Plants and microorganisms have developed active strategies to acquire iron which is essential for their metabolism but has a low availability in soils under oxic environments. The corresponding activities, which include acidification, chelation, and reduction, contribute to increase the dissolu- tion and solubility of iron oxides in the rhizosphere (Figs. 2 and 3). Plants and microorganisms have also evolved strategies for regulating their iron content by homeostasis.
3.1. Iron solubilization in the rhizosphere
Acidification results from proton extrusion and secretion of organic acids by plants and microorganisms leading to proton concentrations up to 100-fold greater in the rhizosphere than in bulk soil (Darrah, 1993; Hinsingeret al., 2003; Nye, 1981). Root and microbial respiration also contribute to soil acidification, due to the resulting elevation of pCO2 and dissociation of carbonic acid (Hinsingeret al., 2003; Nye, 1981). Efficacy of acidification by plants and microorganisms depends on the buffering capacity of the soil which is especially high in calcareous soils as a consequence of the con- sumption of protons by the dissolution of CaCO3(Hinsingeret al., 2003;
Siebner-Freibachet al., 2003).
Protons Organic acids (citric, oxalic, malic acids,...)
Molecules with high iron affinity
(phytosiderophores, siderophores) Reducing compounds Reductases (phenolic, aliphatic acids,...)
Reduction Chelation
Acidification
Iron solubilization
Iron bioavailability
Figure 3 Schematic representations of mechanisms affecting iron availability in the rhizosphere. Plants and microorganisms may increase iron availability by (i) acidifica- tion through proton extrusion and organic acid secretion (and possibly respiration), (ii) chelation through secretion of complexing molecules with variable affinity for iron (phytosiderophores, siderophores, phenolics, and carboxylic acids), and (iii) reduc- tion through secretion of compounds characterized by reducing properties or through the expression of a membrane-bound reductase activity.
194 A. Robinet al.
3.1.1. Acidification
The respiration of living organisms, including plant roots and microorgan- isms, leads to an increasedpCO2in soils, which is commonly several tens or hundreds times higher than that in the atmosphere (Hinsingeret al., 2003).
Accordingly, the concentration of carbonic acid (H2CO3) is expected to be much higher in soils, inducing a significant acidification when dissociating.
Given its rather high pK (6.36), H2CO3 dissociates more at neutral and alkaline than at acidic pH. Therefore, the contribution of respiration to soil acidification is especially significant in calcareous soils known for their low iron availability. The pH of these soils, close to 8 at ambient atmospheric pCO2, is known to decrease by 0.67 pH units for every tenfold increase in pCO2 and may then decrease down to 6.7 at a pCO2 of 0.1 mol mol1 corresponding to values of pCO2 reported to occur in the rhizosphere (Gollanyet al., 1993; Hinsingeret al., 2003 ). Although seldom accounted for, root and microbial respiration can thus have a significant effect on soil pH and ultimately on the solubility of soil iron oxide.Kraemeret al.(2006) calculated that the total concentration of dissolved iron species in equilib- rium with ‘‘soil iron oxide’’ (as defined by Lindsay, 1979) increased from about 1010 M, at ambient atmospheric pCO2 , up to 2 109 M, at a pCO2of 0.1 mol mol1, these values ofpCO2being commonly found in the rhizosphere (Gollanyet al., 1993).
Nongraminaceous plants have developed an active strategy of iron uptake (strategy I) based on (i) the extrusion of protons (Bienfait, 1985;
Guerinot and Ying, 1994), (ii) the reduction of Fe3þ to the more soluble Fe2þspecies by plasmalemma-bound reductases (Robinsonet al., 1999; see paragraph below), and (iii) the absorption, that is, transport, of Fe(II) through the plasmalemma by iron transporters (Curie et al., 2000; Eide et al., 1996; Vert et al., 2001). Proton effluxes from strategy I plants commonly reach 6mmol Hþ h1 (g root biomass)1 (Ro¨mheld et al., 1984). Proton extrusion results from the activity of Hþ-ATPases that is promoted under iron deficiency (Dell’Orto et al., 2000; Ro¨mheld and Marschner, 1981; Santi et al., 2005) in strategy I plant species. The Hþ-ATPase activity is promoted also by humic substances (Pinton et al., 1997; Varaniniet al., 1993) and even more by the root uptake of nutrients (Hinsinger et al., 2003; Marschner, 1995). Indeed, plant root balance cations/anions influx ratio is regulated by plant release of protons when an excess of cations over anions has been taken up (Haynes, 1990; Hinsinger, 1998; Hinsingeret al., 2003; Nye, 1981; Ro¨mheld and Marschner, 1986a).
Protons are not extruded homogeneously along the root. Their greater release has been frequently reported in some specific zones such as subapical and basal zones, especially in strategy I species exposed to Fe deficiency (Jaillardet al., 2002; Plassardet al., 1999; Ro¨mheld and Marschner, 1981).
In tobacco (Nicotiana tabaccum) rhizosphere proton efflux appeared to be
Iron Dynamics in the Rhizosphere 195
significantly higher [7–9 pmol Hþ (m root length)1s1] in the basal than in the subapical zone [3–5 pmol Hþ(m root length)1s1] (Vansuytet al., 2003). Further information is given in several reviews of root-mediated pH changes in the rhizosphere (Haynes, 1990; Hinsinger, 1998; Hinsingeret al., 2003; Nye, 1981).
Microbial metabolism also contributes to pH variations (Latour and Lemanceau, 1997). Observations and in situ measurements indicated reduced pH close to active microbes and biofilms using confocal microscopy (Barker and Banfield, 1998) and micro-electrodes (Yu and Bishop, 2001; Yu et al., 1998). Microbial acidification is especially well documented in bacteria involved in the oxidation of ferrous iron sulfide, such as pyrite, and in that of ammonium leading to the formation of sulfuric and nitric acids, respectively.
Ferrous iron sulfides do not commonly occur in oxic environments and only few bacteria show the ability to oxidize these sulfides. Among them, Thiobacillus ferrooxidans, recently renamed Acidithiobacillus ferrooxidans (Lu et al., 2006), is commonly applied for the treatment (bioleaching) of metal sulfide ores (Fernandezet al., 1995). Conversely, acidification resulting from the oxidation of ammonium in nitrate is common in soils, but again relies on the activity of only few bacteria genera, that is,NitrosomonasandNitrobacter (Holloway and Dahlgren, 2002; Sitaulaet al., 2001).
Carboxylates, such as citrate, oxalate, malate, and many others com- monly found in root exudates, also contribute to pH decrease in the rhizosphere, when their exudation is coupled with proton efflux (Hoffland, 1992; Jones, 1998; Joneset al., 2003). A metabolic link between citrate excretion and proton extrusion has been proposed (Ohwaki and Sugahara, 1997) and the amount of citrate exuded correlated with the capacity of rhizosphere acidification. The concentration of carboxylates in the rhizosphere ranges from 50mM (Jones et al., 1996) to 9000mM (Dinkelaker et al., 1989). This concentration considerably varies among plant species and depends on environmental constraints, notably phospho- rus and iron deficiency (Curl and Truelove, 1986; Hinsinger et al., 2003;
Jones, 1998). The exudation of carboxylic acids from maize (Zea mays) roots contributed to less than 0.3% of rhizosphere acidification according to Petersen and Bo¨ttger (1991). Conversely, cluster roots of white lupin (Lupinus albus) were shown to release massive amounts of carboxylates which accounted for a considerable acidification of the rhizosphere (Dinkelaker et al., 1989; Vance et al., 2003). Small organic acids from bacterial metabolism also contribute to pH decrease (Hoberg et al., 2005;
Liermannet al., 2000). This is the case for oxalic acid, which is produced in large amounts by ectomycorrhizal fungi (Casarin et al., 2004). Microbial organic acids appear to enhance silicate dissolution rates (Rogers and Bennett, 2004). Carboxylic acids, such as malic and citric acids, could be involved in the dissolution of Fe(III) polymers bound to the surface of microbial cells (Winkelmann, 1979, 2007). These acids as well as oxalic acid
196 A. Robinet al.
affect the fate of iron not only via proton-promoted dissolution but also via ligand-promoted dissolution of iron-bearing minerals (see following section).
3.1.2. Chelation and complexation
Ferric iron can be chelated by a range of organic ligands showing a high affinity for this metal cation. Among them, siderophores produced by graminaceous plants and by microbes are involved in their active iron-uptake strategies.
Phytosiderophores such as mugineic acids produced by graminaceous plants efficiently chelate ferric iron due to their amine and carboxyl groups (Fig. 4A and B) (Kraemeret al., 2006; Takagi, 1976). These root exudates are nonproteinogenic amino acids which are synthesized from methionine via nicotianamine. The strategy of phytosiderophore-mediated iron uptake (strategy II) has been described in graminaceous plants including wheat (Triticum aestivum), barley (Hordeum vulgare), rice (Oryza sativa), and maize (Z. mays), and in a range of pasture grasses (Marschner and Ro¨mheld, 1994).
Rice plants have, however, the peculiarity to take up both and Fe3þ- phytosiderophore and Fe2þas nongraminaceous strategy I plants (Ishimaru et al., 2006). Phytosiderophores can reach local concentrations of 1 mM in the soil solution, according to model calculations made byRo¨mheld (1991).
Little information is available onin situmeasurements of phytosiderophore concentrations except the work byShiet al.(1988)reporting the presence of only micromolar concentrations of mugineic acid in the rhizosphere of barley (H. vulgare). As stressed byKraemeret al.(2006), these measurements did not take into account the temporal and spatial patterns of secretion, which indicate that phytosiderophores are secreted at much larger rates near the root apices and between 3 and 6 h after the onset of light (Marschner et al., 1987; Reichman and Parker, 2007; Takagi et al., 1984). At such locations and during these temporal pulses of secretion, phytosiderophore concentration in the rhizosphere may reach that computed by Ro¨mheld (1991).Reichardet al.(2007)recently demonstrated that pulse additions of phytosiderophores led to considerably increased rate of dissolution of goe- thite, relative to a steady supply of phytosiderophores. Phytosiderophores exhibit a high affinity for iron with a stability constant of the corresponding chelate equal to 1017–1018. This stability constant is high enough for phy- tosiderophores to compete efficiently for iron bound to the humic fraction of soils (Cescoet al., 2000).Kraemeret al.(2006)further discussed this process in their review and concluded that ligand-exchange reactions between humic substances and organic ligands such as phytosiderophores (and most microbial siderophores) is fast enough to make a portion of this pool of iron available for uptake. More importantly, the chelating capacities of the phytosiderophores allow them to dissolve iron oxides, including poorly soluble minerals such as goethite (Hiradate and Inoue, 1998, Kraemer
Iron Dynamics in the Rhizosphere 197
Enterobactin Fusarinine
A
E
O O
CH3 N
O OH O O
CH3
N O OH
O O
CH3 N
HO O
H2N NH2
H2N
C HO
HO HN O
O O O
O
O O
HN NH
O OH
OH O
OH OH
D
OH O
OH N+
NH NH H NH O NH2 H N HO O HN
O
HO O HN H3C
H3C L-Ala
D-aThr O HO
HN O
N
O H N OH
L-Lys
D-OHA sp
L-Ala
L-cOHOrn
R
Pyoverdine
F G
Schizokinen
CH2 CH2 CH2 CH3 H2C H2C H2C CH3 O
COOH OH HN
O NH
OH O N OH O
N COOH
COOH HO HN
O
COOH OH HOOC HN
O
Rhizoferrin Fe-DMA
Hydroxymugineic acid 2⬘-deoxymugineic acid (DMA)
Mugineic acid Avenic acid
COO− COO− COOH
OH OH
HO NH2+
OH COO−
COO− COOH
NH+
OH
OH COO−
COO− COOH
NH+
OH
HO NH+ OH
COO−
COO− COOH
CH2 CH2
H2C H2C
H2C
Fe
OH NH O CH
CH
N C
O O
C C
CH O
O
H
B
O C O NH2+
NH2 +
NH2 +
NH2+
Figure 4 Structures of phytosiderophores, avenic acid, 20-deoxygemugineic acid (DMA), mugineic acid, 3-hydroxymugineic acid, (A); the phytosiderophore DMA chelated with iron, (B); fusarinine, a trihydroxamate siderophore produced by the fungiFusarium, (C); enterobactin, a tricatecholate siderophore produced by the bacterium E. coli, (D); rhizoferrin, a polycarboxylate siderophore produced by the fungi Rhizopusspp. and the bacteriumRalstonia pickettii, (E); bacterial monohydroxamate mixed-ligands pyoverdine produced byPseudomonassp.
strain B10, (F); and schizokinen produced byBacillus megaterium, (G).
et al., 2006), by enhancing the rate of ligand-promoted dissolution of these minerals ( Reichard et al., 2005). Phytosiderophores show a poor specificity for iron relative to other metals, since most phytosiderophores can efficiently chelate a number of divalent metal cations, especially zinc, nickel, and copper, and form stable chelates ( Murakami et al., 1989). As an example, the stability of the chelate of mugineic acid with Cu(II) is even slightly higher than that with Fe(III) and therefore phytosiderophores are expected to be more frequently bound to copper than to iron at pH values above 5 accord- ing to the model calculation made by Reichman and Parker (2005). Com- petition between cations may explain the iron–copper antagonism reported in durum wheat (Triticum turgidum durum ) grown in calcareous soils con- taminated by copper (Michaud et al., 2007). In this study, iron deficiency symptoms (interveinal chlorosis in leaves) were related to copper toxicity, since they were associated with an elevated copper content of roots and shoots. Such competition of metals for phytosiderophores is less likely to occur in the case of microbial siderophores, which generally show a greater specificity for ferric iron and a much higher stability constant than for other metal cations (Kraemer et al., 2006; Parker et al., 2005).
The active uptake strategy of iron by microorganisms relies on the synthesis of siderophores and ferri-siderophore membrane receptors (Neilands, 1981). However, bacterial siderophores differ structurally from phytosiderophores ( Fig. 4C–F) and show a higher affinity for ferric iron with stability constants ranging from 1023 to 1052 (Albrecht-Gary and Crumbliss, 1998; Neilands, 1981; Winkelmann, 1991). Microbial siderophores are quite diverse molecules with molecular mass values usually less than 1000 Da (Neilands, 1981), with, however, some of them, the pyoverdines synthesized by the fluorescent Pseudomonas, ranging between 1000 and 1800 Da [1764 for the biggest one reported so far by Meyer et al. (2008)].
More than 500 microbial siderophores have been so far been characterized (Boukhalfa and Crumbliss, 2002), among which are more than 100 different pyoverdines (Budzikiewicz, 2004; Meyer et al., 2008). They are classified according to the functional groups acting as ligands: catecholates, hydro- xamates, hydroxypyridonates, hydroxy- or amino-carboxylates (Fig. 4C–F) (Bossier et al., 1988; Winkelmann, 1991, 2002, 2007). Siderophores can efficiently sequester iron adsorbed on different metal oxides and solubilize iron oxides (Hersman et al., 1996; Kraemer, 2004). Further information on siderophore interactions with metal oxides and solid surfaces can be found in several reviews ( Cocozza et al., 2002; Hersman et al., 1995, 2000; Holme´n and Casey, 1996; Holme´net al., 1999; Kraemer, 2004; Liermannet al., 2000;
Mauriceet al., 2001; Watteau and Berthelin, 1994).
Much attention has been dedicated to the major siderophores of fluores- cent pseudomonads (pyoverdines) and to their membrane receptors, both synthesized under iron deficiency (Hohnadel and Meyer, 1988;
Meyer, 2000; Meyer and Abdallah, 1978). Pyoverdines are chromopeptides
Iron Dynamics in the Rhizosphere 199
composed of a quinoleinic chromophore bound together with a peptide and an acyl side chain ( Fig. 4E ) (Budzikiewicz, 2004). Synthesis of pyo- verdines and related protein membrane receptors correspond to a significant metabolic effort for bacterial cells that is expressed only when required via regulation processes. Their synthesis occurs in response to cellular iron deficiency resulting from a low iron availability of the environment (low supply) but is repressed under noniron stress conditions (Meyer et al., 1987). Similarly, pyoverdine synthesis is regulated by the phenomenon of Quorum Sensing through the production of acyl homoserines lactones (AHLs) when the bacterial density is high and corresponds to a significant demand in iron (Stintzi et al., 1998). This synthesis was shown to occur in the rhizosphere by the use of (i) monoclonal antibodies against ferri- pyoverdine (Buyer et al., 1990) and of (ii) the ice-nucleation reporter gene inaZ (Duijff et al., 1999; Loper and Lindow, 1994). As for pyoverdines, hydroxamate siderophores have been detected in soils with concentrations in soil solution ranging from 10 7 to 10 8 M (Powell et al., 1980).
In addition to these high-affinity ligands, microorganisms and plants also produce a range of lower-affinity ligands such as phenolics and, more abundantly, carboxylates, oxalate, and citrate, for instance ( Jones, 1998;
Jones et al., 1996; Reichard et al., 2005). A large proportion of these carboxylates occur in soil solutions as complex species with a range of metal cations including iron. As an example, 1–40% of citrate in soil solution is present as a Fe–citrate complex ( Jones et al., 1996). The stability constant of the complexes of citric and malic acids with Fe(III) varies from 107 to 10 11 , respectively ( Hue et al., 1986; Jones, 1998; McColl and Pohlman, 1986), which is orders of magnitude less than that of phytosiderophores and even more so for microbial siderophores. Some carboxylates, such as citrate and oxalate, form poorly soluble minerals with Ca and are thus prone to precipi- tate in calcareous soils, thereby reducing their role in complexing metals such as iron ( Jones, 1998). A more thorough discussion of the interactions of carboxylates with iron oxides and their ligand-promoted dissolution can be found in Kraemer et al. (2006).
3.1.3. Reduction
Mechanisms accounting for reduction by plants were recently reviewed (Schmidt, 2003). Fe(III) reduction is an essential and prerequisite compo- nent of iron uptake by strategy I plant species since they are equipped only for taking up Fe(II). Fe(III) reduction in strategy I plant species is essentially mediated by reductases, whose activities are stimulated as a response to iron deficiency. This reductase activity in the plasma membrane is promoted by Hþ extrusion into the rhizosphere (Marschner and Ro¨mheld, 1994;
Marschner et al., 1986) and is suppressed at high level of HCO3 in calcareous soils (Siebner-Freibachet al., 2003). Reductase activity enables
200 A. Robinet al.
strategy I plants to have access to Fe(III) including iron complexed by citrate.
TheFRO2 gene fromArabidopsisis a member of a reductase gene family encoding the root ferric-chelate reductase activity induced in response to iron deficiency (for review, seeCurie and Briat, 2003; Robinsonet al., 1997, 1999). Reducing activity by roots has also been attributed to reducing compounds such as phenolic (caffeic acid) or some carboxylic acids (malic acid) (Brown and Ambler, 1973; Ro¨mheld and Marschner, 1983). Their contribution appears, however, to be of limited ecological significance (Bienfaitet al., 1983; Ro¨mheld and Marschner, 1983).
Microbial reduction of metals such as iron is well documented and has been reviewed by, for example,Lovley (1995). Membrane and extracellular reductases have been described in various pathogenic bacteria (Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella thyphimurium, Staphylococcus aureus) (Coulangeset al., 1997; Cowart, 2002; Deneeret al., 1995; Vartivarian and Cowart, 1999). In these bacteria, extracellular iron reduction contributes to iron acquisition from their hosts and from soils during their parasitic and saprophytic life, respectively (Barchini and Cowart, 1996; Schro¨der et al., 2003). Extracellular reductases and side- rophores would act together to solubilize and transport iron from the environment (Cowart, 2002). Recently, phenazines, known for their antagonistic activities against phytopathogenic fungi (Thomashow and Weller, 1988), were reported to act as electron shuttles contributing to metal cation reduction (Hernandezet al., 2004; Price-Whelanet al., 2006).
Siderophores are also expected to affect the redox status of the rhizo- sphere through their electronegative characteristic or the modification of the Fe(II)/Fe(III) balance resulting from iron chelation (Emery, 1977; Pidello, 2003). Indeed, inoculation of the pyoverdine-producing strainP. fluorescens C7R12 was shown to increase the pe in soil compared to that of the corresponding nonproducing pyoverdine (pvd-) mutant (Pidello, 2003).
In microaerophilic or anaerobic zones (Hojberg et al., 1999), such as waterlogged soils, oxygen can be released by the roots (Amstrong, 1964) allowing iron oxidation as demonstrated by the presence of iron oxide precipitates coating the root surface of many wetland plants, thereby forming the so-called Fe-plaque (Mendelssohnet al., 1995). Microscopic observations have revealed the presence of bacterial cells in this Fe-plaque (St-Cyret al., 1993) and Fe-oxidizing bacteria have been suggested to contribute to its precipitation (Emersonet al., 1999). In return, Fe(III) oxides of this plaque are used as electron acceptors by dissimilatory iron-reducing bacteria (Luu and Ramsay, 2003; Schro¨deret al., 2003). The high Fe(III) reduction potential of the Fe-plaque promotes a microbial-mediated Fe cycle around the roots of wetland plants (Weiss et al., 2004) and their rhizosphere is the site of an unusually active microbial Fe cycling.
Iron Dynamics in the Rhizosphere 201