Plant and Soil

, Volume 327, Issue 1, pp 1–21

Arsenic uptake and toxicity in plants: integrating mycorrhizal influences

Authors

    • Soil and Land Systems, School of Earth and Environmental Sciences, Waite CampusThe University of Adelaide
  • Helle M. Christophersen
    • Soil and Land Systems, School of Earth and Environmental Sciences, Waite CampusThe University of Adelaide
  • Suzanne Pope
    • Soil and Land Systems, School of Earth and Environmental Sciences, Waite CampusThe University of Adelaide
  • F. Andrew Smith
    • Soil and Land Systems, School of Earth and Environmental Sciences, Waite CampusThe University of Adelaide
Marschner Review

DOI: 10.1007/s11104-009-0089-8

Cite this article as:
Smith, S.E., Christophersen, H.M., Pope, S. et al. Plant Soil (2010) 327: 1. doi:10.1007/s11104-009-0089-8

Abstract

Arsenic (As) contamination of soil and water is a global problem that impacts on many areas of biology. This review firstly covers aspects of soil chemistry and soil-plant interactions relevant to the ways plants take up As (particularly arsenate (As(V)) from aerobic soils, with especial attention to As-phosphorus (P) interactions. It then assesses the extent to which studies of plant As tolerance based on short-term uptake of As(V) from nutrient solutions can be extrapolated to longer-term growth in contaminated soil. Mycorrhizal symbioses are then highlighted, because they are formed by ~ 90% of higher plants, often with increased uptake of phosphate (Pi) compared with non-mycorrhizal (NM) counterparts. It is therefore likely that mycorrhizas influence As(V) uptake. Published work shows that arbuscular mycorrhizal (AM) plants (the most common mycorrhizal type) have higher P/As ratios than NM plants, and this would be expected to affect sensitivity to soil As. We discuss ways in which higher P/As selectivity might result from differential operation of P and As uptake pathways in AM compared with NM plants, taking into account new understanding of P uptake mechanisms. We also give suggestions for future research required to increase understanding of mechanisms of As(V) uptake, and its interactions with plant P.

Keywords

ArsenicPhosphateMechanisms of plant toleranceMycorrhizasSoil toxicity

Introduction

Contamination of soil and water by arsenic (As) impacts on many areas of soil biology. It is now recognised as a serious threat to human health, as a consequence of consumption of contaminated plant material (Fitz and Wenzel 2002; Meharg 2004; Meharg and Hartley-Whitaker 2002; Smith et al. 1998). Furthermore, As toxicity towards plants may pose threats to plant establishment, particularly in revegetation of contaminated sites. Previous research aimed at addressing these problems has emphasised two contrasting aspects of As accumulation: 1) minimising As uptake and hence toxicity and contamination of foods, and 2) bioextraction of As in contaminated soils by hyperaccumulation in shoots (McGrath and Zhao 2003; Tripathi et al. 2007). Prediction of outcomes of different plant and soil management practices depends on understanding the forms of As in soil and their availability, as well as mechanisms by which As is absorbed, metabolised and detoxified by plants. Zhao et al. (2008) have recently reviewed what is known about such mechanisms, with a brief section on the role of mycorrhizal fungi. The present contribution gives mycorrhizas more attention, because they are symbioses formed by the vast majority of land plants under both natural and managed (agricultural and forest) conditions. Thus, mycorrhizas – not non-mycorrhizal (NM) roots – are the normal nutrient absorbing organs of most land plants, and it is well established that mycorrhizas play major roles in acquisition, particularly of phosphorus (P) in the form of H2PO4 (Pi) (Smith and Read 2008). Arsenate (As(V)) is an analogue of Pi, so it might be expected that mycorrhizas would enhance uptake of both. Because of the As(V)/Pi analogy it might also be expected that a role of mycorrhizas in As tolerance would be different to heavy metal tolerance, which we therefore do not address here. A clear picture of underlying mechanisms has not yet emerged, so here we summarise the findings so far and discuss useful directions for future research, including measurements that need to be included in future experiments to help ensure better understanding of the roles played by the fungal symbionts. This approach is particularly relevant because recent work has shed new light on the ways in which the fungal and root pathways for nutrient uptake are integrated and regulated (Bucher 2006; Javot et al. 2007; Smith et al. 2003a, b, 2004; Smith and Read 2008).

This article goes beyond Zhao et al. (2008) by covering background information on As availability and speciation in aerobic soils and considering the extent to which experiments with plant material in nutrient solutions can be extended to plants grown in soil. We then review work that has explicitly addressed roles of mycorrhizas in As uptake and metabolism by plants.

Forms of arsenic in aerobic soils

The chemistry of As in soils is complex, and As can be present in both inorganic and organic forms (Quaghebeur and Rengel 2005). Availability to soil microorganisms and plants, particularly relevant to uptake, is influenced by environmental factors that include soil redox potential, pH, composition (including clay mineralogy, organic content, the presence of Fe‐ and Al-oxides and hydroxides, and other elements), and microbial activity generally (Smith et al. 1998; 2006; Ultra et al. 2007a, b; Vetterlein et al. 2007). Importantly, addition of fertiliser-P can mobilise adsorbed As, though the extent of mobilisation is again heavily dependent on underlying soil chemistry (Vetterlein et al. 2007). The inorganic forms of As, As(V) and arsenite (As(III)), usually dominate in As-contaminated soil, and in aerobic soils the oxic conditions favour the presence of As(V) over As(III). The As(III) in herbicides and pesticides is oxidised to As(V) (Smith et al. 1998, 2006).

Measurements of speciation in aerobic soil (or artificial soil-like media) initially containing As(V) made after plants had been grown experimentally have shown in general that As(V) still predominates over As(III) (Ultra et al. 2007b; Vetterlein et al. 2007). However, use of compartmented pots has shown relatively high As(III) levels in rhizospheres compared with bulk soil. Ultra et al. (2007a, b) suggested that rhizosphere accumulation might be caused by enhanced As transformation by a relatively high density of microbes, or by mass flow of As(III) from bulk soil associated with water uptake. The latter suggestion is feasible because the soil used was relatively high in water-soluble As(III) as well as As(V). Vetterlein et al. (2007) suggested that the cause might be reduction of As(V) caused by low redox potential in microsites around roots, or release of As(III) from roots. Xu et al. (2007) showed rapid efflux of As(III) into nutrient solution from roots supplied with As(V). They suggested that this is part of a detoxification mechanism, and that roots and soil microbes are likely to be engaged in ongoing As(V)/As(III) reduction/oxidation, with considerable As(III) cycling between roots and soil. We consider their results in more detail below.

Absorption of arsenic species by plants

Uptake of As(V) and As(III) has been extensively studied in plants, and some attention has been given to the uptake of organic species (Zhao et al. 2008 and references therein). The uptake of As(III), which is predominantly undissociated below about pH 8, is believed to occur passively through membrane aquaporins (Ma et al. 2008; Meharg and Jardine 2003; Zhao et al. 2008). The suggestion that As(III) uptake into rice is active, i.e. energy-dependent, can be discounted, as it was based entirely on the fact that uptake versus concentration showed saturation kinetics (Abedin et al. 2002b). Unsurprisingly, attention on uptake of As from aerobic soils has focused on As(V) rather than As(III).

Physiological and electrophysiological experiments have shown that As(V) competes weakly with Pi for uptake (Asher and Reay 1979; Esteban et al. 2003; Hurd-Karrer 1939; Lee et al. 2003; Pickering et al. 2000; Ullrich-Eberius et al. 1989) (see Fig. 1). These studies were made with excised roots, root pieces and (to a smaller extent) intact plants, all in solution culture. The major disadvantage of using excised roots is the disruption of the signalling from shoots to roots. This is unlikely to be a confounding factor in short-term experiments with material grown under P-starvation, but shoot/root signalling will be significant in longer-term studies. For example, Clark et al. (2000) showed that after 12 h exposure of Arabidopsis to As(V) the biggest effect was on Pi transport to the shoot. They suggested that changes in P-related signals from the shoot might influence high-affinity Pi uptake. Such effects would also be expected in soil-grown plants.
https://static-content.springer.com/image/art%3A10.1007%2Fs11104-009-0089-8/MediaObjects/11104_2009_89_Fig1_HTML.gif
Fig. 1

Diagrammatic representation of arsenic(As)/phosphate (P) interactions related to plant uptake. Active fluxes of Pi and As(V) inwards at the epidermis via a common transporter are indicated by a black square. The suggested active efflux of arsenite [As(III)] is indicated by a grey square. Other fluxes (of unknown mechanisms) are indicated by open circles. As(III)-phytochelatin interactions are also indicated (As(PC)). Methylated forms of As may also be present but are not shown. Pathways that do not occur in (sterile) culture solutions are marked by a dashed line (----)

There are certainly differences in the interactions between Pi and As(V) for uptake by plants grown hydroponically compared with plants grown in soil or soil-like substrates. In soil, the interactions depend on Pi and As(V) availability, as affected by underlying soil chemistry, so that addition of fertiliser-P can increase or lower plant growth (Woolson et al. 1973). Release of adsorbed As(V) can occur following high applications of P, potentially increasing uptake of both (Fitz and Wenzel 2002). However, because growth is often reduced in the presence of As, elevated P concentrations, whether plants are grown in solution or soil, may actually be due to the lower biomass (i.e. so-called ‘tissue concentration’, using ‘concentration’ in a different sense) that occurs in smaller plants.

Although some investigations have shown Pi/As(V) competition for uptake (e.g. Pigna et al. 2009), other investigations give no evidence for competition. Abedin et al. (2002a) found no effect of additional Pi on As(V) uptake by rice in flooded soil (P contents were not provided); these results contrasted with short-term uptake from nutrient solution (Abedin et al. 2002b). Christophersen et al. (2009) found that additional Pi ameliorated As toxicity in soil-grown Medicago truncatula (medic) and Hordeum vulgare (barley) but had no effect on the specific uptake of As(V) (i.e. uptake per g root). In this case the amelioration may have been due to the much higher tissue P concentrations at the higher external P level, with the result that P was able to outcompete As in metabolic reactions. Gunes et al. (2009) showed that increased P supply in soil had no effect on the P/As ratio in chickpea (Cicer arienatum) at low external As and only a small effect at higher As, again indicating little or no competition between As(V) and Pi for uptake from soil.

Only recently have there been molecular investigations of the expression of genes encoding transporters for Pi uptake that are needed to throw light on P/As interactions beyond the physiology of uptake (see Zhao et al. 2008). Work with Arabidopsis mutants has confirmed that Pi transporters also mediate As(V) uptake (Shin et al. 2004). Plants generally possess more than one (commonly two) epidermal Pi transporters involved in uptake from soil. Their relative selectivity for Pi and As(V) warrants attention in the context of interpreting some data obtained before molecular biology of membrane transport was developed.

Efflux of arsenic

Just as Pi can be effluxed into nutrient solutions (Bieleski and Lauchli 1992; Clark et al. 2000), As efflux would also be expected (Fig. 1). Efflux of As(V) is likely to be relatively low when, as is normal, As(V) in roots is rapidly reduced to As(III) which is either converted to organic As or transported to shoots. However, efflux of As(III) can be substantial (Fig. 1). This was shown by Xu et al. (2007) with tomato and rice plants grown hydroponically and supplied with As(V). Extensive As(III) efflux has also been shown with other species, including Holcus lanatus, Arabidopsis thaliana, wheat, barley, and maize (unpublished work cited by Zhao et al. 2008). There is little efflux from the As-hyperaccumulator Pteris vittata (Su et al. 2008). Xu et al. (2007) suggested that As(III) efflux is active (energy-dependent, i.e. not passive though aquaporins) because it was partly inhibited by the uncoupler carbonylcyanide-m-chorophenylhydrazone (CCCP). This is unconvincing, because CCCP also increased efflux of As(V), probably a result of decreased internal reduction to As(III) and hence a lower internal concentration of As(III) available for passive efflux; nevertheless, active efflux cannot be ruled out (Fig 1). Xu et al. (2007) also suggested that rapid re-absorption of As(III) could falsify the assumption that ongoing As uptake from aerobic soils is dominated by As(V) uptake. They provided no evidence for rapid As(III) re-absorption. However, it is possible that re-absorption was decreased by toxic effects that are suggested by relatively high net As(V) efflux after 24 h, presumably due to impaired metabolism resulting in re-oxidation of internal As(III). Given the possibility of As(III) re-absorption in plants that remain healthy, we agree with Xu et al. (2007) that constant As concentrations over 32 d in the sunflower plants used by Raab et al. (2007) do not indicate lack of As(III) efflux, as was claimed. We do not consider that ongoing As(III) re-absorption – if it occurs – will complicate interpretation of mechanisms of As uptake from aerobic soils dominated by As(V), especially because 1) any such re-absorption of As(III) can only occur following previous As(V) uptake, and 2) there is no evidence that, in aerobic soils containing mainly As(V), As(III) comes to dominate the soil As content even in rhizospheres (see Vetterlein et al. 2007). This point is important seeing that while uptake of As(V) and Pi involves competition for common transporters, As(III) and Pi do not compete (Abedin et al. 2002b; Wang et al. 2002). In fact, any such re-absorption of effluxed As(III) could negate As detoxification in plants grown aerobically. Re-absorption of As(III) from soil would of course be prevented if it is rapidly taken up and immobilised by rhizosphere microbes or re-oxidised to As(V).

Tolerance to arsenic involving uptake of arsenate and phosphate

Tolerance of the grass H. lanatus and some other plants to As(V) is known to be associated with relatively low uptake of As(V) compared with uptake into non-tolerant plants (see Abedin et al. 2002a; Meharg and Hartley-Whitaker 2002; Zhao et al. 2008 and references therein). In the pioneering study with H. lanatus, Macnair and Cumbes (1987) showed that unrooted tillers of tolerant (T) clones grew longer roots than non-tolerant (NT) clones when exposed to As(V) in nutrient solution, and after 7 d growth in the absence of As took up less As(V) over the following 7 d. They proposed that As tolerance is caused by an altered Pi uptake system that has very large selectivity against As(V). Meharg and Macnair (1990) showed that influx (i.e. uptake/g root/unit time, in this context) of 32Pi (up to 40 min) into excised T roots in low-P solution (0.1 mM or lower) was considerably lower than influx into NT roots, as was influx of As(V). They concluded that T plants had suppressed high-affinity Pi uptake, and that this was an important mechanism of As tolerance in H. lanatus. This conclusion was supported by the similar Ki for inhibition of As(V) influx by Pi in both NT and T plants (Meharg et al. 1994a). It was proposed that low-affinity Pi uptake could maintain Pi uptake adequate to allow growth of T plants. Overall, however, results have shown that initial short-term 32Pi influxes into excised T roots from plants of different provenance vary widely, and that differences from NT roots can be large, small, or negligible because the gene involved is polymorphic. Thus, Hartley-Whitaker et al. (2001) showed that after 7 d growth in low As(V) (25 μM), there was no difference in As content (amount per plant) between the NT and T plants that they chose. It is also worth noting that although the T plants can accumulate higher As content than NT plants, because the latter become unhealthy and have shorter life-spans, it is not clear from the short-term studies how T plants could accumulate higher concentrations (i.e. As/g tissue) (cf Hartley-Whitaker et al. 2002).

Quaghebeur and Rengel (2003) grew H. lanatus (from the same sources as previously used) with As(V) for 24 d. With 10 μM Pi and only 1.33 μM As(V) (there was no As-free treatment) both NT and T plants grew well. The T plants took up less As(V), as expected from the previous work, but contained about 12% more P than the NT plants so that their P/As ratio was considerably higher than the NT plants. These results suggest that the T plants took up Pi by high-affinity transport with a higher selectivity against As(V) than in the NT plants. The possibility that lower As(V) accumulation by T plants involves a high rate of As(III) extrusion seems ruled out by short-term uptake kinetics of As(V) (Meharg and Macnair 1992c) and by the recent finding that As(III) efflux is proportional to As(V) uptake in both NT and T plants (Logoteta et al. 2009).

Surprisingly, experiments with H. lanatus grown in soil or similar media do not provide evidence that growth of T plants over the long term involves significant suppression of high-affinity Pi transport. Meharg et al. (1994a) showed that P concentrations in Holcus growing in both mine and non-mine sites depended both on extractable soil P and As levels; there was no clear inherent difference between T and NT plants. Gonzalez-Chavez et al. (2002) showed that short-term As(V) influx into T roots taken from plants grown in attapulgite clay for 4 months was lower than influx into NT roots (again as expected), but they did not measure Pi influx. Meharg et al. (1994b) found that T plants grown in sterile As-free and low-P potting compost for up to 13 weeks grew slightly less well than NT plants, but had higher P content. Wright et al. (2000) found that after 2 years growth in As-free soil T plants were larger than NT plants, and again had higher P content. This is all unexpected based on the results of short-term Pi uptake experiments, as Wright et al. (2000) pointed out. They suggested possible limitation of Pi uptake into both T and NT plants by diffusion in soil solution (as also proposed by Meharg and Macnair 1992c); however, it is not clear how this would cause the T plants to grow better than the NT plants. Likewise, it is not obvious that – as was suggested – accumulation of shoot P in NT plants would produce potentially toxic effects on growth, given that shoot P concentrations were lower than in T plants. Wright et al. (2000) also hypothesised that 1) the reproductive output and early flowering in T plants resulted from higher shoot P concentrations, despite the (proposed) absence of high-affinity Pi uptake, and 2) suppression of high affinity uptake itself results from the higher shoot P concentration, by means of a feedback. However, such feedback cannot apply to short-term Pi uptake into P-starved excised roots from T and NT tillers that initially have the same P concentrations (Meharg et al. 1994b). We have dealt with these issues in some detail because suppression of high-affinity Pi uptake in soil-grown T plants could be very relevant to the possible role of AM symbioses in As tolerance.

Measurements with Arabidopsis mutants are starting to add new information about As tolerance mechanisms that are associated with changes in transport capacity of As(V) and Pi. The ars1 mutant shows increased tolerance not by decreased As(V) uptake but by increased Pi uptake, suggesting that higher internal P outcompetes As in metabolic reactions (Lee et al. 2003), as mentioned above for soil-grown barley (Christophersen et al. 2009). Interestingly, ars1 had lower uptake of Zn, which can upregulate expression of high-affinity Pi uptake (Huang et al. 2000). Clearly, any such upregulation in ars1 involves Pi transport that is highly selective against As(V), which seems a possibility in other As-tolerant plants, given that most plants possess several Pi transporters whose functions may overlap.

Catarecha et al. (2007) describe a mutant of the Arabidopsis Pi transporter PHT1;1 which they claim allows the mutant to accumulate more As than the wild-type, while having a lower As(V) influx. However, when measurements were made the wild-type was suffering extreme stress and growth depression, probably resulting in greatly inhibited uptake of Pi and also As(V). Total P contents were not measured (or at least not given). Short-term influxes of Pi and As(V) at an earlier stage were both lower in the mutant (but by only about 20%). In the absence of a more detailed time-courses of As(V) and Pi uptake in the mutant and wild-type it is unfortunately not possible to explain the eventual higher As concentrations in the latter.

Arsenic metabolism in plants, distribution between roots and shoots and hyperaccumulation

This area of research has recently been reviewed (Quaghebeur and Rengel 2005; Zhao et al. 2008). In consequence, we cover these topics only briefly here. After As(V) is taken up, it is rapidly reduced to As(III), most probably enzymically via arsenate reductases (Duan et al. 2007; Ellis et al. 2006). The current consensus is that reduction occurs largely in roots and that As(III) is the major form transported to shoots. In consequence, unlike uptake from soil, there will be no competition between P and As in root-to-shoot transfer. Detoxification mechanisms for As(III) include efflux from the roots (see above), sequestration in cell vacuoles and complexation with thiols such as phytochelatins (PCs), for which As(III), unlike As(V), has very high affinity. Small amounts of methylated As species (monomethylarsonic acid: MMA, dimethylarsinic acid: DMA, and trimethylarsine oxide: TMAO) are also found in plants, originating either in soil or produced internally. Quantitatively the methyated species are less important than either As(III) or PC-As(III) complexes.

Accumulation of As in plants and its toxic consequences are the result of the relative contributions of different processes of avoidance and detoxification. For example, T lines of H. lanatus synthesise higher quantities of PCs, leading to higher PC:As concentration ratios internally (Hartley-Whitaker et al. 2001; 2002). This almost certainly contributes to tolerance to As in long-term growth experiments. There are also marked differences between As hyperaccumulators and non-accumulators. Hyperaccumulators are almost all found in the fern genus Pteris, the best known being P. vittata. These plants are capable of accumulating enormous amounts of As in their above-ground fronds without apparent toxic effects (up to ~ 22 g/kg has been measured in a pot experiment with P. vittata (Ma et al. 2001)). Furthermore, very little As(III) is effluxed by roots (Su et al. 2008) or bound to PCs (Zhao et al. 2003) in hyperaccumulators. These differences are neatly summarised diagrammatically by Zhao et al. (2008, see their Fig. 3). The mechanisms of extreme tolerance in the face of massive concentrations in these plants remains a mystery. Nevertheless, the hyperaccumulating ferns have been suggested to be strong candidates for bioremediation programs, so that the extent to which environmental conditions influence transfer of As from soil to above-ground parts (Bioconcentration Factor: BF) and from roots to shoots (Transfer Factor: TF) has received a good deal of attention (McGrath and Zhao 2003), including in mycorrhizal plants (Trotta et al. 2006).

Arsenic toxicity in plants

The symptoms of As toxicity in plants frequently include poor seed germination and very marked reductions in root growth, with effects influenced both by the concentration and availability of As and P. These effects may relate to rapid disruption of plasma membrane structure, including fluidisation, as shown for algal cells and artificial liposomes, with external As(V) concentrations greater than about 0.1 mM (Tuan et al. 2008). The possibility of direct effects of external As(V) on root growth adds uncertainty to longer-term toxic effects conventionally interpreted as a result of As(V) influx and disruption of P metabolism. Arsenic-related changes in plasma membrane structure do not explain differences in Pi influx between T and NT H. lanatus that occur in the absence of external As(V) (Meharg and Macnair 1990). However, they may help explain large differences in growth of young roots in 0.133 mM As(V) that occur in both T and NT plants, and a rather weak correlation (R2 = 0.35) between such growth and short-term As(V) influx (Meharg and Macnair 1992a, b). If plants survive, they may show reduced growth, nutrient deficiencies and chlorosis, resulting from reduced chlorophyll biosynthesis (Mascher et al. 2002; Singh et al. 2006), as well as reduced photosynthetic oxygen evolution (Ullrich-Eberius et al. 1989). Arsenic-affected rice exhibits ‘straighthead disease’ in which increased sterility reduces yield (Wells and Glilmour 1977).

As already indicated, P supply has frequently been shown to affect As toxicity in soil-grown plants. Thus, As depressed growth of several different plants at high levels of As and low levels of P, but at high levels of P or at low levels of As, growth can actually be stimulated (Anastasia and Kender 1973; Knudson et al. 2003; Mascher et al. 2002). Reductions in root growth impact on uptake of nutrients and water, so that nutrient deficiencies may not be direct effects of As toxicity itself, but a consequence of reduction of the root surface area available for uptake. Mechanisms that increase the capacity of roots to acquire nutrients, such as mycorrhizal symbioses, may compensate for As-induced reductions in root growth.

Once absorbed, As interferes with essential P metabolism because enzymes involved in P conversions sometimes also convert As(V). However products of the latter are usually relatively unstable (Westheimer 1987). Internal As(III) if not effluxed to the soil or converted to organic form (see above), binds to sulfhydryl groups and inactivates some enzymes (Ullrich-Eberius et al. 1989). As(V) reduction can also result in formation of reactive oxygen species and lipid peroxidation, with consequent cellular damage (Geng et al. 2006; Gunes et al. 2009; Hartley-Whitaker et al. 2001; Mascher et al. 2002). Lipid peroxidation is higher in plants that are more sensitive to As (Meharg and Hartley-Whitaker 2002; Singh et al. 2006), possibly reflecting decreased membrane stability (Singh et al. 2006). Such damage can change the selectivity and permeability of plant cell membranes, limit water movement and alter nutrient uptake and transport (Paivoke and Simola 2001), which may, together with effects resulting from poor root growth, explain changes in the plant’s mineral balance. The complexity of P and As interactions in soil and during uptake, and effects of As on metabolism and growth of plants need to be borne in mind when considering possible interactive effects of mycorrhizal symbioses, to which we now turn.

Mycorrhizas - overview

There are four main types of mycorrhiza, all of which have been shown to be involved in plant nutrient acquisition. Overall, approximately 90% of land plants are potentially mycorrhizal, distributed in all major terrestrial biomes (Smith and Read 2008). Most work on As has been done on arbuscular mycorrhizal (AM) plants, because this is the most widespread mycorrhizal type, involving an enormous number of plant species including trees, shrubs and herbs. Almost all emphasis has been on As(V) because it is generally believed that mycorrhizas are rare in wetland plants which would be exposed to As(III). However, there are exceptions (see Ipsilantis and Sylvia 2007; Khan and Belik 1995, and references therein) and more work, for example on paddy and upland rice, would be warranted. Less attention has been paid to ericoid mycorrhizas (typical of many heathland plants) or to ectomycorrhizas (formed by many trees in the Pinaceae, Fagaceae, Dipterocarpaceae and related families). Nevertheless, plants growing on contaminated sites (including crops) or proposed for revegetation programs are likely to be potentially mycorrhizal, so that investigations of the roles of the symbioses in As uptake and interactions with P nutrition are essential.

Arbuscular mycorrhizas - physiological background

AM symbioses are biotrophic, based on bidirectional transfer of nutrients: sugars from plant to fungus and mineral nutrients, particularly P, from fungus to plant. The fungi are obligate symbionts, completely dependent on the supply of recent photosynthate (Smith and Read 2008). The effects of AM colonisation on plant P status and growth are highly variable, ranging from very large positive increases (in so-called ‘responsive’ plants) to neutral or negative (in ‘non-responsive’ plants) (Johnson et al. 1997; Smith et al. 2009; Smith and Read 2008). When a plant is positively responsive to AM colonisation, disparate sizes of AM and NM plants complicate interpretation of changes in concentrations of elements other than P (Table 1). Comparisons of amounts of As taken up by AM and NM plants will be confounded by the size difference, which includes not only total biomass but also root surface available for uptake. In investigations of effects of As, it is not appropriate to manipulate soil conditions by applying extra P to NM plants to create plants that are ‘matched’ with AM counterparts in size and/or internal P concentration; such applications would invalidate comparison with AM plants, because of the different P/As ratios externally. Furthermore, internal As concentrations may be decreased by ‘tissue dilution’ in larger AM plants (Lambert et al. 1979) and will not provide a good surrogate for total As content as has sometimes been assumed (Table 1); content should be calculated from As concentration and plant dry weight and can be used to provide P/As ratios and hence ‘long-term’ selectivity. Specific uptake (based on uptake/unit weight of root) is also useful in providing a complete picture of As(V) and Pi uptake (Table 1). Differences in P availability in soil modify the magnitude of the mycorrhizal responses and influence colonisation of the roots, so it is particularly important to evaluate responsiveness in the absence of As. However, this approach is not possible in experiments using naturally contaminated soils. Such complexities must be taken into account in design and interpretation of experiments to reveal effects of AM colonisation on As uptake and toxicity in plants, particularly when the interactions between As(V) and Pi in soil and during uptake by plants are under investigation.
Table 1

Some issues that need to be taken into account when interpreting data on effects of mycorrhizal colonisation on As uptake, accumulation and toxicity in plants (see text)

 

Issue

Affected by

Comments

1

Large (responsive AM) plants not good comparators for smaller (NM) plants

Plant identity, fungal symbiont and P supply

Be aware of problems, see 2 and 3 below

Large root systems may take up large amounts of P or As (per plant)

Not appropriate to add extra P to produce ‘matched plants’ as this would change P/As ratio in soil

2

Tissue concentrations of P and As not good surrogates for uptake

Mycorrhizal and P responses (‘big and little plants’, see 1) As-induced reductions in growth

Elements may be ‘diluted’ by increased growth Concentrations of elements including P may be increased in small plants

3

Total uptake of P and As useful for comparisons of uptake selectivity

Dry weight and tissue concentrations

Values required for comparisons of total amounts taken up

4

P/As ratio (based on totals in plant)

Pathways and amounts of P and As uptake

Reveals competitive effects and selectivity with different P and As treatments and/or relative contributions of AM to As and P uptake; molar basis helpful

5

Specific uptake or inflow (uptake on basis of root weight or length)

Total uptake and root growth

Values take into account changes in root growth as a result of treatment Values allow comparisons of uptake efficiency of different elements, especially if calculated on molar basis

6

Determine AM responsiveness in absence of As

Relative effects of As on AM and NM plants

Cannot be done in naturally contaminated soils: be aware of potential problems

Effects of As on AM fungal colonisation and function

7

% root length colonised

Fungal and root growth

Reductions in root growth (e.g. by As) may increase % values

Other possible contaminants/P

Increases in root growth (e.g. by P) decrease % values

Inhibition of fungal growth decreases % values

8

Contribution of AM to uptake of P (occurs in both responsive and non-responsive plants)

Development of AM Uptake and transfer processes in fungus and plant

Cannot be determined by calculating (M-NM), as this does not take into account ‘hidden’ uptake or changes in root tissue involved in uptake

Can only be effectively measured using 32/33P in compartmented pots

9

Contribution of AM uptake of As

See 7

As delivery to plants in compartmented pots (As only in hyphal compartment) or use 73As

Mycorrhiza formation in arsenic-contaminated soils

The first question to be asked before considering effects of AM symbiosis on As uptake is: are arbuscular mycorrhizas formed in As-contaminated soils? The answer is ‘yes’. Propagules of AM fungi do survive and persist in soil at As-contaminated sites, resulting in colonisation of plants (Leung et al. 2005; Meharg et al. 1994b; Meharg and Cairney 2000). Gonzalez-Chavez et al. (2002) assessed spore production in trap cultures in As-contaminated mine-spoil, and showed that a reasonably diverse population of AM fungi was present. They studied effects of As on germination of a few selected species. Of these, spores from the mine-site populations were less sensitive to As than those isolated from uncontaminated sites, leading to the conclusion that AM fungi may become adapted to As contamination, partially explaining survival in contaminated soils. However, work of this type needs to be extended to encompass a wider range of sites and AM fungal taxa. Some experiments have used inoculum of AM fungi sourced from contaminated sites, which would presumably have comprised adapted fungi (Al Agely et al. 2005; Gonzalez-Chavez et al. 2002; Knudson et al. 2003; Leung et al. 2005). However, although such inoculum was clearly infective, its effectiveness in comparison with that composed of AM fungi from uncontaminated sites has not been systematically explored and most investigations have used the latter. The fact that non-As-selected fungi survive experimental As application indicates that they are generally fairly resistant to short-term As toxicity, but the mechanisms have not been investigated.

Most experiments are also consistent in showing that presence of As does not reduce the percentage of the root length colonised by AM fungi. H. lanatus became equally colonised in As-contaminated mine soil and uncontaminated soil (Gonzalez-Chavez et al. 2002), and many experiments with a number of different plant species and several different AM fungi have shown no reductions in percent colonisation when As was artificially added to soil (Christophersen et al. in press; Al Agely et al. 2005; Chen et al. 2007; Liu et al. 2005b; Trotta et al. 2006). Development of external mycelium (when measured) is likewise little affected by As application (Leung et al. 2005; Pope 2006). However, there are reports of reduction in colonisation in medic (Pope et al. 2007), apple (Malus sylvestris) (Trappe et al. 1973) and tomato (Liu et al. 2005b) (the last at a very high As application rate), and also an increase in P. vittata (Al Agely et al. 2005). Because mycorrhizal assessments are expressed as a proportion of root length, both increases and decreases may be the result of changes in root growth rather than direct effects of As on the fungi (Table 1). However, despite this proviso, it is clear that As alone does not eliminate AM colonisation, and there is considerable potential for the symbiosis to play a part in the responses of plants to As in contaminated soils.

A note of caution is required with respect to research using contaminated field soil. Such investigations clearly have significant field relevance, but mine-spoils in particular may contain several toxic elements, and observed effects may not be related to the presence of As. Thus, Chen et al. (2005) surveyed a number of sites with multiple contamination (including As) and found that many potentially AM plant species were not colonised or had only low levels of colonisation (see also Meharg and Cairney 2000). The likelihood is that reductions were caused, not by As, but by one or more of the other soil contaminants. The same applies to the work on apple (Trappe et al. 1973), because both As and Pb had been applied to the soil as lead arsenate pesticide. Involvement of AM symbioses in responses to As may be negated where colonisation is very low or absent. Even if As is the only toxic element present, use of contaminated soil does not allow zero-As controls to be included in experimental designs. Nevertheless, differences in P/As ratios in AM versus NM plants are a good indicator that AM colonisation alters the pattern of As(V) uptake from such soils. Design of effective experiments to unravel mechanisms by which arbuscular mycorrhizas alter plant responses to As requires an understanding of how AM colonisation modifies the pathways of plant Pi uptake as well as growth (see below) in the absence of As. This point may seem obvious, but appears to have been overlooked in some investigations.

New insights into phosphate uptake in arbuscular mycorrhizas - potential influence on arsenate uptake

Knowledge of the mechanisms of Pi uptake in AM plants is required in order to predict how the symbioses may interact with As(V) uptake. It is well recognised that AM plants have two pathways through which Pi is absorbed from the soil solution (Fig. 2). In the direct pathway, Pi is taken up by root hairs and epidermis, as in NM plants. Such uptake can result in depletion of Pi in the soil solution close to the roots because the influx is initially greater than the rate of replacement, due to diffusion limitations in soil. The low concentration in the rhizosphere will reduce subsequent Pi influx and may increase competition from As(V). The AM pathway involves uptake by the external hyphae and translocation to the plant through fungal hyphae and transfer across specialised symbiotic interfaces in root cortical cells (Smith and Read 2008) (Fig. 3). This pathway overcomes severe diffusion limitation of Pi uptake, because the external hyphae scavenge Pi at considerable distances from the root surface. In addition, fungal translocation of P is extremely rapid, bypassing the sharp depletion zones close to roots and delivering P direct to root cortical cells (Smith and Read 2008). The mechanism of P translocation is not fully clarified, but almost certainly involves movement of polyphosphate from sites of Pi absorption to sites of breakdown and transfer to plant cells (Ezawa et al. 2002). The mechanism of As translocation (if any) is a significant issue to be considered with respect to As(V) uptake by mycorrhizal plants. However, polyarsenate is not formed (because of the inherent low stability) and therefore As could not be translocated in the same way.
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Fig. 2

Diagrammatic representation (not to scale) of the direct and mycorrhizal (AM) Pi uptake pathways in an AM root. The direct pathway involves uptake of Pi (and also As(V)) via Pi/As(V) transporters in root hairs and epidermis. The mycorrhizal pathway involves uptake of Pi by fungal Pi transporters in external hyphae, translocation of P (as polyphosphate) along the hyphae over considerable distances and transfer to the plant. The mechanism of efflux of P to the interfacial apoplast is not known. Uptake by the plant involves AM-inducible Pi transporters expressed in root cortical cells (see Fig. 3 for more detail). The roles of fungal and AM-inducible Pi transporters in As transfers are not known (see text and Fig 3). Depletion of P and As in the rhizosphere not shown. Plant Pi transporters black rectangles: fungal Pi transporters: grey rectangles

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Fig. 3

Diagrammatic representation (not to scale) of transfer of P from soil to plant cell via the mycorrhizal pathway (black arrows) and possible pathways of As transfer. Pi uptake from soil, via a fungal Pi transporter (1: grey box) and transfer along external mycelium as polyphosphate (2). Efflux of P from the fungus to the interfacial apoplast (mechanism unknown: 3) and uptake by plant cortical cell by an AM-inducible Pi transporter in the plant interfacial membrane (black box: 4). Possible pathways of As(V) uptake via the same fungal Pi transporter (5), reduction to As(III) and efflux to soil (6), transfer of As through hyphae in an unknown form (As?: 7) and efflux to the interfacial apoplast (8) and thence to the plant cell (9) shown by grey broken arrows

The conventional view has been that the AM and direct pathways of Pi uptake act additively to provide P to plants, and that colonisation by the fungi does not affect the amount of Pi absorbed via the direct pathway (Smith et al. 2001). In AM-responsive plants, where there are large increases in total P, the AM pathway has long been recognised to make a large contribution to Pi uptake, providing ‘extra P’ conventionally determined by subtracting totals in NM plants from those in AM plants, grown under the same conditions (AM-NM> > 0). Importantly, values based on total content per plant cannot be used to estimate the relative contribution of direct and AM pathways because of increased size of root systems (and hence tissue available for direct Pi uptake) (Table 1). Comparisons of specific Pi uptake (or inflow based on root length) in AM and NM plants eliminate this problem but also do not indicate how much each pathway is contributing. In non-responsive plants where the total P in plants is unaffected by AM colonisation, the direct pathway has again been assumed to make the same contribution as in NM plants. In other words, the fungi appeared to provide no extra Pi and the AM pathway of uptake was considered to be non-functional (AM-NM = 0). Using these conventional arguments, it would be expected that As(V) uptake via the direct pathway would be the same (per unit weight or length of root) in NM and AM plants regardless of their responsiveness, and that the rate of uptake would depend on 1) the As(V) concentration in solution at the uptake surfaces, 2) the kinetic characteristics of the uptake system(s) (Pi/As(V) transporters), and 3) on any competition between As(V) and Pi for uptake (see above).

New research has altered the interpretation of how the AM and direct Pi uptake pathways are integrated and controlled, and hence on the way that Pi and As(V) uptake may interact (Fig. 2) (Bucher 2006; Smith and Read 2008; Smith et al. 2003b; 2004). It has now been repeatedly demonstrated, using radioactive P (32P or 33P) supplied as Pi to AM fungal hyphae in compartmented pots, that the fungal symbiont makes large contributions to Pi uptake in non-responsive as well as responsive plants (Grace et al. 2009; Li et al. 2006; Smith et al. 2004 and see references in Smith et al. 2009). In non-responsive symbioses P delivered to the plant via the AM pathway remains ‘hidden’ (not apparent as extra P or increased specific uptake) unless radioactive Pi is used as tracer (Table 1). Separate Pi transporters play key roles in the direct and AM uptake pathways, with AM-inducible plant transporters expressed in colonised cells in the root cortex of both responsive and non-responsive plants (Bucher 2006; Javot et al. 2007; Rausch et al. 2001; Smith et al. 2003a) (see Fig. 3). In the latter, where there is no increase in total plant P (and hence in specific uptake, because there is no change in growth), operation of the AM pathway must be accompanied by a decreased contribution of the direct pathway. Quantitative measurements of the fungal contribution demonstrate that reductions in direct uptake also occur in responsive plants (Smith et al. 2003b; 2004). Mechanisms underlying this reduction could include down-regulation of expression of Pi transporters located in the root epidermis and root hairs (direct pathway) and/or Pi depletion in rhizosphere consequent on rapid uptake by the roots. Some investigations do indicate that expression of Pi transporters in the direct pathway is reduced in AM roots (Burleigh et al. 2002; Glassop et al. 2005; Harrison et al. 2002; Liu et al. 1998; Paszkowski et al. 2002; Rausch et al. 2001; Christophersen et al. in press), but other investigations show no such effect (Grace 2008; Grace et al. 2009; Karandashov et al. 2004; Nagy et al. 2005; Poulsen et al. 2005). Reduction in epidermal Pi transporter expression would result in reduced As(V) uptake, but As(V) depletion in the rhizosphere does not occur to any great extent, so is unlikely to be a significant factor in reducing As uptake in AM plants (Ultra et al. 2007b; Vetterlein et al. 2007).

Details of the ways in which the two Pi uptake pathways are functionally integrated and controlled are still unclear, but will have considerable bearing on effects of AM colonisation on As(V) uptake. There are several important questions. First, are reported AM effects on As tolerance the result of compensation by the fungal pathway for poor root growth? Second, are substantial amounts of As taken up into roots via the AM pathway, as is sometimes assumed (Gonzaga et al. 2006) or does this route deliver only P? Last, is As(V) uptake via the direct pathway lower in AM plants, and is any lowering a consequence of reduced expression of Pi transporters? Only partial answers to these questions have been obtained, as follows.

Effects of AM on plant growth in the presence of arsenic and on arsenic uptake and accumulation

Responsive plants

Most investigations of AM colonisation on As(V) uptake and As toxicity have been carried out with AM-responsive plants. In these, the symbiosis consistently ameliorates effects of As toxicity, and plants generally show increases in growth compared with NM controls grown at the same As and P supplies in soil (Ahmed et al. 2006; Covey et al. 1981; Pope et al. 2007; Ultra et al. 2007b; Xia et al. 2007). In some cases, additional measurements have also demonstrated increases in total (µg/plant) and specific Pi uptake in AM plants. In the same experiments effects on total As(V) content in AM plants were (when measured) quite variable, with values higher than in NM plants observed in both roots and shoots of maize (Xia et al. 2007) and lower values in sunflower, medic and lentil (Lens culinaris) (Ahmed et al. 2006; Pope 2006; Ultra et al. 2007b). Chen et al. (2007) point out that increased total As(V) content in responsive AM plants may not solely be the result of transfer via the AM pathway, because more extensive root systems in the larger plants could also make an important contribution on a whole plant basis. Concentrations of As are frequently lower in AM plants, possibly reflecting tissue dilution of As in the larger plants, rather than reductions in uptake per plant (see Table 1). Increases in P/As ratio (relative to NM controls) have been almost invariably observed (as noted by Zhao et al. 2008), which probably result from increased Pi uptake via the AM pathway in the responsive AM plants. Specific As(V) uptake (where this is presented or can be calculated from the data) was either unaffected or lower in AM plants in the investigations quoted above (with the exception of maize grown at high P: Xia et al. 2007). However, the lower values could contribute to increased P/As ratios and As tolerance and agree with findings from short-term uptake experiments which showed lower As(V) influx in excised roots of AM plants compared with those of NM plants of NT H. lanatus (Gonzalez-Chavez et al. 2002). In this experiment the AM pathway would not have been operational in As(V) uptake, because the external mycelium was removed during preparation of the excised roots.

Non-responsive plants

In contrast, AM-non-responsive species show variable outcomes when confronted with As. NM basin wild rye (Lymus cinereus) showed reduced growth in the presence of As(V) at low P supply, which was not alleviated by AM colonisation. Tissue P and As concentrations were similar in AM and NM treatments, so it appeared that AM colonisation had no effect on As(V) uptake or As toxicity (Knudson et al. 2003). Barley grown with As in the root hyphal compartment (RHC) of compartmented pots (and therefore comparable with other investigations mentioned in this section) behaved in the same way as L. cinereus with respect to growth and showed no differences in P concentrations or content regardless of As and AM treatment. However, there were indications that As concentrations in roots were lower in AM plants compared with NM, that specific As(V) uptake was also lower and that P/As ratios in the whole plants were higher (Christophersen et al. in press). Tomato was also non-responsive in the absence of As and had lower tissue P concentrations and growth when AM (Liu et al. 2005b). However, in this case AM plants grew better than NM plants in the As treatments. Both P and As contents were higher in AM tomato when As was applied, particularly in roots. Similar results had been obtained for H. lanatus (T lines) growing in contaminated soil (Gonzalez-Chavez et al. 2002). It was shown clearly that AM T plants grew much larger than NM T plants and that As contents were lower and P contents considerably higher. Responsiveness in uncontaminated soil was not determined in the experiment with H. lanatus, but the plant showed no response to AM colonisation in a field experiment in the absence of As (Wright et al. 2000) and it is therefore appropriate to discuss results along with other non-responsive plants. Furthermore, NT plants of H. lanatus did not survive in the contaminated soil when NM (Gonzalez-Chavez et al. 2002), but AM NT plants did, although growth was very much lower than the T plants, whether the latter were AM or not. Ranking in terms of growth was: NM-NT (did not survive) < AM-NT < NM-T < AM-T) (Meharg 2003; see his Fig. 1). Thus, with the exception of L. cinereus, experiments with non-responsive plants provide some additional evidence for involvement of AM in As tolerance, but the underlying mechanisms are extremely hard to unravel. Where specific As(V) uptake can be calculated, with one exception it is reduced or unaffected in AM plants, whereas specific Pi uptake is increased or unaffected, again suggesting that pathways of As(V) and Pi uptake are different in AM and NM plants. Another common feature is (again with the exception of L. cinereus) the higher P/As ratios in shoots of AM plants, compared with NM plants. Uptake of Pi via the AM pathway of H. lanatus, which is strongly suggested by the results of Gonzalez-Chavez et al. (2002), would solve the potential problem of survival of T plants (if they do have compromised direct high-affinity P uptake) in low-P soil, regardless of contamination by As.

Transfer of As by external mycelium of AM fungi

Several groups have used compartmented pots to assess the roles of external hyphae of AM fungi in uptake of As(V) and Pi separately from the roots. The designs of the pots used in the different experiments were very different, which had a bearing on outcomes. Chen et al. (2007) grew M. sativa (normally a highly AM-responsive plant) in a main (root hyphal) compartment (RHC) containing 750 g soil/sand mix and supplied As(V) and/or Pi in separate hyphal compartments (HCs) of 125 g. Thus, interactions between As and P in the soil, which might have influenced their availability (see above) and uptake by the hyphae, were largely prevented. However, the soil used to fill all the compartments did contain a low level of As (around 0.125 mg/kg soil mix) which meant that roots as well as hyphae had access to it in the RHC. Roots, but not hyphae, were prevented from accessing the ‘extra’ P and/or As in the HCs by 37 µm mesh. There were large AM responses in terms of growth and Pi uptake, regardless of As treatments, reflecting nutrient acquisition by hyphae from both RHC and HCs. All plants took up some As(V) but contents were much greater in AM plants. Chen et al. (2007) point out that this could have been the result of presence of some As in the RHCs, coupled with very much greater root development and hence uptake by the larger AM plants. Provision of additional As in the HCs (at two levels) did increase root As contents slightly, suggesting some uptake and transfer by the hyphae. However, uptake into shoots of AM plants actually decreased with increasing As in the HCs, so there was no evidence that the small amount of As translocated to the roots was transferred across the symbiotic interface to the plants (see Fig. 3).

Similar results have been obtained for non-responsive barley (Christophersen et al. in press). In this case the basic soil mix contained no As, and As(V) was added either to a large RHC (2.3 kg) or a large HC (1.8 kg), or both. Compartments were separated by 30 µm mesh. When As was supplied in the RHC there were marked depressions in growth that were not influenced by AM colonisation. P concentrations in plant tissues were increased when As was present in the RHCs, probably as a result of tissue concentration in the smaller plants, because total and specific Pi uptake did not increase and there were only small variations in shoot/root ratios between treatments. There were no effects of AM on As concentrations in shoots when As was present in the RHC, but concentrations in roots were lower in AM plants, significantly so when As was present in both RHC and HC. Total As content was also reduced in these AM treatments. When As was present only in the HC the total As in the plants was slightly higher in AM than NM plants, although values were generally very much lower than when As was present in the RHCs. The roots contained a small amount of As, again indicating some uptake and translocation by the external hyphae, but no As appeared in the shoots. These results support the indications from the work of Chen et al. (2007) that As is not transferred across the symbiotic interface. Overall, lower As content in AM plants was again observed. In this investigation the expression of barley genes encoding Pi transporters in the direct and AM uptake pathways was determined. Those in the direct pathway (HvPht1;1 and HvPht1;2) were consistently down-regulated in AM plants. This is in agreement with the observations of lower specific As(V) uptake from the soil in the RHCs, and (in a separate investigation) markedly lower short term As(V) influx in excised AM roots of T H. lanatus, compared with NM roots (Gonzalez-Chavez et al. 2002 and see below). The AM-inducible transporter gene (HvPT1;8) was expressed in all AM barley plants. This gene showed significantly reduced transcript accumulation when As was present in RHC, HC or both, but the effect of this down-regulation on P transfer could not be determined. Only concurrent measurements of gene expression and 32/33P transfer will show whether the changes in expression were associated with different contributions of the direct and AM pathways to Pi uptake.

A recent paper (Meding and Zasoski 2008) has provided data which appears at odds with the findings of Chen et al. (2007) and Christophersen et al. (in press). They tested the ability of As (as Na2AsO4) supplied (supposedly as a tracer for Pi) to the cut foliage of donor plants to move down to the roots, through AM hyphae to receiver plants. (Donor and receiver plants were forbs or grasses, in different treatment combinations.) Transfer through soil was prevented by imposition of an air gap (1 cm wide) in the compartmented pots, which was designed to allow growth of hyphae between the plants. Small amounts of As, which could not be satisfactorily quantified, were detected in the shoots of the receiver plants 7 d after application in some treatments. Unfortunately no NM treatments, or treatments in which the hyphal bridge in the air gap was cut, were included as controls. The pathway of transfer, from shoots of a donor to shoots of a receiver was obviously very different from the soil-to-plant pathway examined in the experiments described above. A satisfactory explanation for the difference between the two types of investigation is not forthcoming at present, but it must be noted that potential As uptake from soil via direct and AM pathways was bypassed in the foliar feeding experiment.

Ultra et al. (2007b) used highly As-contaminated soil in all pot compartments, which limited the conclusions that could be drawn. In particular, transfer of As from HCs to plants by AM fungal hyphae could not be followed. The RHC was very small (80 g) and the authors assumed that the plants (sunflower) would be able to access both nutrients and As from the outer (hyphal) compartments (one of which was very large) by diffusion or mass flow, even though the compartments were divided by 27 µm mesh. They found that symptoms of As toxicity were reduced by AM inoculation and increased P supply in the RHC, in line with effects on other AM-responsive plants. Growth of AM plants was higher than NM plants and concentrations of As lower, again probably as a result of tissue dilution. Specific Pi uptake was increased by both AM inoculation and P application (as expected), but specific As(V) uptake was decreased by AM colonisation and increased by P. Analysis of the soil showed that P application increased the water-soluble As, providing some explanation for the latter effect. As(V) predominated (80% of water-soluble As), but there were significant amounts of As(III), particularly close to roots, and of DMA close to roots in AM treatments only. Similar results were obtained with a much simpler compartmented pot (Ultra et al. 2007a).

Conclusions from AM studies

We sought answers to several questions, outlined above. First, are reported AM effects on As tolerance the result of compensation by the fungal pathway for poor root growth? Second, are substantial amounts of As taken up into roots via the AM pathway, as is sometimes assumed (Gonzaga et al. 2006) or does this route deliver only P? Third, is As(V) uptake via the direct pathway lower in AM plants, and is any lowering a consequence of reduced expression of Pi transporters? Overall the findings show that a simple explanation for improved As tolerance in AM plants, based on compensation for poor root growth in terms of Pi uptake, is not adequate. The AM fungi can probably absorb small amounts of As(V) from aerobic soil and some is translocated to the roots. The form in which it moves is unclear, but cannot involve synthesis of polyarsenate (equivalent to polyphosphate) as this is highly unstable. At this stage, most evidence indicates that As is retained in the fungal compartment of AM roots and not transferred to the root cells themselves and thence to the shoots. Any As(V) taken up is likely to be reduced to As(III) in the fungal mycelium, which might then be effluxed to the soil solution. This theoretical possibility has not yet been tested for AM fungi (Fig. 3).

The picture that emerges both for responsive and non-responsive plants challenged by As is that AM plants (in comparison to NM) take up relatively more Pi than As(V); i.e. the P/As ratio in AM plants is higher than NM plants. The change in relative uptake (selectivity) could be achieved in several ways, which are not mutually exclusive and probably operate differently in different plant/fungus combinations, due to differences in responsiveness and in relative amounts of Pi taken up via the direct and AM pathways (Grace et al. 2009; Smith et al. 2003b; 2004): 1) Selectivity of direct uptake of As(V) and Pi through the root epidermis (direct pathway) remains unchanged in AM vs NM plants, but extra P is provided by the AM pathway which does not transport As(V) or has a higher selectivity for Pi over As(V) than the direct pathway (a P bypass). This option would explain higher P/As ratios but not lower specific As(V) uptake by AM plants, where this is observed; 2) AM colonisation results in reduced activity of the direct uptake pathway (possibly by down-regulation of genes encoding epidermal Pi transporters) and hence lower uptake of both Pi and As(V) by this route (with unchanged selectivity towards Pi and As(V)). The AM pathway would require the same higher selectivity towards Pi as in option 1, and both increased P/As ratios and lower As content and specific uptake can be explained; 3) The characteristics of the direct uptake pathway are changed by AM colonisation, for example by differential Pi transporter expression, such that the selectivity towards Pi is increased. In this case it is not a requirement that the AM pathway provides a P bypass by being more selective towards Pi. There is some evidence that As uptake via the direct pathway is reduced in AM plants, as suggested by short-term As(V) fluxes into excised AM and NM roots of H. lanatus. NT lines showed lower As uptake when AM than when NM (Gonzalez-Chavez et al. 2002). This finding is consistent with reduced expression of the genes encoding Pi transporters involved in this pathway in AM barley roots (Glassop et al. 2005; Christophersen et al. in press) and hence reduced specific As(V) uptake from soil. As already noted, operation of the AM uptake pathway as a ‘P bypass’ in field-grown plants (options 1 and 2) would overcome difficulties of survival that occur in As-tolerant plants if high-affinity P uptake is impaired over the longer term (Gonzalez-Chavez et al. 2002; Meharg and Cairney 2000, and see discussion above).

The possibility that increased efflux of As from AM roots would reduce As accumulation and hence increase the P/As ratio has not been seriously considered. Efflux from the external hyphae could occur either before transfer to the plant, a process that would contribute to lower internal concentrations and hence availability for transfer (pathway 6 in Fig. 3), or following transfer from plant to fungus across the symbiotic interface. Efflux of As might also occur across epidermis and root hairs following uptake either directly or via the AM pathway. No evidence for any of these options is available.

Suggestions for further work on arbuscular mycorrhizas

Further work is clearly needed to clarify mechanisms and distinguish between the options outlined. The hypothesis that the AM pathway transfers P (as is normal) but little or no As seems likely to be correct, but requires unequivocal demonstration. Tissue localisation of As and P in external hyphae, fungal structures inside the roots and in root cells themselves would be illuminating with respect to accumulation of As, particularly if As speciation could be determined. Additionally, more sensitive methods of tracking As movement in the AM pathway, such as use of radioactive 73As(V) in compartmented pots would demonstrate the fungal potential (or lack of it) for As(V) transport and As transfer to the plant more conclusively. Coupled with use of 33/32P, such experiments would provide quantitative measures of the relative contributions (selectivity) of the AM pathway to both As(V) and Pi uptake and transfer. These experiments would also show unequivocally whether the AM pathway provides a ‘P bypass’ that would compensate for reduced Pi uptake via the direct pathway if this has reduced activity.

Concurrent characterisation of Pi transporters and their expression patterns in AM- and As-challenged plants would further help to elucidate underlying mechanisms. The Pi/As(V) selectivity of epidermal transporters in As-tolerant and non-tolerant plants has not been determined, as far as we are aware, but would be illuminating with respect to potential changes in selectivity as a result of differential gene expression. Moreover, it is not clear whether the high-affinity AM fungal Pi transporters in external hyphae or the AM-inducible plant transporters at the fungus/plant interface can also transport As(V). It seems likely that the high-affinity fungal Pi transporters do transport some As(V), given the indications of As(V) uptake from the soil and As transfer to roots. However, it is not clear how the fungi avoid consequent toxic effects. We can speculate that As(V) may be reduced to As(III), which would then be effluxed to the soil, as in ericoid mycorrhizal fungi (see Fig. 3 and below). Experiments will be challenging, given the unculturability of AM fungi, but might be achieved in compartmented monoxenic cultures, as used effectively by Maldonado-Mendoza et al. (2001) to study P uptake. The AM-inducible plant Pi transporters have not all been fully characterised and there is no information as far as we are aware of their selectivity for Pi/As(V). High selectivity would decrease relative As(V) transfer across the symbiotic interface if these Pi transporters are indeed involved in that process. In any case, until significant transfer of As from fungus to plant is confirmed, it seems unnecessary to discuss details of alternative possibilities, such as efflux of As(III) from the fungi and passive uptake by the root cortical cells at the interface (see Fig. 3).

AM effects on the As hyperaccumulator fern Pteris vittata

There have been several investigations of the effects of AM colonisation on As(V) uptake in the hyperaccumulator fern, P. vittata¸ with the aim of assessing benefits in terms of bioremediation (Al Agely et al. 2005; Chen et al. 2005; Leung et al. 2005; Liu et al. 2005a; Trotta et al. 2006). This plant is variably reported to be slightly positively responsive or non-responsive to AM colonisation, regardless of presence of As in the soil. AM colonisation was generally unaffected or increased by additional As applications, again regardless of whether the basal soil used was contaminated. The majority of investigations indicated increased growth of AM compared with NM plants in the presence of As, as found for responsive and some non-responsive species discussed above. This would result in increased potential for removal of As in shoot material if there were no changes in As concentrations in shoots. However, these were generally lower in AM plants (probably caused by ‘tissue dilution’ in the larger AM plants, see above), so that values for total above-ground As were often unaffected by the symbiosis. There are also two reports of lower As concentrations in roots, suggesting again tissue dilution or possibly lower uptake (Liu et al. 2005a; Trotta et al. 2006). The shoot and root concentrations are sometimes used to calculate translocation factors (TF) and hence assess the potential of AM in bioremediation strategies (Trotta et al. 2006), so it is important to remember that reductions in As concentrations in roots give higher TF values which may not actually be related to increased accumulation in the shoots. At this stage of research it appears that AM are unlikely to have a major effect on the potential of P. vittata to remove As from soil. It must be remembered that, given the tolerance of AM fungi to As, the AM state will be normal at contaminated sites and manipulation of the symbiosis difficult. This does not however, negate the value of future investigations of AM effects on As accumulation, taking into account knowledge of the pathways by which it is taken up and mechanisms of As tolerance in AM plants.

Arsenic in ericoid and ectomycorrhizal fungi and plants

Although much less extensive, some of the first work on possible involvement of mycorrhizal fungi in As tolerance was carried out with heathland isolates of the ericoid mycorrhizal fungus Rhizoscyphus (Hymenoscyphus) ericae, following the recognition that its host plant, Calluna vulgaris, was a major coloniser of As- and Cu-contaminated mine-sites in the United Kingdom. An ectomycorrhizal fungus (Hebeloma crustuliniforme) was included in some investigations for comparison (Sharples et al. 1999). It was found that growth of both fungi in pure culture was inhibited by increasing As(V) concentration in the culture medium, with H. crustuliniforme much more sensitive. Addition of Pi ameliorated the toxic effects, in line with competition between As(V) and Pi for uptake by fungal Pi transporters (Sharples et al. 1999). Subsequent work demonstrated that strains of R. ericae isolated from the mine-site were much more tolerant to As(V) than ‘normal’ heathland isolates, suggesting adaptation of the fungi to the contaminated environment (Sharples et al. 2000; 2001). Differences in kinetic characteristics of high-affinity Pi transport of the strains from different origins could not explain the different sensitivities, but one mine-site isolate exhibited much faster As(III) efflux than the heathland isolate (Sharples et al. 2000). The authors suggested that As(III) efflux allowed the fungi to eliminate As and maintain Pi uptake and transfer to the plants as also suggested for arbuscular mycorrhizas and shown in Fig. 3. However, all work was done on cultured fungi and the suggestion does not appear to have been tested in symbiotic systems.

Considering the significance of ECM and ERM plants in natural ecosystems (including contaminated sites) the recent neglect of these symbioses in studies of As uptake and toxicity is surprising. There is clearly scope for much new work on both outcomes of presence of As in soil and of mechanisms of tolerance in the symbiotic plants. These may well be different from the mechanisms in AM symbioses, because both anatomical and physiological relationships between fungi and plants are different (Smith and Read 2008).

General conclusions

Arsenic uptake by plants growing in soil involves a range of processes that include 1) aspects of soil chemistry, 2) plant-soil interactions in the rhizosphere and, with mycorrhizal plants, the more extensive mycorrhizosphere, and 3) transport across plasma membranes. Interactions between As and P are fundamentally involved in all these contexts, and emphasised in this review. Use of plants grown in nutrient solutions, or roots excised from such plants, is attractive in allowing focus solely on membrane transport but (as with many mineral elements) inevitably introduces uncertainties in extrapolating results to soil-grown plants. In this respect we have raised some concerns about the conclusion that long-term As tolerance in NM plants including H. lanatus involves significant suppression of high-affinity Pi uptake – a process generally regarded as essential for growth in low-P soil. We believe that to resolve this issue there is need for more detailed measurements of time-courses of both Pi and As(V) uptake into such As-tolerant plants grown in soil amended with As. It must be also now be borne in mind that plants investigated so far have been shown to possess more than one root epidermal high-affinity Pi transporter; these are encoded by different genes (Bucher 2006). These transporters may have different selectivity for As(V) vis-à-vis Pi. If this is the case, development-related changes in expression of these genes may increase Pi/As(V) uptake selectivity during long-term growth of NM plants, or AM plants in which the direct epidermal uptake pathway still provides a large component of total uptake. In the absence of more definitive long-term measurements, we do not believe that demonstration of relatively low uptake of As(V) by plants grown in soil without concurrent measurements of Pi uptake is prima facie evidence for suppression of high-affinity Pi uptake. It is safer to conclude only that Pi/As(V) uptake specificity is higher in the T plants than in the NT plants, as originally proposed (Macnair and Cumbes 1987) and indicated by the data of Quaghebeur and Rengel (2003). Despite our concern, we emphasise that work with H. lanatus has been important in highlighting the way in which mycorrhizal (particularly AM) symbioses may lower As sensitivity in long-term plant growth by reducing the contribution of direct Pi and As(V) uptake, and producing uptake with relatively high Pi/As(V) selectivity. In other words, mycorrhizal symbioses allow improved ‘evasion’ of As toxicity, with internal detoxification remaining the second line of defence in plants whether mycorrhizal or not.

Appreciation of the diversity of AM responses and pathways of P uptake in AM plants should lead to improved design of experiments to unravel mycorrhizal effects on As-P interactions. Measurement of isotopic fluxes combined with As speciation seem necessary to help resolve mechanistic uncertainties in both NM and AM plants. Such experiments will also be necessary to quantify the significance of As(III) efflux from roots or mycorrhizal fungi. Importantly, there is need to bring together molecular approaches (e.g. changes in Pi transporter expression and selectivity of transporters for Pi and As(V)) with physiological measurements under the same conditions. This combined approach should lead to much clearer understanding of basic principles of tolerance to As(V), which can be applied to understanding As tolerance in plants growing in soils that are contaminated with As, whatever the cause.

Acknowledgements

Our research is funded by the Australian Research Council. We are grateful to Rebecca Stonor and Maria Manjarrez who have provided technical support for our project. We thank many friends and colleagues for helpful discussions and the anonymous referees for detailed appraisals of the original version of the manuscript.

Copyright information

© Springer Science+Business Media B.V. 2009