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The evolution of arsenate (the dominant plant-available arsenic species in aerobic soils) resistance has been determined in a range of higher plants (Meharg, 1993, 1994; Fitter et al., 1998). The genetic basis of this adaptation involves a major gene in all species investigated to date (Watkins & Macnair, 1991; Macnair et al., 1992), with this gene encoding for suppression of high-affinity phosphate/arsenate transport in grasses (Meharg & Macnair, 1992a,b). Arsenate is a phosphate analogue and is transported across the plasma membrane via high-affinity phosphate transporters (Meharg & Macnair, 1990, 1992a). The most detailed studies on the mechanisms of arsenate resistance have been conducted on the grass Holcus lanatus (Meharg & Macnair, 1990, 1992a; Hartley-Whitaker et al., 2001b). Constitutive suppression of high-affinity arsenate/phosphate transport results in arsenate resistance in H. lanatus by decreasing the rate of arsenate uptake (Meharg & Macnair, 1992b). This enables the plants to reduce arsenate to arsenite and complex this with phytochelatins (Hartley-Whitaker et al., 2001b). In nonresistant plants, arsenate influx is at too great a rate to achieve detoxification by phytochelatin (PC)-complexation, leading to considerable oxidative stress, which is not observed in resistant plants (Hartley-Whitaker et al., 2001a).
Many plant species adapted to grow on metalliferous soils are colonized by mycorrhizal fungi in these environments (Meharg & Cairney, 1999). Experiments on arsenate resistance in grasses have, to date, not considered the role of arbuscular mycorrhizal fungi (AMF), which are known to colonize H. lanatus roots on highly arsenic-contaminated mine spoils (Meharg & Cairney, 1999). Meharg & Cairney (1999) outlined the role that mycorrhizal fungi may play in adaptations of plant hosts to metal(loid)-contaminated soils. They proposed that mycorrhizal fungi need to be resistant, like their hosts, to the contaminated environment and: (1) that they may just be fulfilling their normal role in the association, not conferring enhanced resistance to the host; or (2) that they also further enhance resistance. Arbuscular mycorrhizal fungi are known to benefit the P nutrition of many plant species by increasing phosphate capture from soil via the enhanced surface area of the fungal hyphae emanating from the mycorrhizas (Smith & Read, 1997). Because AMF have a role in acquiring P for mycorrhizal associations, this may provide a paradox in achieving arsenate resistance as the AMF may potentially enhance arsenate uptake.
In the only detailed physiological explanation of the role of mycorrhizal fungi in adaptation of its hosts to toxic metal(loids), Sharples et al. (2000a,b) found that the ericoid mycorrhizal fungus Hymenoscyphus ericae was responsible for the colonization of arsenic-contaminated mine spoils by its host Calluna vulgaris. Unlike grasses, C. vulgaris does not achieve arsenate resistance through suppression of arsenate uptake (the mechanism of arsenate resistance in uninfected C. vulgaris has not yet been elucidated). However, its fungal symbiont H. ericae is capable of arsenate exclusion and therefore enhances the resistance of C. vulgaris to arsenate. The fungus achieves this resistance by reducing cellular arsenate to arsenite, and then effluxing the arsenite (Sharples et al., 2000a,b), which is the mechanism used to achieve arsenate resistance in bacteria and yeasts (Rosen, 1999). This is an elegant solution as the fungus can obtain P for the association at the exclusion of arsenate.
In this study, we investigated the role of AMF in arsenate resistance in arbuscular mycorrhizal associations. First, the resistance of AMF spores from arsenic mine spoils to arsenate was investigated. Next, the role of arsenate-resistant and nonresistant strains of the arbuscular mycorrhizal fungus Glomus mosseae in the short-term uptake kinetics of arsenate by arsenate-resistant and nonresistant H. lanatus roots was ascertained. Finally, the long-term effects of AMF colonization on arsenate resistance of H. lanatus growing on arsenic-contaminated mine spoil soil was determined.
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Evidence is presented that the AMF strains tested here, regardless of their arsenate resistance, reduced arsenate influx in both arsenate resistant and nonresistant H. lanatus plants. This strong suppression occurred even though plants had low levels of AMF colonization. This is in contrast to a comparable experiment on mycorrhizal and nonmycorrhizal tomato roots, where short-term phosphate influx was enhanced in AMF-colonized roots (Cress et al., 1979). It has been often noted that AMF colonization can improve P nutrition in pot experiments, whereas little difference is observed in colonized native plants under temperate field conditions (Fitter, 1991). Francis & Read (1995) outlined that AMF can have a neutral effect with respect to grass fitness. There is now an increasing body of evidence (Koide, 2000) highlighting an effect of AMF on plant reproduction, growing in natural systems, in P-limiting soils. Natural populations of AMF had no significant effect on the P uptake of field-grown Vulpia ciliata (Carey et al., 1982) but did increase seed production. Furthermore, seeds produced by mycorrhizal Avena fatua were lighter than those of nonmycorrhizal plants but contained more P (Lu & Koide, 1991; Koide & Lu, 1992).
The results presented here show that AMF colonization of H. lanatus suppressed the high-affinity phosphate transport system. This suppression may explain why AMF may not benefit the P nutrition of certain wild plants (Fitter, 1991). AMF can enhance the P-absorbing area of the root surface but in our experiments reported here, it appeared to downregulate phosphate transport in the host at least. It must be stressed that the AMF colonization levels in the experiments presented here were low. Under field conditions higher rates of colonization may improve the phosphate scavenging potential of the association, through increased hyphal biomass in soil, assuming that the AMF phosphate transporters are not downregulated in a similar manner to the plants. In our experiments, we could not distinguish between the fungal and plant contribution to arsenate uptake.
Because these kinetic studies were conducted on excised roots where external AMF hyphae may have been damaged or partly removed, the results of this experiment should be interpreted with this in mind, as more healthy external hyphae may have enhanced arsenate acquisition. Even though plant phosphate transport is downregulated, phosphate transport in AMF hyphae emanating from roots might not be downregulated. Nonresistant plants maintained higher levels of AMF than resistant plants for both arsenate-resistant and nonresistant AMF strains, implying that AMF may have a greater role in phosphate acquisition for nonresistant plants compared with resistant plants.
The mechanism by which AMF colonization downregulates phosphate/arsenate uptake in our experiments is not known. Molecular evidence shows that Mt4 cDNA is a phosphate-starvation inducible gene, which is downregulated in response to phosphate exposure and to AMF colonization (Burleigh & Harrison, 1999). The authors of that study hypothesized that Mt4 encoded for a signalling molecule involved in a phosphate starvation response. Furthermore, Liu et al. (1998) and Burleigh (2001) showed cDNA of MtPT2, which encodes for the high-affinity phosphate transporter, is downregulated in roots in response to AMF colonization.
Evidence for the mechanisms by which AMF downregulates arsenate/phosphate transport is found with the higher P concentrations in colonized roots compared with nonmycorrhizal roots when plants were grown in (contaminated) soil for 16 wk. The higher P levels in the root may have a role in regulating the kinetics of phosphate/arsenate uptake. Shoot phosphate levels, however, were lower and it is thought that shoot P status has an overall role in regulating P accumulation and metabolism in plants (Burleigh & Harrison, 1999). Transport of P to the shoots was decreased in plants colonized by AMF. The AMF may be the direct causal agent of decreased phosphate/arsenate transport through release of signalling molecules, rather than just simply altering the P status of the host. This hypothesis remains untested. The reduction in shoot P in colonized plants in the soil experiment presented here was also observed for field-grown arsenate resistant and nonresistant plants colonized by AMF when compared with nonmycorrhizal plants (Wright et al., 2000).
Arsenate resistance in H. lanatus is intriguing since, unlike other metal(loid) resistances, the gene for resistance and downregulation of phosphate/arsenate resistance is polymorphic in all populations investigated from uncontaminated (i.e. low As levels) soils (Meharg & Macnair, 1992c; Meharg et al., 1993, 1994b). There is evidence that this polymorphism is also present in other species, including both monocotyledons and dicotyledons (Fitter et al., 1998). It has long been considered that the rate-limiting step in phosphate uptake by plant roots is not the rate of transport across the plasma membrane, but diffusion of phosphate to the root surface since phosphate mobility in soil is low (Nye, 1977). This may partly explain why plants with suppressed phosphate uptake systems can compete effectively with those that have inducible uptake systems. Our present results indicate that AMF colonization of H. lanatus may make the nonresistant phenotype more like the resistant phenotype by making the differences in phosphate uptake kinetics less pronounced. Wright et al. (2000) argue that polymorphism is present in H. lanatus populations as a result of resistant and nonresistant plants having differing reproductive strategies, rather than differences in phosphate uptake per se. Resistant plants put more effort into reproductive growth while nonresistant plants put more effort into vegetative growth. However, it is thought that the differences in reproductive strategy in resistant and nonresistant phenotypes of H. lanatus is fundamentally related to P nutrition, with nonresistant plants being more P efficient than resistant plants (Wright et al., 2000).
It appears that AMF and their plant hosts have coevolved in arsenate-contaminated environments. While H. lanatus does not require its fungal symbiont to achieve arsenate resistance, its ability to grow on contaminated soils is greatly enhanced by being mycorrhizal. This is not because of enhanced P nutrition in mycorrhizal plants, as they had lower shoot P level. They did, however, have higher root P concentrations. It appears that AMF benefit the host on mine soils through a considerable reduction in arsenic uptake, particularly in the portion of arsenic translocated to the shoot. AMF colonization further reduces arsenate uptake compared with nonmycorrhizal plants, resulting in enhanced resistance. Furthermore, AMF have a wider role in the health of plants than just arsenate/phosphate uptake, including plant water relations and pathogen resistance of plants (Newsham et al., 1995). There remains the possibility that other benefits result from the mycorrhizal status, which may have helped to improve the health of colonized H. lanatus growing on mine spoil.
In the only comparable experiments on arsenate resistances in mycorrhizal associations, Sharples et al. (2000a,b, 2001) found that the ericoid mycorrhizal fungus H. ericae had a direct role in the arsenate resistance of its host C. vulgaris. This was through the ability of the fungus to efflux arsenite, while at the same time assimilating phosphate. Hymenoscyphus ericae has itself, similar to the AMF fungi under study here, evolved arsenate resistance. This is through reduced uptake of arsenate. The mechanistic basis of how arsenate-resistant AMF achieves arsenate resistance, and how it achieves reduced arsenate uptake by its resistant host is unclear. The fungus may have suppressed phosphate/arsenate uptake across its plasma membrane, which is the mechanism of arsenate resistance employed by most higher plants investigated to date (Meharg, 1993, 1994), or through enhanced efflux of arsenate, which is the mechanism employed by arsenate-resistant H. ericae (Sharples et al., 2000a,b). Unlike many ericoid mycorrhizal fungi, AMF cannot be cultured in the absence of the plant, and physiological studies on transport are restricted to germ tubes using radio-isotopes (Thompson et al., 1990). From the results presented here, it appears that one of the mechanisms employed by resistant AMF to reduce arsenate toxicity to its host is through further suppressing arsenate uptake by the association.