during the first few weeks is as high as that of chelates such as DFOB and EDDHA, and much higher than that of EDTA and MA. We therefore propose that very slow Fe3+ to Ca2+ exchange kinetics enable this metastable complex to act as an efficient Fe source for plants. These assumptions are in accordance with previous reports that indicate that Fe–rhizoferrin functions as a very efficient Fe carrier for plants in nutrient culture at high pH levels (Shenker et al., 1992; Shenker et al., 1995 b; Yehuda et al., 1996).


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  		Table 2 Apparent stability constants (Kapp{dagger}) of rhizoferrin, EDTA, EDDHA, DFOB, and MA and Fe3+, Fe2+, Cu2+, Zn2+ and Ca2+ at pH 7.0 and an ionic strength of 0.1 M

 
Since siderophores are excreted as free ligands into the rhizosphere, and since Ca2+ is usually the major counter ion, the kinetics of Fe3+ extraction from the solid phase and the kinetics of Ca2+ to Fe3+ exchange seem to be of crucial importance to the siderophore's function in the rhizosphere. The ability of rhizoferrin to extract Fe from Fe hydroxide at high pH levels and in the presence of high concentrations of Ca2+, and the long period it holds the Fe3+ in solution at the nonequilibrium state suggest that this siderophore might have an important role in the rhizosphere.

The mechanisms of stabilization of Fe in solution by rhizoferrin in the tested solutions seems to involve an interaction of the siderophore with newly formed microcolloidal Fe(OH)3 or a solid Fe(OH)3 surface to yield higher Fe3+ solubility than that predicted for amorphous Fe(OH)3. Indeed, stability constants given by Schwertmann and Taylor (1977) for poorly crystallized ferrihydrite may yield Fe3+ concentration 30 times higher than that of the amorphous Fe(OH)3 proposed by Lindsay (1979). Furthermore, transformations of poorly crystallized Fe(OH)3 to well crystallized minerals may take several years under certain conditions (Schwertmann, 1988) and can lead to a very slow kinetics until equilibrium is achieved.

Previously Buyer and Sikora (1990) argued for the importance of rate limiting processes like siderophore extraction and ligand exchange, along with degradation, adsorption, and leaching, in order to predict which species of a siderophore will dominate the rhizosphere. They further speculated that nonequilibrium of siderophores speciation is the common case rather than the exception in the rhizosphere. Consequently, they stressed the importance of a nonequilibrium state in determining the bioavailability of Fe3+ complexed by siderophores.

The present study adds metal exchange kinetics as a crucial issue for determining siderophore speciation in the solution. While Buyer and Sikora (1990) based their hypothesis merely on theoretical assumptions, this study brings experimental evidence that shows that in the time scale of biological uptake processes, nonequilibrium of rhizoferrin is indeed the state of this siderophore. Only by including such considerations can the biological function of rhizoferrin as a Fe3+ carrier to plants be understood.


   ACKNOWLEDGMENTS
 
This research was partially supported by a grant from the US–Israel Binational Agricultural Research and Development Fund (BARD) and by a grant from the European Union and the Israel Ministry of Science.

Received for publication July 23, 1998.


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 ABSTRACT
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 Results
 Discussion
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