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Keywords:

  • arbuscular mycorrhizal (AM) fungus;
  • Ri T-DNA transformed roots;
  • uranium (U) uptake;
  • uranium translocation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Uranium (U) uptake and translocation by the arbuscular mycorrhizal (AM) fungus Glomus intraradices were studied under root-organ culture conditions with Agrobacterium rhizogenes -transformed carrot ( Daucus carota ) roots as host.
  • • 
    Two-compartment Petri plates were used to spatially separate a root compartment (RC) and a hyphal compartment (HC); root growth was restricted to the RC while extraradical hyphae grew in both RC and HC. The HC was labelled with 0.1 µM 233 U at different pH conditions. At the end of the experiment, U was measured in the RC and in the HC.
  • • 
    The U absorption by the AM fungus was observed. It included; U uptake by the mycelium developing in the HC, and U translocation from the HC to the RC. The magnitude of this uptake and translocation was highly influenced by the pH of the growth medium, while translocation was highly correlated with the number of hyphae crossing the partition separating the two compartments.
  • • 
    These results are the first to show that an AM fungus can take up and translocate U towards roots.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Mining and milling of uranium (U) ores have produced considerable amounts of radioactive waste materials. In local areas, improper waste-storage practices have led to U contamination of the surrounding environment at levels thereafter requiring remedial actions (Abdelouas et al., 1999; Shahandeh et al., 2001). As well as chemical and physical methods suggested for the management and restoration of U contaminated areas (Abdelouas et al., 1999; Vandenhove et al., 2000), the use of plants and associated microbiota to remediate U-contaminated sites, so called phytoremediation, is a method nowadays considered with great interest, because it is not harmful to the environment (Shahandeh et al., 2001). However, the success of any option of phytoremediation, for instance the phytostabilization, is highly dependent on the knowledge and understanding of all parameters and processes involved in U soil–plant transfer.

Several studies with U involving plants with soils (Sheppard et al., 1989; Mortvedt, 1994; Huang et al., 1998) as well as with nutrient solutions (Ebbs et al., 1998) have been conducted, but often without considering the effects of plant symbiotic microflora on U uptake. However, in the field, roots of most plant species are associated with microorganisms that can have either a direct or an indirect effect on U availability. Among the soil microflora, arbuscular mycorrhizal (AM) fungi are root symbionts forming association with 80–90% of all seed plant species (Harrison, 1997). In this association, the extraradical mycelium plays an important role in the absorption of essential nutrients (Smith & Read, 1997; Martin et al., 2001) as well as nonessential heavy metals (Colpaert & Vandenkoornhuyse, 2001; Leyval & Joner, 2001). AM fungi could also influence radionuclides acquisition by plants and hence radionuclides bio-cycling, as was reported for 137Cs (Entry et al., 1999; Berreck & Haselwandter, 2001) and 90Sr (Entry et al., 1999). The contribution of AM fungi to U bio-availability has rarely been reported. Uranium concentration has been shown to be higher in intraradical structures (vesicles) of an undefined species of AM fungi than in the host root tissues (Weiersbye et al., 1999), but information on the role of AM fungi on U uptake process and particularly on U transport by fungal hyphae to plant roots is lacking.

Whatever the radionuclide considered, the difficulty to maintain experimental systems void of undesirable organisms other than the two symbionts and the interferences with soil particles, which may complicate radionuclide availability, has impeded clear identification of the role of AM fungi in uptake and translocation of radionuclides. Root-organ culture systems, associating excised roots with AM fungi on synthetic growth media have been used to study various aspects of the symbiosis (Fortin et al., 2002). It was demonstrated that the spatial separation of mycorrhizal roots and extraradical mycelium ramifying into a root-free compartment (St-Arnaud et al., 1996) was a convenient area to study the uptake and translocation of phosphorus (Joner et al., 2000b; Koide & Kabir, 2000; Maldonado-Mendoza et al., 2001; Nielsen et al., 2002) and could presumably be applied to radionuclides.

The objective of this study was to assess the AM fungal contribution on U uptake and translocation under root-organ culture conditions and to determine unambiguously the role of the extraradical fungal mycelium on U uptake and translocation under various pH conditions.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Biological material

Glomus intraradices Schenk and Smith (MUCL 41833) was used for the experiment. Root-organ cultures were established in association with Agrobacterium rhizogenes (Ri T-DNA)-transformed carrot ( Daucus carota L.) roots as described by Declerck et al. (1998 ). Routine maintenance of roots and AM fungi was made on the modified Strullu-Romand (MSR) medium ( Declerck et al., 1998 ; Strullu & Romand, 1986 ), but solidified with 3 g l −1 Gel Gro™ (ICN, Biomedicals, Inc., Irvine, CA, USA) instead of 8 g l −1 Bacto agar. After 3 months, several thousand spores were obtained in each Petri plate and used for the experiment.

Experimental design

A system was devised to study the translocation of U from a compartment containing hyphae to a compartment containing roots. The system consisted of a 50-mm Petri plate glued without cover in a 140-mm Petri plate, thus separating a root compartment (RC) and a hyphal compartment (HC); root growth was restricted to the RC while extraradical hyphae grew in both RC and HC (Fig. 1). Carrot roots (approx. 70 mm length) were introduced in the RC containing 15 ml MSR medium, and inoculated with approximately 50 spores of G. intraradices. The Petri plates were incubated horizontally in an inverted position at 27°C in the dark for 3 wk. Thereafter, they were set upright for an additional week and the HC were filled with 100 ml liquid MSR without sucrose and vitamins. Hyphae started to cross the partition between RC and HC and proliferated in the liquid medium. Roots that crossed the partition between the two compartments were trimmed to leave the HC void of roots. At this stage, the cultures were ready for experimental manipulation.

image

Figure 1. Schematic representation of a two-compartment in vitro growth system allowing the spatial separation of a colonized root, developing in the root compartment (RC), from an extraradical mycelium ramifying into a neighbouring hyphal compartment (HC). The modified Strullu-Romand (MSR) medium was gelled in the RC, while the HC contained liquid MSR medium lacking sucrose and vitamins, but labelled with 0.1 µM 233 U (1: front view; 2: upper view). Adapted from Joner et al. (2000b ).

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Uranium treatment

Liquid MSR medium (100 ml) without sucrose and vitamins, but labelled with U at a concentration of 0.1 µM, the main isotope being 233U, was added to the HC after removing the old medium with a pipette. The source of U was a solution of uranyl nitrate (IRMM-040a Spike Isotopic Reference Material) supplied by EC-JRC-IRMM (Mol, Belgium) with a specific activity of 352 KBq α-emission mg−1 U (and < 0.009 KBq βγ-emission) and an isotopic composition with the following mass fraction: 98.02%233U, 0.918%234U, 0.216%235U, 0.024%236U and 0.821%238U. The pH of the solution was adjusted to 4.0, 5.5 and 8.0 with 0.01 M NaOH before sterilization at 121°C for 15 min. These pH values were chosen to evaluate the effects of U speciation on U uptake or translocation by the AM fungus since U speciation is known to be pH-dependent (Grenthe et al., 1992). The total U concentration was fixed at a value where no precipitate at any pH was possible, as predicted by the speciation calculation with the geochemical computer code, The Geochemist's Workbench® (Bethke, 2001). For each pH treatment, metabolic activity of hyphae was inhibited in a half of Petri plates by formaldehyde (2% v/v) added to the solution in the HC, 24 h before U was supplied. This control treatment was included to determine if U uptake and translocation by hyphae was an active process. For each treatment, the hyphae were kept in contact with U-labelled solution during 2 wk. Root-free compartments of non-mycorrhizal cultures were also filled with U-labelled solution as controls for the possible contamination of the RC caused by experimental manipulations. Each treatment was replicated six times.

Assessment of variables

At the end of the experiment the AM fungal development estimated by the total extraradical fungal mycelium length and the number of spores in both RC and HC, and the total root length in the RC were assessed using a 1-cm grid of lines marked on the bottom of each Petri plate to form 1 cm squares. Vertical and horizontal lines were observed under binocular microscope and the presence of root and hyphae recorded at each point where they intersected a line. The total root or hyphae length, expressed in cm, was determined using the formula of Newman (1966). Spores were also counted in each cell formed by the grid to obtain the total numbers in each Petri plate.

The solution was sampled for pH and U activity measurements, and thereafter removed from the HC. The HC was rinsed twice with 100 ml distilled water before the hyphae and spores developing in this compartment were collected. For the RC, roots with intraradical fungal structures (mycorrhizal roots) and the gel with extraradical fungal biomass were separately collected. The different parts were then placed in 20-ml glass scintillation vials, weighed, dried at 60°C for 4 d, calcined at 500°C for 24 h, and ashes were dissolved in 0.1 M HCl. Ten ml of liquid scintillation cocktail were added to 5 ml aliquots of all solutions, and U activity determined by liquid scintillation counting with a counting efficiency of 100% and a detection limit of 0.03 Bq. Counts were corrected by subtraction of background levels of 0.06 ± 0.02.

Samples of dry roots were cut into 10 mm segments length, cleared in 10% KOH, and stained with 0.1% Trypan blue for measurement of root AM fungal colonization (Phillips & Hayman, 1970). Fifty randomly selected segments were examined under microscope. The frequency of AM fungal colonization (%F) was calculated as the percentage of root segments colonized by either hyphae, arbuscules or vesicles. In addition, the intensity of colonization (%I), that is, the abundance of hyphae, arbuscules and vesicles in each mycorrhizal root segment, was determined using the method outlined by Declerck et al. (1996).

Statistical analysis

Statistical analysis of data was performed with the statistical software STATISTICA for Windows (StatSoft, 2001). Significant differences were considered at P= 0.05, and mean values were ranked by Scheffé's multiple-range test when more than two groups of data were compared by ANOVA, or t-test paired method when only two groups of data were compared.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

pH and U speciation

The pH of the solution in the HC did not significantly change during the experiment. All U minerals in the liquid MSR had negative saturation indexes indicating that the system was undersaturated, that is, no precipitation occurred (Fig. 2a). The calculated U speciation was highly pH-dependent (Fig. 2b). Uranyl cation and uranyl-sulfate species were dominant in the solution at pH 4, phosphate and hydroxyl species at pH 5.5, and hydroxyl and carbonate species at pH 8.

image

Figure 2. The Uranium (U) solubility and speciation as a function of pH in the liquid modified Strullu-Romand (MSR) medium assuming an equilibrium with a partial pressure of CO 2 (g) of 10 −3.5 atm. (a) U saturation indexes (log Q / K , where Q is the solubility product and K the solubility constant with precipitation when QK ). (b) Calculated concentration of dominant U species. Total dissolved U, 0.1 µM. The calculation was made with the geochemical computer code, The Geochemist's Workbench ® ( Bethke, 2001 ), using the thermodynamic data compiled by Grenthe et al. (1992 ).

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Root biomass and intra- and extraradical AM fungal structures development

At the end of the experiment, root length and root f. wt in the RC were in the range 173–192 cm and 0.63–0.78 g f. wt per Petri dish, respectively, but did not significantly differ between the pH treatments. A relative root length increase of 94–95% was observed, demonstrating the vigour of root growth on the MSR medium. High root AM fungal colonization was observed (%F = 77–81 and percentageI= 24–28), and hyphae length and number of spores developing in the RC varied in the range 840–965 cm and 3228–3493, respectively, but none of these three parameters significantly differed between the pH treatments. Some hyphae, in the range of 7–13, crossed the partition between the two compartments, and a good branched mycelium with spores developed in the HC. Hyphae length, spore numbers and fungal f. wt in the HC were in the range of 98–113 cm, 311–352 spores, and 3.4–4.2 mg per Petri dish, respectively, but did not significantly differ between pH treatments. With formaldehyde added in the solution, hyphae were killed in the HC, and no further development was observed. Hyphae length and f. wt in the HC were in the range of 68–89 cm, and 1.2–2.2 mg per Petri dish, respectively, and no spores were formed.

233U activity

Uranium activity measured in the AM fungal structures, that is, hyphae and spores, developing in the HC was significantly higher at pH 5.5 than at pH 4 and pH 8 (Table 1). The U concentrations were higher for the fungal biomass than for the labelled solution at both the three pH values, on the f. wt basis and considering a volumic density of the solution of 1. Uranium was also found in the RC, that is, in the gel with fungal biomass and in the mycorrhizal roots. Uranium content was higher in the gel with fungal biomass than in the mycorrhizal roots, but its concentration was higher in the latter due to differences in weight between the two components. In both cases, U activity was significantly higher at pH 4 than at pH 5.5 and pH 8. Uranium translocated from the HC to the RC was positively correlated with the number of hyphae crossing the partition between the two compartments, for all pH treatments with linear regression coefficient r2 of 0.86, 0.83 and 0.58 at pH 4, pH 5.5 and pH 8, respectively. The total U absorption, that is, the sum of U taken up in AM fungal structures in the HC and U translocated to the RC in the gel with fungal biomass and mycorrhizal roots via the hyphae, represented 2.2, 1.4 and 0.9% of initial U supply at pH 4, pH 5.5 and pH 8, respectively. Uranium content in the mycorrhizal roots accounted for 12–25% of the total U absorption. Uranium was not detected in the RC for nonmycorrhizal cultures or in the roots developing in the RC for cultures receiving formaldehyde added to the solution in the HC.

Table 1.  Uranium activity contents (Bq per Petri plate) and concentrations (Bq g −1 f. wt) in the hyphal compartment (HC), that is, the solution, hyphae and spores, and in the root compartment (RC), that is, the gel with fungal biomass and the mycorrhizal roots for Ri T-DNA transformed carrot ( Daucus carota ) roots grown in association with Glomus intraradices in a two-compartment system (see Fig. 1 ) with 0.1 µM 233 U added to the HC set at pH 4, 5.5 and 8
 U contentU concentration
 pH 4pH 5.5pH 8pH 4pH 5.5pH 8
  1. The initial U supply was 833 Bq per Petri plate. Values are averages of six replicates and values in parentheses indicate percentages of initial U supply. nd, not detectable. Within rows, averages followed by the same letter a, b or c for U content, and x, y or z for U concentration are not significantly different (P = 0.05).

Without formaldehyde
 Solution800 (96) a809 (97) a817 (98) a8.0 x8.1 x8.2 x
 Hyphae and spores0.4 (0.05) c6.7 (0.81) a4.2 (0.50) b139 z1855 x839 y
 Gel with fungal biomass14.1 (1.7) a3.3 (0.39) b1.1 (0.14) c1.6 x0.3 y0.2 z
 Mycorrhizal roots4.0 (0.48) a1.4 (0.17) b1.8 (0.21) b5.3 x2.2 y2.6 y
With formaldehyde
 Solution831 (99.8) a830 (99.6) a832 (99.8) a8.3 x8.3 x8.3 x
 Hyphae and spores0.6 (0.07) b0.87 (0.10) a0.9 (0.11) a511 x635 x643 x
 Gel with fungal biomass0.09 (0.01) a0.08 (0.01) a0.07 (0.01) a0.006 x0.005 x0.005 x
 Mycorrhizal rootsndndndndndnd

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The two-compartment in vitro growing system (Joner et al., 2000b) used in the present study was particularly convenient since it avoids interference with soil particles thus allowing to control precisely the form and the concentration of U available to AM hyphae. The results obtained demonstrated, for the first time, that U could be translocated by AM fungal hyphae towards roots, and this translocation was assured only by living hyphae. However, at this stage, we could not determine if U was transferred into the root cells or if it was mainly immobilized in the intraradical fungal structures. Some recent findings suggest a high concentration of U in intraradical AM fungal structures (Weiersbye et al., 1999), probably due to chemical conditions differing between intraradical fungal cells and host root cells. Large differences in P concentration between the intraradical parts of AM fungi (with high concentration) and the host root cells or extraradical hyphae were recently observed (Pfeffer et al., 2001; Solaiman & Saito, 2001). Furthermore, intracellular pH varying between 5.6 and 7.0 was reported for hyphae of G. intraradices (Jolicoeur et al., 1998), and a pH of approx. 6 was reported for the cell wall of rhizodermal cells of roots (Marschner, 1995). Both high P concentration and weakly acidic to neutral pH are factors which may favour the formation of U-phosphate complexes and precipitates in intraradical AM fungal structures, and thus restricting its transfer from the fungus to root cells. A restricted transfer of elements from fungus to root cells due to fungal immobilization was also hypothesized for Cd (Joner & Leyval, 1997), and other metals (Kaldorf et al., 1999), but the mechanisms involved remain unclear and are probably diverse (Colpaert & Vandenkoornhuyse, 2001).

Little information is available in the literature concerning the effect of U speciation on the U bio-availability (Ebbs et al., 1998), although the pH-dependence of U speciation in aqueous systems and in soil has been extensively studied. We did a U speciation calculation using the most up-to-date and coherent U thermodynamic data (Grenthe et al., 1992). Our results support other studies reporting that U speciation is highly pH-dependent (Langmuir, 1978; Mortvedt, 1994; Ebbs et al., 1998). However, considerable uncertainty exists in the value of the formation constant of the UO2(OH)2 (aq) species. Other values for this species may be possible (Silva et al., 1995), and if used may affect the predicted U speciation in the pH range of 6–8. The influence of the U speciation was shown for both U uptake and translocation by the mycorrhizal fungus. It seems that soluble uranyl cations or uranyl-sulfate species that are stable under acidic conditions were translocated to a higher extent through fungal tissues, while phosphate and hydroxyl species dominating under acidic to near neutral conditions or carbonate species dominating under alkaline conditions were rather immobilized by hyphal structures. The effect of pH and U speciation was also reported on U uptake in roots and translocation from roots to the shoots of plants grown in nutrient solution (Ebbs et al., 1998). These authors found highest U content and concentration in shoots at pH 5.0 when U was presumably taken up predominantly as the free uranyl cations, while at this pH, the U content and concentration in roots were the lowest.

The increase of element contents in AM fungi may result from many mechanisms including the metabolism and incorporation in tissues, as demonstrated for essential nutrients such as P and N (Pfeffer et al., 2001), precipitation of nonessential metallic cations in the fungus assumed to occur with PO4 (Turnau et al., 1993) and adsorption on negatively charged constituents of fungal tissues (Joner et al., 2000a). In the present study, most exchange sites of fungal hyphae were probably saturated by H+ at pH 4, and this may result in the low UO22+ adsorption on hyphae. This had probably contributed to the low U content in hyphae at this pH, while its translocation was facilitated. Low rate and extent of bio-sorption of metals at low pH were also reported in another study (Gadd, 1990). By contrast, rising the pH would increase negative charges by deprotonation of constituents of fungal tissues, with enhancement of metallic cation adsorption. However, for U, increasing the pH led to the formation of neutral and even negatively charged species at alkaline conditions. Thus, high pH would impair the bio-adsorption of U. Following this, we could hypothesize that the uptake of U by fungal hyphae in the hyphal compartment was due to its precipitation in fungal structures, especially at pH 5.5 where phosphate species were dominant.

The comparison between data for living and formaldehyde-killed hyphae indicated that the hyphal U concentration was influenced by the metabolic activity of fungal hyphae because killing them resulted in increasing U concentration at low pH and in decreasing U concentration at high pH. The increased U content of the formaldehyde-killed hyphae was exclusively due to passive mechanisms such as the adsorption on exchange sites of hyphae, while for living hyphae, active mechanisms of absorption were also involved. Since the adsorption capacity of hyphae can considerably differ between living and killed hyphae, as it was also reported in another study (Joner et al., 2000a), the respective contribution of absorption and adsorption processes on U content in hyphae could not be determined by simple comparison between data for living and killed hyphae.

The two processes discussed above, that is, U uptake in fungal biomass and translocation by fungal hyphae to roots, are interesting aspects for phytostabilization strategies of U polluted soils. They indicate that AM fungi could contribute to the immobilization of U either in the hyphosphere or in the mycorrhizosphere or in roots, with possible effect in decreasing U dispersion in the environment. Further experiments could quantitatively evaluate the relative importance of this fungal contribution to the U immobilization, for instance by comparing U uptake and translocation by hyphae and by plant roots.

In conclusion, this study has provided, for the first time, fundamental information indicating that the AM fungus G. intraradices can take up and adsorb U, and translocate it to roots. The uptake and adsorption of soluble U by the AM fungus were shown by the amount of U observed in the fungal biomass developing in a U-labelled compartment (HC). The presence of U in a neighbouring compartment initially U-free (RC) demonstrated that U was translocated within the fungal hyphae towards roots, as the contact between the two compartments was mediated only by fungal hyphae. The magnitude of U uptake or translocation by the AM fungus appeared to be influenced by the U speciation which is highly pH-dependent. A next step would be to enhance the sink strength of the mycorrhizal host using entire plants grown in vitro or in vivo, and to determine possible changes in U sequestration by roots or in U transfer to shoots linked to the presence of AM fungus in roots.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the Belgian Nuclear Research Centre (SCK°CEN) and the EU-MYRRH project contract-CT-2000–00014 ‘Use of mycorrhizal fungi for the phytostabilization of radio-contaminated environments’. S. Declerck gratefully acknowledges the financial support from the Belgian Federal Office for Scientific, Technical and Cultural affairs (OSTC, contract BCCM C2/10/007) and thanks the director of MUCL for the facilities provided and for continual encouragement. The authors are grateful to C. Bivort for technical support in root-organ cultivation.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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