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Groundwater in many parts of the world contains elevated concentrations of As, particularly in the Indian subcontinent. This As-contaminated groundwater is of concern because of the risks associated with direct ingestion and also with its use in the irrigation of soils for crop production. Indeed, it is estimated that 0.9–1.36 Gg of As is brought onto arable land each year via groundwater extraction for irrigation (Ali, 2003). Within the Austrian Central Alps, for example, Wenzel et al. (2002) reported up to 2.3 μM As in solutions of soils that had been contaminated by mining and smelter activities. Similarly, in soil solutions of paddy soils, concentrations of As often range up to 5 μM, or even up to 170 μM (Zhao et al., 2009; Khan et al., 2010).
The accumulation of As within rice grains poses a health risk to populations that consume a lot of rice (see Carey et al. (2012) for a review). For As(V), uptake by roots occurs via phosphate transporters (Asher & Reay, 1979), whilst As(III) is assimilated by roots via the silicic acid transport system (Ma et al., 2008). When exposed to As(V), it is observed that the As within root tissues is predominantly As(III), thereby indicating that As(V) is efficiently reduced to As(III) within the root (Xu et al., 2007). Therefore, As(III) is typically the dominant oxidation state of As within roots whether taken up in the As(III) form or formed in planta from the reduction of As(V). Much of this As is complexed with phytochelatins and stored within the vacuoles of the root (Raab et al., 2007; Zhao et al., 2009; Liu et al., 2010; Moore et al., 2011), although some As is translocated to the above-ground biomass. Within the xylem and phloem, As is generally dominated by inorganic forms, mainly As(III) (Carey et al., 2010; Ye et al., 2010). The movement of As, particularly through the phloem, is important for the accumulation of As within rice grains (Zhao et al., 2012). Indeed, within rice grains (for example), As is predominately found in the inorganic form (mainly As(III)) and as methylated As species (particularly dimethylarsinic acid, DMA) (Meharg et al., 2008; Carey et al., 2010).
These changes in As speciation have often been measured in bulk tissues, and few studies have provided data showing the spatial variability of speciation within the root cylinder (Lei et al., 2012). Indeed, there has generally been a division between speciation techniques (such as high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS)) and imaging techniques. The recent development of fast and efficient X-ray fluorescence (XRF) detectors such as the Maia detector (Ryan et al., 2010) has permitted the development of ‘X-ray absorption near-edge spectroscopy (XANES) imaging’ as an analytical technique (Etschmann et al., 2010). With XANES imaging, laterally resolved speciation maps are obtained by scanning the same area repeatedly with X-ray energies ranging across an absorption edge. This approach differs from typical studies where a single elemental map of the sample is acquired and then, as a separate analysis, XANES spectra are collected from specific regions of interest within that map. Rather, in XANES imaging, XANES spectra can be recovered at each location in the image. Although XANES imaging has been used for > 10 yr in scanning transmission X-ray microscopy (STXM), this is done in transmission where measurements in bright field are comparatively rapid (see, e.g. Jacobsen et al., 2000). Although fluorescence measurements are more sensitive, XANES imaging has not been possible in fluorescence until recently, because of the previous low speed and efficiency of fluorescence detectors. Using the Maia detector at the Australian Synchrotron (Melbourne, Australia), Etschmann et al. (2010) employed XANES imaging to examine As speciation in a mineral sample. However, analysis of fresh and highly hydrated plant tissues (such as roots) is considerably more difficult, because of the earlier onset of beam damage in hydrated samples (Lombi & Susini, 2009); the dehydration of the sample during the analysis; possible movement/growth of the sample during analysis; and the much lower concentration in environmental samples. This approach (i.e. XANES imaging) is similar to that utilized in fresh tissues of an As hyperaccumulator (Pteris vittata) grown in 1 mM As by Pickering et al. (2006), although they compared maps at three energies corresponding to the peak absorption values of the three standard compounds investigated, As(III)–glutathione (GSH), uncomplexed As(III), and uncomplexed As(V). Rather, here we collect elemental fluorescence maps at 81 energies across the As absorption edge, allowing extraction of detailed near-edge spectra, this permitting more rigorous chemical analyses.
The aim of the present study was to develop fluorescence-XANES imaging as a technique to allow laterally-resolved speciation of metal(loid)s within hydrated and fresh plant tissues; and use fluorescence-XANES imaging to provide spatial data on the speciation of As within roots of wheat and rice exposed to As(V) or As(III). We hope that the demonstration of fluorescence-XANES imaging as a viable technique in hydrated plant tissues will facilitate further studies in this area.
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In the present study, we utilized an efficient XRF detector to develop fluorescence-XANES imaging as a viable technique for highly hydrated plant tissues to provide fully spatially resolved speciation data, whereby XANES spectra can be reconstructed for each pixel of an area mapped at many energies across the absorption edge of the element of interest. This technique will allow important advances in a range of plant-science fields.
This study has provided laterally resolved information on the speciation of As in situ within fresh and hydrated roots, extending the results of previous studies. It is known that As(V) is generally efficiently reduced to As(III) within roots, and it is thought that the toxicity of this As(III) results from its binding to the -SH group of proteins, thereby altering their structure and function (Meharg & Hartley-Whitaker, 2002). Indeed, spatial analysis of As speciation revealed that uncomplexed As(V) in the root tissues was confined to the rhizodermis (plus the exodermis and sclerenchyma in rice) with a calculated concentration of c. 5–20 μg cm−3 at a distance of 0.9 mm from the apex – calculated concentrations of uncomplexed As(V) in the cortex and stele were below the detection limit (Figs 3, 6, Tables 1, 2). The data support the observations of previous studies that the reduction of As(V) to As(III) is important and that this conversion is rapid. We observed that As(V) had been reduced by the time it had moved through rhizodermis (plus the exodermis and sclerenchyma in rice). Interestingly, this reduction of As(V) to As(III) did not appear to be confined to certain areas of the roots, with As(III)–thiol complexes being the dominant species in all inner tissues observed, including the meristem, stele, and the inner tissues of the root cap (Figs 3, 6). Thus, the reduction of As(V) to As(III) did not depend upon the type of tissue, but rather, it appeared to occur in all tissues of the root apex.
As already mentioned, As(III) has a high affinity for -SH groups, including phytochelatins (Zhao et al., 2009). As a result, much of the As(III) within root tissues is complexed with GSH or phytochelatins (Bluemlein et al., 2008; Liu et al., 2010). Subsequently, for most plant species, it is assumed that the reduction of As(V) and the subsequent complexation of As(III) by thiol peptides is the main mechanism of As detoxification in plants (Ha et al., 1999; Zhao et al., 2009; Liu et al., 2010). Indeed, in the present study, although As(V) was efficiently reduced to As(III) (see earlier), no uncomplexed As(III) was detected in root tissues exposed to either As(V) or As(III) (Table 1). Rather, regardless of whether As(III) was supplied in the bulk solution or whether the As(III) formed in planta from the reduction of As(V), LCF suggested that 100% of the As(III) was complexed with thiol complexes. For example, in As(V)-exposed wheat roots, As(III)–thiol complexes accounted for 100% of the total As within the cortex and stele, and 100% of the As in roots exposed to As(III) (maximum concentration c. 3 μg cm−3) (Tables 1, 2, Fig. 5). This does not, however, preclude the possibility that there is a low concentration of uncomplexed As(III) (at concentrations lower than that which can be detected using this technique; see later), which would allow the radial transport of As(III) via the silicic acid transporters and its loading into the xylem. Following its formation within the roots, it seems that much of the of As(III)–thiol complexes are sequestered within the vacuole where the acidic pH (c. 5.5) is favourable for the stability of these complexes (Zhao et al., 2009). Studying roots of rice, Moore et al. (2011) found that As accumulated in vacuoles (including the vacuoles of the endodermis and pericycle), and that this As appeared to be associated with S. However, in the present study, the subcellular distribution of As could not be assessed using fluorescence-XANES imaging because of insufficient spatial resolution.
These results help to explain why As has a comparatively low mobility from the roots to the shoots (see Raab et al., 2007). Regardless of whether As(III) was supplied in the bulk solution or whether it is formed in planta, uncomplexed As(III) was rapidly complexed to form As(III)–thiol complexes (Fig. 3, Fig. 6, and Table 1). In the present study, the observation regarding the spatial distribution of As species within the root tissues assists in explaining the reduced movement of As into the shoots observed here (Table S1) and in other studies (Raab et al., 2007). Of particular importance, it is known that much of the As(III)–thiol complexes are stored within the vacuoles of the root (Raab et al., 2007; Zhao et al., 2009), including in the vacuoles of the pericycle and xylem parenchyma near the root tip (Moore et al., 2011). However, within the phloem and xylem, As is transported primarily as inorganic As (mainly As(III), and, to a much lesser extent, monomethylarsonic acid and DMA) (Zhao et al., 2009; Carey et al., 2010; Ye et al., 2010). Therefore, given that concentrations of uncomplexed As(III) within all root tissues were below the detection limit (including in the tissues where As would be loaded into the stele), these data suggest that the apparently rapid complexation of As(III) to form As(III)–thiol complexes maintains concentrations of As(III) at a low value in all tissues, thereby restricting its movement through the xylem and phloem.
These laterally resolved data reported here regarding the reduction of As(V) to As(III) and the complexation of As(III) by thiol complexes are consistent with previous studies that have investigated the speciation of As in bulk root tissues. Using synchrotron-based X-ray absorption spectroscopy (XAS) to examine Indian mustard (Brassica juncea) plants exposed to 250 μM As(V) in solution, Pickering et al. (2000) reported that 97% of the As within the root tissues was present as As(III)–thiol complexes (and only 3% as uncomplexed As(V)). Similarly, using both HPLC-ICP-MS/electrospray ionization-MS and XAS, Bluemlein et al. (2008) studied root tissues of Thunbergia alata grown in solutions containing 13 μM As(V) and found that c. 50% of the As within the roots was present as As(III) complexed with thiol compounds (c. 40% was uncomplexed As(III) and 10% was uncomplexed As(V)). Finally, in roots of sunflower (Helianthus annuus) exposed to 66 μM As(V) or As(III), Raab et al. (2005) found that 60% of the As was complexed with thiol groups.
Arsenic has been shown to accumulate in Fe-plaques on the external surface of the root, with these plaques consisting of Fe (oxyhydr)oxides, such as ferrihydrite and goethite (Liu et al., 2006). Although substantial quantities of As can adsorb to these plaques when plants are grown in soils, we did not deliberately induce their formation in this solution culture experiment, because we were focusing on the transformations of As within the root itself (the strong As signal from plaque would probably hinder analysis of the As within the root tissues). Regardless, Fe-plaque appeared to form in some roots (Fig. S7). Analysis of this Fe-plaque on the roots exposed to As(V) identified that 80% of the As was present as uncomplexed As(V) and 20% as As(III)–thiol complexes (Table 1). Liu et al. (2006) reported similar findings when studying Fe-plaque on roots of rice: 71–74% of the As was As(V) and 26–29% was As(III). The finding that As(V) (ad)sorbs strongly to the variable-charge surfaces of the Fe (oxyhydr)oxides is hardly surprising given that arsenic acid has pKa values of pKa1 2.3, pKa2 6.8, and pKa3 11.6, whilst the pKa values of arsenious acid are substantially higher (pKa1 9.2 and pKa2 12.7).
Although fluorescence-XANES imaging has been used in the present study to provide laterally resolved speciation of As in highly hydrated plant roots, this technique (as any other) is not without limitations. First, although roots are three-dimensional structures, the resulting image is two-dimensional. However, our use of the mathematical model described by Wang et al. (2013) to calculate concentrations in the individual tissues of the concentric root cylinder can, at least to some degree, overcome this limitation. Secondly, because of the large number of scans required (in this study, 81 scans were used to collect the XANES spectra for each pixel), the parameters of the scan must be chosen carefully in order to avoid damage to the sample. Finally, because synchrotron-based techniques often only have a comparatively small number of standard compounds to use when fitting the sample spectra, minor constituents can be overlooked or not quantified accurately.
In conclusion, using fluorescence-XANES imaging, we have provided laterally resolved data on the speciation of As within root tissues of wheat and rice exposed to either 2 μM As(V) or 2 μM As(III). When exposed to As(V), the As was comparatively rapidly reduced to As(III), with uncomplexed As(V) observed to be the dominant As species only in the rhizodermis (plus the exodermis and sclerenchyma in rice) of both wheat and rice. However, no uncomplexed As(III) was found in any root tissues, regardless of whether the As(III) was formed in planta or whether the As(III) was supplied in the bulk nutrient solution. Rather, As(III) was efficiently complexed to form As(III)–thiol complexes (or extruded into the external solution), with this As species calculated to account for 100% of the As within the cortex and stele. Given that uncomplexed As(III) is the major form transported within the phloem and xylem, understanding the spatial variation in speciation assists in understanding why the mobility of As from the roots to the shoots is low. Indeed, even though As(V) is rapidly reduced to As(III), because As(III) is efficiently complexed by thiol groups (and previous studies have reported that thiol-complexed As(III) are sequestered within vacuoles) the concentrations of uncomplexed As(III) were below the detection limit, thereby restricting the transport of this As species to the shoots. We hope that the development of fluorescence-XANES imaging to provide fully laterally resolved speciation in situ within hydrated and fresh plant tissues will allow important advances in a range of areas within plant science.