全 文 ：Received 10 Apr. 2003 Accepted 22 Jul. 2003
Supported by the National Natural Science Foundation of China (40232002), the Knowledge Innovation Program of The Chinese Academy
of Sciences (KZCX-401-01), National High-tech Research and Development Program (2001AA640501) and Natural Science Foundation of
Beijing (6990002).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Direct Determination of Arsenic Species in Arsenic Hyperaccumulator
Pteris vittata by EXAFS
HUANG Ze-Chun1, CHEN Tong-Bin1 * , LEI Mei1, HU Tian-Dou2
(1. Laboratory of Environmental Remediation, Institute of Geographical Sciences and Natural Resources Research,
The Chinese Academy of Sciences, Beijing 100101, China;
2. Laboratory of Synchrotron Radiation, Institute of High Energy Physics, The Chinese Academy of Sciences, Beijing 100039, China)
Abstract: Synchrotron radiation extended X-ray absorption fine structure (SR EXAFS) was used to
study the transformation of coordination and redox state of arsenic (As) in a newly discovered As
hyperaccumulator, Pteris vittata L., which is considered to have great potential for phytoremediating the
As contaminated soil. It is shown that the As in this plant was mainly coordinated with oxygen in the
reduced state, As (Ⅲ), and the reduction of As (V) occurred in the root after it was taken up. No oxidation
of As (Ⅲ) was found during the translocation of As, from root to shoot. Only a small amount of As was
coordinated with sulfur in root and petiole, but not distinct in pinna.
Key words: arsenic species; extended X-ray absorption fine structure (EXAFS); hyperaccumulator;
Pteris vittata (Chinese brake); reduction; translocation
Arsenic (As) pollution has received increasing atten-
tion recently (Nordstrom, 2002). Remediation of arsenic con-
taminated soils has become a major environmental issue.
As a economic, efficient and environmental friendly method
for the remediation of contaminated soils and waters, more
and more attention has been paid to the phytoremediation
of arsenic contaminated soils (Chen et al., 2002). Three
arsenic hyperaccumulators, i.e. Pteris vittata (Ma et al.,
2001; Chen et al., 2002), P. cretica (Wei et al., 2002) and
Pityrogramma calomelanos (Visoottiviseth et al., 2002),
have been discovered recently, and provide hopeful candi-
dates for the phytoremediation of arsenic contaminated
soils.
It has been reported that P. vittata has extremely high
trend to take up arsenic and transports it from root to shoot.
However, why this plant has such special character still
remains unknown. Study on species transformation of ar-
senic during root uptake and root to shoot translocation
within this plant might play an important role in under-
standing the hyperaccumulating nature of P. vittata, and
help to understand the arsenic uptake and transport mecha-
nisms in the plant and arsenic detoxification inside the plant
cells. The extended X-ray absorption fine structure (EXAFS)
provides a promising tool to determinate elemental oxida-
tion state and local coordination environments in samples
directly, whereas most other commonly used methods such
as chromatographic are indirect, requiring time consuming
and cumbersome preparation, which may alter elemental
species originally presented in samples and lead to false
results. EXAFS has been newly used in biological and en-
vironmental sciences for studying the transformation of
metal and metalloid ions in plants, and has been consid-
ered as a powerful and unique technique to study the physi-
ological actions of elements in living plant (Pickering et al.,
2000; Parson et al., 2002).
The arsenic uptake and its species in different tissues
of P. vittata treated with two inorganic arsenic species,
arsenate and arsenite, usually found in soils, were studied
in the present study by using EXAFS method.
1 Materials and Methods
1.1 Plant culture and arsenic treatment
Spores of Pteris vittata L. obtained from Hunan Prov-
ince of China were sprinkled onto a moist soil in a seedbed
covered with a plastic cling film to maintain moisture. After
the spores germinated, and grew into sporelings with true
leaves about 2 cm in height. The plant was pre-cultured for
one week in a modified Hoagland nutrient solution con-
taining 2.0 mmol/L of Ca (NO3)2, 0.75 mmol/L of K2SO4, 0.1
mmol/L of KCl, 0.25 mmol/L of KH2PO4, 0.65 mmol/L of
MgSO4, 0.1 mmol/L of EDTA-Fe(Ⅱ), 0.01 mmol/L of H3BO3,
0.001 mmol/L of MnSO4, 0.001 mmol/L of ZnSO4, 0.000 1
mmol/L of CuSO4 and 5.0´10-6 mmol/L of (NH4)Mo7O24.
Two plants were transplanted into a plastic pot containing
0.7 kg quartz sand, which was pretreated with dilute HCl
and washed with deionized water. The plant in individual
Acta Botanica Sinica
植 物 学 报 2004, 46 (1): 46-50
HUANG Ze-Chun et al.: Direct determination of arsenic species in arsenic hyperaccumulator Pteris vittata by EXAFS 47
treatment was cultured in the sandy media containing the
nutrient solution with 10 mg/L of As, either arsenite or
arsenate. The solution was renewed every 3 d to maintain
the arsenic concentration and species during the culture
period. The plant culture was conducted in a growth cham-
ber with the following conditions: 16 h of light period with
a light intensity of 300 mE.m-2.s-1, temperature of 26 ℃/15
℃ (day/night), and average relative humidity of 60%. Each
treatment was repeated triplicately.
After 4-week’s cultivating, the plant was harvested and
washed with tap-water followed by three rinses with deion-
ized water. The plant sample was freeze-dried and ground,
and divided into two parts: one part for the analysis of
arsenic concentration and another for EXAFS measurement.
The grounded sample (about 0.1 g) was weighted and di-
gested with a mixture of HNO3-HClO4-H2SO4 following the
method 3050B of USEPA (1996). The determination of ar-
senic concentration was performed on an atomic fluores-
cence spectrometer (AFS-2202). The sample for EXAFS was
stored in a sealed bag and kept in a refrigerator before the
EXAFS experiment.
1.2 EXAFS measurement
The plant sample was packed into a 3.0 cm× 0.7 cm
sample holder, the solutions of model arsenic compounds
(sodium arsenite, sodium arsenate dibasic and As (Ⅲ)- glu-
tathione (GSH)) were pipetted into a Lucite liquid holder of
the same size. The complex of As (Ⅲ)-GSH was composed
by adding a 10-fold molar excess of glutathione to a solu-
tion of sodium arsenite, following the method mentioned
by Pickering et al. (2000) .
Arsenic K-edge (11 867 eV) X-ray absorption spectra
collection were performed using a double crystal mono-
chromator (Si 111) in fluorescence mode at room tempera-
ture at EXAFS station on Beamline 4W1B of Beijing Syn-
chrotron Radiation Facility (BSRF). The electron storage
ring was operated at 2.2 GeV.
Arsenic K-edge (11 867 eV) X-ray absorption spectra
reduction and analysis were performed using following
procedures. Pre-edge background was removed from the
spectra and then normalized. The resulting data were con-
verted from E space to k space and weighted by k3. EXAFS
spectra obtained by the steps mentioned above were cal-
culated with Cerious2-XAFS software (Fig.1A). Fourier
transformation was then performed to obtain the radial
structural function (RSF)(Fig.1B). Final fitting of the spec-
tra was done on Fourier transformed k3 weighted spectra in
R space to get the value of interatomic distance (R) and
coordination number (N)(Arai et al., 2001).
2 Results
Symptom of arsenic toxicity was found to appear in fronds
of P. vittata treated with arsenic, both arsenite and arsenate,
after three weeks of the culture. The symptom, dark brown
coloration at the tips and the margins of pinnae, was ob-
served mainly in the bottom pinnae of senescent fronds
while young pinnae did not show any symptom of arsenic
toxicity. This toxicity was not too serious that the plants
could survive throughout the whole culture period. No sig-
nificant difference between arsenite and arsenate treatments
was found in both aboveground and root biomass (data
not shown).
Hyperaccumulation of arsenic in P. vittata was demon-
strated clearly in this experiment, with As concentrations in
the pinnae reaching up to more than 3 000 mg/kg DW, the
concentration of As in the shoots was always greater than
that in the roots, and the translocation factors (i.e., shoot/
root ratio of As concentration) were about 10, in both treat-
ments (Table 1). As concentration in the plant treated with
arsenate was significantly higher (P<0.05) than that in the
plant treated with arsenite, for all the tissues except normal
pinna (Table 1).
Figure 1A shows the k3 weighted c functions of several
model compounds and samples from P. vittata. Fine sig-
nal-to-noise arsenic K-edge EXAFS spectra were obtained
in most of the plant samples, except in the root (Fig.1d) and
mature petiole (Fig.1e) in arsenite treatment, with As con-
centration lower than 400 mg/kg (Table 1). It can be con-
cluded that interpretable EXAFS spectrum with K-space
ranging between 3-13 Å could be obtained from samples
containing As concentrations higher than 400 mg/kg.
Figure 1B shows the corresponding Fourier transforms
(radial distribution functions, RDFs) and best fitting of
EXAFS spectra. Final calculated coordination numbers,
interatomic distances, and fitting parameters are presented
in Table 2. Only one coordination shell was discovered in
each of those three arsenic model compounds, As-S shell
Table 1 Arsenic (As) in tissue of Pteris vittata treated with
either arsenite or arsenate*
As concentration (mg/kg DW)
Arsenite added Arsenate added
Root 349.5±119.2a 416.6±165.4b
Mature petiole 216.2±31.8a 390.1±39.5b
Young petiole 884.2±130.1a 1 075±109b
Normal pinna 3 306±932a 3 466±1 178a
Pinna with As-toxic symptom 3 573±1 007a 5 019±1 693b
*, values are Means ± SE (n = 3).Values followed by different letters
are significantly different between treatments at P<0.005.
Acta Botanica Sinica 植物学报 Vol.46 No.1 200448
for As(Ⅲ)-GSH with N=3 and R=2.26, As-O shell for arsen-
ate with N=4 and R=1.70, and As-O shell for arsenite with
N=3 and R=1.78, respectively. The arsenic in all samples
was coordinated with oxygen in the first coordination shell
at a distance of 1.79 ± 0.01Å, which was similar to that of
arsenite, but significant longer than that of arsenate, means
that As (V) was reduced to As (Ⅲ) immediately after it was
taken up by the root of P. vittata and no oxidation from As
(Ⅲ) to As (V) happened during the process of transloca-
tion from root to shoot. A second As-S coordination shell
with interatomic distance of 2.26 ± 0.01Å matching that of
As (Ⅲ)-GSH is found in most of the plant samples. In com-
parison with other samples, the complex of As (Ⅲ)-GSH
was more distinct in root treated with both arsenic species
Fig.1. Arsenic K-edge EXAFS (A) and corresponding Fourier transforms (phase shift uncorrected)(B) of samples from Pteris vittata
and arsenic model compound. a, As (Ⅲ)-GSH; b, sodium arsenite; c, sodium arsenate dibasic; d-h, plant treated with arsenite (d, root;
e, mature petiole; f, young petiole; g, normal pinna; h, pinna with As-toxic symptom); i-m, plant treated with arsenate (i, root; j, mature
petiole; k, young petiole; l, normal pinna; m, pinna with As-toxic symptom). Solid lines show the data and the dashed lines the best fit
(Table 2). The spectra were offset vertically but were plotted with the same relative scale.
HUANG Ze-Chun et al.: Direct determination of arsenic species in arsenic hyperaccumulator Pteris vittata by EXAFS 49
and mature petiole treated with arsenate (Fig.1B, d, i, j). The
coordination number of As-S is significant smaller than that
of As-O in all plant samples (Table 2), indicating that only a
small amount of As (Ⅲ) combined with sulfur in plant.
Additionally, a third undefined coordination shell dashed
with line D in Fig.1B was also found almost in all the plant
samples, which does not appear in the RDFs of all the ar-
senic model compounds including crystalloid sodium ars-
enite (data not shown) in the present study.
3 Discussion
Electron microscopes such as SEM and TEM coupled
with elemental probes are usually employed to observe el-
emental distribution in plant tissues at the scale larger than
several microns (Chen et al., 2003). However, the EXAFS
technology is direct technology with less pretreatment to
investigate the arsenic species at the scale of atomic level.
It is shown by the results of EXAFS that As (Ⅲ) coordi-
nated with oxygen was the predominant species in P. vittata,
the reduction of As (V) to As (Ⅲ) in arsenate treatment
mainly occurred in root after it was taken up, and no oxida-
tion As (Ⅲ) was discovered during its translocation from
root to shoot in both arsenic species treatments. As (Ⅲ)-S
coordination was also noted in the root and a part of peti-
ole of P. vittata, but it was not distinct in pinnae where
arsenic was mainly stored.
Although the arsenic species in P. vittata was studied
with X-ray absorption spectrum by Lombi et al. (2002) and
Webb et al. (2003), none of them obtained any interpret-
able spectrum from plant tissues other than pinna. Therefore,
it is not possible to trace the transformation of arsenic spe-
cies in the process of root uptake and translocation, and to
identify where As (V) was reduced to As (Ⅲ) in plant.
The reduction of As (V) in P. vittata may be an impor-
tant mechanism remained to be elucidated. Arsenic reduc-
tion in higher plant was also observed in Brassica juncea
by other researchers (Pickering et al., 2000). It was also
hypothesized that thiol groups might be the possible re-
ductant for As (V) ( Pickering et al., 2000; Schmöger et al.,
2000). However, all those studies based on arsenic non-
hyperaccumulators and the arsenic appeared to be entirely
combined with thiols in the form of As (Ⅲ)-tris-glutathione
or As (Ⅲ)-PCs (phytochelatins) in those plants, which was
different to arsenic in P. vittata. Whether the thiol com-
pound in arsenic hyperaccumulator playing an important
role in arsenic reduction or not is still a question needed to
be studied in the future.
4 Conclusion
It is found from EXAFS study of P. vittata that As (V)
was reduced to As (Ⅲ) after it was taken up and the ar-
senic was mainly presented as As (Ⅲ)-O in the plant, no
matter arsenate or arsenite was added in sandy culture.
Arsenic was kept as As (Ⅲ) during the translocation from
root to shoot. Different from that arsenic mainly combined
with thiols in some arsenic non-hyperaccumulator, only a
small amount of arsenic was coordinated with sulfur, in
root and petiole of P. vittata, and no distinct As-S was
found in pinna where arsenic was mainly stored.
Acknowledgements: Beijing Synchrotron Radiation Fa-
cility (BSRF) at BEPC is acknowledged for providing the
facility of EXAFS measurement.
Table 2 Results of fitting arsenic K-edge EXAFS of Pteris vittata and model compounds*
Treatment Sample or model compound Type N R (Å) σ2
Arsenite a Root As-O 3.0 1.79 0.010
As-S 0.3 2.27 0.004
Mature petiole As-O 2.9 1.79 0.006
Young petiole As-O 3.0 1.79 0.006
Normal pinna As-O 3.2 1.79 0.008
Pinna with As-toxic symptom As-O 3.3 1.77 0.009
Arsenate b Root As-O 2.9 1.79 0.008
As-S 0.3 2.25 0.006
Mature petiole As-O 3.0 1.79 0.007
As-S 0.3 2.25 0.005
Young petiole As-O 3.1 1.79 0.006
Normal pinna As-O 3.0 1.79 0.007
Pinna with As-toxic symptom As-O 3.3 1.80 0.006
Model compound As (III)-GSH As-S 3.0 2.26 0.007
Arsenate As-O 4.0 1.70 0.002
Arsenite As-O 3.0 1.78 0.007
a, the plant was treated with arsenite; b, the plant was treated with arsenate; σ2, the measure of the static disorder of the shell; N, the
coordination number; R, interatomic distance; Type, the coordination type.
Acta Botanica Sinica 植物学报 Vol.46 No.1 200450
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