MBA, measured by dialysis titration, varied from 160 to 206 mEq/100 g HS according to the nature of the metal, log K values, following the series Pb ~> Cu > Cd.
Biology and Fertility
o Soils
Biol Fertil Soils (1987) 3:165-169
© Springer-Verlag1987
Metal binding properties of high molecular weight soluble exudates from maize (Zea mays L.) roots M. Mench, J.L. Morel, and A. Guekert Department of Plant Science, Ecole Nationale Sup+rieure d' Agronomie et des Industries Alimentaires, 2, Avenue de la Foret de Haye, F-54500 Vandoeuvre, France
Summary. A high molecular weight (MW > 1000) soluble root exudate fraction (HS) was isolated from hydroponic axenic maize cultures in order to investigate its metal-binding properties. Measurements of the maximum binding ability (MBA) and the overall stability constants (log K) for cadmium-, copper-, lead- and zinc-HS associations were obtained from dialysis and ion-selective electrode titrations. All resuits showed the occurrence of organometallic bindings. Data fitted to linear Langmuir isotherms. The MBA, measured by dialysis titration, varied from 160 to 206 mEq/100 g HS according to the nature of the metal, log K values, following the series Pb ~> Cu > Cd 5 Zn, varied from 3.15 to 3.65. Due to these metalbinding properties, soluble root products could play a role in the transfer of metal into the rhizosphere.
Key words: Root exudates - Copper - Cadmium Lead - Zinc - Dialysis - ISE The control of micronutrient fertilization and of risks connected to the accumulation of heavy metals in the soils requires a detailed knowledge of the processes (immobilization, release, transport) which govern cation movement in the soil and especially in the rhizosphere. Physicochemical factors (pH, Eh, ion concentration, etc.) can be significantly altered by the proximity of roots (Marschner and R6mheld 1983; Foster 1983; Schaller and Fischer 1985). In addition to inorganic elements, protons and discarded cells, roots release organic substances including soluble compounds ranging from simple sugars to complex viOffprint requests to: M. Mench
tamins and polysaccharidic gels (Rovira et al. 1979). It has been shown that root mucilages are able to bind metals, suggesting a role of these materials in the retention of some cations in the rhizosphere (Horst et al. 1982; Morel et al. 1986). Also, there is increasing evidence that root exudates may promote iron and manganese solubility (Bromfield 1958; Jauregui and Reisenauer 1982; Uren 1984), but the interactions between metals ahd root products are not yet well elucidated. Their biochemical nature enables some of these compounds to bind heavy metals, as was shown by gel filtration experiments with cadmium and with a high molecular weight fraction of soluble root exudates (HS) released by maize roots (Mench et al. 1985). In this work an HS fraction was isolated from hydroponic and axenic maize cultures and its metalbinding properties for lead, cadmium, copper and zinc were then measured.
Material and methods Maize (Zea mays L.) plants were grown in a hydroponic culture device allowing aseptic conditions for at least 2 weeks (Morel et al. 1986). The nutrient solution's sterility was tested by incubating a sample on nutrient agar for 24 h, Then, the soluble root exudates were collected from the solution by filtration under nitrogen (porosity 0.45 lam). The filtrate was concentrated by evaporation (30°C) under vacuum and then dialysed 4 times against bidistilled water at + 4°C under a nitrogen atmosphere (Spectra/por 6 MW 1000membranes). The non-dialysablecompounds, which constitute the high molecular weight soluble root exudate fraction (HS), were freeze dried and stored under vacuum. The dialysate was stored at -18°C for use in later studies. HS was analysed for carbon and nitrogen (CHN CarloErba analyser), ash content, total reducing sugars (Dubois et al. 1956; glucose as standard), total proteins (Hartree 1972; albumin as standard), total phenolic compounds (Marigo 1973; cafeic
166
M. Mench et al.: Binding of metals with soluble root exudates
acid as standard) and uronic acids (Bitter and Muir 1962; galacturonic acid as standard). The binding of HS with metal ions was studied using the equilibrium dialysis procedure (Zunino and Martin 1977; Blaser et al. 1980), used previously in the study of root mucilage-metal interactions (Morel et al. 1986): 20 ml of a 100 mg HS 1-] solution in 0.1N KNO3 was introduced into a dialysis bag which was immersed into a 200-ml metallic solution adjusted to pH 6 and containing sufficient KNO3 to maintain a constant ionic strength in the system (/z = 0.01). Spectra/Por membranes MW 1000 were used. They were preliminarily purified according to Truitt and Weber (1981); dialysis bags were closed off with Spectra/Por medical enclosures. The concentration of metal in the external medium was measured after a 24-h equilibrium period at + 4°C, giving the free metal concentration (/VIi'). The concentration of metal ions linked with HS (Mb) was expressed as the difference between total (Mt) and free metal ion concentrations. All measurements of metal concentrations were made using atomic absorption (Varian AA6) after, an acidification of aliquots with pure HNO3 (100 /zl/10 ml). The experiment was repeated with increasing concentrations of metal added to a constant HS concentration. This enabled HS to reach saturation in defined pH and ionic strength conditions. Blanks without HS were treated in the same way. In addition to dialysis experiments, ion-selective electrode potenfiometric titrations (ISES) were also conducted to study the HS-Cu binding, according to the method described by Sposito et al. (1979). A 50-ml HS solution (1.55g 1-1) was titrated by a 5.10 -2 M Cu(C104) 2 solution at 25°C in a 0.1N KC104 medium at pH 5 and under a nitrogen atmosphere. A Tacussel ISIS 20000 mVmeter, Tacussel TS/50 N1 pH meter. ISE (pCu 2M Tacussel) and a reference electrode (C18 Tacussel) were used. Prior to each experiment, ISE was standardized by titrating a 0.1MKC104 solution at pH 5, this giving the working Nemst equation for ISE: mV = 166.5961 + 28.9709 pCu (r = 0.99994). A function similar to that of Langmuir was used for the treatment of data collected (Blaser et al. 1980; Morel et al. 1983): (Mr)~ (Mb)
1 K . MBA
carbon pool, corresponding to 0.9% of the total root carbon. Basic analysis of HS was assumed to be C 330/0, H 4.80/0, N 3.40/0 and ash content 1%. Major components found were polysaccharides and uronides (Table 1). As in root mucilages, proteins were also detected, which may indicate the presence of glycoproteins. A few phenolic compounds were also observed. Table 1. Analysis of the high molecular weight soluble root exudate (HS) (g. i00 g-1 dry matter) Total reducing sugars Total proteins Uronic acids Phenols ( ~bl
43.8 16 13 3.4
, Hb
( ~I )
40
40
20
20 HI |
200
I
400
I
( ~M ) ( ~M
40
40
20
20 HI I
I
200
4oo
L
200
( .M
(Mr)~ MBA
where M f = free metal concentration, Mb = bound metal concentration, MBA = maximum binding ability (Zunirio and Martin 1977), K = overall stability constant of the association and a = number of binding sites per unit of ligand. This equation was solved using the distributions of bound metal concentrations as a function of free metal concentrations. It was then possible to calculate the two parameters describing the binding of metal to HS: MBA and K. The energy-binding term obtained is related to n stepwise successive stability constants (kl, k2 ..... kn) and provides an average value among all the energies of the possible linkages.
M|
t
Mb
S I
200
( ~M )
400
I
400
Fig. 1. Binding of Cd 2+, Cu 2+, Pb 2+ and Zn 2+ with a high molecular weight soluble root exudate fraction (HS): distribution isotherms of bound metal concentration (Mb) and free metal concentration (Mr) at equilibrium obtained from dialysis experiments (pH 6,/z = 0.01) I,uM}
Mb
600'
"00
Results
200
HS characterization 1000
After a 15-day hydroponic axenic culture, each maize plant reached the four- to six-leaf stage and had released 0.41 (+/-0.1) mg soluble carbon into the nutrient medium. HS represented 42% of the soluble
2000
30 0
1.000
50 0
6000(,uMI t,-lt
Fig. 2. Binding of Cu z+ with a high molecular weight soluble root exudate fraction (HS): distribution isotherm of bound metal concentration (Mb) and free metal concentration (M0 obtained from ion-specific electrode titration (pH 5, a = 0.1)
M. Mench et al.: Binding of metals with soluble root exudates MflMb
(~M)
( pM )
MI/Mb
167
Metal binding properties of HS
4k."
8
s ji,"
Isotherms curves Mb = f ( M f ) obtained using dialysis and potentiometry show that organometallic-binding 4 occurs between HS and the investigated cations (Cd, Cu Pb 2 Cu, Pb, Zn) (Figs. 1,2). These distributions give HI HI ,1 adsorption-like curves which are similar in shape to b I I I I Langmuir isotherms. The ascending curves show that 200 400 ( ~M ) 200 400 ( pM ) saturation of the ligands was not attained for the HIIHb ~(~M) metal ion concentration range used in our experimen8 tal conditions. 6 When Mf/Mb was plotted against Mfa straight line 6 was obtained ( r > = 0.92) (Figs. 3,4). Thus, assuming 4 a = 1 in Eq. 1, it was possible to calculate MBA and Cd 4 Zn M1 log K (Table 2). MBA obtained from dialysis experiHI ,i i I L I I ments (pH 6, # = 0.01) varied from 160 to 206 200 400 ( pM ) 200 400 ( ~¢H ) mEq/100 g dry matter HS. ISE potentiometric titraFig. 3. Binding of Cd 2+, Cu 2+, Pb z+ and Zn 2+ with a high molecular tion gave an MBA value of 84 mEq/100 g dry matter weight soluble root exudate fraction (HS): distribution isotherms HS for the Cu-HS binding (pH 5, # = 0.1). of (Mf/Mb) (Mf; free metal concentration; Mb, bound metal The curve shapes were dependent on the metal, concentration) and Mfobtained from dialysis experiments indicating different affinities (Fig. 1). Apparent overall stability constants (log K) varied from 3.15 to 3.65 / / • following the sequence: Pb > Cu > Cd > Zn. Mb/Mf Mf/Mb / // = f(Mf) distributions turn slightly from the linearity / / for low metal concentrations, which may indicate / / / more than one class of sites involved (Blaser et al. / / 1980).
y,
6 4
~.~'~"
6
/ //
5,
/ /
/
Discussion
/
•i /
•// /
o/
/
,i
Mf 2000
4000
6000
(ktM)
Fig. 4. Binding of Cu 2+ with a high molecular weight soluble root exudate fraction (HS): distribution isothe .r.m of (Mf/Mb) (Mr free metal concentration; Mb, bound metal concentration) and Mf obtained from ion-specific electrode titration Table 2. Maximum binding ability and apparent overall stability constant (log k) values calculated with the linear Langmuir equation
Method Dialysis
ISE
Pb
Cu
Cd
Zn
Cu
MBA m E . 100 g-1
160
194
198
206
84
Log k
3.65
3.4
3.35
3.15
3.65
0.97
0.99
0.98
0.92
0.99
r, correlation coefficient for the function Mf/Mb = f (Mf)
The soluble root product pool is composed of secretions, exudates and lysates (Rovira et al. 1979). Analytical studies of these compounds are generally relative to simple molecules such as sugars, organic acids and amino acids (Lespinat and Berlier 1975); Hale et al. 1978). Besides these substances, our results confirm that an important macromolecular fraction, made of polysaccharides, uronides and proteins (Juo and Stotzky 1970; Collet 1975) is present, suggesting that molecules within a wide range of molecular weight coexist in soluble root products. Assuming that molecules similar to HS are released into the rhizosphere of plants, axenic hydroponic cultures constitute a well-defined approach to investigate the role of these compounds in mineral nutrition. As shown by carbon-labelling experiments, stable rhizodeposition of maize during the growing season reaches about 2%-3% of the total carbon fixed by the plant. Twenty percent of the labelled root products, of which half have a molecular weight over 10 000-30 000, are soluble in water against only 1% of the stable organic matter (Hetier et al. 1980; Merckx 1983). Dialysis and potentiometry gave results which agreed with the existence of organometallic binding
168 involving HS and heavy metal ions, thus confirming previous results obtained from gel filtration experiments (Mench et al. 1985). MBA values are close to those of fulvic and humic acids (100-300 mEq 100 g-i Andreux 1979; Rainville and Weber 1981). They are higher than MBA values obtained in similar experimental conditions with maize root mucilages (5-24 mEq. 100 g-~; Morel et al. 1986). The higher protein or glycoprotein content and perhaps the lower polymerization level of HS compared with mucilages may be involved in these differences. Maximum binding ability variations between dialysis method and ISE potentiometric titration can be explained by differences in the experimental p H and ionic strength. Changes in these parameters can cause modifications in the conformational properties of macromolecules and the accessibility of binding sites. Moreover, metal ion complexation by natural organic compounds with no definite structure and whose binding sites present a large diversity of nature and o f affinity for metals cannot be described perfectly by one method or a simple equilibrium (Bizri et al. 1984). Affinity o f the different metals for HS-binding sites vary, following a sequence similar to that obtained with pectic substances (Morel et al. 1983), and root mucilages (Morel et al. 1986). The present state o f knowledge about the nature of the reactions between soluble root products and metal ions is rather poor. As humic compounds, HS includes macromolecules, presenting many possibilities in the number and nature of functional groups. Therefore, several mechanisms (adsorption, complexation, chelation, etc.) may be involved in the binding o f metals with HS, which could be due to salification o f u r o n i c acid and protein carboxylic groups. These metal-binding properties combined with a hydrosoluble and diffusable nature support the hypothesis that HS could act as a cation carrier in the vicinity of roots. Thus, HS could be able to increase cation mobility in the rhizosphere as complexing substances do (Elgawhary et al. 1970). Such potential action would be, however, dependent on their possibilities of diffusion in soil. Smaller molecules are probably the most efficient in the transfer of cations and it would be of great interest to determine the global binding properties of the low molecular weight fraction o f soluble products. Microorganisms are not uniformaly distributed into the rhizosphere (Foster 1983). They are particularly abundant along old parts of roots, while apex and elongation zones are relatively free of microbial colonization. Thus, soluble root products released from these zones may be not readily decomposed and can diffuse away from the root surfaces, playing an effective role as a metal carrier. However, immobilization
M. Mench et al.: Binding of metals with soluble root exudates of these substances in the soil by adsorption on clay minerals should be taken into account in the evaluation of the possibilities o f transfer back to the root surface. The metal ion binding properties of HS added to local conditions created by the root, and to potential acid and/or complexing properties of low molecular weight soluble root exudates (organic acids, amino acids, phenols), may strongly influence metal ion availability in the rhizosphere and may help to explain micronutrient solubility modifications in soil, observed after a culture (Linehan et al. 1985). It has be noted that major cations in soil, i.e. calcium and magnesium, decrease the binding of cadmium and zinc with root mucilages (Morel 1985). On the basis o f MBA it can be assumed that HS may compete with other ligands of the rhizosphere, like stable organic matter, mucilages and clay minerals. Further investigations with competitive ligands or metal ions would be o f interest. A better understanding of such processes could be o f great importance in the definition of culture rotations in contaminated environments, such as soil amended with sewage sludges, and for the prediction of quantities o f trace elements available to plants, which is based only on chemical extractability methods, which have frequently been found to be inadequate.
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yen
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Received April 2, 1986
