Science of the Total Environment 475 (2014) 83–89
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Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
Antimony uptake, translocation and speciation in rice plants exposed to
antimonite and antimonate
Jing–Hua Ren a, Lena Q. Ma a,b,⁎, Hong–Jie Sun a, Fei Cai a, Jun Luo a,⁎
a
b
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Jiangsu 210046, China
Soil and Water Science Department, University of Florida, Gainesville, FL 32611, USA
H I G H L I G H T S
•
•
•
•
We examined the impact of iron plaque on different species of Sb uptake by rice.
Iron plaque accumulated more Sb than rice plants and affected SbIII and SbV uptake by rice roots.
Rice was much more efficient in taking up SbIII than SbV, and SbV was the predominant Sb species in rice plants.
Sb was mostly localized to the cell walls of rice plants, resulting in limited translocation from the roots to shoots.
a r t i c l e
i n f o
Article history:
Received 15 October 2013
Received in revised form 20 December 2013
Accepted 20 December 2013
Available online xxxx
Keywords:
Antimony
Iron plaque
Uptake
Speciation
Subcellular distribution
Rice plants
a b s t r a c t
Antimony (Sb) accumulation in rice is a potential threat to human health, but its uptake mechanisms are unclear.
A hydroponic experiment was conducted to investigate uptake, translocation, speciation and subcellular distribution of Sb in rice plants exposed to antimonite (SbIII) and antimonate (SbV) at 0.2, 1.0 or 5.0 mg/L for 4 h. More
Sb was accumulated in iron plaque than in the plant, with both the roots (~10–12 times) and Fe plaque (~28–54
times) sequestering more SbIII than SbV. The presence of iron plaque decreased uptake of both SbV and SbIII. SbIII
uptake kinetics fitted better to the Michaelis–Menten function than SbV. Antimonate (56 to 98%) was the predominant form in rice plant with little methylated species being detected using HPLC–ICP-MS. Cell walls accumulated more Sb than organelles and cytosol, which were considered as the first barrier against Sb entering into
cells. Sb transformation and subcellular distribution can help to understand the metabolic mechanisms of Sb in
rice.
Published by Elsevier B.V.
1. Introduction
Antimony (Sb) is considered a priority environmental pollutant by
the United States Environmental Protection Agency and the European
Union. It has no known biological function and can be toxic at elevated
concentrations. Antimonite (SbIII) and antimonate (SbV) are the common species of Sb in the environment and they can be taken up by
plants from soil, causing adverse health effects to human. As a metalloid,
its environmental behavior has received little attention though it is
gaining interest as a global contaminant (Wilson et al., 2010).
The maximum allowable Sb level in drinking water is 5 μg/L in China
whereas the World Health Organization (WHO) sets safe drinking
water level for Sb at 20 μg/L (He et al., 2012). Anthropogenic activities
such as mining, smelting, fossil fuel combustion and waste incineration
⁎ Corresponding authors. Tel.: +1 25 89680631.
E-mail addresses: lqma@ufl.edu (L.Q. Ma), [email protected] (J. Luo).
0048-9697/$ – see front matter. Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.scitotenv.2013.12.103
have elevated Sb levels in the environment. Landrum et al. (2009) recently reported values of 1.12–4.19 mg/L Sb in water from El Tatio Geyser field in Chile. The Sb concentrations in the seepage water from
leakage of a smelter in Hunan, China are elevated, ranging from 8.4 to
11 mg/L (He, 2007). The Sb concentration in paddy soils near
Xikuangshan Sb mine area in Hunan, China reached 1565 mg/kg (He
and Yang, 1999), which was much greater than 36 mg/kg, the maximum permissible pollutant concentrations for Sb recommended by
WHO in soils (Chang et al., 2002). Rice plants accumulate high concentration of Sb up to 225 mg/kg in the roots and 5.79 mg/kg in the seeds.
Hence, it is important to study Sb behavior in the environment.
Rice is a major food crop for 3 billion people, especially in Asian
countries. Rice has been implicated as a major route for Sb exposure, especially in mining areas. The Sb concentrations in rice near the
Xikuangshan Sb mine were 160–930 μg/kg (Wu et al., 2011b). According to WHO, rice contributes 33% of the total daily intake of Sb, which
is higher than other exposure routes. However, limited data are available regarding Sb uptake and translocation in rice plants. Sb negatively
impacts rice growth, with rice yield dropping by 10% when SbIII and SbV
concentrations are 150 and 300 mg/kg in soils (He and Yang, 1999).
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J.–H. Ren et al. / Science of the Total Environment 475 (2014) 83–89
Feng et al. (2011a) found that more Sb is concentrated in rice shoots
than roots after 14 d exposure to 5 mg/L SbIII under hydroponic conditions. However, SbIII can be oxidized to SbV rapidly in solution. So it is
necessary to study SbIII and SbV uptake by rice in short term to minimize SbIII transformation in solution.
Mechanisms of arsenic uptake have been studied extensively in rice
plants (Meharg and Jardine, 2003; Zhao et al., 2010). Arsenite (AsIII)
and arsenate (AsV) are taken up by aquaporin channels and phosphate
transporters by rice, with AsV being reduced to AsIII in root cells. By contrast, little is known about the mechanism of uptake, speciation, and
transformation of Sb in rice plants. Okkenhaug et al. (2012) found that
SbV is the main species in rice roots and shoots, with N90% of Sb being
SbV in porewater in pots. He and Yang (1999) investigated SbIII and
SbV accumulation in rice without considering Sb speciation. Huang
et al. (2011) studied the influence of Fe plaque on Sb uptake and translocation in rice without considering Sb speciation. It is known that SbIII
is more toxic than SbV, so it is necessary to understand Sb transformation in rice to better assess its toxicity.
In addition, subcellular distribution of toxic elements can help to understand their translocation and detoxification mechanism in plants. Cr
is mainly associated with cell walls in rice plants (Zeng et al., 2011) so is
most of Cd (He et al., 2008). However, information about the subcellular
distribution of Sb in rice has rarely been documented.
As a waterlogged plant, rice grows in anaerobic environment and releases oxygen to its rhizosphere through developed aerenchyma
(Winkel et al., 2013). The oxygen oxidizes ferrous iron (FeII) to form
iron plaque coating on the root surfaces (Zhao et al., 2010). Iron plaque
has been shown to have a high affinity for AsV, playing an important
role in As uptake by rice. Fe plaque may be responsible for AsIII oxidation to AsV, reducing As toxicity (Zhao et al., 2009). Sb and As are chemical analogs so we hypothesized that Sb uptake and speciation in rice
was similar to As. Huang et al. (2011) reported that Sb accumulation
by rice is influenced by Fe plaque on root surface, with 40–80% of total
Sb being accumulated in Fe plaque. However, the direct role of iron
plaque in Sb uptake into rice roots needs further investigation.
The overall goal of this study was to examine the uptake, translocation and speciation of Sb by rice plants exposed to SbIII and SbV. Our
specific objectives were to: 1) evaluate the effects of iron plaque on Sb
accumulation in rice plants; 2) investigate Sb distribution and speciation in rice plants; and 3) study Sb subcellular distribution in rice plants.
2. Materials and methods
2.1. Germination and cultivation of rice plants
Rice seeds (Oryza sativa L., Nanjing 45) were surface sterilized by
soaking them in 30% H2O2 solution for 15 min and then rinsed in
Milli-Q water. They were then soaked in Milli-Q water for 48 h, and germinated on moistened filter papers placed in a petri dish. After germination, they were transferred to a 96-orifice plate. At one-leaf stage, they
were treated with 0.25-strength nutrition solution recommended by International Rice Research Institute (Wu et al., 2011a). At three-leaf
stage, they were treated with 0.5-strength nutrition solution for 1 wk
before using full strength nutrition solution.
The nutrient solution pH was adjusted to 5.5–5.8 with KOH. The solution was changed twice a week. The seedlings were acclimated in nutrient solution for 3 wk before treatment. They were grown in a
greenhouse with 16 h light period and 8 h dark period using sodium
lamps at 180–240 μmol/m2 s. The temperature was kept at ~28 °C during the day and ~20 °C during the night and relative humidity was
maintained at 60–70%.
2.2. Short term uptake of SbIII and SbV by rice plants
After 3-wk acclimation, uniform rice seedlings were washed with
deionized water and placed in a 550 mL solution containing 0.5 mM
CaCl2 and 0, 0.2, 1, or 5 mg/L SbV (potassium hexahydroxoantimonate
(KSb(OH)6)) or SbIII (potassium antimonyl tartrate trihydrate (C8H4K2O12Sb2·3H2O)) for 4 h (pH 6.0). Aliquots of 10 mL of the solution were
taken before and after the experiment for total Sb and Sb speciation
measurement. After 4 h, plants were collected and carefully rinsed
with Milli-Q water. The plants were divided into roots, stems and leaves.
They were flash-frozen in liquid nitrogen and stored at −80 °C for further analysis.
After harvest, all roots were then rinsed with an ice-cold phosphate
buffer solution including 1 mM K2HPO4, 5 mM MES and 0.5 mM
Ca(NO3)2 for 20 min to remove apoplastic Sb. The colored iron plaques
were visible on the root surface. To analyze Fe and Sb concentrations in
the iron plaque, fresh root surfaces were extracted using dithionite–
citrate–bicarbonate (DCB; Liu et al., 2004). Roots were weighed and incubated for 1 h at room temperature in a 40 mL solution containing
0.125 M NaHCO3 and 0.03 M Na3C6H5O7·2H2O with addition of 0.8 g
Na2S2O4. After extraction, roots were rinsed three times with Milli-Q
water, which were then added to the DCB extract. The resulting solution
was made up to 50 mL with Milli-Q water. The Fe and Sb concentrations
were measured by inductively coupled plasma mass spectrometry
(ICP-MS; PerkinElmer NexION 300X, USA) after dilution.
2.3. Impact of Fe plaque on Sb uptake kinetics by excised rice roots
Rice roots were excised at the basal node. Half were cleaned with
Milli-Q water and incubated in DCB solution for 1 h to remove the
iron plaque on the roots. They were then rinsed with Milli-Q water
and blotted with tissue paper. All roots were placed in 50 mL plastic
tubes containing 5.0 mM 2-(N-morpholin) ethansulfonic acid (MES),
0.5 mM Ca(NO3)2, and 0–165 mg/L SbIII or 0–251 mg/L SbV (pH 6.0).
After 30 min of Sb uptake, all roots were rinsed with an ice-cold phosphate buffer solution and roots with iron plaque were rinsed with
50 mL DCB solution. They were then washed with Milli-Q water and
blotted dry. All roots were flash-frozen in liquid nitrogen and stored at
−80 °C for further analysis.
2.4. Antimony speciation in rice plants
Frozen plant samples were ground to powder in a mortar under liquid nitrogen for total Sb analysis. ~ 0.4 g of fine plant powder was
weighed into digestion vials, mixed with 10 mL of 1:1 HNO3:water and
left overnight. The digestion was performed using the Hot Block Digestion System (Environmental Express, USA). They were heated at 105 °C
for 2 h, removed from the block and cooled for 3 min. The samples
were added with 1 mL 30% H2O2 slowly, and then heated for another
15 min. The samples were cooled completely, and then diluted up to
50 mL with Milli-Q water. The final solution was stored at 4 °C before
analysis with ICP-MS. The blank and certified reference material for
rice samples (GSB-21, Chinese geological reference materials) was used
for quality control. The mean ± standard error was 4.68 ± 0.36 μg/kg
for the sample, which was comparable with the certified value of
4.5 μg/kg. The internal standards were carried out to ensure accuracy
and precision. Standard solution at 1 μg/L Sb was measured every 20
samples to monitor the stability of ICP-MS.
The frozen rice samples were crushed in a mortar under liquid nitrogen for Sb speciation (Okkenhaug et al., 2012). ~ 0.4 g of sample was
weighed into a 15 mL polypropylene centrifuge tube and 5 mL of
0.1 M citric acid was added. They were shaken (100 rpm, 25 °C) for
4 h and then sonicated for 1 h. Extracts were centrifuged at 4000 rpm
for 15 min, and then collected in a 50 mL centrifuge tube. The residue
was rinsed twice with 5 mL of 0.1 M citric acid and all extracts were
mixed, and then diluted to 20 mL with Milli-Q water. After filtering
using 0.45 μm nylon filter membrane, Sb speciation was measured by
high performance liquid chromatography (HPLC; Waters 2695, USA)
coupled with ICP-MS. The aqueous solution samples were stored at
4 °C and analyzed within 24 h (Lindemann et al., 2000).
J.–H. Ren et al. / Science of the Total Environment 475 (2014) 83–89
85
15.0
Sb content in rice seedling (mg/kg)
A guard column (Hamilton, UK) connected to a PRP-X100 10 μm
anion-exchange column (Hamilton, UK) was used to separate Sb species. The mobile phase consisted of 10 mM EDTA and 2 mM potassium
hydrogen phthalate adjusted to pH 4.5 using ammonium hydroxide
(Liu et al., 2010). Before flowing into chromatographic columns, they
were sonicated and filtered (0.22 μm). Sample injection volume was
50 μL, flow rate 1.2 mL/min and temperature 40 °C. SbV and SbIII standard solutions at 10 μg/L were run to obtain retention time. Stock solutions of Sb species were prepared from C8H4K2O12Sb2·3H2O (SigmaAldrich, 99%) and KSb(OH)6 (Fluka, 99%) with Milli-Q water. The stock
solution at 1000 mg/L was stored in the dark at 4 °C until use. All standard solutions were diluted from the stock solution with 0.1 M citric
acid on the day of analysis. SbIII and SbV standard solutions were calibrated on ICP-MS using a 100 ppm multielement environmental calibration standard (Perkin Elmer, in 5% HNO3).
12.0
9.0
A
Root
Stem
Leaf
6.0
3.0
1.5
1.2
0.9
0.6
0.3
0.0
0
0.2
1
5
SbIII concentration in solution (mg/L)
2.5. Subcellular distribution of Sb in rice plants
1.50
Sb content in rice seedling (mg/kg)
Fresh rice plants, after exposing to 0.2, 1 and 5 mg/L SbIII or SbV for
4 h, were ground in a mortar using liquid nitrogen. Subcellular distribution was determined by gradient centrifugation technique at 4 °C (Feng
et al., 2011b). About 0.5 g of sample was homogenized in 10 mL grinding solution containing 50 mM Tris–maleate buffer (pH 7.8), 0.25 mM
sucrose, 1 mM MgCl2 and 10 mM cysteine. The homogenate was centrifuged at 300 g for 30 s. The residue in the tube was designated as cell
wall fraction (Fw). The supernatant of the first centrifugation step was
transferred into another tube using a pipette and then centrifuged at
20,000 g for 45 min. The supernatant of the second centrifugation
step was collected as cytosol fraction (Fc) and the residue as cytoplasmic organelle fraction (Fo). Each fraction was then digested using
HNO3 + H2O2 and measured by ICP-MS.
1.20
B
Root
Stem
Leaf
0.90
0.60
0.30
0.12
0.08
0.04
0.00
2.6. Statistical analysis
0
0.2
1
5
SbVconcentration in solution (mg/L)
All experiments were carried out in four replicates. All results were
presented as mean ± standard deviation. Statistical analysis including
one-way ANOVA and the least significant difference was performed
using PASW statistics 18.0.
Fig. 1. Antimony concentrations in the roots, stems and leaves of rice after exposing to 0,
0.2, 1 and 5 mg/L SbIII (A) or SbV (B) for 4 h.
indicating that rice was much more effective in taking up SbIII than
SbV. This was consistent with plant Sb concentrations (Fig. 1). For example, after 4 h exposure to 5 mg/L Sb, Sb concentration in the roots
in SbIII treatment was ~ 9 times higher than that in SbV treatment
(12.5 vs. 1.40 mg/kg). These results are consistent with Sb uptake by
rice (Huang et al., 2011) and wheat (Shtangeeva et al., 2012).
Regardless SbIII or SbV was provided, rice accumulated the highest
Sb in the roots, followed by the stems and leaves (Fig. 1). Take 5 mg/L
SbIII for example, Sb concentrations in the roots, stems and leaves
were 12.5, 1.30 and 0.299 mg/kg. Sb concentrations in all tissues increased with increasing external concentrations. Sb concentration in
the roots in 1 mg/L SbIII treatment was ~ 3 times higher than that in
0.2 mg/L SbIII treatment (3.79 vs. 1.04 mg/kg). We calculated the translocation factor (TF) of SbIII and SbV (the ratio of Sb in the shoots to
roots). Rice was ineffective in translocating either SbIII or SbV as the
highest TF was 0.51. However, translocation of SbV was ~ 3–4 times
3. Results and discussion
Antimony speciation in the growth media was determined after exposing rice plants to 0.2, 1 and 5 mg/L SbIII or SbV for 4 h (Table 1).
After 4 h exposure, 6.20–38.1% of the SbIII was oxidized to SbV while
SbV was stable. This indicated that SbIII was unstable during the 4 h experiment and both SbIII and SbV existed in the SbIII treatment.
3.1. Rice was much more effective in taking up SbIII than SbV and
accumulated Sb in roots
After exposing rice to SbIII or SbV for 4 h, total Sb concentrations in
the media were determined (Table 1). They decreased by 8.53–52.9% in
the SbIII treatment and were unchanged in the SbV treatment,
Table 1
Sb speciation in solution and rice plants after 4 h of exposure to 0.2, 1 and 5 mg/L SbIII or SbV.
Sb species
Initial Sb concentration (mg/L)
Final Sb concentration (mg/L)
SbV (%)
Solutionfinal
Solutionaverage
Root
SbIII
0.208
0.996
5.04
0.225
1.11
5.40
0.098
0.713
4.61
0.228
1.14
5.47
38.1 ± 3.27
18.9 ± 3.90
6.20 ± 0.916
100
100
100
19.1 ± 1.64
9.82 ± 1.95
3.10 ± 0.458
100
100
100
85.4
88.0
95.7
97.2
97.8
97.2
SbV
±
±
±
±
±
±
0.010
0.024
0.014
0.002
0.021
0.291
±
±
±
±
±
±
0.007
0.071
0.143
0.002
0.025
0.071
Stem
±
±
±
±
±
±
1.65
1.53
0.85
1.15
0.35
0.17
89.7
74.9
56.4
78.1
91.2
97.2
±
±
±
±
±
±
Leave
1.69
3.59
4.51
1.89
1.44
0.12
87.0
89.5
92.0
92.0
92.9
98.2
±
±
±
±
±
±
0.76
2.64
0.64
0.40
2.30
0.10
86
J.–H. Ren et al. / Science of the Total Environment 475 (2014) 83–89
higher (0.23–0.51) than SbIII (0.061–0.161), indicating that SbV was
more mobile than SbIII.
To better understand Sb uptake by rice, we examined Sb uptake kinetics using excised rice roots. Sb uptake by rice plants with Fe plaque
showed a hyperbolic pattern, increasing with increasing Sb concentration (Fig. 2). Uptake kinetics with iron plaque were well described by
the Michaelis–Menten function (R2 = 0.974–0.984). The mean Vmax
values (maximum uptake rate) for SbIII and SbV uptake were 38.4 and
26.9 mg/kg fw h, and Km values (Michaelis constant, reflecting affinity
between substrate and carrier) were 0.079 and 6.80, indicating the
much greater affinity of rice roots for SbIII than SbV. SbIII carrier obeyed
saturation kinetics (Fig. 2A), but SbV carrier did not (Fig. 2B).
At pH 6.0 in solution, SbV mainly existed as [Sb(OH)6]− (Wilson
et al., 2010). At low external concentrations, its uptake into the root
symplast probably required anion transporters of low selectivity, such
as Cl− or NO−
3 (Tschan et al., 2009). As a P analog, AsV is transported
through cell membranes by P transporters. But SbV in aqueous solutions
is different from AsV and is not taken up by phosphate channel, which is
still unidentified. When external concentrations are 2–3 orders of magnitude higher than internal concentration, SbV can overcome an electrical potential difference across the membrane and be taken up passively
by plants (Tschan et al., 2009). It was possible that 5 mg/L, the highest
concentration we tested, was not high enough for rice plant to overcome the potential difference. This was because the highest concentration in the roots was 1.40 mg/kg (Fig. 1B). Bell et al. (2003) suggested
apoplastic pathway as an alternative uptake route of SbV, which has
Sb influx (mg/kgFW h)
80
60
10
8
6
4
2
0
Without Fe plaque
With Fe plaque
A
0.0 0.5 1.0 1.5 2.0
40
20
0
0
40
80
120
160
200
SbIII concentration in solution (mg/L)
12
Reddish iron plaque was visible on the surface of rice roots after Sb
treatment (data not shown). DCB was used to extract Sb associated
with Fe plaque, which was 8–20 and 3–4 times higher than those in
plants in SbIII and SbV treatments (Fig. 3). DCB-extractable Sb was
26–40 times higher in SbIII treatment (35.4 to 190 μg) than that in
SbV treatment (0.893 to 7.43 μg). In comparison, Sb in rice roots was
1.72 to 24.8 μg in SbIII treatment and 0.202 to 2.91 μg in SbV treatment.
DCB-extractable Sb accounted for 37–51% of the total Sb in rice in SbV
treatment and was much higher at 86–95% in SbIII treatment. Hence,
substantial amount of Sb was accumulated on Fe plaque, especially for
SbIII. This is consistent with the high DCB-extractable Sb at 70–90% of
the total Sb in rice by Huang et al. (2011).
Uptake kinetics (Fig. 2B) using excised roots showed that rice roots
without iron plaque fit the Michaelis–Menten function too (R2 =
0.988–0.989). The mean Vmax values for SbIII and SbV uptake were
52.8 and 72.5 mg/kg fw h, and Km values were 0.16 and 15.0. All parameters were greater than those in roots with iron plaque (Fig. 2).
The iron plaque significantly decreased SbV uptake into the roots. However, at SbIII concentrations up to 14.2 mg/L, iron plaque significantly
increased SbIII uptake, but it decreased SbIII uptake from 20.8 to
165 mg/L. This may suggest that iron plaque was the source of SbIII
transfer into the roots at low concentration and barrier at high concentration. However, Huang et al. (2011) showed that SbV concentration in
the roots increased with increasing Fe plaque amount, but the formation and amounts of iron plaque on rice roots have no significant effect
on SbIII uptake by rice plants exposed to 2.44 mg/L SbIII for 3 d. So the
exact role of Fe plaque in Sb uptake into rice roots needs further
investigation.
It is known that SbIII sorbs more strongly to mineral surfaces than
SbV, especially to FeIII and MnIV (hydr)oxides (Alvarez-Ayuso et al.
(2013); Leuz et al., 2006b). In pH range of 2–10, SbIII is present as an undissociated Sb(OH)3° and it is less affected by surface charge. SbIII binds
strongly to Fe, Al and Mn (hydroxide) oxides via inner sphere surface
complexation with little pH dependency. On the other hand, SbV is primarily present as [Sb(OH)6]−, a monoprotic acid. Due to its low
Without Fe plaque
With Fe plaque
B
1.6
3.2. Iron plaque accumulated more SbIII than SbV and reduced Sb uptake by
excised rice roots
100
1.2
80
0.8
60
8 0.4
DCB-Sb (mg/kg)
Sb influx (mg/kg FW h)
10
2.0
been confirmed in wheat, maize and sunflower (Tschan et al., 2009).
Unlike SbV, Sb(OH)3° was the main SbIII form in the growth solution
at pH 6 (Wilson et al., 2010). SbIII was probably transferred across
membranes via aquaglyceroporins with the transpiration stream
(Zangi and Filella, 2012). So SbIII influx into rice roots was substantially
faster than SbV.
0.0
0
6
10
20
30
40
4
40
20
4
3
2
2
0
1
0
50
100
150
200
250
300
SbV concentration in solution (mg/L)
Fig. 2. Concentration-dependent uptake of SbIII (A) and SbV (B) by excised rice roots after
30 min exposure.
SbIII
SbV
0
0
0.2
1
5
Sb concentration in solution (mg/L)
Fig. 3. Antimony concentrations in DCB extract of rice roots after exposing to 0, 0.2, 1 and
5 mg/L SbIII or SbV for 4 h.
J.–H. Ren et al. / Science of the Total Environment 475 (2014) 83–89
deprotonation constant, SbV does not possess ligand exchanging ability
and it can only be adsorbed at deprotonated surface sites at acid pH
range. Hence, its adsorption ability is low compared to that of SbIII.
This may have important implication on the higher mobility and bioavailability of SbV in the environment (Leuz et al., 2006b; Vithanage
et al., 2013). This is consistent with our data in that the Fe plaque on
rice roots sequestered ~ 26–40 times more SbIII than SbV (Fig. 3).
These results indicated that Fe plaque had higher affinity to SbIII than
SbV, consistent with results of Huang et al. (2011). They find that 2.7–
10 times more Sb is accumulated in the iron plaques with SbIII than
SbV in three rice cultivars.
under reducing conditions. These results emphasized the need to monitor Sb speciation in the medium even in short-term experiments.
After 4 h exposure to SbIII or SbV, SbV was the predominant species
in rice plants, with little methylated species being detected (Fig. 4 and
Table 1). About 85–96%, 56–90% and 87–92% of total Sb were present
as SbV in the roots, stems and leaves in SbIII treatment. Similar results
but with higher SbV were found in the plant in SbV treatment, with
97–98%, 78–97% and 92–98% as SbV. Okkenhaug et al. (2012) reported
that SbV accounts for 83% of total Sb in the roots after exposing to
1562 mg/kg Sb in a soil for 6 wk. Muller et al. (2012) found that 56–
77% of water-extractable Sb was SbV in the shoots and 80−90% in the
roots of As-hyperaccumulator Pteris vittata.
Sb species present in solution and rice roots were different in SbIII
treatment, with SbV concentration being much higher in the roots
than that in solution (Table 1). Since rice was more efficient in taking
up SbIII than SbV, the amount of SbV taken up by rice was probably limited in the SbIII treatment. Take 5 mg/L SbIII treatment for example,
after 4 h exposure, the SbV and SbIII concentrations were 0.29 and
4.32 mg/L in the growth media, with 6.2% being SbV (Table 1). In comparison, the SbV and SbIII concentrations were 9.07 and 0.408 mg/kg in
the roots, with 96% being SbV (Fig. 4A). The results suggested that SbIII
was probably oxidized to SbV during uptake by the roots. Leuz et al.
(2006a) reported adsorption and oxidation of Sb by amorphous Fe
and Mn oxyhydroxide. It was possible that part of SbIII was oxidized
to SbV on the root surface before entering the roots. Though SbV was
stable in the growth solution, it may be reduced to SbIII in the roots.
3.3. SbV was the dominant species in rice plants
Sb speciation in rice seedlings (mg/kg)
Antimonate remained stable in the solution after 4 h exposure to
rice plants at pH 6.0. By contrast, 6.2–38% of SbIII was oxidized to SbV
in SbIII treatments (Table 1). Oorts et al. (2008) found that N70% of Sb
in a soil is oxidized to SbV within 2 d whereas Leuz and Johnson
(2005) showed no significant oxidation of 98 μg/L SbIII with O2 in
200 d at pH 3.6–9.8. However, iron plaque coated on root surfaces contains ferric oxides. Adsorption of SbIII onto Fe hydroxides may accelerate SbIII oxidation (Leuz et al., 2006a). It is possible that Fe oxides may
have caused the rapid oxidation of SbIII to SbV. Indeed, there is a
study showing that 77% of SbIII at 25 mg/L is oxidized in the presence
of amorphous Fe hydroxides in slurry solution at pH 6.0 (Belzile et al.,
2001). Okkenhaug et al. (2012) confirmed that SbV is stable even
10.0
10.00
Roots
10.00
Stems
8.00
8.00
6.00
6.00
4.00
4.00
2.0
1.0
2.00
0.40
0.20
2.00
0.50
0.30
0.8
0.15
8.0
6.0
4.0
0.20
0.10
0.15
0.4
0.05
0.0
0.00
0
0.2
1
5
A
Leaves
0.25
0.6
0.2
87
0.10
0.05
0.00
0
0.2
1
5
0
0.2
1
5
Sb speciation in rice seedlings (mg/kg)
SbIII concentration in solution (mg/L)
0.90
0.60
0.60
0.30
1.80
1.50
1.20
0.90
0.60
0.30
0.15
0.10
0.08
0.12
0.08
0.06
0.09
0.06
0.06
0.04
0.03
0.02
1.80
1.80
Roots
Stems
1.50
1.50
1.20
1.20
0.90
B
Leaves
0.04
0.02
0.00
0.00
0
0.2
1
5
0.00
0
0.2
1
5
0
0.2
1
5
SbV concentration in solution (mg/L)
Fig. 4. Total Sb concentrations (SbIII + SbV) in the roots, stems and leaves of rice after exposing to 0, 0.2, 1 and 5 mg/L SbIII (A) or SbV (B) for 4 h. Solid bars were SbIII and open bars were SbV.
88
J.–H. Ren et al. / Science of the Total Environment 475 (2014) 83–89
In the SbV treatment, 3.8–42.1 μg/kg SbIII (1.8–21.9%) was observed in
rice roots. The result was consistent with Okkenhaug et al. (2011) and
Zangi and Filella (2012).
protein and polysaccharides, such as lignin and cellulose, which include
ligands like carboxyl, aldehyde, and hydroxyl. It can also actively secrete
calloses to chelate heavy metal (Tang et al., 2009). Cell wall is considered
as the first barrier against heavy metal entering cells. So after being taken
up, Sb was accumulated in cell walls in the rice roots. Similar results were
observed for Cr (53%) and Cd (56–62%) in rice roots (Zeng et al., 2011).
Among different plant parts, Sb in cell wall was the highest in the roots
at 77–86%, with 55–74% in the stems and 56–72% in the leaves whereas
Sb associated with organelle was the lowest being b 14%. In our study,
we did not observe a great difference in Sb localization between the SbV
3.4. Sb was mostly associated with cell walls in rice plants
Sb concentration in subcellular fractions
of roots (mg/kg)
Subcellular distribution of Sb in rice plants was similar in SbIII and SbV
treatments, with Sb being associated with cell wall N cytosol N organelle
(Fig. 5). It is understandable as SbV was the dominant species in rice plant
regardless SbIII or SbV was provided (Fig. 4). Plant cell wall contains
10.00
Cellwall
Cytosol
Organelle
8.00
6.00
A
4.00
2.00
0.25
0.19
0.15
0.10
0.05
0.00
0
0.2
1
5
0
0.2
SbV
1
SbIII
5
Sb concentration in subcellular fractions
of stems (mg/kg)
Sb concentration in solution (mg/L)
1.00
Cellwall
Cytosol
Organelle
0.80
B
0.60
0.40
0.20
0.15
0.12
0.08
0.04
0.00
0
0.2
1
5
0
0.2
SbV
1
5
1
SbIII
5
SbIII
Sb concentration in subcellular fractions
of leaves (mg/kg)
Sb concentration in solution (mg/L)
0.20
Cellwall
Cytosol
Organelle
0.16
C
0.12
0.08
0.04
0.03
0.02
0.01
0.00
0
0.2
1
SbV
5
0
0.2
Sb concentration in solution (mg/L)
Fig. 5. Subcellular distribution of Sb in the roots (A), stems (B) and leaves (C) of rice after exposing to 0, 0.2, 1 and 5 mg/L SbIII or SbV for 4 h.
J.–H. Ren et al. / Science of the Total Environment 475 (2014) 83–89
and SbIII treatments. This was probably due to a rapid oxidization of SbIII
to SbV in root cells. To our knowledge, there was limited report about Sb
subcellular distribution in rice plants (Feng et al., 2011b).
4. Conclusion
In conclusion, SbIII oxidization to SbV in solution was observed after
exposing rice plants for 4 h. More Sb was accumulated on Fe plaque
than rice roots, which affected Sb accumulation in rice. SbIII and SbV uptake kinetics by excised rice roots were described by Michaelis–Menten
function. Rice was more efficient in SbIII than SbV uptake with most of
the Sb being accumulated in the roots as being SbV. Plant cell walls
may have acted as key storage compartment for Sb in rice.
Acknowledgments
This work was supported in part by the National Natural Science
Foundation of China (No. 21277070) and Jiangsu Provincial Innovation
Fund.
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