Environmental Pollution 129 (2004) 69–78
www.elsevier.com/locate/envpol
Low molecular weight thiols in arsenic hyperaccumulator Pteris
vittata upon exposure to arsenic and other trace elements
Yong Caia,*, Jinhui Sua, Lena Q. Mab
a
Department of Chemistry and Biochemistry and Southeast Environmental Research Center, Florida International University, Miami, FL 33199, USA
b
Soil and Water Science Department, University of Florida, Gainesville, FL 32611-0290, USA
Received 30 May 2003; accepted 25 September 2003
‘‘Capsule’’: Arsenic induces synthesis of low molecular weight thiols in the arsenic hyperaccumulator Pteris vittata.
Abstract
Low molecular weight thiol-containing compounds have been reported to play an important role in metal detoxification and
accumulation in some higher plants. The formation of these low molecular weight thiols in the recently discovered arsenic hyperaccumulator, Chinese Brake fern (Pteris vittata) upon exposure to arsenic and other trace metals was investigated. In addition to
cysteine and glutathione, an unidentified thiol was observed in the plants exposed to arsenic, which was not found in the control.
The concentration of the unidentified thiol showed a very strong and positive correlation with arsenic concentration in the leaflets.
The unidentified thiol was low in rachises and undetectable in the roots for As-treated plants. Total and acid-soluble thiols were
also measured and the results indicated that arsenic mainly stimulated the synthesis of acid-soluble thiol in Chinese Brake. The
investigations of other trace elements (Cd, Cu, Cr, Zn, Pb, Hg, and Se) showed that these elements were not accumulated in Chinese
Brake to high levels and the synthesis of the unidentified thiol in the plant was not observed. Our study suggests that the unidentified thiol was induced specifically by arsenic and the distribution patterns of the unidentified thiol and arsenic in the plant were
consistent, indicating that the synthesis of this compound was related to As exposure.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Arsenic hyperaccumulator; Thiols; Low molecular weight thiols; Phytochelatins; Arsenic-contaminated soils
1. Introduction
Arsenic (As) is a highly bioactive and toxic element,
and its presence at elevated levels in soils and drinking
water threatens human health (Cullen and Reimer,
1989; Nriagu, 1994a,b). Arsenic-contaminated soils may
be cleaned up via phytoremediation, i.e. the use of
plants to extract the As from the soil and transport it
into above-ground tissues, which may then be harvested
and removed (Terry and Banuelos, 2000). Until
recently, no As hyperaccumulator was available to
make this process technically feasible. This situation has
now changed with the discovery of Chinese Brake fern
(Pteris vittata), which is a staggeringly efficient hyperaccumulator of As (Ma et al., 2001; Tu and Ma, 2002;
Cai and Ma, 2002). A recent survey in Thailand has
* Corresponding author. Tel.: +1-305-348-6210; fax: +1-305-3483772.
E-mail address: cai@fiu.edu (Y. Cai).
0269-7491/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2003.09.020
identified another As hyperaccumulator fern, Pityrogramma calomelanos (Visoottiviseth et al., 2001). Under
laboratory-controlled conditions with elevated soil As
supply, Zhao et al. (2002) identified three new species of
As hyperaccumulators in the Pteris genus. These reports
suggest that As hyperaccumulation is a constitutive
property in P. vittata.
Understanding the As detoxification mechanism of
Pteris vittata is of critical importance from the practical
standpoint of optimizing As phytoremediation using the
plant (Meharg and Hartley-Whittaker, 2002; Meharg,
2002; Cai and Ma, 2002). Plants that accumulate of
toxic metals all share one common characteristic, the
ability to tolerate high metal concentrations without
suffering toxic consequences. Some plants have evolved
the ability to avoid or exclude metals in order to reduce
their cellular incorporation while others have developed
mechanisms to detoxify metals and can therefore tolerate them at otherwise toxic concentrations. Mechanisms
that are based on tolerance rather than avoidance of
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Y. Cai et al. / Environmental Pollution 129 (2004) 69–78
metals are of interest for phytoremediation (Verklaij
and Schat, 1990; Goldsbrough, 2000). The proposed
mechanisms for metal detoxification and hyperaccumulation in plants include chelation of the metal by
ligands and/or sequestration of metals away from sites
of metabolism in the cytoplasm, notably into the
vacuole or cell wall (Salt et al., 1998; Baker et al., 2000).
Other possible adaptive responses include activation of
alternative metabolic pathways less sensitive to metal
ions, modification of enzyme structures, or alteration of
membrane permeability by structural reorganization or
compositional changes (Strange and Macnair, 1991;
Murphy et al., 1997; Baker et al., 2000).
Based on our previous results, it seems that chelation
of As by ligands and/or sequestration of As away from
sites of metabolism in the cytoplasm, notably into the
vacuole or cell wall, is the most likely As detoxification
mechanism in Pteris vittata (Ma et al., 2001; Zhang et
al., 2002a, 2002b; Tu et al., 2003; Zhao et al., 2002).
Such mechanisms have been proposed for the detoxification of many metals in a variety of plants (Salt et
al., 1998; Rauser 1999; Baker et al., 2000). Organic
ligands containing sulfur donor centers form stable
complexes with many elements, including trivalent As.
Plants can produce low molecular weight thiols that
have high affinity to some toxic metals (Fraústo da Silva
and Williams 1991; Baker et al., 2000). Phytochelatins
(PCs), a family of sulfur-rich peptides with the general
structure (g-GluCys)n-Gly, where n=2 to 11 (Grill et
al., 1987, 1989; Goldsbrough, 2000), are rapidly induced
in vivo by exposure to cations (Cd, Cu, Ni, Zn, Ag, Hg
and Pb) and anions (arsenate and arsenite) (Rauser
1995). Also, phytochelatins are considered to be a part
of As detoxification mechanisms in higher plants since
some authors have reported the accumulation of PCs
following exposure to As (Grill et al., 1987; Zenk 1996;
Maitani et al., 1996; Sneller et al., 1999; Koch et al., 2000;
Pickering et al., 2000; Schmöger et al., 2000). Glutathione and phytochelatin form AsIII-tris-thiolate complexes through thiolate bonds (Pickering et al., 2000;
Schmöger et al., 2000). However, in some plants heavy
metal tolerance was independent of phytochelatin
synthesis (De Knecht et al., 1992; Schat and Kalff, 1992).
Although it has been demonstrated that PCs may play
an important role in metal detoxification and hyperaccumulation in some higher plants, the role of these
low molecular weight thiols in As hyperaccumulation
and detoxification in Pteris vittata remains to be determined. In addition, the current research results show
that PCs seem to be general products induced by exposure to metals and metalloids for many tolerant as well
as non-tolerant plant species. Therefore, it remains to be
determined if low molecular weight thiols can be induced
in Pteris vittata upon exposure to other metals, and if
so, to what extent. The objectives of this study were to
(1) investigate the formation of thiols by exposing plants
to As; (2) determine the relationship between the distributions of As and thiols in the plants under different
experimental conditions; and (3) investigate the specificity of As uptake by Pteris vittata and the formation of
thiols during exposure to As and other metals (Cd, Cu,
Cr, Zn, Pb, Hg) and a metalloid (Se), which have been
shown to induce the formation of thiols in other plant
species.
2. Materials and methods
2.1. Materials and chemicals
Chinese Brake ferns used in this study were originally
from University of Florida, where they were propagated
(Ma et al., 2001; Tu et al., 2002; Zhang et al., 2002).
Potting soil (Lambert peat moss) was purchased from a
local Home Depot store (Miami, FL). The soil pH was
5.5. The concentrations of P, N, As, Ca, K, and Mg in
the soil were 23.0 0.5, 578.5 0.003, 0.007 0.0003,
436.3 28.8, 61.9 1.7; and 146.1 40.4 mmol kg 1,
respectively. Na3AsO.47H2O, Pb(NO3)2, CuCl2, CdCl2,
Hg(CH3COO)2, Na2SeO4, Na2SeO3, glutathione
(GSH), l-cysteine (Cys), n-acetyl-l-cysteine (NAC), trin-butylphosphate (TBP), N,N-dimethyl-formamide
(DMF), trifluoroacetic acid (TFA), ammonium-7fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) and
5,5-Dithiol-bis(2-nitrobenzoic acid) (DTNB), and
(NH4)2SO4 were purchased from Sigma (St. Louis,
MO). Acetonitrile, EDTA, sodium borate (Na2B4O7),
analytical grade sodium hydroxide (NaOH), trace metal
grade hydrochloric acid (HCl) were purchased from
Fisher. Working standards for Cd, Cr, Cu, Zn, Pb, and
Se were prepared by diluting 1000 mg l 1 ICP/MS grade
stock solutions purchased from GFS Chemicals
(Powell, OH) with either 2% trace metal grade HNO3
(for Cd, Cr, Cu, Zn, and Pb) or 30% trace metal grade
HCl (for Se). Mercury stock solution at 1000 mg l 1 was
purchased from Fisher and the working solution was
prepared with 1% trace metal grade HCl. Distilled
deionized water (DDW) was prepared using a Barnstead Fistream II Glass Still System (Barnstead Thermolyne Corp., Dubuque, Iowa) and was used in all
standard and sample preparations. All chemicals were
used as received without further purification. Standard
reference material (SRM) Dorm-2 was purchased from
National Research Council Canada (Ontario, Canada).
2.2. Methods
Young fern plants with 2–3 fronds were transplanted
into 0.5-gallon plastic pots containing ca. 500 g of potting soil (one fern per pot). After a 3-month growth in
the greenhouse, the plants were used for the following
experiments. To investigate the relationship between As
Y. Cai et al. / Environmental Pollution 129 (2004) 69–78
accumulation and thiol formation in Pteris vittata,
plants were divided into five groups (three plants for
each group). Soils were supplemented with As using
sodium arsenate at different concentrations. Arsenic
spiking solutions were prepared by dissolving appropriate amount of Na3AsO4 (AsV) in distilled deionized
water (DDW). The As solutions (300 ml) at concentrations of 0, 100, 200, 500, and 1000 mg l 1 were slowly
applied into the soils, which translated to As concentrations of 0, 60, 120, 300, and 600 mg kg 1 or 0, 0.8,
1.6, 4.0, 8.0 mmol kg 1 in soils. Care was taken to avoid
any water leaching from the pots. No fertilizer was used
during the greenhouse experiments.
The plants were grown in a greenhouse and watered
as needed (daily most of the time). Fronds, separated
into leaflets and rachises (stems), were collected from
the fern after As treatment at 1, 3, 7, 14, and 21 days,
and analyzed immediately for low molecular weight
(LMW) thiols and As contents. Efforts were made
ensure that the samples from different groups were
similar. After 21 days, the plants were harvested and
rinsed thoroughly first with tap water, then with DDW.
The roots, rachises and leaflets were immediately analyzed for the presence of low molecular weight thiols.
Aliquots of these samples were used for As analysis.
For evaluating the elemental accumulation and thiol
formation in Chinese Brake in response to exposure to
other trace elements, a number of environmentally
important elements, including Cd, Cr, Cu, Zn, Pb, Hg
and Se were investigated. Spiking solutions of different
elements were prepared by dissolving appropriate
amount of CdCl2, K2CrO4, CuCl2, ZnSO4, Pb(NO3)2,
Hg(CH3COO)2, Na2SeO4, and Na2SeO3 in DDW. Final
concentration of 200 mg l 1 working solution for each
element was prepared. The soils were supplemented
with 300 ml of these solutions. The final concentrations
of the elements in soils were 120 mg kg 1 or 1.07, 2.38,
1.89, 0.75, 0.58, 0.60, and 1.52 mmol kg 1 for Cd, Cr,
Cu, Zn, Pb, Hg, and Se, respectively. Similar greenhouse experimental procedures as described before were
then followed. To avoid the potential contamination
from ions adsorbed on the root surface, roots were first
washed with DDW. For treatments with Cd, Cu, Zn,
Pb, and Hg, they were placed in a CaCl2 (10 mM) solution for 30 min in an ice bath, and then washed with
DDW again. For the roots treated with Cr and Se, they
were placed in a solution containing KNO3 (0.5 mM)
and K2HPO4 (1.0 mM) for 10 min in an ice bath, and
then rinsed with DDW.
2.3. Total As analysis
Samples were digested using an open vessel digestion
following a procedure reported previously (Cai et al.,
2000; Zhang et al., 2002a). Briefly, fresh plant samples
were first ground to powder in the presence of liquid
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nitrogen; then 0.2 g leaflets, 0.05 g rachises or 0.2 g
roots were transferred into 15-ml glass tubes. Concentrated HNO3 (10 ml) was added to each test tube
and the samples were then digested with a heating plate
at 120 C for 1 h. Next, H2O2 (1 ml) was slowly added
to test tubes to digest for another 20 min. The solution
was transferred to a 100-ml volumetric flask and DDW
was added to exact 100-ml. Samples were then transferred to 125-ml polyethylene bottles. Prior to the ICP/
MS analysis, digested samples were further diluted in
15-ml polyethylene test tubes, the final HNO3 concentration being 2%. Internal standards (In, Y, Sc) at 50 mg
l 1 were added to all the standards and samples. Analysis was carried out using a Hewlett-Packard HP 4500
ICP/MS system. Detailed experimental parameters for
the ICP/MS analysis can be found elsewhere (Cai et al.,
2000).
2.4. Determination of low molecular weight thiols using
pre-column derivatization with SBD-F and HPLC analysis
Several methods are available for detecting thiolcontaining compounds in biological samples using
HPLC coupled with various detectors. Generally, derivatization reactions are required before or after HPLC
separation when UV/vis or fluorescence detection is
utilized (Shimada and Mitamura, 1994; Dias et al.,
1998). HPLC with fluorescent detection is widely used.
Derivatization with ammonium-7-fluorobenzo-2-oxa1,3-diazole-4-sulfonate (SBD-F) has an advantage over
other fluorescence methods, such as the o-phthalaldehyde, and the monobromobimane (mBBr), because
SBD-F is highly specific to SH groups and does not
react with alcohols, phenols or amino groups (Imai et
al., 1983). SBD-F has no fluorescence itself. In addition,
the thiol-SBD-F adducts are stable more than 1 week at
less than 4 C, and there is no reagent interference peak
for HPLC analysis. The SBD-F derivatization method
has been widely used to measure low-molecular weight
biological thiols, such as cysteine, homocysteine and
GSH, in human plasma (Araki and Sako, 1987; Fermo
et al., 1998), rat tissues (Toyo’Oka et al., 1988), natural
water sample (Tang et al., 2000), human and mice
serum (Ubbink et al., 1991). Application of this method
for determining thiols present in plant samples has been
thoroughly investigated (Su, 2002).
Plant samples were collected and rinsed with DDW
and ground to powder with a mortar and pestle in presence of liquid nitrogen. The analysis of the LMW thiols
in the plants were carried out using pre-column derivatization with SBD-F and followed by HPLC. The
homogenized samples (0.2 g) were extracted with 0.1 M
HCl (in a ratio of 1 g sample/10 ml HCl). The mixture
was centrifuged at 12,000 rpm at 4 C for 10 min. The
supernatant (200 ml) was transferred to a 2-ml plastic
centrifuge vial and 10% TBP (10 ml) in DMF was
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Y. Cai et al. / Environmental Pollution 129 (2004) 69–78
added. TBP was used as the reducing reagent to break
the disulfide bonds in samples. The reduction was
allowed to develop at room temperature for 10 min.
NaOH (20 ml, 1.0 M) was added to neutralized HCl
used in the extraction, and then borate buffer (200 ml,
pH 9.5) and SBD-F (30 ml, 1 mg ml 1) were added for
derivatization. Reaction was allowed to proceed at 60 C
in water bath for 60 min. After this period of time, HCl
(10 ml, 4.0 M) was added to stop the reaction, and samples were then transferred to 300-ml glass HPLC autosampler vials, and stored in refrigerator at 4 C prior to
analysis.
The analysis of the derivatized thiols was performed
using a Shimadzu HPLC system with fluorescence
detection. This system consists of a FCV-10AL lowpressure gradient flow control valve, a SCL-10A
controller module, a RS-232C interface for SCL-10A
controller, a SIL-10AXL automatic sample injector, a
LC-10AT solvent delivery module, a RF-551 spectrofluorometric detector, and a GT-104 degasser. Experiments were carried out at room temperature using a
flow rate of 1.0 ml/min. Reversed-phase HPLC separation was carried out on a Zorbax SB C18 (4.6 250 mm
with 5 mm particle size) analytical column with 50 ml
injection. A gradient elution using acetonitrile (A) and
0.1% TFA (B) was used for better separation and column cleaning prior to the subsequent injections. The elution profile was: 0 – 20 min, isocratic 10% B; 20 – 25 min,
10 – 15% B; 25 – 35 min, 15 – 35% B; 35 – 50 min, 35 –
90% B; 50 – 65 min, isocratic 90% B; 65–90 min, 10%
B. In all steps, the gradients used were linearly changed,
except for the last step, in which the mobile phase B
instantaneously dropped from 90 to 10%. The excitation and emission wavelengths of the fluorescence
detector were set at 385 and 515 nm, respectively.
2.5. Total thiol and acid-soluble thiol analysis
A Shimadzu UV–Vis spectrophotometer (UV-2101
PC) was used for the total and acid-soluble thiol measurements (Zhang et al., 2002b). For total thiol analysis,
fresh plant samples were ground to powder and homogenized with 0.02 M EDTA (10 ml per gram of plant
sample), and then centrifuged at 12,000 rpm at 4 C for
10 min. The supernatant (0.5 ml) was transferred to a
polyethylene test tube and mixed with 0.2 M Tris buffer
(pH 8.2, 1.5 ml), 0.01 M DTNB (100 ml), and methanol
(7.9 ml). Reaction was allowed to develop for 10 min at
room temperature before absorbance was measured. A
sample blank (without adding DTNB) and a reagent
blank (without sample) were prepared and measured in
the same manner.
For the acid-soluble thiol analysis, 1.5 ml aliquots of
supernatant were mixed with 10% TCA (1.5 ml) to
precipitate proteins. The mixture was then centrifuged
at 12,000 rpm and 4 C for 10 min. The supernatant
(2.0 ml) was transferred to a 15 ml polyethylene test
tube and mixed with 0.4 M Tris buffer (pH 8.9, 4.0 ml)
and 0.01 M DTNB (0.1 ml). The reagent blank and
sample blank were prepared at the same time. The
absorbance at 412 nm was measured within 5 min after
adding DTNB. Thiol concentrations were calculated by
using an extinction coefficient of 13,100 (Zhang et al.,
2002b).
2.6. Analysis of Se, Hg, and other elements
A hydride generation atomic fluorescence spectrometer (HG-AFS) (PSA 10.055 Millennium Excalibur
System, P S Analytical Ltd, Kent, England) was used
for the selenium analysis. Fresh plant samples (0.1 g)
were digested in a 125-ml Erlenmeyer flask with 10 ml
concentrated HNO3. The digestion was conducted at
100 C for 60 min using a heating plate with a sand
bath. Then, H2O2 (2 ml) was slowly added (0.5 ml at a
time) followed by further heating for another 20 min.
HCl (6 N, 50 ml) was added to the sample to reduce the
HNO3 concentration and ensure that all SeVI had been
converted to SeIV for hydride generation, and heating at
100 C for another 60 min. The digested sample was
then transferred to a 100-ml volumetric flask and diluted to 100 ml using DDW. The detailed instrumental set
up for selenium analysis using HG-AFS can be found
elsewhere (Irizarry et al., 2001).
The digestion and analytical procedures for mercury
were adapted from the method for fish analysis reported
by Jones et al. (1995). Fresh plant samples (0.5 g of
leave samples, 0.2 g of stems or roots) were weighed and
placed in 10-ml glass ampoules. DDW (1 ml) and concentrated HNO3 (2 ml) were added into each ampoule.
The ampoules were left standing for 20 min. The
ampoules were sealed and autoclaved at 121 C for 1 h.
After autoclaving, the ampoules were left standing to
reach room temperature. The samples (1 ml) were
pipetted from the ampoule to a 20-ml polyethylene
scintillation vial containing 20 ml of 1% HCl. Samples
were then ready for cold vapor atomic fluorescence
spectrometer (CVAFS) analysis (Jones et al., 1995).
For the analysis of Cd, Cr, Cu, Zn, and Pb, fresh
samples (0.2 g) were transferred to 15-ml glass test tubes
for digestion. The analyses were performed with ICP/
MS using a digestion and analytical procedure similar
to that for As (Cai et al., 2000).
3. Results and discussion
3.1. Total arsenic in Chinese Brake fern
Changes in As concentration in plant leaflets with
time after As treatment, calculated based on fresh
weight, are showed in Fig. 1. In agreement with our
Y. Cai et al. / Environmental Pollution 129 (2004) 69–78
previous findings (Ma et al., 2001; Zhang et al., 2002a),
As uptake by the fern was rapid as can be observed by
the obvious As accumulation in leaflets after just 1 day
after treatment. Regardless of the treatment times, As
concentrations in leaflets increased with the soil As
concentration. There were clearly positive correlations
between As concentrations in the soil and in the leaflets.
The highest As concentration in the leaflets (7395 mg As
g 1) was from the soil treated with 600 mg As g 1 after
14 days of growth. For the same time period, As concentration in the plant from soil containing 60 was 1162
mg As g 1. It was observed that As concentrations
increased with time for up to 14 days, while decreases
occurred for the 21-day of growth, especially for these
at high soil As levels. The decrease in As concentration
after 14 days could be attributed to the dilution factor
resulting from an increase in the plant biomass after 2
weeks.
Arsenic concentrations in rachises were also analyzed
(Fig. 2). As with leaflets, As in the rachises increased
with soil As concentration. It should be noted that As
accumulation in the rachises was much lower than that
in the leaflets (Figs. 1 and 2). The highest As concentration in rachises was around 20% of that in the
leaflets. Fig. 3 shows the changes in As concentrations
in the roots with As supplemented into soil after 21 days
of treatment. Effects of time were not evaluated since
the roots were only analyzed 21 days after treatment.
Of the three parts of the plant investigated, roots
contain the lowest As, which was consistent with our
previous results (Ma et al., 2001; Zhang et al., 2002a).
73
3.2. Low molecular weight thiols
Thiol synthesis in the plant growing in As-contaminated soil after 1, 3, 7, 14, and 21 days was investigated. Several LMW thiols were observed in the plant
tissues. Fig. 4 shows chromatograms obtained for thiol
analysis in plant leaflets with or without As exposure.
Two thiols, Cys and GSH, were found in both controls
and As-treated samples. An unidentified thiol peak that
appeared around 31 min was observed in the leaflets of
the As-treated plants, whereas this was not detectable in
the controls. Several minor peaks were also observed
in leaflets of the As-treated samples. These peaks were
found to be uncorrelated with As treatment during the
course of experiments.
Because of the lack of an appropriate standard for
quantifying the unidentified thiol, peak height was used
for the following discussion. Fig. 5 shows the changes in
the peak height of the unidentified thiol in leaflets with
plant growth time for all As treatment levels. The unidentified thiol contents in the leaflets generally increased
with soil As concentration for all growth periods, suggesting that the synthesis of thiols was triggered upon
As exposure. It appears that the thiol synthesis in the
plant leaflets reached a maximum after 7 days of As
exposure, and then decreased. The reason for this to
occur is not very clear at this time. However, the dilution factor resulting from the increase in the biomass
after a week with limited As left in the soil may contribute, to some extent, to the decrease in thiol concentration in the plant.
Fig. 1. Changes in As concentrations in leaflets with time for different As treatment levels.
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Y. Cai et al. / Environmental Pollution 129 (2004) 69–78
Fig. 2. Changes in Arsenic concentrations in Rachises with time for different As treatment levels.
Fig. 3. Arsenic concentrations in roots for different soil As levels after 21 days.
Rachises and roots were also analyzed for thiols. The
concentration of As-induced unidentified thiol in the
rachises was much lower than that in the leaflets (data
not shown). The concentrations of the unidentified thiol
in rachises were slightly increased with As content in
soil for the high As treatment groups (300 and 600 mg
kg 1 As), whereas no significant changes in thiols concentration were observed for others. Overall, the As
concentrations in soil and the unidentified thiol peak
height in rachises did not show a strong relationship.
The unidentified thiol was not detected in the roots after
21 days of As treatment.
3.3. Relationship between As and the unidentified thiol
in leaflets
In order to evaluate the role of the unidentified thiol
in the As uptake by Chinese Brake, As accumulation
and thiol synthesis in the leaflets after 21 days of As
treatment were plotted (Fig. 6). The result clearly shows
Y. Cai et al. / Environmental Pollution 129 (2004) 69–78
75
Fig. 4. Chromatograms obtained for analysis of thiols in leaflets of Pteris vittata with SBD-F as derivatizing reagent. A: Control; B: arsenic-treated.
a very strong positive correlation between As uptake
and thiol synthesis in the leaflets, indicating that the
unidentified thiol was involved in the As hyperaccumulation and detoxification in the fern. The exact
role played by the thiol, however, was not clear based
on the current results.
3.4. Total thiols and acid-soluble thiols
In order to assess the role of the LMW thiols (in
addition to the unidentified thiol observed using HPLC)
in the As accumulation by Chinese Brake, the concentrations of total and acid-soluble thiols in the plant
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Y. Cai et al. / Environmental Pollution 129 (2004) 69–78
Fig. 5. Changes in the concentration of the unidentified thiol in leaflets with plant growth time.
Fig. 6. Arsenic accumulation and formation of the thiol in Pteris vittata with increased soil arsenic concentration.
with (600 mg kg 1 As spiked in soil) and without As
treatments were measured and compared. The fern
leaflets were collected after 21 days of As exposure.
Arsenic concentration in the leaflets was 7395 mg g 1.
For the control plants, low concentrations of total and
acid-soluble thiols in leaflets were detected
(0.041 0.007 and 0.199 0.020 mmol kg 1 for acidsoluble and total thiols, respectively). For the As-treated
plants, both acid-soluble and total thiols increased dramatically in leaflets with 1.26 0.13 and 1.66 0.20
mmol kg 1 for acid-soluble and total thiols, respectively. Among the thiols synthesized in the As-treated
plants, acid-soluble thiols were the major component,
suggesting that small thiol-containing compounds, such
as sulfur-containing polypeptides, may be involved in
As accumulation and detoxification in Chinese Brake.
However, there was a large difference between the concentrations of As and the acid-soluble thiols detected in
the plant leaflets. The highest As concentration detected
in this study was 7395 mg g 1 (98.6 mmol kg 1) in leaflets,
Y. Cai et al. / Environmental Pollution 129 (2004) 69–78
while the acid-soluble thiol was only 1.26 mmol kg 1.
The molar ratio of the acid-soluble thiol to As was
0.0128. Concentration of the unidentified thiol observed
using HPLC fluorescence was also estimated. Since
standard was not available for the unidentified thiol,
GSH was used as a reference for the estimation. The
concentration was found to be around 0.24 mmol kg 1,
about 20% of the acid-soluble thiols in the leaflets.
While the results confirmed the formation of LMW
thiols upon exposure to As, it seems that the acid-soluble thiols synthesized are not sufficient to complex all As
accumulated in the fern. The mechanism of As detoxification in Chinese Brake may be more complex than
simply chelation of As anions by the thiols. For example, the LMW thiols may only play a transport role by
facilitating the transport of As into the vacuole where
As may form a more stable aggregation with sulfide and
organic acids (Cobbett, 2000).
3.5. Response to other trace elements
Several trace metal and metalloid ions, including Cd,
Cr, Cu, Zn, Pb, Hg, selenite (SeIV), and selenate (SeVI),
were selected in this study to (1) determine if the formation of the unidentified thiol in Chinese Brake was
induced specifically by As, or was a common phenomenon for all trace metals and (2) to investigate if these
elements can be accumulated by the fern, and if so, to
what extent. These results will be useful fro assessing the
specificity of this fern plant for As hyperaccumulation.
Concentrations of these elements in roots, rachises,
and leaflets in the control and As-treated plants were
measured (data not shown). Compared with the controls, the concentrations of all the elements in the roots
seem to be moderately elevated. Relatively high standard deviations were observed, which could be attributed in part to the cleaning processes applied prior to
the analysis. In other words, the elements adsorbed on
the root surface may not be efficiently removed. Nevertheless, the bioaccumulation factors for all elements
tested, calculated as the ratio of metal concentrations in
plant roots to those in soils (120 mg kg 1), were much
less than 1. No significant differences were observed for
concentrations of Cd, Cr, Cu, Zn, Hg, and Pb in rachises and leaflets between controls and metal-treated
plants, indicating that Chinese Brake does not translocate these elements to rachis and leaflet. Slight increases
in Se concentration were observed in the plant tissue for
Se treatments. The plant accumulated up to 9.1 and 49.0
mg g 1 of Se in leaflets for SeIV and SeVI treatments,
respectively, while there was 0.39 mg g 1 in the control.
As for the synthesis of LMW thiols, GSH and Cys
were detected in plant tissue for all elements tested.
However, no significant differences were observed
between the controls and the treated plants (data not
shown). The unknown thiol found in As-treated plants
77
was not detected. These results indicate that the hyperaccumulation ability of Chinese Brake fern was highly
specific to As, and the formation of the unidentified
thiol compound was also highly specific to As exposure.
4. Conclusions
Results from this study confirmed that Chinese Brake
fern, Pteris vittata, accumulates As to extremely high
concentration. The majority of As was translocated to
the aboveground biomass and most of it were stored in
leaflets. An unidentified thiol was observed in the plants
exposed to As, which was not found in the control. The
concentration of the unidentified thiol showed a very
strong and positive correlation with As concentration in
the leaflets. The unidentified thiol concentration was
low in rachises and undetectable in the roots of Astreated plants. Our investigations of other trace elements showed that Chinese Brake did not accumulate
these elements to high levels and the unidentified thiol
was not observed when plants were exposed to these
elements. This result suggests that the unidentified thiol
was induced specifically by As. The distribution of the
unidentified thiol was consistent with As distribution
patterns in the plants, confirming that the formation of
this compound was related to As exposure.
The results from the analysis of total thiol and acidsoluble thiol show that As stimulated the formation of
acid-soluble thiol in Chinese Brake. The LMW thiol
may play an important role in As detoxification in the
plants, although details on the detoxification mechanism remain unknown.
Acknowledgements
This research is partially supported by NSF grants
(BES-0086768 and BES-0132114). We would like to
thank the advanced mass spectrometry facility (AMSF)
at FIU for the access to the ICP/MS. We thank Weihua
Zhang for useful discussions and assistance with the
experiments and David Chatfield for his assistance with
preparation of the manuscript. This is contribution #
213 of the Southeast Environmental Research Center
(SERC) at FIU.
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