Metallophytes—a view from the rhizosphere

kar bernal

Some plants hyperaccumulate metals or metalloids to levels several orders of magnitude higher than other species. This intriguing phenomenon has received considerable attention in the past decade. While research has mostly focused on the above-ground organs, roots are the sole access point to below-ground trace elements and as such they play a vital role in hyperaccumulation. Here we highlight the role of the root as an effective trace element scavenger through interactions in the rhizosphere. We found that less than 10% of the known hyperaccumulator species have had their rhizospheres examined. When studied, researchers have focused on root physical characteristics, rhizosphere chemistry, and rhizosphere microbiology as central themes to understand plant hyperaccumulation. One physical characteristic often assumed about hyperaccumulators is that their roots are small, but this is not true for all species and many species remain unexamined. Transporters in root membranes provide avenues for root uptake, while root growth and morphology influence plant access to trace elements in the rhizosphere. Some hyperaccumulators exhibit unique root scavenging and direct their growth toward elements in soil. Studies on hyperaccumulator rhizosphere chemistry have examined the role of the root in altering elemental solubility through exudation and pH changes. Different interpretations have been reported for mobilization of non-labile trace element pools by hyperaccumulators. However, there is a lack of evidence for a novel role for rhizosphere acidification in hyperaccumulation. As for microbiological studies, researchers have shown that bacteria and fungi in the hyperaccumulator rhizosphere may exhibit increased metal tolerance, act as plant growth promoting microorganisms, alter elemental solubility, and have significant effects on plant trace element concentrations. New evidence suggests that symbiosis with arbuscular mycorrhizae may not be rare in hyperaccumulator taxa, even in some members of the Brassicaceae. Although there are several reports on the presence of mycorrhizae, a cohesive interpretation of their role in hyperaccumulation remains elusive. In summary, we present the current state of knowledge about how roots hyperaccumulate and we suggest ways in which this knowledge can be applied and improved.

Plant Soil DOI 10.1007/s11104-010-0482-3 REVIEW ARTICLE Metallophytes—a view from the rhizosphere Élan R. Alford & Elizabeth A. H. Pilon-Smits & Mark W. Paschke Received: 9 March 2010 / Accepted: 23 June 2010 # Springer Science+Business Media B.V. 2010 Abstract Some plants hyperaccumulate metals or metalloids to levels several orders of magnitude higher than other species. This intriguing phenomenon has received considerable attention in the past decade. While research has mostly focused on the above-ground organs, roots are the sole access point to below-ground trace elements and as such they play a vital role in hyperaccumulation. Here we highlight the role of the root as an effective trace element scavenger through interactions in the rhizosphere. We found that less than 10% of the known hyperaccumulator species have had their rhizospheres examined. When studied, researchers have focused on root physical characteristics, rhizosphere chemistry, and rhizosphere microbiology as central themes to understand plant hyperaccumulation. One physical characteristic often assumed about hyperaccumulators is that their roots are small, but this is not true for all species and Responsible Editor: Fangjie Zhao. É. R. Alford (*) : E. A. H. Pilon-Smits : M. W. Paschke Graduate Degree Program in Ecology, Colorado State University, Fort Collins, CO 80523-1472, USA e-mail: elan.reine@gmail.com É. R. Alford : M. W. Paschke Department of Forest, Rangeland, and Watershed Stewardship, Colorado State University, Fort Collins, CO 80523-1472, USA E. A. H. Pilon-Smits Biology Department, Colorado State University, Fort Collins, CO 80523-1878, USA many species remain unexamined. Transporters in root membranes provide avenues for root uptake, while root growth and morphology influence plant access to trace elements in the rhizosphere. Some hyperaccumulators exhibit unique root scavenging and direct their growth toward elements in soil. Studies on hyperaccumulator rhizosphere chemistry have examined the role of the root in altering elemental solubility through exudation and pH changes. Different interpretations have been reported for mobilization of non-labile trace element pools by hyperaccumulators. However, there is a lack of evidence for a novel role for rhizosphere acidification in hyperaccumulation. As for microbiological studies, researchers have shown that bacteria and fungi in the hyperaccumulator rhizosphere may exhibit increased metal tolerance, act as plant growth promoting microorganisms, alter elemental solubility, and have significant effects on plant trace element concentrations. New evidence suggests that symbiosis with arbuscular mycorrhizae may not be rare in hyperaccumulator taxa, even in some members of the Brassicaceae. Although there are several reports on the presence of mycorrhizae, a cohesive interpretation of their role in hyperaccumulation remains elusive. In summary, we present the current state of knowledge about how roots hyperaccumulate and we suggest ways in which this knowledge can be applied and improved. Keywords Metallophyte . Hyperaccumulation . Trace elements . Rhizosphere bacteria . Arbuscular mycorrhiza . Root structure Plant Soil Introduction Metallophytes can be defined by their ability to survive and reproduce on metal-rich soils without suffering toxicity (Baker et al. 2010). Often these plants have been identified by the habitats in which they grow, but high levels of trace elements (defined here as elements other than the 8 most abundant rockforming elements: Al, Ca, Fe, K, Mg, Na, O, and Si) in plants can also be achieved by growth in amended or contaminated substrates (Reeves 1988; Lombi et al. 2000; Szarek-Lukaszewska and Niklinska 2002). Some metallophytes have a specialization to concentrate elements at levels that would be toxic to nonaccumulators. This innate ability to hyperaccumulate trace elements in plant leaves has been observed in species that grow naturally on metal-rich substrates (Brooks et al. 1977; Baker and Brooks 1989; Wenzel and Jockwer 1999), but the trace element accumulation potential in hyperaccumulators may not be solely a matter of their habitat, when Thlaspi caerulescens was subjected to amended substrates ecotypes originating from low-metal soils accumulated more than ecotypes originating from metalliferous soils (Escarré et al. 2000; Dechamps et al. 2005). Clearly hyperaccumulators collect large quantities of soil trace elements and sequester them in their leaves, yet we do not have a clear understanding of the ways roots achieve this. Some hyperaccumulators amass large amounts of essential elements (Cu, Mn, Ni, Zn), but other species store non-essential elements (As, Co, Cd, Se). The leaf has been the focus of much study because it serves as a storage site for elements where they are available to livestock and human consumers. Meanwhile, the root, the site of initial and continuous element acquisition, has received far less attention. Plant access to trace elements is mediated by bioavailability and root location in relation to the element. The rhizosphere, defined as the root-soil interface, where microorganisms, roots, and soil come together (Hiltner 1904), is the micro-ecosystem where roots access soil trace elements. Multiple gradients co-occur in the rhizosphere (bulk density, elemental concentrations, root exudation, microorganisms, moisture, pH, redox potential); thus the rhizosphere is a dynamic system in time and space. Within the rhizosphere additional differences are caused by variation in root activity among root classes (i.e. lateral, primary roots, etc.), root ages, and location (i.e. tip, elongation zone, maturation zone) (Doussan et al. 2003). Given the vast complexity of the rhizosphere, there is much to explore to explain the belowground aspects of plant hyperaccumulation. Hyperaccumulators have adapted to life rooted in high-metal soils. Over geologic time the quantity and type of trace elements found in soils change, and in turn, local flora can adapt. Naturally-occurring metalliferous soils foster unique assemblages of tolerant and/or hyperaccumulating plant taxa (Beath et al. 1937; Reeves 2002) and human-influenced metalliferous soils may develop similar types of metal tolerant plant populations (Malaisse and Brooks 1982). Five different explanations for why hyperaccumulators may have evolved have been proposed (Boyd and Martens 1992; Boyd 2007). The hypotheses put forward are (i) plants may hyperaccumulate trace elements because storing large quantities of metals may be a means of metal tolerance and disposal, (ii) hyperaccumulating plants may use metals as elemental allelopathy against nearby competitors, (iii) metals may serve as osmotic resistance to drought, (iv) accumulated metals may defend the plant against herbivores or pathogens, and (v) metal accumulation may be accidental. Our current review addresses how hyperaccumulators work, rather than why they hyperaccumulate. We review here what is known about the novel and conventional ways in which hyperaccumulators access and accumulate trace elements. There is a general lack of ecological knowledge about hyperaccumulators (Boyd and Martens 1992; Whiting et al. 2004), particularly with respect to rhizosphere processes (Abou-Shanab et al. 2003a). Metallophytes are botanical curiosities. Their rhizosphere may have unique properties, yet also will retain similarities to nonaccumulating plant species. Hyperaccumulators will continue to have exciting applications if we are able to identify their unique hypertolerance and hyperaccumulation mechanisms, including how they manipulate their rhizospheres. Trace element uptake depends upon plant roots; therefore this region merits extensive study in the context of hyperaccumulation. Current information on rhizosphere conditions in hyperaccumulating plants is based on a small subset (less than 10%) of known hyperaccumulators. Below we review what is known about hyperaccumulation rhizosphere processes, grouped into three categories—root physical characteristics, rhizosphere chemistry, and rhizosphere micro- Plant Soil organisms. Figure 1 illustrates several of the interactions occurring in the rhizosphere that have been described in the literature and are reviewed below. Root physical characteristics Root system development determines the rhizosphere size, configuration, and plant access to soil-borne elements. The root surface area plays a large role in 6. O Growth OH 1. 7. N H CH 2 TE TE C H2 Acid Production _ H+ Mycorrhizae DOC 8. 2. 3. 9. TE Exudation 4. _ TE Soil _ _ _ 5. TE Transporters Fig. 1 This conceptual diagram shows the many ways that processes in the rhizosphere influence trace element (TE) uptake in hyperaccumulators. Roots search for trace elements and grow towards them by chemotropism (1). Plant growth promoting bacteria can alter root growth by altering hormones such as indole-3 acetic acid or degrade ethylene in the rhizosphere or as endophytes living within the plant (2). Trace element solubility can be altered by acid production (H+) by bacteria (3) or roots (4). Hydrogen ions can then displace trace element cations adsorbed to soil particles. Bioavailable trace elements can enter the plant through transporters located in the root (5). Mycorrhizae can increase plant access to trace elements by increasing belowground surface area (6) where trace elements can enter transporters in the fungus (7). Finally, trace element solubility can be increased in the rhizosphere by complexation with dissolved organic carbon (DOC) that is deposited in the rhizosphere by roots (8) or microorganisms (9) nutrient uptake (Comerford 2005) and it therefore has a significant role in trace element uptake by hyperaccumulators. Researchers have commented on the small root systems characteristic of some hyperaccumulators (Ernst 1996). While it is true some hyperaccumulators are slow-growing and small compared to many annual crop species proposed for phytoremediation, there are perennial hyperaccumulators with well-developed root systems (Kutschera and Lichtenegger 1992; Ernst 1996). Root depth and morphology are important traits related to uptake, yet there are very few reports in the literature that compare root morphology between species and how it affects hyperaccumulation. However, it must be noted that comparisons may be challenging to conduct because root structure and length are constrained by soil physical characteristics. Rather than conducting comparisons between species, studies have focused on root length, depth, and surface area within a single species. Several hyperaccumulators have been described with small, shallow (<0.5 m) root systems and a high proportion of fine roots that contribute to trace element accumulation (Keller et al. 2003; Himmelbauer et al. 2005). Although these reports featured shallowrooted plants, deep-rooted (2 m) herbaceous species such as Biscutella laevigata (Cd-hyperaccumulator) exist (Kutschera and Lichtenegger 1992), and roots of many hyperaccumulator tree species remain unexamined but could be large and deep. At the root surface, membrane transporters provide uptake sites for soil borne elements to enter the symplast. Alternatively, trace elements may enter in the plant apoplast and enter the symplast through transporters in the endodermis that surrounds the root vascular cylinder. Several hyperaccumulated metals are essential (Cu, Mn, Ni, and Zn), therefore specific transporters for these elements should be located in root membranes of hyperaccumulators. Zinc (Lasat et al. 2000; Assunção et al. 2001) and Ni (Gendre et al. 2007) transporters have been described for hyperaccumulator species, but transporters for Mn (Mizuno et al. 2008) and Cu have not. Transporters for these elements have been described for nonhyperaccumulator species (Grotz et al. 1998; Clemens 2001; Pittman 2005; Burkhead et al. 2009); although Ni transport is not well understood (Gerendás et al. 1999). The difference in accumulation of some trace elements between hyperaccumulators and non- Plant Soil hyperaccumulators may be related to a higher expression level of the same transporters in hyperaccumulators than in non-hyperaccumulators, or the presence of transporters with different kinetic properties; there is more evidence so far for the former (Pence et al. 2000; Li et al. 2005a). The constitutive upregulation of a root Zn transporter in hyperaccumulator Thlaspi caerulescens has been suggested to be caused by the roots sensing the plant to be continuously Zn-starved (Talke et al. 2006) and increased expression of HMA4 causes a similar response in Arabidopsis halleri (Hanikenne et al. 2008). Besides transporters at the root surface, hyperaccumulators have other ways to promote metal accumulation in the shoots, such as reducing sequestration in root vacuoles so metals can enter xylem transport pathways (Lasat et al. 1998; Papoyan and Kochian 2004), and a highly lignified endodermis that prevents metal efflux out of the root vasculature (van de Mortel et al. 2006). In addition, plant species vary in their levels of metal chaperones and chelators such as glutathione and phytochelatins, organic acids, histidine, and nicotianamine (Krämer et al. 1996; Salt et al. 1999; Freeman et al. 2004; Kupper et al. 2004; Raab et al. 2004; Weber et al. 2004; Mari et al. 2006); some of these may facilitate metal transport within hyperaccumulator root cells and root xylem or serve roles in plant metal tolerance. Rather than discussing these processes within the plant in more detail, we turn our attention back to the rhizosphere and the root surface. Non-essential trace elements can enter roots through transporters for essential elements that have similar valence states and ionic diameters. For instance, As, Cd, and Se resemble the essential elements P, Cu/Fe/Mn/Zn, and S, respectively (Cataldo et al. 1983; Marschner 1995). Given these less specific avenues for entry, there may be ways in which hyperaccumulating species acquire larger amounts of non-essential trace elements than non-hyperaccumulating species. Hyperaccumulators may have higher expression levels for essential element transporters. For example, it was shown that higher S levels occurred in Se-hyperaccumulators than non-accumulators (Galeas et al. 2007), if those transporters do not discriminate against Se this could contribute to higher Se levels in plants. In addition, the transporters in hyperaccumulators could have higher affinities for non-essential elements than transporters in non- accumulators (Bell et al. 1992; Zhao et al. 2002b; Poynton et al. 2004; White et al. 2007). Once inside the hyperaccumulator, trace elements may be complexed and stored in vacuoles. This has been suggested for thiol reductants in the As-hyperaccumualtor Pteris vittata (Webb et al. 2003), although the amount of As complexed in this manner is small (Zhao et al. 2003; Raab et al. 2004). In the soil, root transporters need to be in the vicinity of trace elements, so roots should be located in areas of bioavailable elements. Directed root growth towards trace elements in soil (chemotropism) has been reported in some hyperaccumulating species or ecotypes, but the mechanisms are not understood. This response demands an adequate ecological interpretation because current thought on chemotropism relates solely to nonhyperaccumulators that can alter root growth to dynamically compensate for spatial variability in soil nutrients (Jackson and Caldwell 1996; Robinson 1996; Bloom et al. 2002; Doussan et al. 2003). Many hyperaccumulators exhibit similar root proliferation in soil, but in response to elevated levels of trace elements including Cd, Ni, Se, and Zn (Schwartz et al. 1999; Whiting et al. 2000; Haines 2002; Goodson et al. 2003; Dechamps et al. 2008). Nickel and Zn are essential, but Cd and Se are not; therefore the hypothesis of root-proliferation yielding increased plant nutrition only holds if non-essential elements are co-localized with nutrients. In contrast, there are known negative effects of high concentrations of some trace elements on root growth in non-hyperaccumulators (Robertson 1985; Barceló and Poschenrieder 1990; Paliouris and Hutchinson 1991; El Kassis et al. 2007), but some hyperaccumulators can direct their root growth, and thus rhizosphere development, in a positive response toward trace elements in ways that non-hyperaccumulators could not (Whiting et al. 2000; Goodson et al. 2003). Additionally, some hyperaccumulating plant ecotypes respond positively to trace elements while other ecotypes within the same species did not (Whiting et al. 2000; Haines 2002; Li et al. 2005a, 2009; Dechamps et al. 2008). Positive chemotropism may partially explain the higher trace element content in some hyperaccumulating plants, but not all hyperaccumulators have chemotropic responses to hyperaccumulated elements (Moradi et al. 2009). These ecotype-, population-, and species-specific responses indicate that when possible, care should be taken in choosing seed sources and species for experimental studies. Similar to the ideas of using local ecotypes Plant Soil genetically adapted to specific restoration settings (McKay et al. 2005), selecting appropriate hyperaccumulator ecotypes that exhibit chemotropic root growth may be important in some phytoremediation settings. Even though positive root chemotropism is not observed in all hyperaccumulators, these plants may provide a new framework to explain root behavior. Instead of nutrition, what is directed growth in some hyperaccumulators used for? One explanation may be that chemotropic growth causes increased access to trace elements that enhance plant herbivore defense. There is no experimental evidence for this, but chemotropism could be an extension of the elemental defense hypothesis, where hyperaccumulators can reduce herbivore and pathogen attack because their tissues contain toxic amounts of trace elements (Boyd et al. 1994; Martens and Boyd 1994). Preferential root scavenging could increase access to elements and consequently increase concentrations within plants— leading to increased herbivore defense. Physical characteristics of hyperaccumulator roots have received some attention in the literature, but further work focusing on transport mechanisms, root turnover, trace element partitioning, and root morphology is needed. Investigating more species of hyperaccumulators is important to further our understanding of how roots respond to trace elements. In addition, further studies should be conducted on root uptake and transport by hyperaccumulators; for example Co transport mechanisms have not been described. Further, the study of root turnover in response to trace elements may be useful for studies in carbon sequestration, but it would be particularly important for phytoremediation where root death could significantly reduce plant uptake. Notably, leaf senescence and turnover from metal toxicity has received attention (Jana and Choudhuri 1982; Fuhrer 1983; Ryser and Sauder 2006), but corresponding analyses belowground are rare (Helmisaari et al. 1999). Finally, describing trace element partitioning in roots can help us understand how hyperaccumulators work and relate to their environment. For example, the fine and coarse roots of Alyssum murale (Ni and Zn-hyperaccumulator) have specific Ni and Zn localization where the metals were in fine root vasculature and on the coarse root exterior (McNear et al. 2005). One reason for this type of pattern may be xylem transport in the vasculature, but there could be various explanations for rhizoplane localization, such as an herbivore or pathogen defense strategy, deposition by root exudation, or association within a sheath of rhizosphere microorganisms. Further research on root physical traits should be conducted. If we learn the genetic mechanisms of chemotropic responses in hyperaccumulators there may be potential to transfer this trait to agricultural species to improve plant nutrition or herbivore defense. Currently, we have a better understanding of root growth in hyperaccumulator species than trace element allocation patterns. Root uptake of large amounts of trace elements is responsible for hyperaccumulation. With such high uptake comes high demand for soluble trace elements in the rhizosphere. How then do hyperaccumulators manage trace element dynamics in the rhizosphere? Rhizosphere chemistry There are four distinctive soils in which most hyperaccumulating plants have been discovered: serpentine soils (Ni), seleniferous soils (Se), calamine soils (Zn), and soils of the African copper belt (Co, Cu, Cr, Ni, Zn) (Reeves 2002). While there are characteristic soils in which hyperaccumulators typically evolve, some hyperaccumulators can be found in an array of soil conditions. As an example, Pteris vittata (As-hyperaccumulator) has been found in multiple soils with one thousand fold differences in their As concentrations (Liao et al. 2004). Additionally, accessions of Pteris vittata from uncontaminated soils accumulate similar As concentrations as accessions from contaminated soils (Zhao et al. 2002a). Soil descriptions should be included in hyperaccumulator literature when studies occur in the field, particularly if new hyperaccumulators are described. Once background soil conditions are known we can examine how roots modify the rhizosphere. We know roots alter soil chemistry in many ways, via exudation and uptake. Roots may change soil chemical concentrations, pH, redox conditions, form organic acid complexes with nutrients, and chelate metals (Hinsinger 1998). Hyperaccumulators somehow manage to acquire large amounts of trace elements, likely at least in part through the same mechanisms as their non-hyperaccumulating relatives. Although novel ways of accessing trace elements in the rhizosphere may exist in hyperaccumulators, this remains in debate. There is some evidence for Plant Soil enhanced access of hyperaccumulators to non-labile soil pools, as will be reviewed in the next section. Root exudation effects on trace element solubility Some reports suggest potentially unique trace element solubilization by hyperaccumulators. For example, it was shown that populations of Thlaspi caerulescens grown in pots amended with ZnS accumulated more Zn in their shoots than the calculated total water and ammonium-nitrate extractable Zn in pots, indicating that the plants were accessing less available pools of Zn (Whiting et al. 2001d). Non-hyperaccumulators were not used in this study, so we cannot know whether or not these findings are indeed unique to hyperaccumulators, but due to the lack of high Zn accumulation in the ZnS treatment compared to the other Zn treatments, the mobilizing ability of these plants may not be very strong (Whiting et al. 2001d). Another study found that the mobile Zn fraction in soil accounted for less than 10% of Zn within shoots in a Thlaspi caerulescens, while a related nonhyperaccumulator obtained 55% of shoot Zn from the mobile fraction (McGrath et al. 1997). The authors suggested that the hyperaccumulator is therefore better able to access Zn from the non-mobile fraction; the route by which the remaining Zn was acquired remains unknown. It is possible that the unaccounted Zn in both species was accumulated by the same undetermined mechanism(s), which could include microbiological activity. In both of these studies it is possible that no novel mobilizing mechanism exists, rather plant depletion of the mobile pool could shift the equilibrium from the less mobile to the mobile pool. Although unique mechanisms of hyperaccumulators can be challenging to demonstrate, some reports of general changes in rhizosphere chemistry exist. A Nihyperaccumulator decreased the residual Ni pool in rhizosphere soil and increased the reducible and oxidizable fractions while a non-hyperaccumulator did not (Kidd et al. 2007). Root exudation should be examined further because it may play a key role in metal solubilization. For example, root exduates (and/ or microbial activity) were proposed to be an integral part of Ni accumulation in Thlaspi goesingense where organic acids may participate in dissolution of Nibearing mineral surfaces (Puschenreiter et al. 2005). Other studies have focused specifically on root exudate amounts and composition. Plant exuded reductants can reduce Mn and Fe oxides that have adsorption sites for metals, increasing metal solubility, but the Ni-hyperaccumulator Alyssum murale was found to produce less reductant than a nonhyperaccumulator (Bernal et al. 1994). Twice as much dissolved organic carbon (DOC) and a different composition of exudates was produced by the Ashyperaccumulator Pteris vittata than a related nonhyperaccumulator (Tu et al. 2004). However, in another study with Pteris vittata, DOC in the rhizosphere was shown to be similar in quantity to a non-accumulating plant (Cattani et al. 2009). DOC may have different functions in different plant species, e.g. DOC may immobilize and detoxify metals (Römkens et al. 1999), or rather increase trace element availability (Fitz and Wenzel 2002). Field data coupled with modeling indicated that DOC may create ligand-induced mobilization of Ni, thus improving hyperaccumulation potential (Wenzel et al. 2003). Water-soluble root exudates from hyperaccumulating plants increased trace element extraction (Tu et al. 2004; Li et al. 2005b) and non-water soluble exudates or mucigel production may influence metal desorption in the rhizosphere as well (Ingwersen et al. 2006). Further investigation may provide better insight into differences between species in exudate composition and their importance for hyperaccumulation, for example the role of histidine in Nihyperaccumulation in Alyssum lesbiacum has been better understood because of complementary studies with the non-accumulator Alyssum montanum (Krämer et al. 1996). Several studies so far have shown no indication that the root activity of Cd/Zn-hyperaccumulator Thlaspi caerulescens or Ni-hyperaccumulator Alyssum murale have a unique capability to mobilize trace elements in soil (Hutchinson et al. 2000; Zhao et al. 2001; Massoura et al. 2004; Hammer et al. 2006). If hyperaccumulators have a novel mechanism for mobilizing trace elements in soil, they may be expected to increase accumulation in co-cropped plants (if competition is not a factor). This effect was indeed seen in Hordeum vulgare co-cropped with Thlaspi caerulescens (Gove et al. 2002), but most evidence so far points to the contrary (Whiting et al. 2001b, c; Liu et al. 2005a; Ingwersen et al. 2006). Lowering rhizosphere pH has also been proposed as a mechanism for increasing metal hyperaccumulation in plants because it generally increases cation Plant Soil bioavailability in soil (Delorme et al. 2001). Wenzel et al. (2004) reviewed this topic and presented a table showing that many studies that investigated this mechanism rejected this hypothesis, e.g. Cd/Znhyperaccumulation in Thlaspi caerulescens (Knight et al. 1997; Hutchinson et al. 2000; Luo et al. 2000); and Ni-hyperaccumulation in Alyssum murale (Bernal and McGrath 1994), Alyssum serpyllifolium subsp. lusitanicum (Kidd et al. 2007), and Thlaspi goesingense (Puschenreiter et al. 2003; Wenzel et al. 2003). In contrast, a recent study suggested rhizosphere acidification is important for the Mn-hyperaccumulator Chengiopanax sciadophylloides (Mizuno et al. 2006). It must be noted that there are limitations in the interpretation of some of these results. Without the use of microelectrodes or plants growing in agar containing pH indicators rhizosphere sampling can be poor and some cases may have reported differences in bulk soil pH rather than rhizosphere pH. In addition, suitable controls must be used to determine if the amount of acidification by the hyperaccumulator is novel, or the result of typical rhizosphere acidification. When Thlaspi caerulescens was compared to the non-hyperaccumulator Thlaspi ochroleucum, pH differences did not account for increased Zn uptake by the hyperaccumulator because both plant types had a similar rhizosphere pH (McGrath et al. 1997). Although metal mobility in the rhizosphere can increase because of lower pH, from the data collected so far it appears hyperaccumulators are not novel drivers of this effect and this may not be a satisfactory explanation for hyperaccumulation (Wenzel et al. 2004). Amidst differing results and the small number of species investigated, much more work is required to understand the mechanisms by which hyperaccumulators differ from non-accumulators with respect to trace element mobilization. We may find mechanisms used by hyperaccumulators for trace element extraction differ by plant species and elemental species. The function and composition of root exudates should be examined in more detail. rhizosphere microorganisms playing out in the soils beneath hyperaccumulator populations? In general, soil microorganisms influence the rhizosphere by altering nutrient cycling and availability (Gobran and Clegg 1996), but these mechanisms could also influence nonnutrient trace element availability. Table 1 summarizes several studies that have shown that hyperaccumulators grown with inoculated or non-sterilized soil often accumulated different amounts of trace elements aboveground than plants grown in uninoculated or sterilized soil. Of the twenty-six effects shown in Table 1, in sixteen instances plants accumulated more trace elements when they were inoculated; compared to seven instances where there was no effect and three instances where inoculation reduced the trace element concentration in plants. Although these effects can be strong, seven of the effects reported increased plant concentrations by >95%, the mechanisms responsible for these microorganism-associated changes often remain unknown. One possibility could be the effects of sterilization procedures on soil chemistry (Salonius et al. 1967; McNamara et al. 2003), however recent work on Ni-hyperaccumulation has shown that rhizosphere microorganisms affected plant gene expression, as evident from differences in shoot proteome (Farinati et al. 2009). These findings warrant further studies of the interactions between rhizosphere microorganisms and plant hyperaccumulators. Three specific mechanisms of how microorganisms may increase plant hyperaccumulation have been suggested: they may increase root surface area and hair production, increase element solubility, or increase soluble element transfer from the rhizosphere to the plant (Whiting et al. 2001a). In the next section we examine recent findings from the literature regarding rhizosphere bacteria and fungi, and discuss them in the context of these three mechanisms. Knowing more about these mechanisms and their consequences will improve our understanding of plant hyperaccumulation. Rhizosphere bacteria Rhizosphere microorganisms Increasing root surface area and root hair production While we know very little about how roots work compared to aboveground organs, we know even less about other components of the soil ecosystems. What is the co-evolutionary legacy between roots and Bacteria did increase root hair production and root surface area when the non-hyperaccumulator Brassica juncea was grown with Se (de Souza et al. 1999). Rhizosphere bacteria that improve plant growth are Plant Soil known as plant growth promoting rhizobacteria (PGPR) (Benizri et al. 2001). PGPR can increase root growth by restricting rhizosphere accumulation of ethylene, which inhibits plant growth; the bacterial mechanism involves ACC-deaminase activity. Also, some bacteria produce plant growth regulators that cause root cell elongation, such as indole-3-acetic acid (IAA, an auxin). Both PGPR mechanisms have been found in association with Ni-hyperaccumulators Alyssum serpyllifolium and Thlaspi goesingense (Idris et al. 2004; Ma et al. 2009). Another, highly specialized PGPR interaction occurs where legume growth is increased by symbiotic root nodule bacteria. Although these bacteria have the largest effect on legumes when the bacteria infect the plants and Table 1 The magnitude of the effect of microorganism inoculation on plant aboveground trace element concentrations Plant species Microorganism Effecta Reference Microbacterium arabinogalactanolyticum ↑ 32% [Ni] (Abou-Shanab et al. 2003a) Microbacterium arabinogalactanolyticum AY509225 Microbacterium liquefaciens ↑ 46% [Ni]b (Abou-Shanab et al. 2006) ↑ 24% [Ni] (Abou-Shanab et al. 2003a) Microbacterium oxydans AY509222 ↑ 41% [Ni]b (Abou-Shanab et al. 2006) Microbacterium oxydans AY509223 ↑ 35% [Ni]b (Abou-Shanab et al. 2006) Non-sterile soil ↑ 95% [Ni] (Abou-Shanab et al. 2003b) Sphingomonas macrogoltabidus ↑ 17% [Ni] (Abou-Shanab et al. 2003a) Rhizosphere derived inoculant ↑ 100% [Cd]b (Farinati et al. 2009) Rhizosphere derived inoculant ↑ 100% [Zn]b (Farinati et al. 2009) Berkheya coddii Glomus intraradices Native AMF ↑ 167% [Ni] ↑ 45% [Ni] (Turnau and MesjaszPrzybylowicz 2003) Pityrogramma calomelanos Soil derived bacteria No effect [As] (Jankong et al. 2007) Soil derived fungi ↓ 31% [As] (Jankong et al. 2007) Alyssum murale Arabidopsis halleri Pteris vittata Sedum alfredii Thlaspi caerulescens Thlaspi praecox Gigaspora margarita No effect [As] (Trotta et al. 2006) Glomus mosseae No effect [As] (Trotta et al. 2006) Glomus mosseae No effect [As] (Liu et al. 2009) Glomus mosseae ↓ 33% [As] (Liu et al. 2005b) Glomus mosseae ↑ 31% [As] (Wu et al. 2009) Soil derived inoculant ↑ 42% [As]b (Al Agely et al. 2005) Burkholderia cepacia ↑ 243% [Cd] (Li et al. 2007) Burkholderia cepacia ↑ 96% [Zn] (Li et al. 2007) Mixed inoculant containing Enterobacter cancerogenes, Microbacterium sapherdae, and Psuedomonas monteilii ↑ 100% [Zn]b (Whiting et al. 2001a) No effect [Zn] (Whiting et al. 2001a) Soil inoculant predominantly containing Glomus etunicatum, Glomus fasciculatus, and Glomus mosseae ↓ 28% [Cd]b (Vogel-Mikuš et al. 2006) No effect [Pb] (Vogel-Mikuš et al. 2006) No effect [Zn] (Vogel-Mikuš et al. 2006) a The equation ([TE]inoculated−[TE]control)/[TE]control expressed as a percentage was used to determine the magnitude of the effect Different control conditions were used as described in the original publications b Estimates from graphically presented values in the original publications were used to calculate the magnitude of the effect Plant Soil live within the plant nodule, rhizobia also live in the rhizosphere. Root nodules or nodule scars have been observed in several leguminous hyperaccumulators such as Se-hyperaccumulating species of Acacia cana (Beadle 1964), Astragalus bisulcatus and Astragalus pectinatus (Wilson and Chin 1947), as well as the Ni-hyperaccumulator Pearsonia metallifera (Corby 1974); but the metal content of the soil was not recorded at the time. A study of two legumes growing on a copper mine containing 461 μg g−1 Cu indicate that nitrogen fixation occurred at similar levels in a copper tolerant population of Lotus purshianus compared to a population from a control meadow site, but Cu-tolerant Lupinus bicolor had a lower rate of N2 fixation than the control meadow population (Wu and Kruckeberg 1985). Sinorhizobium fredii and S. meliloti have been shown to be Se-tolerant (Kinkle et al. 1994), but further work is necessary to determine the trace element tolerance of rhizobia associated with metallophytes growing on highmetal soils and the effects of the symbiosis on plant metal accumulation. Other PGPR may also become endophytes within the plant xylem by infecting the plant roots (Gagné et al. 1987), although this infection route has never been verified in hyperaccumulators (Rajkumar et al. 2009). Bacterial genera inside the roots and shoots of hyperaccumulators have been found to be both similar to soil bacteria in Alyssum bertolonii (Barzanti et al. 2007). Yet others have found that endophytes in Thlaspi caerulescens and Thlaspi goesingense were different from soil bacteria (Lodewyckx et al. 2002; Idris et al. 2004). The difference between habitats of plant roots, shoots, and the soil as well as infection modes may explain some of these observed differences (Idris et al. 2004; Barzanti et al. 2007). The presence of endophytic bacteria in hyperaccumulators may increase plant growth because they can have ACC-deaminase activity (Idris et al. 2004). Other mechanisms may exist to promote plant growth but there are not many studies that focus on hyperaccumulators (Rajkumar et al. 2009). The survival of endophytes within a hyperaccumulator with its high levels of metals indicates that those bacteria may have distinct adaptations for metal tolerance (Idris et al. 2004; Mengoni et al. 2010). Further studies on PGPR could be an effective way to expand our knowledge of the role of bacteria in hyperaccumulation (Glick 2010); research in this area may prove fruitful because many modes of PGPR action have been described in non-hyperaccumulator systems (Glick 1995). Increasing element solubility Microorganisms have the ability to improve trace element solubility in the rhizosphere, and thus may affect hyperaccumulation. Indeed, increased acid production and metal solubility was described in the presence of rhizosphere bacteria from hyperaccumulating plants (Abou-Shanab et al. 2003b). Besides acids, bacteria can produce other exudates that solubilize metals. Whiting et al. (2001a) found that bacteria solubilize Zn in soil without a change in pH, but specific exudates were not identified. Although the bacteria increased water soluble Zn, only the Zn-hyperaccumulator Thlaspi caerulescens achieved higher Zn concentrations with the inoculation while the non-accumulator Thlaspi arvense did not (Whiting et al. 2001a). Bacteria can also produce iron-chelating organic molecules called siderophores that may affect availability of Fe and perhaps other trace elements (Lodewyckx et al. 2002); the presence of such bacteria alleviated Fe deficiency in Ni-stressed plants (Mishra and Kar 1974). Rhizosphere isolates examined from the Nihyperaccumulators Alyssum serpyllifolium (Ma et al. 2009) and Thlaspi goesingense contained siderophores (Idris et al. 2004). Endophytic bacteria of Alyssum bertolonii and Thlaspi goesingense may also have the potential to produce siderophores (Idris et al. 2004; Barzanti et al. 2007). Bacterial siderophore production can be induced by metals other than Fe (Abou-Shanab et al. 2006 and references therein), so there is opportunity for more investigation here. Another mechanism to increase solubility in the rhizosphere is bacterial phosphatase-mediated dissolution of metal phosphates. However, when examined in Ni hyperaccumulator Alyssum murale there was no difference in siderophore or phosphatase activity between bulk soil and rhizosphere isolates (Abou-Shanab et al. 2003b). Finally, trace element solubility may be affected by redox changes mediated by bacteria. Di Gregorio et al. (2005) have identified Se-reducing bacteria from the rhizosphere of Astragalus bisulcatus that reduce selenite to elemental Se, this could influence plant Se uptake because reduced Se forms are less soluble than Plant Soil oxidized forms. Although mechanisms of solubilization may appear straightforward, it remains to be determined how much of the solubilized trace elements are then encountered by roots or leached away. Brassica juncea (a Se accumulating species but not a hyperaccumulator) via a combination of enhanced root hair growth (as mentioned above) and higher rhizosphere levels of the amino acid serine/O-acetylserine. O-acetylserine is a known upregulator of plant sulfate/ selenate uptake and assimilation (de Souza et al. 1999). Other mechanisms Increasing soluble element transfer In the rhizosphere bacteria are more abundant than in bulk soil, owing to root-released carbon compounds (Rouatt 1959; Grayston et al. 1998; Badri et al. 2009). Hyperaccumulator rhizospheres may provide a niche for specialized, metal-resistant bacteria, since hyperaccumulator roots typically harbor bacteria that are more resistant to metals than bacteria in the bulk soil (Mengoni et al. 2001; Lodewyckx et al. 2002; Abou-Shanab et al. 2003b; Aboudrar et al. 2007; Becerra-Castro et al. 2009). Higher resistance to Cd and Zn was found in rhizosphere bacteria and fungi isolated from Thlaspi caerulescens than from a non-hyperaccumulator, even though the hyperaccumulator had fewer rhizosphere microorganisms overall (Delorme et al. 2001). Evidence of rhizosphere bacteria that exhibit increased trace element resistance is noteworthy, and the mechanisms driving these observations are unknown and deserve further study. One evolutionary explanation for the observed increased microbial resistance around hyperaccumulating plants is accumulation of trace elements in soil under hyperaccumulator litter, and subsequent adaptation by the microorganisms (Schlegel et al. 1991). Another explanation may be root release of accumulated elements via excretion and/ or root turnover. The mechanisms of bacterial resistance to elevated trace elements in the rhizosphere of hyperaccumulators have not been studied much. Whether they are similar to (Mengoni et al. 2010 and references therein) or differ from known plasmid-borne metal efflux pumps in other bacteria (Idris et al. 2006) remains to be determined in many cases. Bacteria also have the potential for metal uptake (Pal et al. 2007). To learn more from these observations it is important to also determine the effect of these resistant microorganisms on plant hyperaccumulation. Altered levels of regulatory metabolites may also affect uptake of certain hyperaccumulated elements. Bacteria have been shown to enhance selenate uptake in Although microorganisms affect trace element accumulation in hyperaccumulators, no evidence has been found that bacteria increase trace element movement towards the plant root in the rhizosphere (Whiting et al. 2001a). Hyphal foraging may make fungi better candidates for this. Mycorrhizae, for instance, are well-known to transport P and other elements toward plant roots (Bolan 1991). Fungi are discussed in the following section. Rhizosphere fungi Arbuscular mycorrhizae are very important in plant nutrition. Fungal hyphae can supply large portions of essential elements to plants, including up to 60% of plant Cu, 80% of plant P, 25% of plant Zn, and have been implicated in transport of S to plants as well (Marschner and Dell 1994). The similarity in ionic radius and charge to P, Cu/Zn, and S make the non-essential elements As, Cd, and Se candidates for hyphal transport also. However, many hyperaccumulators are from the Brassicaceae, which is generally considered to be a nonmycorrhizal family (Leyval et al. 1997). Recently, arbuscular mycorrhizae have been found in several non-Brassicaceae hyperaccumulator species (Turnau and Mesjasz-Przybylowicz 2003; Perrier et al. 2006; Trotta et al. 2006; Amir et al. 2007; Wu et al. 2007) and several metallophytes of the Brassicaceae (Orłowska et al. 2002; Regvar et al. 2003; Vogel-Mikuš et al. 2005; Pongrac et al. 2007). Although several hyperaccumulating species are woody, no associations with ectomycorrhizae have been reported yet. We now know arbuscular mycorrhizal symbiosis occurs in hyperaccumulators, but generally root colonization was found to be low (Trotta et al. 2006; Amir et al. 2007; Pongrac et al. 2007; Wu et al. 2007), and stronger hyperaccumulators have been reported to be less colonized by mycorrhizae (Amir et al. 2007). These findings are ecologically intriguing. Does the incidence of low levels of mycorrhizal Plant Soil colonization in some hyperaccumulators point to a cost of trace element tolerance in these species, where plants can only weakly support mycorrhizal networks? Although some hyperaccumulator species have low colonization, in others moderate to high colonization rates were found (Turnau and MesjaszPrzybylowicz 2003; Vogel-Mikuš et al. 2006). The amount of mycorrhizal colonization depends upon both plant and fungus; fungal isolate identity affected root colonization rates in the As-hyperaccumulator Pteris vittata (Wu et al. 2009). The factors determining mycorrhizal colonization rates in hyperaccumulators are largely unknown, but in Thlaspi praecox and other Brassicaceae it may be restricted by plant glucosinolates (Vierheilig et al. 2000; Pongrac et al. 2008), although this evidence does not exclude other factors. In fact, percent root colonization by mycorrhizae has also been found to be related to metal content, but all types of correlations have been found. Negative correlations between metal concentration and root colonization have been observed in some Ni-hyperaccumulators including Geissois pruinosa, Phyllanthus favieri, Psychotria douarrei, and Sebertia acuminata (Amir et al. 2007). Positive correlations have been observed in some species where higher metal concentrations were associated with higher root fungal colonization in the As-hyperaccumulator Pteris vittata and the Cd/Pb/Zn hyperaccumulator Thlaspi praecox (Al Agely et al. 2005; Vogel-Mikuš et al. 2006; Pongrac et al. 2007). No correlation between metal content and mycorrhizal colonization was found in the As-hyperaccumulator Pteris vittata and the Cd/Zn-hyperaccumulator Sedum alfredii (Trotta et al. 2006; Wu et al. 2007). The differences reported in the direction of these correlations may be related to how mycorrhizae respond to metals or plants. Reduced spore germination and counts have been observed in the rhizospheres of several hyperaccumulators (Pawlowska et al. 2000; Amir et al. 2007), but this is not true for all hyperaccumulators (Turnau and Mesjasz-Przybylowicz 2003). Just like their plant hosts, some mycorrhizae have developed tolerance to metals (Adriaensen et al. 2006). This could help them colonize hyperaccumulators, which are found in highmetal soils and contain high levels in their roots (Trotta et al. 2006; Vogel-Mikuš et al. 2006; Amir et al. 2007). In several hyperaccumulators roots meet the concentration criteria (0.01% Cd; 0.1% for As, Co, Cu, Ni, and Se; and 1% for Mn and Zn on a dry weight basis) used to evaluate aboveground parts of plants as hyperaccumulators (Amir et al. 2007; Barzanti et al. 2007; Wu et al. 2007). Like the differences reported in rates of colonization and trace element tolerance, mycorrhizae have varying effects on hyperaccumulator trace element uptake (Table 1). In some cases organs from mycorrhizae-inoculated plants had lower trace element concentrations than uninoculated plants; As concentrations were lower in inoculated Pityrogramma calomelanos, Pteris vittata and the Cd/Pb/Zn hyperaccumulator Thlaspi praecox had less Cd in all plant parts as well as less Zn in roots when inoculated (Liu et al. 2005b; Vogel-Mikuš et al. 2006; Jankong et al. 2007). In other cases higher trace element concentrations were found in inoculated hyperaccumulators, including the Ni-hyperaccumulator Berkheya coddii and the As-hyperaccumulator Pteris vittata (Turnau and Mesjasz-Przybylowicz 2003; Al Agely et al. 2005; Wu et al. 2009). In contrast to those effects, no differences were found between inoculated and uninoculated plants with regard to trace element concentrations with As in Pteris vittata or Pb and Zn in Thlaspi praecox (Trotta et al. 2006; Vogel-Mikuš et al. 2006; Liu et al. 2009). One possible mechanism for increased trace element accumulation in inoculated plants is increased absorptive surface area from mycorrhizae, but this mechanism has not been investigated in hyperaccumulators. Differences in trace element concentration from inoculated and uninoculated plants may be explained by the increased growth rate and biomass in inoculated plants (Al Agely et al. 2005; Trotta et al. 2006). Another variable playing a role in accumulation and plant allocation patterns is the translocation factor between roots and shoots in mycorrhizal plants. This type of effect has been observed with Cd and Zn in Thlaspi praecox where the shoot/root translocation factor was higher in mycorrhizal plants (Vogel-Mikuš et al. 2006) and in Pteris vittata where the As translocation factor increased at least five times in inoculated plants (Trotta et al. 2006), however this did not increase the concentration of those elements in plants. From these studies we know mycorrhizal fungi can alter plant trace element accumulation patterns in different ways, but the mechanisms remain obscure. Future research may shed more light on the role of mycorrhizae in trace element hyperaccumulation. This is achievable if a single fungal species is studied, its Plant Soil metal tolerance is noted, and the mechanisms by which it affects plant uptake are identified. Specifically, we note that mycorrhizae are not all alike; like plants, they differ in metal tolerance (Adriaensen et al. 2006). Mycorrhizal species also occupy different niches in the rhizosphere; some have a majority of their hyphae within the root, while others have a majority outside the root (Maherali and Klironomos 2007). Fungal species not identified as mycorrhizae have also been found in hyperaccumulator rhizospheres (Jankong et al. 2007; Wangeline and Reeves 2007). The role of these organisms in hyperaccumulation has yet to be determined, but some have the ability to accumulate and volatilize trace elements (Wangeline 2007). Information on hyperaccumulators and their microorganisms is continuing to amass, and findings thus far suggest significant roles for bacteria and fungi in hyperaccumulation (Table 1). A mechanistic approach, as used by Whiting et al. (2001a) where alternative microorganism-driven modes of action were investigated, will greatly enhance the applicability of these findings. Reporting inoculation effects with accompanying mechanistic information will be beneficial for advancing phytoremediation because it will enable replication of processes in other systems. Conclusions Hyperaccumulators make use of conventional rhizosphere mechanisms to improve their trace element accumulation (i.e. the same mechanisms as other plants) but also may have novel ways in which they manipulate their rhizospheres. Root surface area and nutrient transporters are known to be important in plant nutrition and these traits are important in hyperaccumulation as well. Some hyperaccumulators possess unique root physical characteristics and are able to exhibit chemotropism towards non-nutrient trace elements. Some have altered expression levels or substrate specificities of trace element transporters. Chemical characteristics in the rhizosphere are similar between hyperaccumulators and non-accumulators in that both types of plants manipulate the solubility of trace elements by root exudates, access labile trace elements in the rhizosphere, and alter rhizosphere pH. In addition, hyperaccumulators may be able to mobilize trace elements that are non-labile. Like the majority of land plants, hyperaccumulators make use of exudates produced by microorganisms, interact with PGPR, and support arbuscular mycorrhizal symbioses, all of which can affect plant trace element uptake in significant ways; yet there are distinctive characteristics of hyperaccumulators because they harbor rhizosphere microorganisms that are very tolerant to metals. In closing we wish to ignite interest in hyperaccumulators and call for increased research on these remarkable plants that have served as an impetus for recent remediation technologies. Studies addressing a larger diversity of hyperaccumulating species are needed. While we attempt to generalize patterns, studies of the roots and rhizospheres of less than 10% of known hyperaccumulators have been conducted. From observations on this scant number of plants we find that one cannot assume hyperaccumulator roots are all alike because results differ depending on trace element and plant species. Some hyperaccumulators are quite rare and live in very specialized niches. A greater understanding of how root mechanisms help these plants survive and thrive in such unique soils will contribute greatly to their utilization. Unfortunately, habitat destruction proceeds at alarming rates near some species (Whiting et al. 2004) and extremely valuable information could be lost. If hyperaccumulator species disappear, so will our chance to elucidate and harness their unique capabilities. Acknowledgements Funding for É. R. 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