JOURNAL OF PLANT NUTRITION
Vol. 27, No. 7, pp. 1227–1242, 2004
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Comparison of Arsenic and Phosphate Uptake
and Distribution in Arsenic Hyperaccumulating
and Nonhyperaccumulating Fern
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S. Tu and L. Q. Ma*
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Soil and Water Science Department,
University of Florida, Gainesville, Florida, USA
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ABSTRACT
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Uptake of arsenic (As) and its distribution in Chinese Brake fern
(Pteris vittata L.), an As hyperaccumulator, and Boston fern
(Nephrolepis exaltata L.), a nonhyperaccumulator, in the presence
of phosphorus (P), were characterized by employing a hydroponic
experiment with a complete three-factorial design. Two levels of As
(100 and 1000 mM) and four levels of P (0, 100, 500, and 1000 mM)
were used in this study. Arsenic uptake rates on the basis of root
fresh weight for the two ferns were similar at low As concentration
(100 mM). At high As concentration (1000 mM), however, As uptake
rates (373–987 nmol g1 f wt h1) of P. vittata were significantly
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*Correspondence: L. Q. Ma, Soil and Water Science Department, University
of Florida, Gainesville, FL 32611-0290, USA; Fax: 1-352-392-3902; E-mail:
[email protected].
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DOI: 10.1081/PLN-120038545
Copyright & 2004 by Marcel Dekker, Inc.
0190-4167 (Print); 1532-4087 (Online)
www.dekker.com
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greater than those of N. exaltata (164–459 nmol g1 f wt h1). In both
ferns, addition of P reduced their As uptake rate as well as
accumulation. Pteris vittata had a greater As TF (Translocation
factor ¼ concentration ratio of fronds to roots) than N. exaltata. On
the contrary, N. exaltata displayed a greater P TF than P. vittata. As
a result, high P/As ratio was observed in the roots of P. vittata,
whereas high P/As ratio was observed in the fronds of N. exaltata.
The study illustrated that As hyperaccumulation by P. vittata may
be facilitated by its high As influx rate and its high molar P/As ratio
in the roots resulting from both high As TF and low P TF.
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Key Words: Arsenic; Distribution; Hyperaccumulator; Kinetics;
Nephrolepis exaltata L.; Phosphorus; Pteris vittata L.; Uptake.
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INTRODUCTION
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Arsenic (As) is a naturally occurring metalloid in the earth’s crust; its
levels, however, have been elevated primarily by anthropogenic activities,
resulting in thousands of As-contaminated sites worldwide.[1] Commonly,
physical and chemical processes are employed to cleanse As-polluted
sites.[2] The major downside of such strategies is that they are very expensive, environmentally-unfriendly, and most importantly, they render
the site unsuitable for future use. An alternative approach is to use plantbased remediation technology, known as phytoremediation, which is
environmentally-benign and economical.
The paradigm of phytoremediation is based on hyperaccumulators,
plants that take up toxic elements from soil and water, and sequester high
concentrations in their aboveground parts.[3] There are about 417 known
metal hyperaccumulators, most of which, however, belong to nickel
(Ni).[4] Ma et al., discovered that a fern (Pteris vittata L.), commonly
known as Chinese Brake fern, hyperaccumulated as much as 2.3% As in
its aboveground parts.[5] Furthermore, it accumulated 744 mg kg1 As in
its aerial tissues growing in uncontaminated soils, indicating that the
fern is equipped with efficient As uptake and translocation systems. This
is in contrast to arsenic concentration in most plants, which is less
than 10 mg kg1 primarily accumulated in the roots.[6] Because of its
extraordinary propensity for As, P. vittata, the first known As hyperaccumulator, seems promising in the phytoremediation of As-contaminated sites. In order to harness its phytoremediation potential, one must
first identify the mechanism of As hyperaccumulation by elucidating
As uptake kinetics and its distribution characteristics.
Comparison of Arsenic Uptake and Distribution
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Arsenic occurs in different forms in soils. Depending on redox
potential and pH, As can exist as As(3), As(0), As(þ3), and As(þ5) in
both inorganic and organic forms. Under aerobic conditions, arsenate
(AsO3
4 ) is the predominant form, mostly bound to clay minerals, iron
(Fe) and manganese (Mn)-oxi/hydroxides, and organic substances.[6]
Plants take up As mainly as arsenate.[7,8] Since arsenate and phosphate
are chemical analogues, their interactions have been investigated in both
higher and lower plants. For instance, studies that used both hydroponic
and soil systems found that the relationships between As and P could be
either positive or negative,[9,10] depending on the nutritional composition
of the growth medium. However, it has been clearly documented that
arsenate uptake into cytoplasm is mediated by the phosphate carrier
present in the plasma membrane.[11,12]
Tu and Ma[13] examined the interactions of As and P in P. vittata in a
20-week potting experiment. Phosphorus (0.83.2 mmol kg1) had little
effect on As uptake and plant growth when the soil is spiked with
As < 2.67 mmol kg1; however, it increases As uptake and plant growth
when the As is 5.34 mmol kg1. On the other hand, moderate amount of
As (< 2.67 mmol kg1) increases plant P uptake but at As level of
5.34 mmol kg1, P uptake is reduced probably due to plant As toxicity.
This result clearly demonstrates the importance of P in As detoxification
of P. vittata. A hydroponic experiment is needed to clearly elucidate the
interactive effects of P and As on their plant uptake since it is difficult to
separate the effects of As and P competition for sorption sites in the soil
using a soil system.
In a hydroponic experiment, Wang et al. examined As uptake
kinetics as well as As interactions with P in P. vittata.[14] Increasing P
supply reduces plant As uptake markedly with the effect being greater on
the roots’ As concentrations than the fronds’. Adequate P decreases plant
arsenate influx, whereas P starvation increases the influx by 2.5-fold.
Such an effect is not observed for arsenite, indicating that arsenate and
arsenite are taken up by the plant via different uptake systems and
arsenate is taken up by P. vittata via the P uptake system. However, the
effects of P the kinetics of As uptake and relative As and P distribution in
P. vittata in comparison with a nonhyperaccumulator were not included
in the study. Such knowledge is crucial to understand the mechanisms
of As hyperaccumulation in P. vittata. Therefore, our study should
complement the study of Wang et al.,[14] in an effort to understand
the potential role of P in As detoxification by hyperaccumulators. The
present study took these factors into consideration using the As
hyperaccumulator, P. vittata, and Nephrolepis exaltata L. (Boston fern),
a nonhyperaccumulator.[15] It is expected that a better understanding of
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As uptake and, As and P distribution in plants would emerge from this
study, which could further elucidate the mechanism of plant As
hyperaccumulation. Most importantly, such knowledge may be of great
importance to commercialize the phytoremediation technology for As
polluted soils and groundwater. The overall aim of this study was to
determine As uptake and As and P distribution characteristics as
influenced by P in P. vittata in comparison with N. exaltata.
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MATERIALS AND METHODS
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Two ferns, P. vittata (an As hyperaccumulator) and N. exaltata
(a nonhyperaccumulator) were used in this experiment. The 4-month old
P. vittata used in this study were propagated in a growth room whereas
the 4-month old N. exaltata was procured from a nursery (Milestone
Agriculture, Inc., Florida, USA). To prepare these plants for the kinetic
study, both ferns were transferred to hydroponic system in a greenhouse
with temperatures ranging from 23 to 28 C and humidity of 70%. A
14-h photoperiod with a daily photosynthetic photon flux of
350 mmol m2 s1 was supplied by cool-white fluorescent lamps. Both
ferns were allowed to grow for three weeks to initiate new roots.
Hoagland-Arnon nutrition solution[16] at 0.2-strength with vigorous
aeration was used to maintain plant growth. The nutrition solution was
replenished twice a week.
A complete three-factorial experimental design was used to study the
uptake of As and P and their distribution as influenced by fern species
(P. vittata and N. exaltata) at two levels of As (100 and 1000 mM) and
four levels of P (0, 100, 500, and 1000 mM). Four days before the
experiment, all plants were transferred to hydroponic tanks containing
0.2-strength P-free Hoagland solution. Prior to the uptake study, the
ferns were removed from the hydroponic tanks and their roots washed
carefully first with tap water followed by deionized water. Thereafter,
uptake study was initiated by placing one plant in a 250 cm3 brown jar
containing 200 mL of 0.2-strength Hoagland solution, which contained
100 or 1000 mM arsenate (Na2HAsO47H2O), and 0, 100, 500, or 1000 mM
phosphate (NaH2PO4), resulting in 16 treatments (including both ferns).
The solution pH was adjusted to 6.25 with dilute HCl and NaOH, and
the solution was aerated vigorously.
The experiment was divided into two parts with three replicates. Part
I was a short-term As uptake experiment to compare kinetic rate of As
uptake between two fern species. In part I, nine aliquots (0.50 mL) of
solution were taken every 30–60 min for up to 6-h. The solution samples
Comparison of Arsenic Uptake and Distribution
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were filtered through 0.42 mM filter, diluted and acidified with
concentrated HNO3 and stored for total As determination. Water
losses via transpiration and sampling were replenished by frequent
additions of deionized water by maintaining a constant volume of
solutions. Upon terminating experiment part I, fresh weights of the roots
and the whole plants were recorded.
Experiment part II was aimed to study the uptake of As and P and
their distribution in two fern species in a long time. However, since
N. exaltata is sensitive to As, they were allowed to grow in As medium for
only two days. Then, plant roots were washed with tap water followed by
rinsing in ice-cold phosphate buffer containing 1 mM Na2HPO4, 10 mM
MES, and 0.5 mM Ca(NO3)2 to ensure desorption of As from material
surface and the root free space.[11] Thereafter, the plants were rinsed in
tap water followed by deionized water. The fern plants were separated
into roots and fronds, and dried at 65 C for determining total As and P.
Total As in the solution samples (acidified by concentrated HNO3)
from the short-term kinetic experiment was determined directly by a
graphite furnace atomic absorption spectrophotometer (GFAAS; Perkin
Elmer SIMMA 6000, Norwalk, CT). Plant samples were digested using
H2SO4/H2O2[17] and total As in the digestion solution was determined by
GFAAS. Since arsenate interferes with P determination,[18] plant P was
determined by a modified method.[19] Briefly, the pH of the digestion
solution was adjusted to around 5 with NaOH and HCl. Ten milliliters of
the solution was pipetted into a 20 mL-glass test tube; to this 0.5 mL of
l-cysteine (5% w/v in 0.6 M HCl) was added. The test tube was capped
tightly to allow complete arsenate reduction for 5 min at 80 C. The
solution was then cooled to room temperature and P concentration was
determined by molybdenum blue method.
Variance analysis was carried out with ANOVA procedure of SAS
Software. Fisher’s Least Significant Difference (LSD) test was used to
compare significant differences of means at P < 0.05.
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RESULT AND DISCUSSION
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Plant Arsenic Uptake Rate
Plant As uptake at low substrate concentration is generally operated
by a high affinity system (HAS), whereas at high substrate concentration, it is controlled by a low affinity system (LAS).[20] Regarding
As uptake, HAS and LAS operate at concentrations of <100 mM and
100 mM–10 mM, respectively.[21]
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To understand plant As hyperaccumulation, it is important to
examine its As uptake rate. In this experiment, As uptake by As
hyperaccumulator P. vittata during a course of 6-h period was compared
with N. exaltata, an arsenic nonaccumulator. Both plants absorbed As
efficiently from the solution containing 100 and 1000 mM of arsenate
during first 6-h. However, the uptake isotherms of the two ferns differed
significantly between the two As concentrations (Figs. 1 and 2).
At the low arsenate concentration of 100 mM without P, both ferns
absorbed As efficiently from the solution during first 6-h. The calculated uptake rates at 6-h were 142 15 nmol g1 root f wt h1 and
123 13 nmol g1 root f wt h1 for P. vittata and N. exaltata, respectively
(Fig. 1). The similarity in uptake rates between the two ferns demonstrated
that both ferns took up As efficiently possibly via the LAS on a unit
root biomass basis. At high arsenate concentration (1000 mM) without P,
the As uptake rate of P. vittata was 987 66 nmol g1 root f wt h1
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Figure 1. Cumulative As uptake rates in 100 mM As (arsenate) solution on the
basis of root fresh weight (f wt) in (a) P. vittata and (b) N. exaltata as influenced
by four levels of P (phosphate, P in the legends). Both ferns were precultured with
0.2-strength Hoagland nutrition solution for 3 weeks to grow new roots and then
were starved of P in 0.2-strength P-free Hoagland nutrition solution for 4 days
before the 6-h uptake experiment. The error bars indicate standard error of the
mean of three replications.
Comparison of Arsenic Uptake and Distribution
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Figure 2. Cumulative As uptake rates in 1000 mM As (arsenate) solution on the
basis of root fresh weight (f wt) in (a) P. vittata and (b) N. exaltata as influenced
by four levels of P (phosphate, P in the legends). Both ferns were precultured with
0.2-strength Hoagland nutrition solution for 3 weeks to grow new roots and then
were starved of P in 0.2-strength P-free Hoagland nutrition solution for 4 days
before the 6-h uptake experiment. The error bars indicate standard error of the
mean of three replications.
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significantly greater than N. exaltata (459 42 nmol g1 root f wt h1)
(Fig. 2). Such high affinity for As at high As concentration by P. vittata
indicated that it was equipped with detoxification mechanisms that
enabled it to accumulate additional As. Analogous results were reported
previously in a Ni hyperaccumulator plant. Kramer et al.[22] found that
the rates of Ni uptake was the same in both Ni hyperaccumulator Thlaspi
goesingense and nonhyperaccumulator Thlaspi arvense at low Ni concentration (both species were not affected by Ni toxicity). At high Ni
concentration, T. goesingense was much more tolerant to Ni than T.
arvense, which enabled it to hyperaccumulate Ni. On the contrary, other
hyperaccumulators such as Zn hyperaccumulator, Thlaspi caerulescens,
usually exhibits greater rate of Zn uptake than the nonhyperaccumulator
T. arvense.[23] These results indicated that the rate of metal/metalloid
uptake by hyperaccumulators is a function of plant species and metal/
metalloid concentrations.
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Similar to the results of Want et al.,[14] addition of P to the solution
reduced As uptake rate in both ferns. Upon increasing P concentrations
from 0 to 1000 mM at As concentration of 100 mM, the decline in As
uptake rate in P. vittata and N. exaltata was about 61–82% and 53–77%,
respectively. Similar results occurred at As concentration of 1000 mM.
This was not surprising since As (arsenate) and P (phosphate) are
chemical analogues, the suppression of As uptake by P was expected as it
is a common phenomenon in many plant species. For instance, plant As
reduction by P has been observed in barley seedlings, where P greatly
inhibited As uptake ( 80%).[11] A hydroponic experiment by Meharg
and Macnair[12] showed that P at 5000 mM (10 times greater than As)
could reduce As uptakes by 75% in both tolerant and nontolerant plant
genotypes of Holcus lanatus L. In Indian Mustard (Brassica juncea L.
Czern), addition of P at 1000 mM resulted in the reduction of As uptake
by 55–72% over the control at As concentration of 500 mM.[24] Based on
these results, therefore, to enhance plant As accumulation, the quantity
of P in the growth medium should be limited.
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Plant Accumulation of Arsenic
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Pteris vittata accumulated 5.8 times more As in the fronds than the
N. exaltata after exposing to As for 2 d, suggesting that P. vittata grown
hydroponically could also hyperaccumulate As (Fig. 3). It was interesting
to note that the As concentrations in the roots of N. exaltata were greater
than those of the P. vittata at both As concentrations. However, As
concentrations in the roots of N. exaltata are usually lower than those of
P. vittata in the control, i.e., uncontaminated soil (unpublished data). The
possible explanations to the high As concentrations in the roots
of N. exaltata could be that N. exaltata, usually maintaining high P
concentration in the plant, was starved of P in this experiment (4 days’
growth in P-free solution before the uptake experiment). Such treatment
may have stimulated it to take up an additional As (arsenate) through its
P (phosphate) uptake system due to the chemical similarities between As
and P. Although N. exaltata had a high concentration of As in the roots,
the total amount of As uptake by N. exaltata was less than that of
P. vittata due to its low root biomass and low As concentration in fronds
(data not shown).
Translocation factor (TF), used as an index to measure the
effectiveness of plant metal translocation,[25] is defined as the ratio of As
concentrations in fronds to those in the roots. This research revealed that
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Figure 3. Arsenic concentrations in different parts (fronds and roots) of P. vittata and N. exaltata grown
hydroponically under low As (arsenate, 100 mM) and high As (1000 mM) conditions as influenced by
different levels of P (phosphate) after 2-d of growth. Both ferns were precultured with 0.2-strength
Hoagland nutrition solution for 3 weeks to grow new roots and then were starved of P in 0.2-strength Pfree Hoagland nutrition solution for 4 days. The error bars indicate standard error of the mean of three
replications. Significance differences between means were determined by using Fisher’s Least Significant
Difference at P < 0.05 (LSD0.05).
Comparison of Arsenic Uptake and Distribution
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Figure 4. Translocation Factors (TFs, ratio of As or P concentrations in
fronds to roots) of (a) As and (b) P in the P. vittata and N. exaltata grown
hydroponically at low As (arsenate, 100 mM) and high As (1000 mM) levels
as influenced by P (phosphate) levels for 2-d. Both ferns were precultured
with 0.2-strength Hoagland nutrition solution for 3 weeks to grow new roots
and then were starved of P in 0.2-strength P-free Hoagland nutrition solution
for 4 days. The error bars indicate standard error of the mean of three
replications.
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were 0.06–0.23
Fs
N.100
exaltata
mM A
s and 0.37–0.48
at
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1000 mM As. In contrast, As TFs for P. vittata were 1.16–1.47 at 100 mM
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As and 1.14–1.72 at 1000 mM (Fig. 4a), indicating that N. exaltata, a
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nonhyperaccumulator of As, transferred much less As from its roots to
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fronds in comparison with P. vittata. As expected, increased As levels
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enhanced As concentration in both fronds and roots of the two ferns.
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However, addition of P to the solution suppressed As uptake significantly
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(Fig. 3). When P concentrations were increased from 0 to 1000 mM in
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solutions with low As (100 mM), the decline in As uptake in the fronds and
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roots was 45–84% and 55–86% in the P. vittata and 32–39% and 45–86%
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in the N. exaltata, respectively. Similar trends were observed for the
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reductions in As uptake when P was added to solutions with high As
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(1000 mM) (Fig. 3). The competitive inhibition of As uptake by P further
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corroborated that both As (arsenate) and P (phosphate) utilize the same
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uptake systems.
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f:/MDI 2004/Pln/27(7)/120038545_PLN_027_007_R1.3d JOURNAL OF PLANT NUTRITION (PLN)
Comparison of Arsenic Uptake and Distribution
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Plant Accumulation of P
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Unlike As, P is a key plant nutrient element because it is a constituent
of macromolecular structures and functions in energy transfer reactions.
In most plants, P is generally concentrated in upper parts or reproductive
organs.[20,26] Such a pattern was also observed in the N. exaltata as
evidenced by the TF values of P, which were 1.64–2.60 at 100 mM As and
2.21–3.27 at 1000 mM As (Fig. 4b), indicating that P concentrations in the
fronds were much greater than those in the roots (Fig. 4). On the
contrary, in P. vittata, the TFs for P were around 1, which implied an
even distribution of P in the fronds and roots (Fig. 4b). The fact that the
root P concentrations of P. vittata were greater than those of N. exaltata
although N. exaltata accumulated twice as much as P in the fronds
compared to the P. vittata (Fig. 5) may imply that its ability to keep high
P concentrations in the roots constitutes one of its As detoxification
mechanisms in P. vittata.
With regard to P accumulation, the ferns responded differently to As
levels. The P concentrations in fronds and roots of P. vittata were similar
at the two As levels. However, at high As (1000 mM) level, accumulation
of P tended to decline in the N. exaltata (Fig. 5). Such a P accumulation
pattern between the two ferns implied that high As levels had no effect on
P uptake system of P. vittata, but it caused serious damage in N. exaltata
as reflected by its inability to accumulate P. In fact, the symptoms of As
toxicity such as wilting and necrosis of leaf tips and leaf margins
appeared in the N. exaltata two days after they were exposed to 1000 mM
As. Thus, a significant increase in P accumulation occurred only at low
As (100 mM) in N. exaltata. Addition of P did not enhance P accumulation in P. vittata (Fig. 5). And such result was also found in soil potting
experiment using P. vittata.[13]
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Molar Ratio of As to P
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Molar ratio of P to As was calculated by determining the ratio of
molar concentrations of P to As in plants. Generally, the molar ratios
increased with P level and declined with As level in our experiment.
Clearly, the molar ratios of P/As in the roots of P. vittata were greater
than those in the fronds, especially at low As levels. Whereas, greater
P/As ratios were observed in the fronds of N. exaltatas than those of
P. vittata (Fig. 6). Studies on the molar ratios of P/As in fronds and
roots of a several As hyperaccumulating or nonhyperaccumulating
fern species[27] yielded the similar results (unpublished data). Such
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Figure 5. Phosphorus concentrations in different parts (fronds and roots) of P. vittata and N. exaltata
grown hyrdoponically under low As (arsenate, 100 mM) and high As (1000 mM) conditions as influenced by
different levels of P (phosphate) after 2-d of growth. Both ferns were precultured with 0.2-strength
Hoagland nutrition solution for 3 weeks to grow new roots and then were starved of P in 0.2-strength Pfree Hoagland nutrition solution for 4 days. The error bars indicate standard error of the mean of three
replications. Significance differences between means were derermined by using Fisher’s Least Significant
Difference at P < 0.05 (LSD0.05).
]
JOURNAL OF PLANT NUTRITION (PLN)
Tu and Ma
Comparison of Arsenic Uptake and Distribution
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Figure 6. Molar ratio of P/As (ratio of molar concentrations of P to As in plant
parts) in fronds and roots of P. vittata and N. exaltata for the low AS (arsenate,
100 mM) and high As (1000 mM) levels as influenced by P (phosphate) levels for
2-d. Both ferns were precultured with 0.2-strength Hoagland nutrition solution
for 3 weeks to grow new roots and then were starved of P in 0.2-strength P-free
Hoagland nutrition solution for 4 days. The error bars indicate standard error
of the mean of three replications.
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differential accumul
ribution patterns
ation
of and
As and
dist P accreted
P. vittata as an As hyperaccumulator equipped with As detoxification
mechanisms involving accumulation of P in the roots, which facilitated
As translocation from its roots to fronds resulting in high P/As ratios in
the roots. Since phosphate and arsenate are analogues and P addition
inhibits As toxicity,[21] a high root molar ratio of P/As may indicate that
As toxicity is less in the roots. On the contrary, like most common plant
species,[28–30] N. exaltata, being an As nonhyperaccumulator, accumulated less P in roots and hence transported little As from its roots to
fronds yielding a low ratio of P/As in the roots. Such distribution pattern
deprived N. exaltata of As detoxification mechanism, which eventually
led to the death of whole plant.
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CONCLUSIONS
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Pteris vittata and N. exaltata exhibited a similar As influx rate on the
basis of root fresh weight at low As concentration (100 mM). However,
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P. vittata surpassed N. exaltata significantly in As influx rate at high As
concentration (1000 mM). Phosphorus addition suppressed As influx rate
in both ferns and thus decreased As uptake and accumulation confirming
that As (arsenate) uptake was carried out by the P (phosphate) absorption system. Contrary to N. exaltata, P. vittata translocated relatively
high amount of As and low amount of P to its aerial parts and hence,
displayed a high molar ratio of P/As in roots. An increased P and reduced
As concentration in the roots (high molar ratio of P/As) facilitated
As detoxification in P. vittata. Furthermore, a unique distribution of P
and As patterns may constitute the mechanistic basis of As hyperaccumulation in P. vittata.
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ACKNOWLEDGMENTS
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This research was supported in part by the National Science
Foundation (Grant BES-0086768 and BES-0132114). The authors
gratefully acknowledge Dr. Mrittunjai Srivastava and Dr. Bhaskar
Bondada for proofreading the manuscript.
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