Environmental Pollution 186 (2014) 110e114
Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Antimony uptake, efflux and speciation in arsenic hyperaccumulator Pteris vittata Rujira Tisarum a, Jason T. Lessl b, a, Xiaoling Dong a, Letuzia M. de Oliveira a, Bala Rathinasabapathi c, Lena Q. Ma b, a, * a b c
Soil and Water Science Department, University of Florida, Gainesville, FL 32611, USA State Key Lab of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Jiangsu 210046, China Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 September 2013 Received in revised form 26 November 2013 Accepted 29 November 2013
Even though antimony (Sb) and arsenic (As) are chemical analogs, differences exist on how they are taken up and translocated in plants. We investigated 1) Sb uptake, efflux and speciation in arsenic hyperaccumulator Pteris vittata after 1 d exposure to 1.6 or 8 mg/L antimonite (SbIII) or antimonate (SbV), 2) Sb uptake by PV accessions from Florida, China, and Brazil after 7 d exposure to 8 mg/L SbIII, and 3) Sb uptake and oxidation by excised PV fronds after 1 d exposure to 8 mg/L SbIII or SbV. After 1 d exposure, P. vittata took 23e32 times more SbIII than SbV, with all Sb being accumulated in the roots with the highest at 4,192 mg/kg. When exposed to 8 mg/L SbV, 98% of Sb existed as SbV in the roots. In comparison, when exposed to 8 mg/L SbIII, 81% of the total Sb remained as SbIII and 26% of the total Sb was effluxed out into the media. The three PV accessions had a similar ability to accumulate Sb at 12,000 mg/kg in the roots, with >99% of total Sb in the roots. Excised PV fronds translocated SbV more efficiently from the petioles to pinnae than SbIII and were unable to oxidize SbIII. Overall, P. vittata displayed efficient root uptake and efflux of SbIII with limited ability to translocate and transform in the roots. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Antimony Pteris vittata Uptake Translocation Speciation
1. Introduction Antimony (Sb) is a toxic metalloid widely distributed in the lithosphere, with average soil background concentrations being 0.3e8.6 mg/kg (Tschan et al., 2009). Antimony is a chalcophile, commonly associated with sulfur-rich minerals (Anawar et al., 2011; Liu et al., 2010). Recently, global Sb concentrations have been increasing at an alarming rate. For example, Sb accumulation in arctic snow and ice has increased 50% during the last 30 years (Krachler et al., 2005) and 6.3 tons of Sb are dispersed annually from the aerosols in Tokyo (Iijima et al., 2007). Since the Industrial Revolution, use of Sb has drastically increased due to its use in car brake linings and fire retardants (Maher, 2009), and as a hardening agent in bullet alloy (2e5% Sb) (Steely et al., 2007). Sb has no known biological function and displays carcinogenic properties (Krachler et al., 2001). Its inorganic form is more toxic than the organic form, with SbIII being 10 times more toxic than SbV (Smichowski, 2008). Dusts and ashes containing Sb can induce
* Corresponding author. E-mail address: Lqma@ufl.edu (L.Q. Ma). 0269-7491/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.envpol.2013.11.033
keratitis, dermatitis, conjunctivitis and gastritis and SbIII oxide causes lung cancer in rats (Smichowski, 2008). Though Sb and As are chemical analogs, their mechanisms of uptake and translocation in plants differ. For example, Ashyperaccumulator Pteris vittata (PV) translocates As to the fronds while Sb remains in the roots (Müller et al., 2013). Rice and tomato reduce arsenate (AsV) to arsenite (AsIII) in the roots and rapidly efflux AsIII out of the roots (Xu et al., 2007). On the other hand, maize and As-hyperaccumulator P. cretica translocates Sb to the shoots regardless they are treated with SbIII or SbV (Feng et al., 2011; Pan et al., 2011; Tschan et al., 2008). The interactions between As and Sb in P. cretica are similar to PV with increasing SbV uptake upon AsV addition (Feng et al., 2011; Müller et al., 2013). However, the presence of Sb does not affect As uptake by PV (Müller et al., 2013; Nagarajan and Ebbs, 2007). Antimonite enters Arabidopsis via AsIII transporters, nodulin 26like intrinsic proteins (Kamiya and Fujiwara, 2009) whereas it is unclear how SbV enters plants. On the other hand, arsenate enters plants via phosphate transporters. Though adding phosphate inhibits As uptake by plants, it has no effect on SbV uptake by maize and sunflower (Tschan et al., 2008). In addition, plants usually take up metals and accumulate them in the roots. Metal translocation to
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the shoots is rare and is hypothesized to play a role in defense against herbivores and pathogens (Rascio and Navari-Izzo, 2011). P. vittata is unique because it has high ability to load As in the xylem and translocate it to the fronds. Translocation of other metals has not been observed in PV, as they mostly accumulate in the roots including Sb (Cai et al., 2004; Mathews et al., 2011). For example, PV accumulates 49 mg/kg Sb in the roots after cultivated in quartz substrate with 5 mg/kg SbV for 7 weeks (Müller et al., 2013). In addition to SbV, we know PV can accumulate AsV in the roots and it is reduced to AsIII in the rhizomes, which is rapidly translocated to the fronds (Wang et al., 2002), but little is known about the fate of SbV. The objectives of this study were to investigate 1) Sb uptake, efflux and speciation in arsenic hyperaccumulator PV during short term exposure, 2) Sb uptake by PV accessions from Florida, China, and Brazil during long term exposure, and 3) Sb uptake, translocation and oxidation of excised PV fronds. Knowledge of how PV takes up, transports, and metabolizes Sb will be helpful to better understand arsenic uptake and metabolism in PV.
Sb in plant tissues was extracted with a modified method of Okkenhaug et al. (2012), with Sb extraction efficiency of 70e92% in plants (Table 2). Briefly, plants were harvested and washed thoroughly in DI water before being separated into roots and fronds and then freeze-dried for 2 d. They were then ground with liquid nitrogen to fine powder in a ceramic mortar and freeze-dried for an additional 2 d. Samples of 50 mg of the powdered tissues were shaken at 100 rpm with 10 ml of 0.1 M citric acid for 4 h and then sonicated for 1 h. Extracts were diluted to 50 ml with DI water and filtered (45 mm filter) before separation of Sb species. Samples were further diluted until Sb concentrations were 99% of Sb in the roots (Fig. 3). PV takes up 5 times more AsIII than AsV (Wang et al., 2010), so the difference was not as striking as SbIII and SbV (23e32 times). Besides PV, rice also prefers to take up SbIII over SbV (Huang et al.,
2012). Three rice cultivars accumulated 1.3e2.3 times more Sb in the roots after exposed to 20 mM SbIII than SbV for 3 d (Huang et al., 2012). Thus, PV effectively accumulated Sb in its roots with a clear preference to SbIII, which was 23e32 times more than SbV (Fig. 2A). Though PV was effective in taking up SbIII, Sb translocation to the fronds was limited (Table 2). Sb accumulation in PV roots (95% of Sb in the plant) was also observed by Feng et al. (2009). However, this was not the case with As-hyperaccumulator P. cretica (Feng et al., 2011). Following 2 w of exposure to 20 mg/L SbV (potassium pyroantimonate) in a hydroponic system, P. cretica accumulated 800 mg/kg Sb in the fronds, with only 23% of Sb in the plant being in the roots. The difference in Sb accumulation between P. cretica and PV could be due to the
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R. Tisarum et al. / Environmental Pollution 186 (2014) 110e114
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Pteris vittata accessions Fig. 3. Sb contents in the roots and fronds of three accessions of P. vittata after exposing to 8 mg/L SbIII for 7 d. Data represent the mean of three replicates with standard error, and bars with the same letters are not significantly different.
difference in exposure time (1 d vs. 14 d). Unlike P. cretica, PV showed little ability in Sb translocation even after 7 d. Our data indicated that PV had a different Sb translocation from P. cretica (Feng et al., 2011). However, a similar pattern of PV Sb accumulation in the roots was noticed in P. cretica grown in 20 mg/L SbIII (potassium antimonyl tartate) for 2 w (Feng et al., 2009). Different Sb accumulation pattern in P. cretica came from different Sb species: SbIII (Feng et al., 2009) and SbV (Feng et al., 2011). Different species of plants have been shown to differ in their Sb uptake capacities and tissue storage targets. Many plants including PV accumulate Sb in the roots (Mathews et al., 2011; Müller et al., 2009; Shtangeeva et al., 2012; Telford et al., 2009), while maize and radish accumulate more Sb in the shoots (He, 2007; Tschan et al., 2008). Similar to PV, wheat and rice take up more SbIII than SbV (Huang et al., 2012; Shtangeeva et al., 2012), but rye takes up more SbV than SbIII (Shtangeeva et al., 2012). SbIII might enter into plant roots via aquaglyceroporin channels due to its neutral charge. PV likely uses separate channels for SbIII and AsIII uptake as up to 100 mM SbIII had no impact on PV uptake of 0.1 mM AsIII (Mathews et al., 2011). Up until now, there has been no report on SbIII uptake pathway in PV. Phosphate, AsV, and SbV are chemical analogs and AsV enters the cells via phosphate channels (Tamás and Wysocki, 2001; Wang et al., 2002). Similarly, SbV uptake pathway into plants is also unknown (MaciaszczykDziubinska et al., 2012). Maize reportedly has a different SbV pathway from phosphate because its SbV uptake is independent of phosphate uptake (Tschan et al., 2008). SbV is octahedral with larger size than AsV and phosphate whereas the latter two are tetrahedral (Tschan et al., 2009). The results from Müller et al. (2013) support the idea that SbV is taken up by a different pathway from AsV in PV. Similarly, PV has different AsIII uptake channel from SbIII while rice and Arabidopsis use the same channels for both AsIII and SbIII (Bhattacharjee et al., 2008). Further study on SbV-phosphate interactions is necessary to examine the SbV channel in PV. 3.3. P. vittata roots were able to efflux SbIII but not SbV
Table 2). With high Sb concentrations in the roots, in lieu of translocation, PV may reduce Sb exposure by effluxing it out to the media. To test this hypothesis, PV was first exposed to 8 mg/L SbIII for 1 d, which was chosen because SbIII was stable in the presence of PV for up to 1 d (Fig. 2B). At the end of 1 d, PV roots accumulated 4,192 mg/kg Sb with 81% being SbIII (Table 2). After the roots were rinsed, it was transferred to Sb-free DI water for 1 d to measure Sb efflux from the roots (Fig. 4). Sb efflux in PV roots increased rapidly during the first 4 h, reaching a plateau at 92 mg/kg (f.w.) at 8 h (Fig. 4). After 1 d, the Sb remaining in the roots consisted of SbIII (74%), with 26% of the total Sb in the roots being effluxed out by PV as SbIII (Table 2). SbIII concentration in the roots decreased from 3,119 to 2,113 mg/kg while SbV concentration remained unchanged. Both Sb speciation in the growth media and reduction in SbIII concentration in the roots indicated SbIII was effluxed out of PV roots. Since PV was more efficient in taking up SbIII than SbV, it was possible that SbIII was also easier to be effluxed out of PV roots than SbV. Efflux is a general detoxification mechanism in plants. For example, Chlorella vulgaris effluxed 60% SbIII and 40% SbV after the cells were transferred to Sb-free medium (Maeda et al., 1997). Maeda et al. (1997) suggested C. vulgaris detoxifies Sb by effluxing it out in addition to convert SbIII to the less toxic SbV. In comparison, PV effluxed 18% of the total As in the roots after exposure to AsV for 1 d (Huang et al., 2011; Xu et al., 2007). The slightly lower efflux of As comparing to Sb by PV might be due to high As translocation efficiency. Since PV was inefficient in translocating Sb to the fronds, effluxing Sb out of the roots may help PV to minimize toxicity. 3.4. Excised PV fronds translocated SbV from petioles to pinnae but were unable to oxidize SbIII PV efficiently took up SbIII, but was unable to translocate Sb from the roots to the fronds (Table 2). Thus, we wanted to demonstrate if Sb translocation could occur in excised fronds after exposure to 8 mg/L SbIII or SbV for 1 d (Fig. 5). PV fronds took up SbIII and distributed it evenly in the petioles and pinnae (99 and 114 mg/kg) whereas SbV was mainly translocated to the pinnae (12 and 158 mg/kg). Again, PV fronds were more effective in taking up SbIII than SbV. However, under these artificially induced conditions, PV fronds were much more efficient in translocating SbV
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Fig. 4. Sb concentration (mg/L) in the media during P. vittata uptake from 8 mg/L SbIII for 1 d and Sb concentration in the media (mg/kg roots fw) during subsequent P. vittata efflux in DI water for 1 d. Sb was present as SbIII in both loading media and efflux media as there was no SbIII oxidation during treatment. Data represent the mean of three replicates with standard error.
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from the petioles to the pinnae with translocation factor of 13 (Fig. 5). It suggested a Sb translocation barrier in the roots or rhizomes and further study is required for the location of Sb translocation barrier in PV. To test whether excised pinnae were capable of oxidizing SbIII, PV pinnae were incubated in 100 mg/L SbIII solution under light for 2 d, which was long enough to detect Sb speciation. After 2 d, the pinnae took up 814 148 mg/kg Sb with 599 114 mg/kg being SbIII. SbIII was unchanged after 2 d, indicating no oxidation occurring in excised PV pinnae. It was possible that SbIII was not as mobile with equal concentration in the petioles and pinnae. On the other hand, PV efficiently loaded SbV from the petioles to pinnae or pinnae contained a strong SbV sink, which allowed higher Sb content in the pinnae compared to the petioles. Together these results (Figs. 3 and 5) suggest that when the roots were exposed to SbIII, there is a constraint in SbIII reaching from the roots to the petiole. In short, PV was more effective taking up SbIII than SbV but was inefficient in Sb translocation and transformation. Almost all Sb taken up by PV was retained in the roots, with Sb species in the roots being closely related to Sb species supplied. PV roots were efficient in SbIII efflux (26%) but not SbV. Based on Sb speciation, we have shown that the uptake and translocation mechanisms of Sb in PV were different from arsenic. These results will be valuable for probing and understanding the nature of Sb uptake, translocation and metabolic processes in arsenic-hyperaccumulator P. vittata. Acknowledgments This research was supported in part by the National Natural Science Foundation of China (No. 21277070), UF/IFAS and the Royal Thai Government. References Anawar, H., Freitas, M., Canha, N., Santa Regina, I., 2011. Arsenic, antimony, and other trace element contamination in a mine tailings affected area and uptake by tolerant plant species. Environ. Geochem. Health 33, 353e362. Belzile, N., Chen, Y.-W., Wang, Z., 2001. Oxidation of antimony (III) by amorphous iron and manganese oxyhydroxides. Chem. Geol. 174, 379e387. Bhattacharjee, H., Mukhopadhyay, R., Thiyagarajan, S., Rosen, B.P., 2008. Aquaglyceroporins: ancient channels for metalloids. J. Biol. 7, 33. Cai, Y., Su, J., Ma, L.Q., 2004. Low molecular weight thiols in arsenic hyperaccumulator Pteris vittata upon exposure to arsenic and other trace elements. Environ. Pollut. 129, 69e78.
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Contents lists available at ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Antimony uptake, efflux and speciation in arsenic hyperaccumulator Pteris vittata Rujira Tisarum a, Jason T. Lessl b, a, Xiaoling Dong a, Letuzia M. de Oliveira a, Bala Rathinasabapathi c, Lena Q. Ma b, a, * a b c
Soil and Water Science Department, University of Florida, Gainesville, FL 32611, USA State Key Lab of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Jiangsu 210046, China Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 September 2013 Received in revised form 26 November 2013 Accepted 29 November 2013
Even though antimony (Sb) and arsenic (As) are chemical analogs, differences exist on how they are taken up and translocated in plants. We investigated 1) Sb uptake, efflux and speciation in arsenic hyperaccumulator Pteris vittata after 1 d exposure to 1.6 or 8 mg/L antimonite (SbIII) or antimonate (SbV), 2) Sb uptake by PV accessions from Florida, China, and Brazil after 7 d exposure to 8 mg/L SbIII, and 3) Sb uptake and oxidation by excised PV fronds after 1 d exposure to 8 mg/L SbIII or SbV. After 1 d exposure, P. vittata took 23e32 times more SbIII than SbV, with all Sb being accumulated in the roots with the highest at 4,192 mg/kg. When exposed to 8 mg/L SbV, 98% of Sb existed as SbV in the roots. In comparison, when exposed to 8 mg/L SbIII, 81% of the total Sb remained as SbIII and 26% of the total Sb was effluxed out into the media. The three PV accessions had a similar ability to accumulate Sb at 12,000 mg/kg in the roots, with >99% of total Sb in the roots. Excised PV fronds translocated SbV more efficiently from the petioles to pinnae than SbIII and were unable to oxidize SbIII. Overall, P. vittata displayed efficient root uptake and efflux of SbIII with limited ability to translocate and transform in the roots. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Antimony Pteris vittata Uptake Translocation Speciation
1. Introduction Antimony (Sb) is a toxic metalloid widely distributed in the lithosphere, with average soil background concentrations being 0.3e8.6 mg/kg (Tschan et al., 2009). Antimony is a chalcophile, commonly associated with sulfur-rich minerals (Anawar et al., 2011; Liu et al., 2010). Recently, global Sb concentrations have been increasing at an alarming rate. For example, Sb accumulation in arctic snow and ice has increased 50% during the last 30 years (Krachler et al., 2005) and 6.3 tons of Sb are dispersed annually from the aerosols in Tokyo (Iijima et al., 2007). Since the Industrial Revolution, use of Sb has drastically increased due to its use in car brake linings and fire retardants (Maher, 2009), and as a hardening agent in bullet alloy (2e5% Sb) (Steely et al., 2007). Sb has no known biological function and displays carcinogenic properties (Krachler et al., 2001). Its inorganic form is more toxic than the organic form, with SbIII being 10 times more toxic than SbV (Smichowski, 2008). Dusts and ashes containing Sb can induce
* Corresponding author. E-mail address: Lqma@ufl.edu (L.Q. Ma). 0269-7491/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.envpol.2013.11.033
keratitis, dermatitis, conjunctivitis and gastritis and SbIII oxide causes lung cancer in rats (Smichowski, 2008). Though Sb and As are chemical analogs, their mechanisms of uptake and translocation in plants differ. For example, Ashyperaccumulator Pteris vittata (PV) translocates As to the fronds while Sb remains in the roots (Müller et al., 2013). Rice and tomato reduce arsenate (AsV) to arsenite (AsIII) in the roots and rapidly efflux AsIII out of the roots (Xu et al., 2007). On the other hand, maize and As-hyperaccumulator P. cretica translocates Sb to the shoots regardless they are treated with SbIII or SbV (Feng et al., 2011; Pan et al., 2011; Tschan et al., 2008). The interactions between As and Sb in P. cretica are similar to PV with increasing SbV uptake upon AsV addition (Feng et al., 2011; Müller et al., 2013). However, the presence of Sb does not affect As uptake by PV (Müller et al., 2013; Nagarajan and Ebbs, 2007). Antimonite enters Arabidopsis via AsIII transporters, nodulin 26like intrinsic proteins (Kamiya and Fujiwara, 2009) whereas it is unclear how SbV enters plants. On the other hand, arsenate enters plants via phosphate transporters. Though adding phosphate inhibits As uptake by plants, it has no effect on SbV uptake by maize and sunflower (Tschan et al., 2008). In addition, plants usually take up metals and accumulate them in the roots. Metal translocation to
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the shoots is rare and is hypothesized to play a role in defense against herbivores and pathogens (Rascio and Navari-Izzo, 2011). P. vittata is unique because it has high ability to load As in the xylem and translocate it to the fronds. Translocation of other metals has not been observed in PV, as they mostly accumulate in the roots including Sb (Cai et al., 2004; Mathews et al., 2011). For example, PV accumulates 49 mg/kg Sb in the roots after cultivated in quartz substrate with 5 mg/kg SbV for 7 weeks (Müller et al., 2013). In addition to SbV, we know PV can accumulate AsV in the roots and it is reduced to AsIII in the rhizomes, which is rapidly translocated to the fronds (Wang et al., 2002), but little is known about the fate of SbV. The objectives of this study were to investigate 1) Sb uptake, efflux and speciation in arsenic hyperaccumulator PV during short term exposure, 2) Sb uptake by PV accessions from Florida, China, and Brazil during long term exposure, and 3) Sb uptake, translocation and oxidation of excised PV fronds. Knowledge of how PV takes up, transports, and metabolizes Sb will be helpful to better understand arsenic uptake and metabolism in PV.
Sb in plant tissues was extracted with a modified method of Okkenhaug et al. (2012), with Sb extraction efficiency of 70e92% in plants (Table 2). Briefly, plants were harvested and washed thoroughly in DI water before being separated into roots and fronds and then freeze-dried for 2 d. They were then ground with liquid nitrogen to fine powder in a ceramic mortar and freeze-dried for an additional 2 d. Samples of 50 mg of the powdered tissues were shaken at 100 rpm with 10 ml of 0.1 M citric acid for 4 h and then sonicated for 1 h. Extracts were diluted to 50 ml with DI water and filtered (45 mm filter) before separation of Sb species. Samples were further diluted until Sb concentrations were 99% of Sb in the roots (Fig. 3). PV takes up 5 times more AsIII than AsV (Wang et al., 2010), so the difference was not as striking as SbIII and SbV (23e32 times). Besides PV, rice also prefers to take up SbIII over SbV (Huang et al.,
2012). Three rice cultivars accumulated 1.3e2.3 times more Sb in the roots after exposed to 20 mM SbIII than SbV for 3 d (Huang et al., 2012). Thus, PV effectively accumulated Sb in its roots with a clear preference to SbIII, which was 23e32 times more than SbV (Fig. 2A). Though PV was effective in taking up SbIII, Sb translocation to the fronds was limited (Table 2). Sb accumulation in PV roots (95% of Sb in the plant) was also observed by Feng et al. (2009). However, this was not the case with As-hyperaccumulator P. cretica (Feng et al., 2011). Following 2 w of exposure to 20 mg/L SbV (potassium pyroantimonate) in a hydroponic system, P. cretica accumulated 800 mg/kg Sb in the fronds, with only 23% of Sb in the plant being in the roots. The difference in Sb accumulation between P. cretica and PV could be due to the
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Treatment (mg/L) Fig. 2. Sb concentrations in the roots (A) and the growth media (B) of P. vittata after exposing to 1.6 or 8 mg/L SbIII or SbV for 1 d. Data represent the mean of three replicates with standard error, and bars with the same letters are not significantly different.
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Pteris vittata accessions Fig. 3. Sb contents in the roots and fronds of three accessions of P. vittata after exposing to 8 mg/L SbIII for 7 d. Data represent the mean of three replicates with standard error, and bars with the same letters are not significantly different.
difference in exposure time (1 d vs. 14 d). Unlike P. cretica, PV showed little ability in Sb translocation even after 7 d. Our data indicated that PV had a different Sb translocation from P. cretica (Feng et al., 2011). However, a similar pattern of PV Sb accumulation in the roots was noticed in P. cretica grown in 20 mg/L SbIII (potassium antimonyl tartate) for 2 w (Feng et al., 2009). Different Sb accumulation pattern in P. cretica came from different Sb species: SbIII (Feng et al., 2009) and SbV (Feng et al., 2011). Different species of plants have been shown to differ in their Sb uptake capacities and tissue storage targets. Many plants including PV accumulate Sb in the roots (Mathews et al., 2011; Müller et al., 2009; Shtangeeva et al., 2012; Telford et al., 2009), while maize and radish accumulate more Sb in the shoots (He, 2007; Tschan et al., 2008). Similar to PV, wheat and rice take up more SbIII than SbV (Huang et al., 2012; Shtangeeva et al., 2012), but rye takes up more SbV than SbIII (Shtangeeva et al., 2012). SbIII might enter into plant roots via aquaglyceroporin channels due to its neutral charge. PV likely uses separate channels for SbIII and AsIII uptake as up to 100 mM SbIII had no impact on PV uptake of 0.1 mM AsIII (Mathews et al., 2011). Up until now, there has been no report on SbIII uptake pathway in PV. Phosphate, AsV, and SbV are chemical analogs and AsV enters the cells via phosphate channels (Tamás and Wysocki, 2001; Wang et al., 2002). Similarly, SbV uptake pathway into plants is also unknown (MaciaszczykDziubinska et al., 2012). Maize reportedly has a different SbV pathway from phosphate because its SbV uptake is independent of phosphate uptake (Tschan et al., 2008). SbV is octahedral with larger size than AsV and phosphate whereas the latter two are tetrahedral (Tschan et al., 2009). The results from Müller et al. (2013) support the idea that SbV is taken up by a different pathway from AsV in PV. Similarly, PV has different AsIII uptake channel from SbIII while rice and Arabidopsis use the same channels for both AsIII and SbIII (Bhattacharjee et al., 2008). Further study on SbV-phosphate interactions is necessary to examine the SbV channel in PV. 3.3. P. vittata roots were able to efflux SbIII but not SbV
Table 2). With high Sb concentrations in the roots, in lieu of translocation, PV may reduce Sb exposure by effluxing it out to the media. To test this hypothesis, PV was first exposed to 8 mg/L SbIII for 1 d, which was chosen because SbIII was stable in the presence of PV for up to 1 d (Fig. 2B). At the end of 1 d, PV roots accumulated 4,192 mg/kg Sb with 81% being SbIII (Table 2). After the roots were rinsed, it was transferred to Sb-free DI water for 1 d to measure Sb efflux from the roots (Fig. 4). Sb efflux in PV roots increased rapidly during the first 4 h, reaching a plateau at 92 mg/kg (f.w.) at 8 h (Fig. 4). After 1 d, the Sb remaining in the roots consisted of SbIII (74%), with 26% of the total Sb in the roots being effluxed out by PV as SbIII (Table 2). SbIII concentration in the roots decreased from 3,119 to 2,113 mg/kg while SbV concentration remained unchanged. Both Sb speciation in the growth media and reduction in SbIII concentration in the roots indicated SbIII was effluxed out of PV roots. Since PV was more efficient in taking up SbIII than SbV, it was possible that SbIII was also easier to be effluxed out of PV roots than SbV. Efflux is a general detoxification mechanism in plants. For example, Chlorella vulgaris effluxed 60% SbIII and 40% SbV after the cells were transferred to Sb-free medium (Maeda et al., 1997). Maeda et al. (1997) suggested C. vulgaris detoxifies Sb by effluxing it out in addition to convert SbIII to the less toxic SbV. In comparison, PV effluxed 18% of the total As in the roots after exposure to AsV for 1 d (Huang et al., 2011; Xu et al., 2007). The slightly lower efflux of As comparing to Sb by PV might be due to high As translocation efficiency. Since PV was inefficient in translocating Sb to the fronds, effluxing Sb out of the roots may help PV to minimize toxicity. 3.4. Excised PV fronds translocated SbV from petioles to pinnae but were unable to oxidize SbIII PV efficiently took up SbIII, but was unable to translocate Sb from the roots to the fronds (Table 2). Thus, we wanted to demonstrate if Sb translocation could occur in excised fronds after exposure to 8 mg/L SbIII or SbV for 1 d (Fig. 5). PV fronds took up SbIII and distributed it evenly in the petioles and pinnae (99 and 114 mg/kg) whereas SbV was mainly translocated to the pinnae (12 and 158 mg/kg). Again, PV fronds were more effective in taking up SbIII than SbV. However, under these artificially induced conditions, PV fronds were much more efficient in translocating SbV
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Sb has no known biological function and can be toxic to plants, so upon uptake, Sb needs to be detoxified. In term of As, once taken up by PV, it is reduced to AsIII in the rhizomes and then efficiently translocated to PV fronds (Mathews et al., 2011). This was not the case with Sb as almost all Sb was accumulated in PV roots (Fig. 2A,
Fig. 4. Sb concentration (mg/L) in the media during P. vittata uptake from 8 mg/L SbIII for 1 d and Sb concentration in the media (mg/kg roots fw) during subsequent P. vittata efflux in DI water for 1 d. Sb was present as SbIII in both loading media and efflux media as there was no SbIII oxidation during treatment. Data represent the mean of three replicates with standard error.
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from the petioles to the pinnae with translocation factor of 13 (Fig. 5). It suggested a Sb translocation barrier in the roots or rhizomes and further study is required for the location of Sb translocation barrier in PV. To test whether excised pinnae were capable of oxidizing SbIII, PV pinnae were incubated in 100 mg/L SbIII solution under light for 2 d, which was long enough to detect Sb speciation. After 2 d, the pinnae took up 814 148 mg/kg Sb with 599 114 mg/kg being SbIII. SbIII was unchanged after 2 d, indicating no oxidation occurring in excised PV pinnae. It was possible that SbIII was not as mobile with equal concentration in the petioles and pinnae. On the other hand, PV efficiently loaded SbV from the petioles to pinnae or pinnae contained a strong SbV sink, which allowed higher Sb content in the pinnae compared to the petioles. Together these results (Figs. 3 and 5) suggest that when the roots were exposed to SbIII, there is a constraint in SbIII reaching from the roots to the petiole. In short, PV was more effective taking up SbIII than SbV but was inefficient in Sb translocation and transformation. Almost all Sb taken up by PV was retained in the roots, with Sb species in the roots being closely related to Sb species supplied. PV roots were efficient in SbIII efflux (26%) but not SbV. Based on Sb speciation, we have shown that the uptake and translocation mechanisms of Sb in PV were different from arsenic. These results will be valuable for probing and understanding the nature of Sb uptake, translocation and metabolic processes in arsenic-hyperaccumulator P. vittata. Acknowledgments This research was supported in part by the National Natural Science Foundation of China (No. 21277070), UF/IFAS and the Royal Thai Government. References Anawar, H., Freitas, M., Canha, N., Santa Regina, I., 2011. Arsenic, antimony, and other trace element contamination in a mine tailings affected area and uptake by tolerant plant species. Environ. Geochem. Health 33, 353e362. Belzile, N., Chen, Y.-W., Wang, Z., 2001. Oxidation of antimony (III) by amorphous iron and manganese oxyhydroxides. Chem. Geol. 174, 379e387. Bhattacharjee, H., Mukhopadhyay, R., Thiyagarajan, S., Rosen, B.P., 2008. Aquaglyceroporins: ancient channels for metalloids. J. Biol. 7, 33. Cai, Y., Su, J., Ma, L.Q., 2004. Low molecular weight thiols in arsenic hyperaccumulator Pteris vittata upon exposure to arsenic and other trace elements. Environ. Pollut. 129, 69e78.
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