Adequacy of the root-organ culture system
The ROC system allowing the physical separation of a nonlabelled mycorrhizal root compartment from an extraradical mycelium ramifying in a neighbouring labelled root-free compartment has successfully been used in a number of experiments to study the uptake and translocation of phosphorus (Joner et al., 2000b; Nielsen et al., 2002), as well as uranium (Rufyikiri et al., 2002b). In most experiments, the hyphal biomass produced in the labelled compartment was low, < 5 mg f. wt, caused by a limited number of hyphae crossing the partition between the two compartments (Joner et al., 2000b; Rufyikiri et al., 2002b). This rather restricted hyphal development could have resulted in an underestimation of the effect of hyphae on element uptake and translocation and could have masked the effect of hyphae on the chemical characteristics of the growth medium such as pH. Indeed, Rufyikiri et al. (2002b) reported that the pH of the solution was not significantly affected by the hyphal development.
In the present study, the ROC system was improved by extending the gelled medium 2 mm above the physical separation between the CC and the EC’s. Around 147 hyphae crossed the partition between the two compartments increasing by sixfold the hyphal biomass reported in previous studies (Joner et al., 2000b; Rufyikiri et al., 2002b).
Impact of mycelium and root development on pH
The considerable mycelium development observed in this study resulted in a marked alkalinization of the solution, while roots induced an acidification under the same growth conditions. The pH changes of growth media are a common phenomena related to imbalances in the uptake of cations and anions (Marschner, 1995). In hydroponic culture conditions, it was shown that a net proton excretion resulting in an acidification occurs when excess cations were absorbed over anions, while alkalinization resulted from a net OH− excretion because of an excess uptake of anions over cations (Rufyikiri et al., 2001). The root-induced pH modification of growth media is well documented for both nonmycorrhizal and mycorrhizal plants (Li et al., 1991; Rufyikiri et al., 2000; Hinsinger, 2001), but few data are available on the effects of extraradical hyphal on the pH of the growth medium. Recently, Bago et al. (1996) used a technique of pH indicator bromocresol purple and observed a similar pH increase induced by the extraradical hyphae of G. intraradices in the presence of NO3−-N as source of N, but not in media lacking this N-form. It was suggested that the pH increase was a consequence of the active uptake of NO3−-N involving the NO3−/H+ symport or NO3−/OH− antiport mechanisms used by the fungus for nitrate uptake. These mechanisms would mask any other hyphal-promoted acidification, resulting in a net alkalinization. In the present study, the modification of pH was also an active process for both roots and hyphae because such an effect was not observed when their metabolic activity was inhibited by formaldehyde added to the solution. Because U speciation in aqueous systems as well as in soil is pH-dependent (Grenthe et al., 1992) and considering differences between U species in their uptake by plants (Ebbs et al., 2000), and uptake and translocation by fungal hyphae (Rufyikiri et al., 2002b), the modification of pH is a physiological process by which growing mycelium and roots can influence the U bio-availability.
Comparison of hyphae and roots on U uptake and translocation
The findings of higher CEC in fungal mycelium than in roots observed in this study corroborated previously reported values (Joner et al., 2000a). The larger U concentration in fungal mycelium than in roots suggests that the exchange sites significantly contributed to the U uptake, as shown by the differences of Cu-extractable U between the mycelium and roots. However, sequential extractions of U with CuSO4 and HCl showed that only a small fraction of U taken up was released from roots and hyphae, while these procedures allowed us to extract most of the contents in Ca and Mg. The reason might be that U fixed on exchange sites served as starting point for precipitation or complexation reactions with various anions. The formation of stable complexes or precipitates was likely the main mechanism of U accumulation in both roots and fungal hyphae in contact with U in the external compartments. This is assumed to contribute to the low translocation of U as especially observed for roots. However, the measurements carried out in this study did not allow us to identify these nonextractable U forms. Similar root accumulation in nonexchangeable dominant forms was reported for Al in roots of banana (Musa spp.) plants (Dufey et al., 2001; Rufyikiri et al., 2002a) and roots of Abies amabilis (Dahlgren et al., 1991). These authors suggested that various mechanisms may be involved including a high affinity with the root exchange sites, complexation and precipitation with organic compounds such as oxalate or with inorganic compounds such as phosphate, and polynuclear hydroxyl formation.
The larger U concentration in the mycorrhizal roots grown in the RHC vs the nonmycorrhizal roots grown in the RC could probably be explained by U uptake mechanisms more active in the mycorrhizal roots than in the nonmycorrhizal ones, and/or a marked contribution of the intraradical hyphae to the accumulation of U in the host roots. A high concentration of U in intraradical fungal hyphae, of an undefined AM fungal species, than in the host root tissues was previously reported (Weiersbye et al., 1999), probably as a result of particular chemical conditions prevailing in the intraradical fungal cells. Large P concentration in the intraradical parts of AM fungi was recently observed (Pfeffer et al., 2001; Nielsen et al., 2002), while intracellular pH varying between 5.6 and 7.0 was reported for hyphae of G. intraradices (Jolicoeur et al., 1998). Both high P concentration and weakly acidic to neutral pH are factors which can promote the formation of U-phosphate complexes and precipitates in the intraradical hyphae, and thus favouring the U accumulation in mycorrhizal roots.
The improvement of the culture system with a large hyphal biomass production in the external compartments resulted also in a large U translocation by the extraradical fungal hyphae in comparison with the results reported previously (Rufyikiri et al., 2002b). Whatever the mechanisms involved in the U translocation, being active or passive, increasing the number of hyphae crossing the partition between the two compartments would result in a proportionally higher U translocation (Rufyikiri et al., 2002b). However, the flux of elements in hyphae may depend, not only on absorbing hyphae, but also on other factors such as metabolic control related to the demand of the host plant and may differ from one element to another (Cooper & Tinker, 1978).
Considering hyphae and roots as cylinders with an average diameter of 11 ± 2 µm for a hyphae (Nielsen et al., 2002) and 1000 µm for a root (measured in this experiment), the total cross section area at the partition between the two compartments (A) was calculated as A= (diameter/2)2 × π × number of hyphae/roots. The A was c. 0.014 mm2 for the average 147 hyphae and 3.93 mm2 for the five roots. Although the total section area of roots was 281-fold larger than what was observed for hyphae the U translocation by roots was lower than its translocation by hyphae. This indicates that U flux rate was larger in hyphae than in roots, perhaps as a result of differences in interactions or exchange reactions between U and cell components as well as in further transport mechanisms involved.
The effect of formaldehyde added to the solution to control capillary action was similar to the experiment reported in our previous paper (Rufyikiri et al., 2002b). The comparison between data for living and formaldehyde-killed hyphae or roots indicated that the hyphal and root U concentration and translocation were influenced by the metabolic activity of fungal hyphae and roots. Killing them has resulted in increasing U concentration and in limiting U translocation from the EC to the EC. As previously reported for hyphae (Rufyikiri et al., 2002b), the U accumulation by the formaldehyde-killed hyphae and roots was caused by only passive mechanisms such as the adsorption on exchange sites of hyphae and roots, while for living hyphae, active mechanisms were also involved to control the U absorption. High capacity of U adsorption of hyphae for killed hyphae than for living ones was also reported in another study (Joner et al., 2000a). The absence of detectable amounts of U in roots developing in the CC and its presence in negligible amounts in the gel, although killed hyphae and roots in the EC remained attached to the CC, as observed at harvest, indicate that if capillary action existed, its contribution was certainly negligible.
In conclusion, these results are the first to determine the uptake and translocation of U by roots under strictly controlled ROC conditions and to compare them to those of the AM fungal hyphae. They demonstrated that the extraradical hyphae of G. intraradices have a relatively higher capacity to take up and to translocate U than carrot roots, while the intraradical hyphae may play an important role in the U immobilization by mycorrhizal roots.