Accumulation of arsenic (As) within plant tissues represents a human health risk, but there remains much to learn regarding the speciation of As within plants.
We developed synchrotron-based fluorescence-X-ray absorption near-edge spectroscopy (fluorescence-XANES) imaging in hydrated and fresh plant tissues to provide laterally resolved data on the in situ speciation of As in roots of wheat (Triticum aestivum) and rice (Oryza sativa) exposed to 2 μM As(V) or As(III).
When exposed to As(V), the As was rapidly reduced to As(III) within the root, with As(V) calculated to be present only in the rhizodermis. However, no uncomplexed As(III) was detected in any root tissues, because of the efficient formation of the As(III)–thiol complex – this As species was calculated to account for all of the As in the cortex and stele. The observation that uncomplexed As(III) was below the detection limit in all root tissues explains why the transport of As to the shoots is low, given that uncomplexed As(III) is the major As species transported within the xylem and phloem.
Using fluorescence-XANES imaging, we have provided in situ data showing the accumulation and transformation of As within hydrated and fresh root tissues.
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.
Materials and Methods
Seeds of wheat (Triticum aestivum L. cv Sunbrook) and rice (Oryza sativa L. cv Nipponbare) were germinated in trays lined with paper towel moistened using tap water. The seeds were maintained at 25°C in a laboratory at The University of Queensland (St Lucia, Australia) for either 3 d (wheat) or 4 d (rice). The seedlings were then transferred to a growth cabinet containing 50 l of nutrient solution (see later). A total of 32 seedlings were placed in the container (16 wheat and 16 rice) suspended using shade cloth, with polypropylene beads used to limit entry of light into the nutrient solution. Temperatures were maintained at c. 26°C during the day and 24°C during the night (16 h photoperiod).
A nutrient solution was prepared with the following final composition (μM): NO3−-N, 11 200; K, 4080; NH4+-N, 4000; S, 1610; Mg, 1600; Na, 500; Si, 200; Fe (supplied as Fe(III)EDTA), 100; P, 80; B, 50; Cl, 10; Zn, 10; Mn, 5; Cu, 0.5; and Mo, 0.1 . The pH value of the continuously aerated nutrient solution was not adjusted. The solution was renewed once after 5 d and then every 2 d thereafter. The plants were grown in this nutrient solution for 2 wk before transfer to the synchrotron for analysis. Once at the Australian Synchrotron, plants were placed in individual glass beakers (650 ml) filled with the continuously aerated nutrient solution (as previously) before the addition of As.
Using appropriate volumes of stock solutions of NaAsO2 (As(III)) or Na2HAsO4.7H2O (As(V)), the As was added with a final concentration of either 2 μM As(III) or 2 μM As(V). As already mentioned, As is found at similar concentrations in soil solutions of contaminated areas (Wenzel et al., 2002; Zhao et al., 2009). Phosphorus was excluded from all As-containing solutions. Owing to the potential efflux of As into the nutrient solution (Xu et al., 2007), all solutions were replaced at least once every 24 h (typically every 12 h) after the As had been added. Plants were grown in these solutions for 24–36 h before analysis by μ-XRF (see later). Two additional experiments were conducted to assist in characterizing the effects of As on the growth of wheat and rice (Supporting Information, Notes S1).
Sample and standard preparation
Following exposure to As, roots were harvested with the apical 5–10 mm of each root cut off and placed between two pieces of 8-μm-thick Kapton polyimide film forming a tight seal around the roots to limit dehydration. At least three replicate roots were examined for each treatment, with the replicate roots positioned vertically in the sample holder and scanned simultaneously. The size of each scan was varied according to a range of factors, but they were sufficiently large that they encompassed all replicates in a single scan. After analysis, the roots were examined using light microscopy to visually check for beam damage.
Four standard compounds were also prepared for analysis using fluorescence-XANES imaging: 1 mM NaAsO2 (FSBS/2320/48, Thermo Fisher Scientific, Waltham, MA, USA), 1 mM Na2HAsO4.7H2O (A6756, Sigma Aldrich), 1 mM As(V)–DMA (PS-51, Sigma Aldrich), and 1 mM As(III)-GSH. The As(III)–GSH was used as a model compound for As(III)–thiol complexes, and was prepared from a solution containing1 mM As(III) and 15 mM L-glutathione reduced (G4251, Sigma Aldrich). For each standard solution, 0.2 ml was placed on a strip of filter paper c. 3 mm wide. The four pieces of filter paper were wrapped in polyimide film and placed vertically on a sample holder before analysis using fluorescence-XANES imaging as outlined in the following section.
The movement of samples during analysis (e.g. as a result of continued growth of the roots) is of concern during XANES imaging. Specifically, the underlying assumption of XANES imaging is that each point of the sample is scanned repeatedly with increasing energy in order to obtain XANES spectra for each pixel. In previous experiments, we have observed that roots of cowpea sometimes continue to grow during the XRF analyses (Kopittke et al., 2011). This did not happen in this experiment, and it is interesting to elucidate the reasons why. First, it is noteworthy that roots of wheat and rice elongate more slowly under toxicant-free conditions (c. 0.8–1.0 mm h−1, Fig. 1) than do roots of cowpea (c. 2 mm h−1). Secondly, this previous problem with cowpea (growth during scanning) was far more pronounced for roots not exposed to toxicants – in the present study, all roots were exposed to As for c. 24 h. Dose–response curves (see Supporting Notes S1 and Fig. 1) showed that after 24 h, 2 μM As(V) reduced root elongation by c. 80–85%, although 2 μM As(III) caused no notable reduction in elongation (Fig. 1). Finally, to limit potential growth of roots during the analysis, a container was filled with ice and a Petri dish with deionized water was placed on the surface. When the roots were harvested, they were placed in the chilled deionized water for 5 min before mounting in the sample holder for analysis.
Description of beamline, elemental mapping, and fluorescence-XANES imaging
Samples were prepared (see the previous section) and analyzed at the X-ray fluorescence microscopy (XFM) beamline (Australian Synchrotron), where an in-vacuum undulator is used to produce a brilliant X-ray beam. An Si(111) monochromator and Kirkpatrick–Baez mirrors delivered a monochromatic focused beam onto the specimen (Paterson et al., 2011). The X-ray fluorescence emitted by the specimen was collected using the 384-element Maia detector placed in a backscatter geometry (Kirkham et al., 2010). For all scans, samples were analyzed continuously in the horizontal direction (‘on the fly’).
Three consecutive measurements were conducted for each individual sample: ‘scan 1’ was a survey map to identify the area of interest and to obtain elemental distributions; ‘scan 2’ was fluorescence-XANES imaging (with the XANES stack itself consisting of 81 individual maps at increasing energies, see description later); and ‘scan 3’ was a repeat survey map to allow comparison to the first scan to identify potential beam damage as evident by changes in elemental distributions.
For the fluorescence-XANES imaging, the focused beam size was 2 μm wide by 20 μm high. The specimen was scanned through the focus in the horizontal direction and the fluorescence event stream divided into ‘virtual pixels’ 20 μm in size. At the end of each line, the specimen was stepped by 20 μm in the vertical direction and data were acquired across another row. The 20 μm beam height and the 20 μm sampling interval therefore ensured that the specimen was well probed in the vertical direction with 20 μm resolution. The continuous horizontal transit with 20 μm ‘binning’ in the horizontal direction also ensured complete coverage, with 20 μm resolution. For all survey maps, the beam was likewise focused to either 2 × 2 μm2 or 2 × 10 μm2 to obtain 2 or 10 μm pixel size, respectively. Hereafter, the horizontal sampling interval and the vertical step size are referred to as the ‘pixel size’ for brevity.
Survey mapping (i.e. scan 1 and scan 3) was conducted at 15.4 keV. The transit time per 20 μm pixel was 9.76 ms (2.048 mm s−1 scanning velocity), and hence survey maps in the present study could generally be collected within 10–15 min (although this varied substantially depending upon the size of the area of interest).
The fluorescence-XANES imaging (i.e. scan 2) consisted of forming ‘stacks’ of μ-XRF maps by scanning the entire area of interest 81 times (corresponding to a series of 81 energies across the As K-edge) while progressively increasing the energy from 11.802 to 12.017 keV across the As K-edge. The energies of these 81 progressive scans were selected to match the energy location of the features of the As XANES spectra of the different As species, and were selected as follows: 11.802–11.852 in 0.01 keV increments (six energies), 11.852–11.867 in 0.001 keV increments (15 energies), 11.867–11.887 in 0.0005 keV increments (40 energies), 11.887–11.917 in 0.003 keV increments (10 energies), and 11.917–12.017 in 0.01 keV increments (10 energies). The selection of these energies allows not only separation of inorganic As(III) and inorganic As(V) (whose white lines differ by c. 0.0035 keV; see Carey et al., 2010), but also identification of other bonding configurations or coordinations. For example, the white lines of inorganic As(V) and DMA (As(V)) differ by c. 0.0015 keV (Carey et al., 2010). Although the parameters (step size and velocity) for the 81 scans within each individual XANES stack were kept constant, these parameters were initially varied between samples in order to investigate measurement fidelity, as discussed later. The parameters that were used for fluorescence-XANES imaging of hydrated plant tissues in the present study were as follows: 20 × 20 μm2 pixel size, dwell of either 4.88 or 9.76 ms per 20 μm pixel (i.e. velocity of 4.096 or 2.048 mm s−1), with the beam focused to 2 × 20 μm2. Across the entire XANES stack (81 scans), this resulted in a total irradiation time of 0.395 or 0.791 s pixel−1. In comparison, single point XANES spectra (typically referred to as ‘μ-XANES’) collected using conventional detectors take substantially longer. For example, each μ-XANES spectrum collected by Kachenko et al. (2010) across the As K-edge when examining an As hyperaccumulator (Pityrogramma calomelanos var. austroamericana) took c. 133 s (i.e. 1 s per energy scan).
The X-ray photon event stream was analyzed with GeoPIXE using the Dynamic Analysis method (Ryan & Jamieson, 1993; Ryan, 2000). For the fluorescence-XANES stacks, the GeoPIXE ‘energy association’ module was used to identify and select areas (pixels) where the speciation varied, by comparing the concentration ratios between two energies for each pixel (Fig. S1). Pixels were excluded where the concentration of As was low and corresponded only to background noise (Fig. S2). The XANES spectra were extracted from pixels within the selected areas in the stack series and subsequently background- and baseline-corrected using Athena v. 0.8.061 (Ravel & Newville, 2005). For these data, linear combination fitting (LCF) was performed using Athena with the combination of standards yielding the lowest residual parameter chosen as the most likely set of components. Areal concentrations of the various As species were calculated by multiplying their relative proportion (determined by LCF) by the areal As concentration determined from the survey maps using GeoPIXE. Root thickness profiles were estimated by assuming the root to be cylindrical and using the width as the diameter. Projected volumetric concentrations of the As species were obtained by multiplying the areal concentration by the thickness of the root at each point of the scan. Using these data, the concentration of each As species within the individual tissues of the concentric root cylinder were calculated using the model described by Wang et al. (2013).
Developing the fluorescence-XANES imaging technique in highly hydrated plant tissues
In the present study, analysis using fluorescence-XANES imaging (with an 81 energy stack) took 81 times longer than would have been required to obtain a single elemental map with the same scan parameters. Therefore, although the Maia detector is faster than traditional detectors and has been used to examine intact and hydrated plant roots for both elemental mapping (Kopittke et al., 2011) and single-slice computed tomography (Lombi et al., 2011; Kopittke et al., 2012), the substantially longer times required for fluorescence-XANES imaging have the potential to damage highly hydrated plant tissues.
Initially, three XANES stacks were collected for a comparatively small area (1 mm in the horizontal direction by 0.06 mm in the vertical direction) across a wheat root to check for the onset of beam damage. For the three scans, dwell was increased (i.e. velocity was decreased) progressively, being 0.244, 0.976, or 2.604 ms pixel−1 (2 × 2 μm2). Given that the total photon flux was c. 1 × 109 photons s−1, these three velocities corresponded to c. 6.1 × 104, 2.4 × 105, or 6.5 × 105 photons μm−2 per scan. Following the measurements, for the two scans with longer dwell time, beam damage could be observed using light microscopy and/or by examining elemental distribution (Fig. S3). By contrast, for the fastest of the three scans (c. 6.1 × 104 photons μm−2 s−1 (0.244 ms pixel−1, 8.192 mm s−1)), no damage was observed using the light microscope and the redistribution of As appeared to be minimal (Fig. S3).
To test these parameters for a full fluorescence-XANES imaging scan (i.e. pixel size 2 × 2 μm2, dwell 0.244 ms pixel−1, velocity 8.192 mm s−1), two wheat roots were placed horizontally in the sample holder and examined using fluorescence-XANES imaging (Fig. S4). Owing to the large scan area, the XANES stack collection took c. 7 h to collect, the roots were observed to dehydrate during this time (Fig. S4). In addition, because of the orientation of the roots within the scan, the beam caused substantial damage to the roots at the edge of the scan area (although scans are ‘on the fly’ in the horizontal direction, there is a period of extended dwell at the end of each horizontal line whilst the vertical direction is repositioned).
In order to overcome the issue of dehydration, we took two approaches. First, a small amount of free water was placed in the sample holder surrounding the roots. Whilst this reduced dehydration, it increased the likelihood that a root would move by slipping during a scan. Secondly, it was necessary to reduce the time taken to complete the scan. In response we reduced the size of the area being scanned and increased the pixel size to 20 × 20 μm2, but concomitantly decreased the velocity from 8.192 to 2.048 mm s−1 in order to improve sensitivity. By scanning a smaller area with these parameters (20 × 20 μm2 pixel size, beam focus of 2 × 20 μm2, and velocity of 2.048 mm s−1, corresponding to c. 2.4 × 104 photons μm−2 per energy scan), XANES stacks took c. 2–3 h to collect and no evidence of beam damage was observed.
Mapping speciation of As in highly hydrated plant tissues using fluorescence-XANES imaging
The use of fluorescence-XANES imaging allows collection of laterally resolved data for the in situ investigation of metal(loid) speciation within fresh and intact samples of highly hydrated plant tissues. Here we used fluorescence-XANES imaging to investigate the speciation of As in roots of wheat and rice grown in solutions containing 2 μM As(V) or As(III). Although much is known about the speciation of As within plant root tissues (e.g. its uptake, metabolism, and transport; Zhao et al., 2009), we are unaware of any studies that have provided laterally resolved information in situ using hydrated and fresh plant tissues.
Analyses of bulk tissues revealed that concentrations were c. 10-fold lower for As(III)-exposed roots than for roots exposed to As(V) (Table S1) owing to the absence of P from the exposure solutions, this being in general agreement with the findings from previous studies (see, e.g. Asher & Reay, 1979). Regardless of this difference, concentrations in both As(V)- and As(III)-exposed roots were sufficient to allow fluorescence-XANES imaging.
For As(V)-exposed wheat roots, it was observed that the outer c. 10–100 μm of the root (i.e. rhizodermis and outer cortex, Fig. 2) was dominated by uncomplexed As(V), which accounted for an average of 68% of the total As within these outer tissues, with As(III)–thiol complexes accounting for the remaining 32% of the As (‘region i’ in Figs 3c, 4, S1 and Table 1). However, the proportion of uncomplexed As(V) within the root tissues decreased as the distance from the root surface increased, and beyond the rhizodermis and outer cortex (i.e. > c. 10–100 μm from the root surface), uncomplexed As(V) was no longer the dominant species (Figs 3c, 4, and Table 1). Rather, for these inner tissues, As(III)–thiol complexes were the dominant species accounting for an average of 94% of the total As within this region, with only 5.6% present as uncomplexed As(V) (‘region ii’ in Figs 3c, 4, and Table 1). However, these analyses (Fig. 3c) only provide data on the relative proportions of As species. Also, the apparent presence of low concentrations of uncomplexed As(V) in the inner tissues (5.6% of the total As) probably results, at least in part, from their detection in the overlying tissues in this two-dimensional scan. Therefore, to provide quantitative data on speciation, a transect was examined across the root at a distance of 0.9 mm from the apex (corresponding to the portion of the root with the highest As concentrations) (Fig. 3b) with the model of Wang et al. (2013) then used to calculate concentrations for the individual tissues of the root cylinder. For this transect, it was calculated that uncomplexed As(V) was present in the rhizodermis at a concentration of 34 μg cm−3, with no uncomplexed As(V) in either the cortex or stele (Table 2). Indeed, As(V) was efficiently reduced to As(III) and complexed to form As(III)–thiol complexes, with concentrations of As(III)–thiol complexes in cortex (79 μg cm−3) and stele (23 μg cm−3) reaching a maximum value of c. five- to 10-fold higher than observed for uncomplexed As(V) in the outer tissues (Table 2, Fig. 3b).
Table 1. Results of linear combination fitting (LCF) of K-edge X-ray absorption near-edge spectroscopy (XANES) data to roots of wheat (Triticum aestivum) or rice (Oryza sativa) exposed to either As(III) or 2 μM As(V) for c. 24 h
Four standard compounds were examined: uncomplexed As(V), dimethylarsinic acid (As[V]–DMA), uncomplexed As(III), and As(III)–thiol complexes. For As(III)-exposed roots, no spatial variability was found in speciation, so data are presented for the entire root (‘All’). For the As(V)-exposed roots, data are presented for the ‘outer’ and ‘inner’ tissues (see Figs 3, 5-7) and averaged across the entire root (‘All’). Data are rounded to two significant figures, means ± SD.
R factor = Σi(experimental – fit)2/Σi(experimental)2, where the sums are over the data points in the fitting region.
68 ± 2.2
5.6 ± 2.3
80 ± 3.0
63 ± 1.8
6.0 ± 2.2
100 ± 0
32 ± 2.2
94 ± 2.3
20 ± 3.0
100 ± 0
37 ± 1.8
94 ± 2.3
Table 2. Volumetric concentrations of uncomplexed As(V) and As(III)–thiol complexes in root tissues of wheat (Triticum aestivum) and rice (Oryza sativa) exposed to either As(III) or As(V)
As(III)–thiol (μg cm−3)
As(V) (μg cm−3)
As(III)–thiol (μg cm−3)
As(V) (μg cm−3)
As(III)–thiol (μg cm−3)
As(V) (μg cm−3)
As(III)–thiol (μg cm−3)
As(V) (μg cm−3)
Concentrations were calculated using the mathematical model of Wang et al. (2013) from the projected volumetric concentrations across transects (e.g. see Fig. 3d). The endodermis was not included in the calculations as it would not have formed this close to the tip. Given that the pixel size was 20 μm, it was not possible to differentiate among the rhizodermis, exodermis, and sclerenchyma in rice, and hence these were examined as a single tissue (see Fig. 2).
In contrast to these observations for As(V)-exposed wheat roots, when wheat roots were exposed to As(III), no spatial variations in As speciation were observed (Fig. 5c), with LCF of the XANES spectra suggesting that 100% of the As within the root tissues was present as As(III)–thiol complexes (region iii in Figs 4, 5c, Table 1). Similarly, when examining a transect at a distance of 0.9 mm from the apex for these As(III)-exposed roots, LCF suggested that 100% of the tissue As was present as As(III)–thiol complexes, with a maximum calculated concentration of 2.7 μg cm−3 in the rhizodermis (Table 2, Fig. 5d).
The results for roots of rice exposed to As(V) were similar to those discussed earlier for wheat. Within the apical tissues, when rice roots were exposed to As(V), the rhizodermis, exodermis, sclerenchyma (i.e. within c. 10–50 μm of the surface; Fig. 2) were dominated by uncomplexed As(V) (63%), with lower concentrations of As(III)–thiol complexes (37%; region iv in Figs 4, 6c, Table 1). In the inner tissues of rice, an average of 94% of the As was present as As(III)–thiol complexes and only 6.0% was present as uncomplexed As(V) (region v in Figs 4, 6c, Table 1). Examining a transect across these As(V)-exposed roots of rice at a distance of 0.6 mm from the apex, As(V) was efficiently reduced to As(III) and complexed to form As(III)–thiol complexes, with no uncomplexed As(V) calculated to be present in the cortex or stele but with concentrations of As(III)–thiol complexes in the cortex and stele reaching a maximum value of c. 90 μg cm−3 (Table 2, Fig. 6d).
Again, the pattern of response for As(III)-exposed roots of rice was similar to that described earlier for wheat. Specifically, for roots of rice exposed to As(III), only a single species of As was observed within the root tissues, with As(III)–thiol complexes accounting for 100% of the As (region vi in Figs 4, 7c, Table 1). Similarly, use of LCF across the transect showed that 100% of the As was present as As(III)–thiol complexes, reaching a maximum concentration of c. 4 μg cm−3 in the cortex and stele (Table 2, Fig. 7d).
For the outer tissues of wheat and rice, both uncomplexed As(V) and As(III)–thiol complexes were found to be present (Table 1). To assist in confirming that the detection of this As(III) species was not an experimental artefact (i.e. to confirm that the As(III) species had not resulted from the photoreduction of As(V) during the analysis), the derivative of the normalized XANES spectra were examined (Fig. S5). It was found that the shoulder of the spectra for the root tissues did indeed correspond to the peak of As(III)–thiol complexes rather than uncomplexed As(III). Importantly, had the fluorescence-XANES imaging caused the photoreduction of As(V), it is expected that this would have resulted in the formation of uncomplexed As(III) rather than As(III)–thiol complexes. Thereby, we consider that the detection of As(III)–thiol complexes in the outer tissues was not a result of photoreduction. Interestingly, it is noteworthy that in long preliminary scans, photooxidation was observed for the As(III) standard (Fig. S6) – photooxidation of As(III) has been reported previously by other authors (see, e.g. Foster et al., 1998). However, in the final scans (when the parameters used were the same as for the samples), no evidence of photooxidation was observed and the magnitude of the difference in the white line peaks between As(III) and As(V) was as expected.
For roots exposed to As(V), it was noted that some small areas of the root surface had high concentrations of As (Fig. S7). Upon closer examination, it was observed that these high concentrations of As corresponded to high concentrations of Fe on the root surface, presumably Fe-plaque. The XANES spectra were extracted for these pixels containing high concentrations of both Fe and As. Using LCF, it was identified that the As associated with the Fe-plaque was 80% uncomplexed As(V) and 20% As(III)–thiol complexes (Table 1).
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.
P.M.K. is the recipient of an Australian Research Council (ARC) Future Fellowship (FT120100277), P.W. is the recipient of an ARC Discovery Early Career Researcher Award (DE130100943), and E.L. is the recipient of an ARC Future Fellowship (FT100100337). This research was mainly undertaken on the XFM beamline at the Australian Synchrotron, Victoria, Australia (AS123/XFM/5181).