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Arsenic (As) is toxic to all forms of life and is a potent carcinogen, which poses a risk to human health (Tripathi et al., 2007; Ali et al., 2009; Zhao et al., 2010a). In general, the inorganic forms (AsIII and AsV) are more prevalent than the organic forms in terrestrial environments, and also more toxic, although this depends on the exact As species. Arsenite (AsIII) has an affinity for sulfhydryl groups found in cysteine residues and, as such, affects protein structure and function. Arsenate (AsV) is a phosphate analogue which can substitute for phosphate at binding sites and can be incorporated into ATP (Meharg & Hartley-Whitaker, 2002).
Humans are exposed to As mainly from drinking water and via the food chain. It has been reported that c. 150 million people are exposed to water containing As above the World Health Organization (WHO) recommended limit of 0.01 ppm (Nordstrom, 2002; Brammer & Ravenscroft, 2009). Dietary intake is especially problematic amongst populations with high rice consumption, because this cereal accumulates relatively large amounts of As (Zhao et al., 2010a).
In plants, AsV is taken up by phosphate transporters (Ullrich-Eberius et al., 1989; Wu et al., 2011), whereas AsIII enters roots via a subgroup of aquaporins, the nodulin-like intrinsic proteins (NIPs) (Isayenkov & Maathuis, 2008; Ma et al., 2008; Kamiya et al., 2009). In Arabidopsis thaliana, two specific NIP isoforms (AtNIP1;1 and AtNIP7;1) have been shown to be involved in the uptake of AsIII (Isayenkov & Maathuis, 2008; Kamiya et al., 2009). In rice, the silicon transporter Lsi1 (OsNIP2;1) is a major uptake pathway for AsIII (Ma et al., 2008).
Although some clarity exists about the uptake mechanisms of AsV and AsIII in plants, it largely remains an open question whether plants contain dedicated As efflux systems. This is in stark contrast with prokaryotes, unicellular eukaryotes and mammals: in bacteria, the ArsAB operon encodes As efflux transporters in the form of antiporters and ABC transporters to pump AsIII out of the cell; in yeast, the ACR3 antiporter removes AsIII from the cytosol to the external medium; and the ABC transporter MRP2 is believed to deliver complexed AsIII into the bile of mammals for subsequent secretion via the faeces (Wysocki et al., 1997; Rosen, 2002; Maciaszczyk-Dziubinska et al., 2011).
As yet, no specific As efflux pathway has been identified in plants, but several studies have shown that plants release AsV and AsIII into the external medium (Xu et al., 2007; Zhao et al., 2010b). Recent work on NIPs has shown that many may be capable of bidirectional transport (Bienert et al., 2008; Isayenkov & Maathuis, 2008; Zhao et al., 2010b), and thus, in certain conditions, may contribute to As removal from the symplast, but the physiological relevance of this process is unknown.
To assess whether As efflux in plants can be augmented, and whether this has positive effects on plant growth, we decided to heterologously express ScACR3 from the yeast Saccharomyces cerevisiae in A. thaliana. ScACR3 is a plasma membrane-located, H+ gradient-driven antiporter, which forms the primary AsIII efflux mechanism in yeast (Wysocki et al., 1997; Maciaszczyk-Dziubinska et al., 2011). No ACR3 homologues have been found in higher plants but, interestingly, the As hyperaccumulator fern Pteris vittata expresses several ACR3-type proteins, which are expressed at the tonoplast and mediate the vacuolar sequestration of AsIII (Indriolo et al., 2010). We found that heterologous expression of ScACR3 improved plant tolerance in response to AsIII and AsV, at both the cellular and whole plant level. ACR3 activity increased As efflux to the external medium and affected the root–shoot partitioning of As.
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Arsenic is toxic to all living organisms, including plants. Consequently, agricultural production is inhibited in areas in which As toxicity is prevalent, such as South-East Asia (Panaullah et al., 2009). Crop plants, especially rice, are also an important route for the transfer of environmental As to humans (Meharg et al., 2009; Zhao et al., 2010a). A lower overall As load in plants is therefore likely to reduce As-induced growth inhibition and would also limit potential human exposure.
The toxicity of As manifests itself mainly in the cytoplasm and, to achieve a reduction in As in this compartment, As influx must be restricted, its efflux must be increased or a mixture of the two will be required. Cytoplasmic efflux can be directed to the vacuole or the apoplast. Recently, the molecular identity of several tonoplast transporters has been reported which are involved in the vacuolar sequestration of As: the ABC transporters AtABCC1 and AtABCC2, which mediate the vacuolar deposition of AsIII–phytochelatin complexes (Song et al., 2010), and antiport mechanisms similar to the yeast ACR3, which sequester free AsIII in the vacuoles in lower plants (Indriolo et al., 2010). As is the case for most harmful substances, the majority of symplastic As remains in the root, and vacuolar sequestration is therefore ultimately limited by the relatively small volume of the root. Not surprisingly, plants possess many clearly defined transporters in root cells for the movement of harmful substances to the apoplast (for a review, see Meharg, 2005). For the removal of cytoplasmic As to the external medium, no dedicated mechanisms have been identified in plants. Nevertheless, AsIII efflux has been reported in many plant species (Xu et al., 2007; Su et al., 2010, Zhao et al., 2010b), but the physiological relevance of this process remains to be assessed. In an attempt to increase As efflux, we used a transgenic approach based on the heterologous expression in Arabidopsis of a well-characterized yeast AsIII efflux system, ScACR3.
ACR3 increases As tolerance in cells and intact plants
Protoplasts from all transgenic lines showed greater viability and As extrusion relative to their respective controls. Thus, at the cellular level, ACR3 expression improves tolerance, presumably through larger As efflux, in all transgenics. In intact plants, three of four of the Arabidopsis lines that expressed the yeast ACR3 under the control of a strong promoter showed better growth in the presence of As stress, whether in the form of AsV, which is rapidly reduced to AsIII in the cytosol, or in the form of AsIII (Figs 4, 5). In addition, the As efflux capacity of transgenic plants was increased significantly in transgenic lines in the wild-type background, but not in transgenic lines in the nip7;1 background (Fig. 6). These findings show that the presence of ACR3 can positively affect plant tolerance, but the level of tolerance may vary depending on the transgenic line, and may be based on different underlying mechanisms.
The overall growth phenotype is probably the result of multiple aspects, and so there may be several factors that contribute to the above-described variation. For example, increased efflux would lower the cytoplasmic As content and, as such, reduce As toxicity and promote growth. However, constitutive As efflux could lead to local apoplastic accumulation around tissues and cells that are particularly sensitive, and thus have a negative impact on systemic tolerance. Enhanced AsIII efflux could increase As loading into the xylem vessels in roots, resulting in increased translocation of AsIII from roots to shoots (Fig. 7). Altered distribution between tissues could either benefit or harm growth and tolerance. For example, protoplasts from line ACR3-W2 showed better As resistance and higher As efflux, but intact ACR3-W2 plants did not show any improvement in growth, irrespective of growth method and As stress. Indeed, occasionally, this line performed worse than the wild-type controls (Fig. 4a). Of all the transgenic lines, line ACR3-W2 showed the lowest ACR3 expression level (c. five- and two-fold lower, respectively, than lines ACR3-W1 and ACR3-N; Supporting Information Fig. S1). This could affect expression patterns or cause local differences in ACR3 functioning, preventing an overall net positive impact of ACR3 function in intact plants. In ACR3-W2 protoplasts, As extrusion is into an ‘infinite’ external medium, and higher level interactions between different compartments, cells or tissues do not occur.
The transgenic lines ACR-N1 and ACR-N2 showed a growth advantage, but no significant increases in As efflux in intact plants. The latter may be a result of an already greater As efflux in nip7;1 plants relative to wild-type plants (Fig. 6). As it has been suggested that NIPs are bidirectional with respect to their As transport (Bienert et al., 2008; Isayenkov & Maathuis, 2008), a loss of function would be expected to reduce AsIII release, but As efflux from nip7;1 control protoplasts was similar to that of wild-type protoplasts, whereas, curiously, As efflux from nip7;1 plants was significantly greater than that from wild-type plants (Fig. 6). This suggests that the loss of function in NIP7;1 alters As release from the symplast, but does so in a cell- and/or tissue-dependent manner. The observation that As efflux in the nip7;1 transgenic plants was not increased significantly suggests that either improved tolerance of these plants relates to phenomena other than As efflux or that efflux is in fact higher in these plants, but the extruded As is largely retained in the apoplast.
ACR3 alters the partitioning of As to root and shoot tissue
In yeast, ACR3 has been shown to be a plasma membrane-localized AsIII efflux system. Our localization study showed that, in plants, too, the yeast ACR3 is localized in the plasma membrane (Fig. 1). In combination with the increased AsIII efflux in transgenic protoplasts (Fig. 3) and from roots of several transgenic lines (Fig. 6), it is likely that ACR3 in plants functions in a similar manner as in yeast, that is, the removal of cytoplasmic AsIII to the apoplast. The lower total As in roots of some transgenic lines is also in agreement with this notion. However, the reduction in total tissue As levels was moderate, which may point to retention of extruded As in the apoplast. However, there are also other processes affected by ACR3 activity: the increased shoot As levels suggest that ACR3 expression enhances As translocation from root to shoot. In general, excess (heavy) metals and metalloids are prevented from entering the shoot, possibly to protect photosynthetic tissues. For As, this strategy is obvious from the very low (c. 0.005) shoot : root ratios that are obtained for total As (Fig. 6), although these would increase after more prolonged exposure to As. The expression of ACR3 in xylem parenchyma cells could contribute to xylem AsIII loading and, as such, alter the shoot : root ratio. The fact that lines ACR-W1, ACR-W2 and ACR-N1 not only have increased shoot : root ratios, but also show better growth, suggests that, within the measured range of c. 0.005–0.008, increased movement of As to the shoot is not detrimental to overall tolerance, possibly because ACR3 expression in shoot tissue contributes to apoplastic deposition of shoot As.
The present study shows that the expression of the yeast ACR3 arsenite export system in Arabidopsis improves tolerance at both the cellular and whole-plant level. The underlying mechanism is a result, at least in part, of greater AsIII efflux into the apoplast and/or external medium, which detoxifies the cytoplasm. Interestingly, a recent study in which ScACR3 was expressed in rice (Duan et al., 2011) also showed that ScACR3 increases efflux. Unfortunately, these authors did not show any growth data, and hence it is not clear whether any increased rice tolerance was achieved, but tissue levels of As were reduced, as was the level of As in the grain for two of three overexpressing lines. Duan et al. (2011) found a greater increase in As efflux in transgenics than reported here, but no change in root–shoot partitioning. Thus, the effects of the heterologous expression of ScACR3 depend on many factors, including plant species, developmental stage and tissue complexity, but, overall, the use of well-characterized As transporters from bacteria and fungi could be a useful tool for the future improvement of crops. This approach would enhance the growth potential of crops in environments in which As is present at toxic levels, but further fine tuning may be required, for example in the form of tissue-specific promoters, to avoid potentially harmful effects, such as greater root to shoot translocation of As. The latter may lead to increased As accumulation in grain, which is undesirable for food crops. However, increased root to shoot translocation provides a means to increase shoot As content, which could greatly benefit phytoremediation applications.