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. 2010 Jan;152(1):309–319. doi: 10.1104/pp.109.146126

Grain Unloading of Arsenic Species in Rice1,[W]

Anne-Marie Carey 1, Kirk G Scheckel 1, Enzo Lombi 1, Matt Newville 1, Yongseong Choi 1, Gareth J Norton 1, John M Charnock 1, Joerg Feldmann 1, Adam H Price 1, Andrew A Meharg 1,*
PMCID: PMC2799365  PMID: 19880610

Abstract

Rice (Oryza sativa) is the staple food for over half the world's population yet may represent a significant dietary source of inorganic arsenic (As), a nonthreshold, class 1 human carcinogen. Rice grain As is dominated by the inorganic species, and the organic species dimethylarsinic acid (DMA). To investigate how As species are unloaded into grain rice, panicles were excised during grain filling and hydroponically pulsed with arsenite, arsenate, glutathione-complexed As, or DMA. Total As concentrations in flag leaf, grain, and husk, were quantified by inductively coupled plasma mass spectroscopy and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy. The roles of phloem and xylem transport were investigated by applying a ± stem-girdling treatment to a second set of panicles, limiting phloem transport to the grain in panicles pulsed with arsenite or DMA. The results demonstrate that DMA is translocated to the rice grain with over an order magnitude greater efficiency than inorganic species and is more mobile than arsenite in both the phloem and the xylem. Phloem transport accounted for 90% of arsenite, and 55% of DMA, transport to the grain. Synchrotron x-ray fluorescence mapping and fluorescence microtomography revealed marked differences in the pattern of As unloading into the grain between DMA and arsenite-challenged grain. Arsenite was retained in the ovular vascular trace and DMA dispersed throughout the external grain parts and into the endosperm. This study also demonstrates that DMA speciation is altered in planta, potentially through complexation with thiols.

Paddy rice (Oryza sativa) is particularly effective, compared to other cereals, at accumulating arsenic (As) in shoot and grain (Williams et al., 2007b). Rice is the staple food for over half the world's population (Fageria, 2007) and rice represents a significant dietary source of inorganic As, a class 1, nonthreshold carcinogen, particularly in Southeast Asia (Meharg et al., 2009). Inorganic As levels in rice grain are problematic even where soil As is at background levels, derived from geogenic sources (Lu et al., 2009; Meharg et al., 2009). However, widespread pollution of paddy soils with As, leading to further elevation of grain As, has occurred in some regions due to base and precious mining (Liao et al., 2005; Zhu et al., 2008), irrigation of paddies with As-elevated groundwaters (e.g. Meharg and Rahman, 2003; Williams et al., 2006), and the use of arsenical pesticides (Williams et al., 2007a). Unlike other cereal grains, paddy rice cultivation is dependent of soils being anaerobic, and it is this anoxia that gives rise to elevated As concentrations in the plant. Anaerobic soil conditions lead to the mobilization of As as arsenite, where under aerobic systems arsenate dominates (Xu et al., 2008). Arsenite is efficiently assimilated by rice roots through silicic acid transport pathway (Ma et al., 2008).

Knowledge of As metabolism and partitioning within plants, particularly rice, is still developing rapidly (Zhao et al., 2009). Several studies have now shown that As in rice vegetative tissue and grain is predominantly speciated as inorganic As and the methylated species dimethylarsinic acid (DMA), with variable, though low, levels of monomethyl arsonic acid (MMA; Abedin et al., 2002a; Williams et al., 2005, 2006; Norton et al., 2009). Arsenate is an analog of phosphate and competes with phosphate for rice root uptake (Abedin et al., 2002a) while arsenite is taken up by rice roots via silicic acid transporters (Ma et al., 2008). Abedin et al. (2002b) demonstrated that the methylated species DMA and MMA are also taken up by rice plants although at a much slower rate than inorganic As, with the protonated neutral forms also transported through silicic acid pathway (Li et al., 2009). Arsenate is reduced to arsenite within the rice root (Xu et al., 2008; Zhao et al., 2009), which then enters the xylem via a silicic acid/arsenite effluxer (Ma et al., 2008; Zhao et al., 2009). Arsenite may be detoxified through complexation with thiol-rich peptides including phytochelatins (PCs) and glutathione followed by sequestration into vacuoles (Bleeker et al., 2006; Raab et al., 2007b; Zhao et al., 2009). Raab et al. (2007a) found that while methylated As species are taken up by rice roots much less efficiently than inorganic species, they appear to be translocated within the plant more efficiently. The comparative contributions of xylem and phloem transport, in translocation of As to the grain, are unknown.

The main species within rice grain, along with DMA, are inorganic As, particularly arsenite, which may be complexed with thiols (Williams et al., 2005; Lombi et al., 2009). Nutrients are unloaded into the grain from the ovular vascular trace (OVT) into the nucellar tissue and from there are uploaded, via the apoplast into the filial tissue (the aleurone and the endosperm; Krishnan and Dayanandan, 2003). Lombi et al. (2009) recently suggested that this may represent a physiological barrier that As species cross with differential efficiency. However, the transport and unloading of As to/into the grain, which are key processes in terms of human exposure to this contaminant, are far from being fully understood.

This study investigated the differential efficiency with which important As species are translocated and unloaded into the rice grain and the comparative contributions of phloem and xylem transport. Rice panicles were excised below the flag leaf node during grain development, 10 DPA, and treated to a hydroponically administered 48-h pulse of arsenite, arsenate, arsenite glutathione, or DMA. Total As concentrations in flag leaf, grain, and husk samples for each treatment were quantified by inductively coupled plasma mass spectroscopy (ICP-MS), and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy (XANES) analysis. To evaluate the contributions of phloem versus xylem transport, a stem-girdling treatment was applied, using steam to destroy phloem cells in a second set of panicles prior to a pulse of either DMA or arsenite. The spatial unloading of As species into the developing grain was examined by synchrotron x-ray fluorescence (XRF) mapping, and fluorescence microtomography for the DMA and arsenite treatments.

RESULTS

Total As Concentrations in Tissues—Experiment A

Mean total As concentrations in flag leaf samples were 26.8 ± 14.2, 7.56 ± 1.31, 30.8 ± 11.4, and 45.9 ± 14.6 mg kg−1 for when excised plants were exposed to 13.3 μm arsenite glutathione, arsenite, arsenate, and DMA treatments, respectively (Fig. 1A). One-way ANOVA revealed As treatments had a highly significant effect on flag leaf total As levels (P < 0.001) and As levels were significantly lower for control leaves (0.107 ± 0.004 mg kg−1) compared to all other As treatments. The DMA and arsenate treatments gave significantly higher flag leaf As than the arsenite treatment, however, differences between the DMA, arsenate, and arsenite glutathione [As(GS)3] treatments, and between the arsenite and arsenite glutathione treatments, were determined not significant. The mean flag leaf As concentration for the 133 μm arsenite treatment was 123.7 ± 24.8 mg kg−1, more than 10-fold that of the 13.3 μm arsenite treatment.

Figure 1.

Figure 1.

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Total As concentrations in flag leaf (A), grain (B), husk for rice and ratios of flag leaf As concentration to grain As concentration (C and D), and husk As concentration to grain As concentration for rice panicles excised at 10 DPA and hydroponically fed, over a 48-h period, a nutrient solution amended with one of five As treatments at 13.3 μm exposure (E). Note that in D and E the ratios for the 133 μm arsenite treatment are included as they are ratios rather than absolute concentrations. Error bars represent ±se of three replicates.

Mean grain As concentrations (Fig. 1B) were, generally, an order of magnitude lower than flag leaf concentrations, with the exception of the DMA treatment. ANOVA revealed that panicle As treatments had a highly significant effect on grain As levels (P < 0.001) and grain As concentrations were significantly lower for the controls (0.027 ± 0.001 mg kg−1) than for all other As treatments. Mean total As concentrations in the grain were similar for the As(GS)3, arsenite, and arsenate treatments (0.16 ± 0.015, 0.18 ± 0.098, and 0.29 ± 0.029 mg kg−1, respectively). The mean grain As level for the DMA treatment was significantly higher than all other treatments (12.5 ± 3.84 mg kg−1). Mean grain As for the 133 μm arsenite treatment was 1.31 ± 0.04 mg kg−1, about 10-fold higher than that that of the 13.3 μm arsenite treatment.

As treatments had a highly significant effect on husk total As (P < 0.001) and all As treatments led to higher mean husk As levels than the control mean of 0.012 ± 0.0008 mg kg−1 (Fig. 1C). The arsenite glutathione, arsenite, and arsenate treatments led to comparatively similar husk As levels (0.28 ± 0.06 mg kg−1, 0.21 ± 0.14 mg kg−1, and 0.73 ± 0.24 mg kg−1, respectively), with significant differences determined between arsenate and arsenite only. However, as with the grain, the DMA treatment led to significantly higher husk As levels, 6.6 ± 1.15 mg kg−1, than all other treatments. Husk As for the 133 μm arsenite treatment was 2.1 ± 0.1 mg kg−1, about 10-fold higher than that of the 13.3 μm arsenite treatment.

The ratio of flag leaf As to grain As for each treatment is reported in Figure 1D. The ratios of leaf As to grain As for the As(GS)3, arsenite, and arsenate treatments were significantly higher than the control and DMA treatments (P = 0.001). Notably, the mean flag leaf to grain As ratios for the arsenite 13.3 μm treatment and 133 μm treatments were not significantly different. The ratio of husk As to grain As is shown in Figure 1E. ANOVA revealed no significant differences between treatments.

Total Element Concentrations in Stem-Girdled Grain—Experiment B

Mean grain concentrations of As, rubidium (Rb), and strontium (Sr) for the stem-girdling experiment are reported in Figure 2. Based on the findings from experiment A, the arsenite treatment of 133 μm was used to give high grain levels for tomography. As DMA was unloaded very efficiently into grain, the 13.3 μm solution was utilized for tomography. One-way ANOVA determined that differences in mean water uptake for treated panicles were not significantly different (23 ± 2 mL, 18.3 ± 1.7 mL, and 23 ± 1 mL for the control, 133 μm arsenite, and 13.3 μm DMA treatments, respectively, P = 0.123). Two-way ANOVA revealed that the As and stem-girdling treatments had highly significant effects on mean grain As concentrations (P < 0.001 for both) and there was a highly significant interaction between treatments (P < 0.001). There was no significant difference between mean grain As levels in stem-girdled control (no As) panicles and mean grain As in non-stem-girdled control panicles (0.074 ± 0.027 and 0.1 ± 0.032 mg kg−1, respectively, P = 0.9975). Stem girdling had a highly significant effect on grain treated with 13.3 μm DMA, leading to a 55% decrease in grain As concentration, 12.8 ± 0.54 mg kg−1 for stem-girdled panicles compared with 28.6 ± 1.1 mg kg−1 in nonstem girdled (P < 0.001). For panicles treated with 133 μm arsenite, stem girdling had an even greater effect, with grain As concentrations 90% lower in stem-girdled plants compared with non-stem-girdled plants, 0.19 ± 0.03 mg kg−1 in stem-girdled plants compared with 1.67 ± 0.41 mg kg−1 in nonstem girdled (P = 0.0012).

Figure 2.

Figure 2.

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Total grain concentrations of As, Rb, and Sr in rice grain hydroponically fed, over a 48-h period, nutrient solution amended with either 133 μm arsenite or 13.3 μm DMA and 1 mm Rb and Sr, for panicles which have been subjected to a ± stem-girdling treatment. Error bars represent ±se of three replicates.

Two-way ANOVA revealed that stem girdling had a highly significant effect on grain concentrations of the phloem marker, Rb (P < 0.001), with stem-girdled plants having significantly less Rb in the grain than non-stem-girdled plants. For the As control plants, mean grain Rb concentrations were 18.3 ± 4.85 and 221 ± 84.9 mg kg−1 for stem-girdled and non-stem-girdled panicles, respectively (P < 0.001). For plants treated with 133 μm arsenite, mean grain Rb concentrations were 34.9 ± 5.4 mg kg−1 for stem-girdled panicles and 166 ± 50.3 mg kg−1 for non-stem-girdled plants (P = 0.048), and for DMA-treated grain, Rb concentrations in the grain were also significantly lower for stem-girdled plants than for non-stem-girdled plants (21.6 ± 4.9 mg kg−1 and 372 ± 27.2 mg kg−1, P < 0.001). As treatments had no significant effect on grain Rb levels (P = 0.310) and there was no significant interaction between the As and stem-girdling treatments (P = 0.093). Two-way ANOVA revealed that the As and stem-girdling treatments had no significant effect on grain levels of the xylem transport marker, Sr (P = 0.234 and P = 0.963, respectively), and there was no significant interaction between the two treatments (P = 0.555). These results provide evidence that Rb and Sr are efficient tracers for phloem and xylem transport (Kuppelwieser and Feller, 1991) and that the stem-girdled treatment produced the desired result.

Spatial Unloading of As into the Grain

Fluorescence microtomography images for experiment B of a virtual transverse slice, scanned across the grain about halfway down the embryo, illustrate grain unloading of As, Rb, and Sr for rice grain treated with 133 μm arsenite and 13.3 μm DMA, respectively. These images are reported in Figure 3 for non-stem-girdled panicles. The equivalent tomograms showing manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) distributions for both As treatments are shown in Supplemental Figure S1. Tomography imaging reveals a remarkable contrast in As unloading into the grain, between the DMA and arsenite treatments. The arsenite-treated grain exhibits strong localization of As coincident with Sr, Mn, and Fe at the site of the OVT, the source of nutrient influx into the grain. Conversely, the image of As distribution for the DMA treatment shows that As has not remained in the OVT but has spread throughout the external parts of the grain with some migration into the endosperm. This pattern is also evident in the elemental XRF maps of As distribution for experiment A, presented in Supplemental Figure S2, for slices cut across the middle of fresh rice grains. Tomography showed that neither As treatment led to visible As accumulation in the embryo (Fig. 3). Stem girdling appeared to have no effect on the As distribution pattern, with tomograms showing identical element distributions (data not shown) to those of their non-stem-girdled counterparts. For both As and stem-girdling treatments the phloem marker, Rb, is dispersed throughout the outer layers and is particularly enriched in the scutellum of the embryo while Sr is restricted to the OVT (Fig. 3). Tomograms and XRF maps for the arsenite and DMA treatments show that Mn and Fe are localized primarily in the OVT. Zn and Cu, conversely, exhibit no such localization in the OVT. Cu appears enriched in the outer layers and the embryo of the grain. XRF maps show Zn is dispersed around the outer layers of the grain, and enrichment in the embryo is evident in the arsenite tomogram. This enrichment is less clear in the tomogram for the DMA-treated grain, probably due to the lower relative intensity.

Figure 3.

Figure 3.

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Fluorescence microtomography images showing distributions of As, Rb, and Sr for virtual cross section of rice grain pulsed with 133 μm arsenite (top) and 13.3 μm DMA (bottom).

XANES Speciation

For experiment A, XANES spectra collected for fresh grain are reported for each As treatment in Figure 4A, together with those for standards for several As species. Additional standards, acquired from another synchrotron source, are presented in Figure 5 to enable further evaluation of the results for DMA speciation. Absorption edge peaks (white line energies) for As standards increased in the order As(GS)3 < MMA(III) < AsIII < DMA(V) < MMA(V) < AsV, in agreement with Smith et al. (2005) and Meharg et al. (2008). All spectra exhibit features that indicated that more than one species of As was present in the samples analyzed. Spectra for the control, arsenate, and 13.3 μm arsenite treatments all exhibit features that can be ascribed to the presence of reduced As species as well as a peak very slightly to the left (lower energy) or concurrent with the arsenate standard, indicating a substantial proportion of arsenate was present. It was observed (data not shown) that the beam caused reduction of arsenate over several replicate spectra despite usage of a sample cold stage, and so arsenate may be underestimated. The 133 μm arsenite treatment exhibited a markedly different speciation pattern within the grain than the 13.3 μm treatment, with an absorption edge corresponding to arsenite.

Figure 4.

Figure 4.

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Bulk As XANES for fresh rice grain hydroponically delivered a 48-h pulse of nutrient solution spiked with 13.3 μm of different As species and XANES for separated grain parts of the DMA treatment. A, Analysis of bulk tissue; B, analysis of separated tissue.

Figure 5.

Figure 5.

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XANES spectra from different trivalent and pentavalent As standards.

The spectrum for arsenite glutathione-treated rice, on the other hand, is shifted to the right, with respect to the As(GS)3 standard, suggesting the complex has dissociated. Although the peak appears coincident with the DMA(V) standard, its broad nature indicates that it represents a mixture of species.

DMA, added as DMA(V), behaved very differently to the inorganic As species, with a striking shift of their absorption edge to the left of the DMA(V) standard, albeit while maintaining a shoulder concurrent with the DMA(V) standard. This indicates that a proportion of added DMA(V) had altered speciation in the grain. This could occur, potentially, through some demethylation and reduction to arsenite, or reduction to DMA(III), with or without thiol coordination. No DMA(III) standards could be run due to the high instability of this compound. Also, DMA(GS)3 is unstable (Raab et al., 2007c). However, XANES data collected at the Daresbury, United Kingdom synchrotron shows that arsenite shares a similar spectra with that for dithiol-complexed DMA(V) and for the DMA(V)-DMA(III) sulphide dimer (Fig. 5). This highlights the limitations of XANES speciation in distinguishing As species and how the choice of standards can significantly influence interpretation of the data (Beauchemin et al., 2002), and indicates that methylated species may have been dithiol complexed rather than reduced or demethylated.

As grain As levels were so high for the DMA treatment, XANES could be collected for the different grain fractions, by manual separation (the pericarp, it is unclear whether this included the aleurone, can be slid off fresh grain) of this treatment (Fig. 4B). For the endosperm and developing embryo fractions, the spectrum, while having a peak corresponding to DMA(V), exhibited a shoulder/s peak at a lower energy than the DMA standard. The pericarp had a spectrum concurrent with, though broader than, the DMA(V) standard. The lower energy peak may be explained by reduction and, potentially, further complexation with thiols, corresponding better with the DMA(III)-DMA(V) dimer thiol.

DISCUSSION

As Speciation and Accumulation in Grain

Translocation of DMA to the rice grain was over an order of magnitude higher than for inorganic species. Abedin et al. (2002b) found that both MMA and DMA influx into rice roots was low compared to arsenate and arsenite. A similar result was reported for maize (Zea mays) by Abbas and Meharg (2008) for DMA. Raab et al. (2007a) found that for DMA and MMA plant uptake over 24 h in hydroponics, for a wide range of plant species tested, including rice, was much lower than for arsenate and arsenite, but that translocation from root to shoot was much more efficient for the methylated species. It was postulated by Raab et al. (2007b) that this was due to poorer thiol complexation of DMA and MMA, leading to less retardation when translocated through the plant. When As-PC complexes were analyzed in Helianthus annus, MMA-PC complexes could be detected, but only free DMA was found (Raab et al., 2005).

Grain As speciation is dominated by inorganic As and DMA (Williams et al., 2005, 2007a). A survey by Williams et al. (2005) of As speciation in rice grain found that inorganic As accounted for between 64% and 81% of As in rice grain from Europe, Bangladesh, and India, and for 42% in U.S. rice grain, with DMA largely accounting for the remainder. DMA content of rice shoots is low, accounting for only 0% to 5% of As species (Abedin et al., 2002a). It has not yet been resolved how rice obtains DMA. While total plant accumulation and/or in planta production of DMA is low (Zhao et al., 2009), export to grain is highly efficient. The results presented here confirm that rice is remarkably efficient in the unloading of DMA into grain, with DMA distributed rapidly around the pericarp/aleurone/subaleurone zone. Furthermore, the results reported here for the stem-girdling experiment show that phloem transport is responsible for the majority of arsenite transport to the grain, with phloem interruption reducing grain As by 90%. DMA is much more mobile than arsenite in both the phloem and the xylem. This explains why husk concentrations of As in the first experiment were also markedly higher for the DMA treatment than those for inorganic arsenite. It has been suggested that as a nontranspiring tissue developing grain will be fed assimilates primarily via phloem transport, and the actively transpiring husk primarily via the xylem (Tanaka et al., 2007; Lombi et al., 2009). This appears evident in the high concentrations of the phloem marker, Rb, compared with the comparatively low grain levels Sr.

At 13.3 μm exposure, the retardation of arsenate and arsenite into the grain, as compared to DMA, was probably not due to thiol complexation, as bulk XANES analysis showed that for both arsenite and arsenate-exposed plants, speciation was dominated by arsenate, i.e. the arsenite had been oxidized. As arsenate normally has a low affinity for thiol groups (Raab et al., 2007c), thiol complexation, and, therefore, retardation in thiol-rich compartments of the grain cannot explain the comparatively poor unloading of inorganic species from the OVT at low exposure concentrations. It may be possible that there is redox cycling of the inorganic As species in the grain, with the reduced form being thiol complexed; however, this is unlikely, as at higher arsenite exposure concentrations of 133 μm, arsenite remains stable. In contrast, when mature grain was analyzed, Lombi et al. (2009) found that much of the inorganic As was thiol complexed. However, the Lombi et al. (2009) study was for field-grown grain, where plants had been continuously exposed to As through the root system, while in this study, the As was pulsed in hydroponically directly through the stem. The concentration-dependent alteration of arsenite speciation in this study may indicate that a threshold, to induce reductases or other As-reducing cellular processes such as reduced glutathione production, is present in grain tissue, as arsenate is the thermodynamically stable arsenate species, and arsenite is oxidized to arsenate in the low arsenite exposure treatment. When plants were fed with an As-PC analog, As(GS)3, transfer into the grain was limited, and the thiol complex dissociated.

With respect to the DMA-exposed treatments presented here, the situation is more complex as the DMA-exposed plants had As XANES absorption edge shifted to lower energies than the DMA(V) standard, albeit with a shoulder on the DMA(V) standard absorption edge. This indicates that most of the DMA(V) added has altered speciation. This finding confirms the observation of Meharg et al. (2008) for rice endosperm (i.e. polished rice) that was known to have a high DMA content by HPLC-ICP-MS detection following extraction, yet the XANES analysis of this tissue found a much lower DMA content than would be expected from the chromatographic quantification, again with the absorption edge shifted toward lower energies than the DMA(V) standard, toward that of the arsenite standard (Meharg et al., 2008). It should be noted however, that the Meharg et al. (2008) study was based on μ-XANES analyses, which may not be representative of the overall speciation, and not bulk XANES. However, this difference between XANES and HPLC-ICP-MS quantification of DMA was also observed by Lombi et al. (2009), with the percentage of DMA in the endosperm found to be 36% via HPLC-ICP-MS analysis and only 26% through linear combination analysis of XANES spectra. In this study, there is a possibility that DMA underwent demethylation to arsenite in the grain tissue, though this could not be confirmed by HPLC-ICP-MS as tissue weights were too small to perform speciation analysis. However, the complementary studies of Meharg et al. (2008) and Lombi et al. (2009), which had both XANES and HPLC-ICP-MS speciation on the same sample, suggest strongly that demethylation is not occurring in this study, but that DMA(V) speciation is altered either by reduction and/or thiol coordination. DMA thiol complexes are not observed in plant extracts due to the oxidizing nature of extraction conditions (Raab et al., 2007a). The advantage of XANES analysis is that it determines speciation in situ. It should, however, be borne in mind that questions regarding the reliability of quantification of XANES data have been raised and, as demonstrated here, XANES analysis relies on the analysis of all relevant standards, and indeed those standards giving significantly resolved spectra from each other (Lombi and Susini, 2009; Williams et al., 2009).

As Localization in Grain

The tomography and XRF images reveal strikingly different As distributions for inorganic and organic As, showing that for the arsenite-exposed grain, As has remained in the OVT (situated opposite the embryo in tomography images and identifiable by the strong localization of Mn and Fe in the XRF maps), while for the DMA-pulsed grain, As has dispersed into, and throughout, the nucellar epidermis region, with migration into the outer parts of the endosperm evident.

The coincidence of As with Mn and Fe at the site of the OVT (for arsenite) agrees with previous studies (Meharg et al., 2008; Lombi et al., 2009), where mature filled grains have been mapped by synchrotron XRF, and the localization of As in the outer grain layer (for both arsenite and DMA) also agrees with findings of previous studies that rice bran contains higher As compared with white rice (Ren et al., 2006; Sun et al., 2008). Indeed, the tomograms and XRF maps for arsenite and DMA presented here, equate to high As in the bran polish of the grain, although it must be borne in mind that the grains in this study are still developing, and metabolically active, and do not necessarily reflect the final localization and speciation within the mature filled grain (Ren et al., 2006; Rahman et al., 2007; Sun et al., 2008; Lombi et al., 2009). The absence of As (and Mn and Fe) within the embryo disagrees with Lombi et al. (2009), however, again, it must be noted that the results presented here represent a developing caryopsis (and embryo) experimentally, and directly, pulsed with As over a very short time period. The enrichment of Zn and Cu in the embryo does agree with Lombi et al. (2009).

It is interesting that, although phloem interruption by girdling the stem with steam substantially affected grain As concentrations for both the arsenite and the DMA treatments, it did not affect the spatial unloading of As within the grain in either case. Therefore, while the ratio of phloem to xylem transport may be important in the rate of As transport to the grain, it may not be important with regard to As mobility within the grain. Lombi et al. (2009) noted that there appears to be a physiological barrier in the transfer of As from the maternal (OVT and nucellar tissue) to filial tissues (aleurone, endosperm, and embryo). It must be remembered, however, that the Lombi et al. (2009) study had mixed grain speciation, dominated by inorganic As. The OVT comprises both phloem and xylem elements and is the only supply of nutrients (and contaminants) into the developing grain (Krishnan and Dayanandan, 2003). Nutrients are unloaded symplastically into the chalazal tissue immediately below the OVT and from there apoplastically into the nucellar epidermis that encircles the grain (Krishnan and Dayanandan, 2003). Nutrients unloaded into the nucellar epidermis spread throughout the nucellar epidermis, encircling the endosperm (as is evident in Fig. 3 and Supplemental Figs. S1 and S2) and are then transported, via the apoplast, radially inwards into the filial tissue (Krishnan and Dayanandan, 2003). The results presented here support the existence of such a barrier and suggest that DMA is more efficient at crossing it. If DMA is indeed more readily unloaded into the nucellar tissue and uploaded into the aleurone (and ultimately deposited in the endosperm) this would explain why levels of DMA are higher in the endosperm compared with inorganic species and why inorganic As is so high in the bran polish of rice grain. The similarities in As distribution between girdled and nongirdled treatments may suggest that either the mechanism responsible for xylem/phloem unloading is similar or that the limiting step in arsenite redistribution in the grain is the uptake from the apoplast into the filial tissues.

It is necessary to revisit and update interpretation of the As XRF data of Meharg et al. (2008) and Lombi et al. (2009), as the new results presented here may explain why for some rice grain samples, As is very much limited to the region of the OVT while in others it is more dispersed. Where the percentage of DMA is high there is more dispersal through the grain. In Meharg et al. (2008) a range of rice grain was analyzed. The percentage of DMA determined by HPLC-ICP-MS for the Bangladesh, Chinese, and U.S. brown grains in Meharg et al. (2008) was very similar (27%–28%). XRF maps showed that all exhibited a mixture of high localization of As in the OVT region with some dispersal around the pericarp/aleurone region (and inwards), which would be concurrent with the presence of a mixture of DMA and inorganic As (with higher concentrations of inorganic As). Although, interestingly, there does appear to be some variation in the degree of dispersal throughout the grain with the Bangladeshi grain in particular showing relatively little As in the interior parts of the grain with respect to the outer layer (Meharg et al., 2008). In Lombi et al. (2009) an XRF map was generated for a sample that had a particularly high proportion of inorganic As and was correspondingly low in organic As. This showed As to be very much localized in the OVT region with limited dispersal around the pericarp region (Lombi et al., 2009).

CONCLUSION

It should be noted that this system of pulsing the grains directly with As species is considerably different to the whole-plant system, having bypassed the initial entry point for whole plants. Nonetheless, this study demonstrates that As species differ in the efficiency with which they are translocated from the shoot to the grain, and that DMA in particular is translocated to the grain much more efficiently than inorganic species. Additionally, this study shows the spatial unloading of As into the rice grain following a pulse of a known As species, and demonstrates marked differences in As unloading into the grain between DMA and inorganic arsenite-fed grain. The results presented here also demonstrate the comparative contributions of xylem and phloem transport in grain unloading of As for arsenite and DMA, representing a key step forward for the development of strategies required to lower grain As content, essential since rice is a predominant source of inorganic As, a class 1, nonthreshold carcinogen, to the human diet (Meharg et al., 2009).

This study also represents evidence that DMA(V) speciation is altered in planta, potentially through complexation with thiols, and highlights a key advantage of in situ XANES speciation versus extraction and chromatographic separation and detection of As speciation. It also highlights the disadvantages of XANES; not all potential As species in plants are known, and as XANES analysis is dependent on the analysis of all relevant standards, and indeed those standards giving significantly resolved spectra from each other, the presence of unknown phases may go unrecognized. These limitations were only determined and overcome in this study by pulsing known As species into the grain in isolation.

MATERIALS AND METHODS

Plant Growth and Panicle Treatments

A quick flowering rice (Oryza sativa) variety, Italica carolina, originally obtained as part of the Rice HapMap (diversity) project (www.ricehapmap.org), was used. Seeds were sown directly into trays of John Innes number 2 potting compost. Plants were transferred into 1 L pots after several weeks and grown under tropical greenhouse conditions. Supplemental lighting was provided at 150 μmol m−2 s−1 by sodium lamps and the temperature range was kept within 23°C to 35°C. Plants were fed weekly with Sangral soluble fertilizer (Sinclair), a 1:1:1 nitrogen:phosphorus:potassium plus 1 MgO mix, diluted 1 in 100 (w/v). Rice panicles were marked by colored thread at anthesis and at 10 DPA; similar sized, healthy looking panicles were excised below the flag leaf node as described in Chen et al. (2007). All plants and panicles were placed in darkness for 2 h prior to both the initial panicle excision and transfer to nutrient solutions. Immediately following excision from the main plant, the bottom 5 cm of the panicle stem was removed, cutting under water. Excisions and panicle transfer were conducted in low red light conditions. These procedures reduced transpiration, limiting potential xylem air bubbles at the base of the cut stems. Excised panicles were immediately transferred to 100 mL Pyrex glass tubes each containing 50 mL of autoclaved nutrient solution. The composition and pH (6.4 ± 0.05) of the nutrient solution was as described by Chen et al. (2007). As markers for phloem and xylem transport, respectively, Rb and Sr were added to the treatment nutrient solutions at a final concentration of 1 mm. Panicle stems were held in position in the tubes, with a sterilized polyurethane foam bung (sliced vertically to wrap around the plant stem) that was then covered with tinfoil to prevent contamination of the solution and limit solution evaporation losses through the bung. Panicles were then transferred to a growth chamber with a 12-h photoperiod, a day/night temperature of 28°C/23°C, relative humidity of 80/60, and light intensity of 1,200 μmol m−2 s−1. After 24 h panicles were removed, rinsed with milliQ deionized water, and transferred to fresh tubes of sterilized phosphate-free nutrient solution (50 mL) that had been spiked with the appropriate As treatment. Treatment stock solutions were prepared by dissolving the relevant amount of the appropriate salt in milliQ deionized water. In every experiment there were three replicate panicles for each treatment including three controls. For each treatment and control three replicate panicles were used.

Two experiments were performed on rice panicles: A, where a range of As species unloading into grain of non-stem-girdled plants was investigated, with As speciation and localization investigated in fresh sliced grains, and B, where arsenite and DMA unloading into ± stem-girdled plants was investigated, with As localized in intact grain through fluorescence microtomography. In experiment B, panicles were also exposed to Rb and Sr as markers of phloem and xylem flow, respectively (Kuppelwieser and Feller, 1991).

Experiment A consisted of six treatments including the control treatment of no As. As treatments were a 48-h pulse of either 13.3 μm arsenate, arsenite, arsenite glutathione, or DMA(V) or 133 μm of arsenite, and were assigned randomly to panicles as they reached 10 DPA. Following the As pulse the flag leaf of each treated panicle was cut at the base of the leaf blade (above the ligule). Grains were selected from the top and middle regions of the panicle and husks were separated manually. A subsample of the grains from each panicle was immediately chilled to 4°C for the synchrotron analyses and the remainder grains were oven dried, together with the flag leaves and husks, for ICP-MS analysis. All samples were analyzed via ICP-MS for total As as described below. For XANES speciation, one grain from each of the three replicates of each treatment was selected and bulked together into a powder. XRF elemental maps were collected for a single grain from one replicate of the 13.3 μm DMA and 133 μm arsenite treatments, respectively.

For the experiment B, As treatments were a 48-h pulse of 133 μm arsenite or 13.3 μm DMA(V) delivered to both stem-girdled and non-stem-girdled panicles. Stem-girdled panicles were subjected to a 30 s jet of steam to a 1 cm area of the stem between 1 and 2 cm below the panicle head, prior to excision from the plant. This destroys the phloem cells, preventing further phloem transport in to the rice grain while xylem vessels remain functional (Martin, 1982; Kuppelwieser and Feller, 1991; Chen et al., 2007). Grains were selected and separated for analysis as described above, with a portion of the grain from each panicle being kept chilled for synchrotron analysis and the remainder oven dried for ICP-MS analysis. One fresh grain from one replicate of each treatment was then randomly selected for fluorescence tomography.

Total As Concentrations in Flag Leaf, Grain, and Husk

Total As concentrations were determined for digested samples by ICP-MS (7500 Agilent Technologies) as described in Sun et al. (2008). Samples were microwave digested in a scaled-down digestion volume of that described in Sun et al. (2008) with 0.4 mL nitric acid added to approximately 0.04 g sample, left overnight to predigest, and 0.4 mL hydrogen peroxide added immediately prior to digestion. Following digestion the sample was made up to 10 mL with 0.1 mL internal standard, 1,000 μg L−1 indium. Quality control measures were as described in Sun et al. (2008).

As Speciation of Rice Grain

As speciation was determined using bulk XANES. Rice grains were frozen in liquid nitrogen and then powdered with pestle and mortar (for each treatment 2–3 grains from each replicate were combined to form a bulk powder). Powders were applied directly to Kapton polyimide tape that was folded over to form a seal. For grain components of the DMA-treated grain, the pericarp was pulled off the fresh grain and the rudimentary embryo was identifiable and removed with a razor blade, leaving the endosperm. Each part was frozen in liquid nitrogen, powdered, and directly applied to Kapton polyimide tape as described above. The samples were inserted into a multisample holder that was positioned on a cryostat stage (−20°C) to limit beam damage. The XANES spectra were collected at sector 20-ID (Pacific Northwest Consortium Collaborative Access Team) at the Advanced Photon Source, Argonne National Laboratory. The electron storage ring operated at 7 GeV with a top-up fill status. The sector 20 undulator beamline includes a liquid nitrogen-cooled Si (111) double crystal monochromator and this was calibrated using the first inflection point of the silver LIII absorption edge (11,919 eV) for measurements at the As K edge (11,868 eV), as in Smith et al. (2005). Spectra were obtained in fluorescence mode with a solid-state 13-element Ge(Li) detector. As standards were analyzed as powders (diluted in BN) and included: arsenite (NaAsO2), arsenate (NaH2AsO4), MMA(V), and DMA(V). Spectra for MMA(III) and As(GS)3 were kindly provided the Environmental Sciences Group of the Royal Military College of Canada and details can be found in Smith et al. (2005).

DMA Derivatives XANES Spectroscopy

XANES absorption spectra at the As K edge were collected, for a range of trivalent and pentavalent As standards, on station 16.5 at the Council for the Central Laboratory of the Research Councils Daresbury Synchrotron Radiation Source operating at 2 GeV with an average current of 150 mA, using a vertically focusing mirror and a sagitally bent focusing Si(220) double crystal monochromator detuned to 70% transmission to minimize harmonic rejection. For the two model compounds data were collected in transmission mode at ambient temperature. Data for the other samples were collected with the station operating in fluorescence mode using an Ortec 30 element solid state Ge detector. Data collection was performed in a liquid nitrogen-cooled cryostat. Several scans were collected and summed for the each sample.

Synchrotron-Based Mapping of As Distribution in Grain

Elemental maps obtained by synchrotron radiation XRF are projections of all the elements in the specific path of the x-ray beam so it is necessary to prepare thin sections for in situ analyses (Lombi and Susini, 2009). This was achieved taping two razor blades together, flash freezing grains in liquid nitrogen, then slicing transversely across the middle of the grain. While this technique did produce sections of even thickness, their thickness (approximately 1 mm), together with the penetration of the beam, led to the distortion evident in Supplemental Figure S2. Grain sections were enclosed in Kapton polyimide tape and, to limit beam damage, mounted on a cryostat (−20°C) sample holder. Laterally resolved XRF analysis of the thin sections was conducted at sector 20-ID of the Advanced Photon Source (APS) using a beam size of approximately 2 μm and step size of 15 μm.

Fluorescence Microtomography

To overcome the sample preparation issues described above, fluorescence microtomography was conducted on fresh rice grain pulsed with 13.3 μm DMA and 133 μm arsenite and subjected to a ± stem-girdling treatment at sector 13 at the APS. As with XANES and XRF described above, the electron storage ring operated at 7 GeV with a top-up fill mode. Fresh rice grain was suspended from a rotation-translation stage in the path of a 3-μm x-ray beam and translated across in 10-μm steps with a dwell time of 0.25 s per step. The grain was then rotated 1° and the scan process repeated until a rotation of 0° to 180° was complete. XRF intensities were collected using a 16-element Ge detector. The resulting two-dimensional sine wave plots were reconstructed as described in McNear et al. (2005).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure S1. Fluorescence microtomography images showing distributions of Zn, Cu, Mn, and Fe for a virtual cross section of rice grain pulsed with 133 μm arsenite (top) and 13.3 μm DMA (bottom).

  • Supplemental Figure S2. XRF elemental maps for a cross section of a rice grain pulsed with 13.3 μμm DMA (top) and 133 μm arsenite (bottom) for 48 h.

Supplementary Material

[Supplemental Data]
pp.109.146126_index.html (667B, html)

Acknowledgments

Spectra for MMA(III) and As(GS)3 were kindly provided by the Environmental Sciences Group of the Royal Military College of Canada. We would like to express our sincere gratitude to Claire Deacon, Norman Little, and Dave Hadwen of the University of Aberdeen and to all the staff of sectors 13 and 20 at the Advanced Photon Source, particularly Robert Gordon, Dale Brewe, and Nancy Lazarz, for their support.

1

This work was supported by a Biotechnology and Biological Sciences Research Council Doctoral Training Grant. Portions of this work were performed at GeoSoilEnviroCARS (sector 13) and PNC/XOR (sector 20), at the Advanced Photon Source, Argonne National Laboratory. GeoSoilEnviro Consortium for Advanced Radiation Sources is supported by the National Science Foundation-Earth Sciences (grant no. EAR–0622171) and Department of Energy-Geosciences (grant no. DE–FG02-94ER14466). Pacific Northwest Consortium Collaborative Access Team Advanced Photon Source, Sector 20 facilities at the Advanced Photon Source, and research at these facilities, are supported by the U.S. Department of Energy-Basic Energy Sciences, a major facilities access grant from the Natural Sciences and Engineering Research Council, the University of Washington, Simon Fraser University, and the Advanced Photon Source. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences (under contract no. DE–AC02–06CH11357). The U.S. Environmental Protection Agency, through its Office of Research and Development, funded and managed a portion of the research; it has not been subject to Agency review and, therefore, does not necessarily reflect the views of the Agency, no official product endorsement should be inferred.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Andrew A. Meharg (a.meharg@abdn.ac.uk).

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The online version of this article contains Web-only data.

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Associated Data

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Supplementary Materials

[Supplemental Data]
pp.109.146126_index.html (667B, html)
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