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Simultaneous hyperaccumulation of arsenic and antimony in Cretan brake fern


MICROC-01222; No of Pages 6
Microchemical Journal xxx (2010) xxx–xxx

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Microchemical Journal
j o u r n a l h o m e p a

g e : w w w. e l s e v i e r. c o m / l o c a t e / m i c r o c

Simultaneous hyperaccumulation of arsenic and antimony in Cretan brake fern: Evidence of plant uptake and subcellular distributions
Renwei Feng a,b,c, Chaoyang Wei a,?, Shuxin Tu c, Shirong Tang b, Fengchang Wu d
a

Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China Centre for Research in Ecotoxicology and Environmental Remediation, Institute of Agro-Environmental Protection, The Ministry of Agriculture, Tianjin 300191, China College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070, China d Chinese Research Academy of Environmental Sciences, Beijing 100012, China
b c

a r t i c l e

i n f o

a b s t r a c t
Arsenic (As) and antimony (Sb) show similar chemical properties and often present together in sul?de ores. Currently, phenomenon of co-contamination of As and Sb at some sites of the world has been increasingly emerged. The present study was conducted to explore the potential of Pteris cretica L. (Cretan brake fern), an arsenic (As) hyperaccumulator, to simultaneously accumulate As and Sb under hydroponic conditions. Arsenic was imposed at medium and high levels of 5 mg L? 1 and 20 mg L? 1, while Sb was imposed either single or co-presence with As at medium and high levels of 10 mg L? 1 and 20 mg L? 1, with no As and Sb addition as the control. The single and interactive effects of As and Sb on their uptake and subcellular distributions were analyzed. Cretan brake fern could accumulate high concentrations of As and Sb, with the highest concentrations of As and Sb been recorded as 1677.2 mg kg? 1 and 1516.5 mg kg? 1 in the fronds, respectively. Arsenic and Sb were found mainly in cytosol, while less in cell wall and cytoplasmic organelles. Sb uptake by Cretan brake fern was enhanced with increasing As levels, which was accompanied with an increase of Sb but a decrease of As in cytosol fractions. Arsenic uptake was slightly enhanced whereas suppressed when Sb was co-present in a medium and high level, respectively; however, in both conditions, As was found to be decreased in cytosol of the above ground parts as fronds and stems of Cretan brake fern. The results demonstrate Cretan brake fern can simultaneously hyperaccumulate As and Sb, thus is valued in phytoremediation of As and Sb co-contamination. ? 2010 Elsevier B.V. All rights reserved.

Article history: Received 15 December 2009 Received in revised form 7 May 2010 Accepted 25 May 2010 Available online xxxx Keywords: Arsenic accumulation Antimony accumulation Hydroponic experiment Interaction Subcellular fractions

1. Introduction Arsenic (As) and antimony (Sb) are both naturally occurring elements. They belong to the same group of 15 in the element periodic table, and share some similar chemical properties. The contamination of As and Sb at some sites of the world have been observed as a result of increasing anthropogenic activities, such as mining and incineration [1,2]. In sul?de ores, high Sb concentrations are often accompanied with high As concentrations [3], which unavoidably results in cocontamination of As and Sb. Intensive accumulation of As and Sb at some locations have been documented [1,2,4,5]. Both As and Sb are not essential but toxic elements to plants. However, plants can readily take up As and Sb to some extent. The accumulation of As and Sb in plants may retard plant root elongation and inhibit plant growth, and occasionally may render human health risks via food chain [6,7]. To date, methods for remediation of As and Sb co-contamination are lacking. The technology of phytoremediation using hyperaccu-

? Corresponding author. Tel.: +86 10 64889465; fax: +86 10 64851844. E-mail address: weicy@igsnrr.ac.cn (C. Wei). 0026-265X/$ – see front matter ? 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2010.05.010

mulating plant may be a feasible and desirable way because of its remarkable traits in environment harmony and cost-effectiveness [8]. Since the ?rst identi?cation of As-hyperaccumulator Pteris vittata L. (Chinese brake fern) [9,10], a few other As-hyperaccumulators have been reported in succession, for example: Pityrogramma calomelanos, Pteris cretica, Pteris longifolia, Pteris umbrosa, Pteris biaurita L., P. quadriaurita Retz, P. ryukyuensis Tagawa, etc. [11–15], and most of them belong to the fern species. Our previous report has shown that an As-hyperaccumulating fern, Pteris cretica (Cretan brake fern), also exhibited a relatively high tolerance to antimonite under hydroponic culture conditions [16]. Some other Sb accumulating plants have also been reported, such as Trifolium pratense L. [17], Achillea ageratum, Plantago lanceolata and Silene vulgaris [18], however, the mechanisms of Sb accumulation in these plants are still left to be resolved. Accumulations of As and Sb in fern plants may suggest particular mechanisms for their uptake, translocation and distribution. Compartmentation of heavy metals in cytosol fraction of cells is proposed as a major tolerant and accumulating mechanism in plants [19]. Plants tend to sequester heavy metals largely in cytosol, so as to reduce the toxicities of heavy metals. For example, 78% frond As in Chinese brake fern, occupying 61% of the total accumulated As in a whole plant, was

Please cite this article as: R. Feng, et al., Simultaneous hyperaccumulation of arsenic and antimony in Cretan brake fern: Evidence of plant uptake and subcellular distributions, Microchem. J. (2010), doi:10.1016/j.microc.2010.05.010

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R. Feng et al. / Microchemical Journal xxx (2010) xxx–xxx

found in cytosol fraction of frond [19]. Similar distribution patterns for other heavy metals were also seen in other hyperaccumulating plants, e.g., cadmium (Cd) and zinc (Zn) were found to be mainly sequestered in the leaf vacuoles in Thlaspi caerulescens, a Cd and Zn hyperaccumulator [20,21]. However, little concern was raised on such behaviors of Sb in plants, and information about the subcellular distribution of Sb in plants has rarely been documented. Since previous studies on As or Sb in plants have only focused on either of them alone, investigation on their interactive effects on accumulations and subcellular distributions in As-hyperaccumulating fern plants may be valued in understanding the relevant mechanisms and potentials of the fern plants for remediation of As and Sb cocontamination. Accordingly in this study, the As-hyperaccumulating plant, Cretan brake fern, was selected to characterize: 1) the interactive effects of As and Sb on their uptake and accumulation; and 2) the subcellular distribution patterns with their relationships with As and Sb tolerance and accumulation. 2. Materials and methods 2.1. Plant materials and treatments

and Sb in fraction I, II and III were measured as standing for their concentrations in cell wall, cytoplasmic organelles and cytosol, respectively. 2.3. Determination of arsenic and antimony The separated portions of fern tissues were washed, oven dried and pulverized for the determination of As and Sb. The powders of plant tissues and the above three subcellular fractions were digested with concentrated HNO3 and HClO4 (9:1, v:v), and the concentrations of As and Sb were determined using a Hydride Generation Atomic Fluorescence Spectrometer (HG-AFS) (Beijing Titan Co Ltd.) [16,26]. Accuracy of elemental analysis was checked using the standard reference material from the Center for Standard Reference of China. 2.4. Data analysis All data were subjected to two-way ANOVA analysis combined with Tukey's multi-comparisons test (P ≤ 0.05). Statistical analyses were performed using SPSS 13.0 software. 3. Results

Healthy and uniform one year seedlings of Cretan brake fern were obtained from the Fern Garden of the Institute of Botany, Chinese Academy of Sciences. After being thoroughly washed with tap and deionized water, the ferns plants were acclimatized for three weeks in a hydroponic culture system containing 0.2 strength Hoagland nutrient solution [22]. Three weeks later, the fern plants were treated with various levels of As and Sb. Sb was supplied at levels of 10 and 20 mg L? 1 in the form of K2H2Sb2O7·4H2O, while As was supplied at levels of 5 and 20 mg L? 1 in the form of Na2HAsO4·7H2O, with no As and Sb added in the solution as the control (As0, As5 and As20 stand for the treatment levels of As as 0—low, 5—medium and 20— high mg L? 1, while, Sb10 and Sb20 denote the treatment levels of Sb as 10—medium and 20—high mg L? 1, respectively). Seven treatments containing varied levels of As and Sb were arranged as follows: control (CK), As0 + Sb10, As0 + Sb20, As5 + Sb10, As5 + Sb20, As20 + Sb10 and As20 + Sb20. Each treatment had three replications and was diluted with HCl or NaOH to adjust the pH value to 6.5. The solution was aerated vigorously and replaced twice a week. The greenhouse conditions were described elsewhere [23]. 2.2. Fractionation of fronds, stems and roots Fern plants were harvested after completion of experiments, rinsed clean by water and deionized water and then carefully separated into four portions as fronds, stems, rhizomes and roots. Some fresh tissues of fronds, stems and roots (excluding rhizomes) in each replication were collected for immediate separations of subcellular fractions. Separations of subcellular fractions were performed according to the methods in literatures [19,24,25] with some modi?cations. Brie?y, about 0.5 g fresh tissues (fronds, stems or roots) were sampled for subcellular fractionation. The fresh samples were homogenized using a pre-cooled mortar and pestle in a medium containing 20 mL grinding solution (pH 7.8) of 0.25 mM sucrose, 50 mM Tris–maleate buffer (pH 7.8), 1 mM MgCl2 and 10 mM cysteine. The fractionation processes were operated at a constant temperature of 4 °C. After homogenization, the homogenate was transferred into a 50 mL centrifuge tube and centrifuged at 300 × g for 30 s. The residue in the centrifuge tube was collected as cell wall fraction (fraction I). The supernatant was carefully transferred into another clean centrifuge tube using a 50 mL syringe needle, then centrifuged at 20,000 × g for 45 min at 4 °C. The residue of the second centrifugation step was taken as cytoplasmic organelle fraction (fraction II), and the resultant supernatant was designated as cytosol fraction (fraction III). Arsenic

3.1. Effects of As and Sb on the growth of Cretan brake fern After 14 days growth under hydroponic conditions, Cretan brake fern grew well. As addition from low to high levels in the presence of medium level of Sb (10 mg L? 1) slightly decreased the biomass by 1.8%, 8.3% and 5.1%, respectively, as compared with the control. With co-exposure of high level of Sb in the solution, medium level of As gently increased, but high level of As signi?cantly decreased, the biomass up to 6.8% and to 31%, respectively, as compared with the control. At the exposure level of As20 + Sb20, the fern had slightly toxic symptoms, appearing chlorosis and curling in the fronds (Fig. 1). 3.2. Effects of As and Sb on their uptake in different tissues of Cretan brake fern 3.2.1. Effect on uptake of As When As was imposed at medium level (5 mg L? 1), addition of high level of Sb (As 5 + Sb20) enhanced As uptake in the fronds, stems, roots and rhizomes of Cretan brake fern by 2.33%, 13.42%, 24.59% and 59.86% as compared to addition of medium level of Sb

Fig. 1. Effects of arsenic and antimony on the biomass of Pteris cretica L. As0, As5 and As20 stand for the treatment concentrations of As as 0, 5 and 20 mg L? 1, respectively; Sb10 and Sb20 denote the treatment levels of Sb as 10 and 20 mg L? 1, respectively.

Please cite this article as: R. Feng, et al., Simultaneous hyperaccumulation of arsenic and antimony in Cretan brake fern: Evidence of plant uptake and subcellular distributions, Microchem. J. (2010), doi:10.1016/j.microc.2010.05.010

R. Feng et al. / Microchemical Journal xxx (2010) xxx–xxx

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(As5 + Sb10), respectively. However, when As was imposed at high level (20 mg L? 1), the increased addition of Sb from medium to high suppressed As uptake respectively in the fronds, stems and roots by 2.32%, 28.65% and 7.03% (Fig. 2a). The highest As concentration as 1677.2 mg kg? 1 was recorded at the treatment of As20 + Sb10 (Fig. 2a). In general, when As and Sb were both present in the solution, Cretan brake fern took up As the most in fronds, followed in decreasing order by stems, roots and rhizomes (Fig. 2a).

1516.5 mg kg? 1 and 176.3 mg kg? 1 in the fronds and stems of Cretan brake fern, respectively (Fig. 2b). 3.3. Distributions of As and Sb in subcellular fractions 3.3.1. Distributions of As and Sb in subcellular fractions of fronds Increasing As levels evidently increased As concentrations in all the subcellular fractions of cell wall, cytoplasmic organelle and cytosol. When As was imposed at medium and high levels, addition of Sb from medium to high more or less increased As concentrations in cytoplasmic organelle and cell wall fractions, but decreased that in cytosol fraction (Fig. 3a). Reduction of As concentration in cytosol by the addition of Sb was more evident when As was imposed at its high level (Fig. 3a). When As was imposed at certain level, increased exposure to Sb from medium to high also enhanced Sb concentrations in the three subcellular fractions of cell wall, cytoplasmic organelle and cytosol (Fig. 3b). Sb concentration in cytosol was positively increasing with co-exposure of As levels to Cretan brake fern (Fig. 3b). When Sb was imposed at medium and high levels, high level of As addition increased Sb concentrations in cytosol fraction by 222.9% and 94.2% as compared with the treatments of As0 + Sb10 and As0 + Sb20, respectively (Fig. 3b). When Sb was imposed at medium level, the addition of As increased Sb concentrations in cell wall and

3.2.2. Effect on uptake of Sb Signi?cant interactive effects of As and Sb on Sb uptake in various parts of Cretan brake fern were observed, except in stems. Sb addition from medium to high increased the uptake of Sb in the fronds, rhizomes and roots (Fig. 2b). Generally, the concentration of Sb in the fronds registered the highest record, in succession, followed by the roots, stems and rhizomes (Fig. 2b). When Sb was imposed at medium level, the addition of As from low to high levels all enhanced plant Sb uptake in various parts of Cretan brake fern by 210.11%, 35.93%, 70.78% and 80.68% in the fronds, stems, roots and rhizomes, respectively. However, when Sb was imposed at high level, Sb in the fronds and stems were found to be increased at low, but decreased at high, levels of As. The highest Sb concentration was recorded at a co-exposure level of As5 + Sb20, as

Fig. 2. Arsenic and antimony concentrations in different tissues of Pteris cretica L. As0, As5 and As20 stand for the treatment levels of As as 0, 5 and 20 mg L? 1, respectively; Sb10 and Sb20 denote the treatment levels of Sb as 10 and 20 mg L? 1, respectively; CK indicates the control without the additions of As and Sb. Bars are means and standard error for the mean of three replications (n = 3). Lowercases, capital letters, (lowercases) and (capital letters) above bars indicate signi?cant differences among different treatments for the frond, stem, root and rhizome of Pteris cretica L., respectively (P ≤ 0.05).

Fig. 3. Arsenic and antimony concentrations in different subcellular fractions of fronds of Pteris cretica L. As0, As5 and As20 stand for the treatment levels of As as 0, 5 and 20 mg L? 1, respectively; Sb10 and Sb20 denote the treatment levels of Sb as 10 and 20 mg L? 1, respectively; CK indicates the control without the additions of As and Sb. Lowercases, (lowercases) and capital letters above bars indicate signi?cant differences among different treatments for the cell wall, cytosol and cytoplasmic organelle fractions from the fronds of Pteris cretica L., respectively (P ≤ 0.05).

Please cite this article as: R. Feng, et al., Simultaneous hyperaccumulation of arsenic and antimony in Cretan brake fern: Evidence of plant uptake and subcellular distributions, Microchem. J. (2010), doi:10.1016/j.microc.2010.05.010

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R. Feng et al. / Microchemical Journal xxx (2010) xxx–xxx

cytoplasmic organelle fractions; whereas with high level of Sb present in the solution, low level of As increased, but high level As decreased, Sb concentrations in these two fractions (Fig. 3b). 3.3.2. Distributions of As and Sb in subcellular fractions of stems When exposure to either high or medium Sb level, As addition increased As concentration in the cell wall fraction of the stems; however, in cytoplasmic organelles and cytosol the As concentrations were found to be increased at low but then decreased at high, As levels (Fig. 4a). When As was imposed at medium and high levels, elevated Sb slightly increased As concentrations in cell wall fraction, while reduced them in cytoplasmic organelle and cytosol fractions (Fig. 4a). When Sb was imposed at 10 mg L? 1, addition of As increased Sb concentrations in all fractions of stems in Cretan brake fern; however, at high Sb level of 20 mg L? 1, medium level of As increased, but high level of As appeared to decrease the concentrations of Sb in all fractions of the stems (Fig. 4b). 3.3.3. Distributions of As and Sb in subcellular fractions of roots When Sb was imposed at medium or high levels, As addition generally increased As concentrations in all subcellular fractions of the roots in Cretan brake fern. When As was imposed at medium level, Sb addition from medium to high levels increased As concentrations by

37.5%, 125.3% and 52.2% in cell wall, cytoplasmic organelle and cytosol fractions, respectively. However, when As was imposed at high level, As concentrations decreased in cell wall and cytoplasmic organelle fractions whereas increased in cytosol fraction with increasing Sb addition (Fig. 5a). Generally, the addition of As showed positive effects on Sb accumulation in all fractions when Sb was added at either medium or high level (Fig. 5b). With co-exposure to certain level of As (low, medium or high), Sb addition from medium to high levels generally increased Sb concentrations in cell wall, cytoplasmic organelle and cytosol fractions (Fig. 5b).

3.4. Changes in the ratios of As and Sb in subcellular fractions 3.4.1. Changes in the ratios of As in subcellular fractions All As in the fronds of Cretan brake fern was found to be stored in cytosol in the control, while in the stems the cytosol sequestered 62.3% of the total. With exposure to either medium or high level of Sb, the ratios of As decreased in cytosol fraction while increased in cell wall fraction both in the fronds and roots with As addition; however, with regard to As proportions in cytoplasmic organelle fractions of the roots and fronds, they were increased in former, but no apparent changes were found in the latter (Table 1).

Fig. 4. Arsenic and antimony concentrations in different subcellular fractions of stems of Pteris cretica L. As0, As5 and As20 stand for the treatment levels of As as 0, 5 and 20 mg L? 1, respectively; Sb10 and Sb20 denote the treatment levels of Sb as 10 and 20 mg L? 1, respectively; CK indicates the control without the additions of As and Sb. Lowercases, (lowercases) and capital letters above bars indicate signi?cant differences among different treatments for the cell wall, cytosol and cytoplasmic organelle fractions from the stems of Pteris cretica L., respectively (P ≤ 0.05).

Fig. 5. Arsenic and antimony concentrations in different subcellular fractions of roots of Pteris cretica L. As0, As5 and As20 stand for the treatment levels of As as 0, 5 and 20 mg L? 1, respectively; Sb10 and Sb20 denote the treatment levels of Sb as 10 and 20 mg L? 1, respectively; CK indicates the control without the additions of As and Sb. Lowercases, (lowercases) and capital letters above bars indicate signi?cant differences among different treatments for the cell wall, cytosol and cytoplasmic organelle fractions from the roots of Pteris cretica L., respectively (P ≤ 0.05).

Please cite this article as: R. Feng, et al., Simultaneous hyperaccumulation of arsenic and antimony in Cretan brake fern: Evidence of plant uptake and subcellular distributions, Microchem. J. (2010), doi:10.1016/j.microc.2010.05.010

R. Feng et al. / Microchemical Journal xxx (2010) xxx–xxx Table 1 Proportions of arsenic and antimony in different subcellular fractions from different tissues of Pteris cretica L. (%). Element Tissue Subcellular fractions Treatments Control As Fronds Cell wall Cytoplasmic organelle Cytosol Cell wall Cytoplasmic organelle Cytosol Cell wall Cytoplasmic organelle Cytosol Cell wall Cytoplasmic organelle Cytosol Cell wall Cytoplasmic organelle Cytosol Cell wall Cytoplasmic organelle Cytosol ND ND 100.0 21.6 16.1 62.3 ND ND ND 24.0 ND 76.0 ND ND ND ND ND ND As0 + Sb10 5.9 4.4 89.7 19.9 49.6 30.6 ND ND ND 4.2 2.1 93.7 19.3 39.9 40.8 50.9 14.1 35.0 As0 + Sb20 19.7 7.8 72.5 64.3 35.7 ND ND ND ND 11.3 3.7 85.0 20.6 32.1 47.3 53.7 14.4 31.9 As5 + Sb10 36.6 4.1 59.3 4.6 6.3 89.1 25.4 1.8 72.8 3.0 1.8 95.2 19.0 37.4 43.6 50.8 12.2 37.0 As5 + Sb20 45.7 6.0 48.3 6.9 6.9 86.2 23.3 2.7 74.0 13.1 2.8 84.1 17.6 34.4 48.0 54.5 3.4 42.1 As20 + Sb10 44.2 5.7 50.1 6.8 3.8 89.3 43.3 12.1 44.6 5.1 0.6 94.3 23.1 33.8 43.1 36.0 12.3 51.7 As20 + Sb20 52.8 7.0 40.2 10.3 4.4 85.3 39.3 10.9 49.9 4.7 1.3 94.0 20.8 45.5 33.7 39.5 16.8 43.7

5

Stems

Roots

Sb

Fronds

Stems

Roots

Data are presented as percentages of As or Sb concentration in one of the subcellular fractions to the corresponding total As or Sb concentrations of the three subcellular fractions. ND = not detected.

When Sb was imposed at medium and high levels, the addition of medium level of As remarkably increased the As proportions in cytosol fractions but decreased them in cell wall and cytoplasmic organelle fractions in the stems as compared to the treatments of As0 + Sb10 and As0 + Sb20, respectively; such changes became nonevident with As addition being at high level, especially for As proportions in cytosol fraction, where they were been kept almost constant (Table 1). When As was imposed at a certain level, Sb addition decreased As ratios in cytosol fraction, with accompanied increases of them in cell wall and cytoplasmic organelle fractions in the fronds and stems. However, no such signi?cant changes were found among subcellular fractions in the roots (Table 1). 3.4.2. Changes in the ratios of Sb in subcellular fractions Sb was mainly stored in cytosol fraction in the fronds of Cretan brake fern, however with exposure to certain As level, elevated Sb decreased such proportions, causing associated increases in cell wall and cytoplasmic organelles. Sb was relatively evenly distributed in the three subcellular fractions in stems and roots as compared to that in fronds across various treatments of As and Sb. Sb occupied 17.6%– 23.1%, 32.1%–45.5% and 40.8%–48.0% of the total in cell wall, cytoplasmic organelle and cytosol fractions in the stems, respectively; while those were 36.0–54.5%, 3.4–16.8% and 31.9–43.7% in the roots (Table 1). When Sb was imposed at medium level, Sb ratios increased in cell wall while decreased in cytoplasmic organelles in the fronds and stems with As addition from low to high, whereas in the roots evident increases vs decreases in Sb ratios happened in cytosol and cell wall, respectively. When Sb was imposed at high level, Sb ratios increased in cytosol while decreased in cell wall fractions in the fronds with As addition from low to high levels; and in the stems and roots such increases were observed in cytoplasmic organelles but decreases appeared differently as in cytosol of stems and cell wall of roots (Table 1). 4. Discussion The present study was carried out to investigate the uptake and subcellular distributions of As and Sb in Cretan brake fern. The results suggested that Cretan brake fern possessed great ability in tolerance and accumulations for both As and Sb. In the presence of both As and

Sb in the solution, Cretan brake fern could effectively take up As and Sb and transfer them from roots to fronds, the highest As concentration was recorded as 1677.2 mg kg? 1 and 1046.4 mg kg? 1 in the fronds and roots, respectively. These results further substantiate that Cretan brake fern is an As-hyperaccumulating and an Sbaccumulating plant [12,16]. In addition, the presence of As with Sb could dramatically enhance Sb uptake by Cretan brake fern, especially at a low As level. The highest Sb concentration in the fronds of Cretan brake fern was recorded as 1516.5 mg kg? 1, while the associated Sb concentration in roots was 839.3 mg kg? 1(Fig. 2), demonstrating that Cretan brake fern is an Sb-hyperaccumulating plant at this condition, according to the popular standard suggested for hyperaccumulator [27]. The results indicate that Cretan brake fern has great potential in phytoremediation of Sb and As co-contamination. Without As and Sb addition, all frond and 62.3% stem As were sequestered in cytosol fraction, suggesting cytosol is the main position for storing As. This did not agree with the ?ndings of Liao et al. [28], who found that Chinese brake fern, an As-hyperaccumulator, stored As mainly in cell wall and cytoplasmic organelles when grew in a nonAs substrate. In this study, with the single exposure of Sb, 85.0%–93.7% frond Sb and 40.8%–47.3% stem Sb were transferred into individual cytosol fractions (Table 1), indicating large cytosol compartmentation to Sb in the above ground parts of Cretan brake fern. This is consistent with the results of As compartmentation in Chinese brake fern [19,29], suggesting a similar mechanism for plant accumulation between As and Sb. It is believed that Chinese brake fern takes up As as arsenate, which is reduced to arsenite, and ?nally sequestered in cytosol fraction in the fronds [30]; With regard to Sb, the mechanisms about its uptake and transformation in plants are still unknown. In the roots of Cretan brake fern, only 31.9%–35.0% Sb was restrained in cytosol fraction, compared to 50.9–53.7% Sb storing in cell wall fraction (Table 1), the increasing Sb proportions in cell wall fractions of the roots suggested cell wall in root tissues played a major role in Sb tolerance in Cretan brake fern. The stimulated uptake of Sb by addition of As in Cretan brake fern was consistent with more transfer of Sb into cytosol fraction (Table 1 and Fig. 2b). In the presence of medium and high levels of Sb in the solution, As addition increased Sb concentrations in all tissues and subcellular fractions of Cretan brake fern (Table 1; Figs. 2b, 3b, 4b, and 5b); in addition, the increased Sb uptake was associated with more transfer of Sb into cytosol fraction (Table 1).

Please cite this article as: R. Feng, et al., Simultaneous hyperaccumulation of arsenic and antimony in Cretan brake fern: Evidence of plant uptake and subcellular distributions, Microchem. J. (2010), doi:10.1016/j.microc.2010.05.010

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The stimulation of As on the uptake of Sb in Cretan brake fern may be partially related with Sb methylation. Previous study had shown that in the presence of As and Sb, trivalent As addition induced more production of tri-methyl-antimony compounds whereas trivalent Sb addition inhibited As-methylation process in Scopulariopsis brevicaulis [31]. The addition of Sb had two opposite effects on the uptake of As, i.e., stimulation and antagonism, depending on the As dosages in the presence of Sb. Supplementation of Sb increased As concentrations in all tissues of Cretan brake fern when As was imposed at low level (Fig. 2a), indicating a stimulation effect, which was consistent with the increases of As ratios in cell wall and cytoplasmic organelles fractions (Table 1, Figs. 3a and 4a). Simultaneously, decreased As uptake in the fronds and stems of Cretan brake fern at high exposure level of As with elevated levels of Sb was accompanied with the decreased As ratios in their individual cytosol fractions (Table 1, Fig. 2a), suggesting As and Sb might compete for entering cytosol, thereafter showing certain antagonistic effects of on the uptake of Sb to As. 5. Conclusions In the presence of As and Sb, Cretan brake fern could simultaneously hyperaccumulate both of the two elements, this fern may be applied to phytoremediate As and Sb co-contamination. The compartmentation of Sb in cytosol was signi?cantly involved in the mechanisms for Sb accumulation in Cretan brake fern. Arsenic addition enhanced the accumulation of Sb in Cretan brake fern and with the increased Sb uptake, the associated increases of Sb concentrations in subcellular fractions were also seen, accompanied with more transfers of Sb into cytosol fractions. The effect of Sb on As uptake and subcellular distribution was different with that of As on Sb, Sb only slightly stimulated As uptake when Cretan brake fern was exposed to low level of As, while when the fern exposed to high level of As, Sb decreased its As uptake. Such decrease was accompanied with associated reduction of As ratios in cytosol fractions in the fronds and stems, suggesting again cytosol played a vital role in As accumulation in Cretan brake fern. Acknowledgements We appreciate the kindly donation of fern seedlings of Prof. Shi Lei from the Institute of Botany, Chinese Academy of Sciences. This research was supported by the National Natural Science Foundation of China (40632011, 40971264, 10979052), and the National Key Technologies R&D Program of China during the 11th Five-Year Plan Period (2006BAJ05A08). We also thank group member Ms. Yanming Zhu for her assistance in chemical analysis. References
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Please cite this article as: R. Feng, et al., Simultaneous hyperaccumulation of arsenic and antimony in Cretan brake fern: Evidence of plant uptake and subcellular distributions, Microchem. J. (2010), doi:10.1016/j.microc.2010.05.010


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