Annals of Botany 99: 869–875, 2007
doi:10.1093/aob/mcm038, available online at www.aob.oxfordjournals.org
Sorghum Roots are Inefficient in Uptake of EDTA-chelated Lead
YON G X U 1 , N AO KI YA M AJ I 1 , R E N FAN G S H E N 2 and J IA N F E N G M A 1 , *
1
Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan and
2
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences,
Nanjing 210008, China
Received: 24 October 2006 Returned for revision: 9 January 2007 Accepted: 30 January 2007
† Background and Aims Ethylene diamine tetraacetic acid (EDTA)-assisted phytoremediation has been developed to
clean up lead (Pb)-contaminated soil; however, the mechanism responsible for the uptake of EDTA – Pb complex is
not well understood. In this study, the accumulation process of Pb from EDTA – Pb is characterized in comparison to
ionic Pb [Pb(NO3)2] in sorghum (Sorghum bicolor).
† Methods Sorghum seedlings were exposed to a 0.5 mM CaCl2 ( pH 5.0) solution containing 0, 1 mM Pb(NO3)2 or
EDTA– Pb complexes at a molar ratio of 1 : 0.5, 1 : 1, 1 : 2 and 1 : 4 (Pb : EDTA). The root elongation of sorghum at
different ratios of Pb : EDTA was measured. Xylem sap was collected after the stem was severed at different times.
The concentration of Pb in the shoots and roots were determined by an atomic absorption spectrometer. In addition,
the roots were stained with Fluostain I for observation of the root structure.
† Key Results Lead accumulation in the shoots of the plants exposed to EDTA – Pb at 1 : 1 ratio was only one-fifth of
that exposed to ionic Pb at the same concentration. Lead accumulation decreased when transpiration was suppressed.
The concentration of Pb in the xylem sap from the EDTA– Pb-treated plants was about 1/25 000 of that in the external solution. Root elongation was severely inhibited by ionic Pb, but not by EDTA– Pb at a 1 : 1 ratio. Root staining
showed that a physiological barrier was damaged in the roots exposed to ionic Pb, but not in the roots exposed to
EDTA– Pb.
† Conclusions All these results suggest that sorghum roots are inefficient in uptake of EDTA-chelated Pb and that
enhanced Pb accumulation from ionic Pb was attributed to the damaged structure of the roots.
Key words: Complex, EDTA, form, Pb, phytoremediation, uptake system, sorghum (Sorghum bicolor).
IN TROD UCT IO N
Since the dawn of the Industrial Revolution, heavy metals
have been used widely by mankind and have accumulated
in the environment at an exponential rate (Nriagu, 1996).
Lead (Pb) is one of the most common toxic metals and
exhibits specific toxicity, especially for young children
(Mushak, 1993). Soils are a major and terminal sink for
lead deposition in the environment. Unlike organic contaminants, Pb cannot be degraded once the soil has been
contaminated by this element. Various soil treatments
may be employed to reduce the in situ bioavailability of
Pb to plants and animals (e.g. Brown et al., 2004);
however, for long-term sustainability it is preferable to
remove Pb from contaminated soils.
Phytoremediation has been proposed as an environmentally friendly and cost-effective plant-based technology
for cleaning up contaminated soil (Salt et al., 1998;
McGrath et al., 2002). However, there are two limitations
for the phytoremediation of Pb. First, Pb has an extremely
low solubility in soils due to complexation with organic
matter, sorption on oxides and clays, and precipitation as
carbonates, hydroxides and phosphates (McBride, 1994).
Low availability of Pb limits its uptake by plants. Second,
there are few natural hyperaccumulators of Pb for use in
phytoextraction.
To increase Pb availability in soils, some synthetic chelates have been used and EDTA is among the most effective
* For correspondence. E-mail: [email protected]
to solubilize Pb in soil and to enhance plant uptake
(Blaylock et al., 1997; Huang et al., 1997; Wu et al.,
1999). However, inconsistency in the literature exists on
the effect of chelates on metal uptake or accumulation.
Some authors reported that EDTA application did not
enhance or even decrease heavy metal uptake (Athalye
et al., 1995), while others observed a significant increase
after EDTA application (Blaylock et al., 1997; Huang
et al., 1997; Vassil et al., 1998). This inconsistency may
be attributed to many factors including different plant
species, experimental conditions, concentrations, and
ratio of EDTA to Pb. In the present study, chelated Pb
(EDTA– Pb) was compared with ionic Pb for their effects
on Pb accumulation, Pb toxicity and root structure in
sorghum. The results show that the plant roots of
sorghum are inefficient in uptake of EDTA– Pb complex.
M AT E R I A L S A N D M E T H O D S
Plant materials and growth conditions
Seeds of sorghum (Sorghum bicolor L. ‘Early Sumac’) were
rinsed in deionized water and transferred to a Petri dish with
moist filter paper overnight at 25 8C. Germinated seeds were
placed on nets floating on a solution containing 0.5 mM
CaCl2 (pH 5.0) in 1.5-L plastic containers and incubated
for 4 d. The solution was renewed daily. Six-day-old
seedlings of similar size were selected and pre-cultured in
an aerated nutrient solution containing one-fifth-strength
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870
Xu et al. — Uptake of EDTA – Pb by Sorghum
Hoagland solution ( pH 5.6). The nutrient solution contained
the following macronutrients (mM): KNO3 (1), Ca(NO3)2 (1),
MgSO4 (0.4), and (NH4)H2PO4 (0.2); and micronutrients
(mM): Fe-EDTA (20), H3BO3 (3), MnCl2 (0.5), CuSO4
(0.2), ZnSO4 (10) and (NH4)6Mo7O24 (1). After 15– 21 d
of culture, the seedlings were used for the following
experiments. All experiments described below were
conducted in a growth chamber with 16 h light and 8 h
dark at 25 8C. Each experiment was repeated at least twice,
with each treatment being replicated three times in an
experiment.
Pb uptake from different chemical forms
Seedlings, as prepared above, were exposed to a 0.5 mM
CaCl2 ( pH 5.0) solution in 50-mL plastic bottles containing
1 mM Pb and different concentrations of Na2EDTA, resulting in different molar ratios (Pb:EDTA) of 1 : 0.5, 1 : 1, 1 : 2
and 1 : 4. Two days later, shoots and roots were harvested
separately and rinsed three times with a 0.5 mM CaCl2 solution to avoid contamination by the culture solution. The
samples were oven dried at 70 8C for 3 d and then their
dry weights were recorded. Dried plant material was
ground and digested in concentrated nitrate acid.
Concentration of Pb was determined using the method
described below.
A time-course experiment was performed by transferring
the seedlings to a 0.5 mM CaCl2 solution ( pH 5.0) containing Pb(NO3)2 and EDTA –Pb (1 : 1) complex at 1 mM. At 0,
3, 6, 9, 12 and 24 h, shoots and roots were sampled as
described above.
Xylem sap collection
Seedlings (21-d-old) were exposed to a solution containing 1 mM Pb(NO3)2 or 1 mM EDTA– Pb (1 : 1) complex at
pH 5.0. At 0, 0.5, 1, 2, 4 and 8 h after exposure, the
stems were severed at 1 cm above the roots, and the
xylem sap was collected for 30 min with a micropipette.
The Pb concentrations in the xylem sap and in the external
solution were determined immediately as described below.
2.0 and 4.0 mmol EDTA– Na2 – 2H2O and 1 mmol
Pb(NO3)2 in 1 L of 0.5 mM CaCl2 solution, respectively.
The pH was adjusted to 5.0 by 1 N NaOH or HCl. Root
length was measured before and after treatments. After
the treatment, the roots were washed with 0.5 mM CaCl2
solution three times and the root segments (0– 1 cm,
1 –2 cm and 2 –3 cm back from the root tip) were then
excised by a razor blade in a 0.5 mM Ca solution in a
Petri dish. Ten root segments were combined and placed
into the tubes containing 2 N HNO3. The tubes were left
to stand for at least 1 d with occasional shaking. The Pb
concentration in the solution was then determined by graphite furnace atomic absorption spectrophotometer (Hitachi
Z-5000, Tokyo, Japan).
Staining of intact roots with Fluostain I
Seedlings of sorghum (6 d old) were transferred to a
0.5 mM CaCl2 solution ( pH 5.0) containing 0, 1 mM
Pb(NO3)2 or 1 mM EDTA– Pb 1 : 1 complex in the presence
of 0.01 % Fluostain I in a 50-mL beaker. Root length was
recorded to check the effect of Fluostain I on the root
growth at this concentration. At 6 h and 24 h after exposure,
the roots were rinsed with deionized water several times.
Root was hand-sectioned 1 mm back from the tip with
a blade, and then subjected to microscopic observation
using an ultraviolet filter set (excitation filter BP 365,
dichroitic mirror FT 395, barrier filter LP 397) (Zeiss
Axioplan2, Oberkochen, Germany).
Effect of pretreatment with ionic Pb on Pb accumulation
Seedlings (21 d old) were exposed to a 0.5 mM CaCl2 solution (pH 5.0) containing 1 mM Pb(NO3)2 or EDTA –Pb 1 : 1
complex for 1 d. Half of the seedlings were then transferred
to a fresh solution containing 1 mM EDTA– Pb (1 : 1) for a
1 d. The plants were harvested at day 1 and 2, respectively,
as described above. The Pb accumulation during the
second day was calculated by subtracting Pb accumulation
of plants treated for 1 day from those exposed for 2 d.
Pb uptake from soil amended with EDTA and HNO3
Effect of transpiration on Pb accumulation
The effect of transpiration on Pb accumulation was investigated by adding abscisic acid (ABA) to the solution. The
seedlings (15 d old) were exposed to 1 mM Pb(NO3)2 or
EDTA – Pb complex (1 : 1) at pH 5.0 in the absence or presence of 0.1 mM ABA in a 50-mL bottle. The weight of the
bottles was recorded before and after the treatment. After
12 h, the plants were sampled as described above and the
Pb concentration in the shoots was determined as described
below.
Bioassay of Pb toxicity
Six-day-old seedlings were exposed to a 0.5 mM CaCl2
solution containing ionic Pb or EDTA– Pb complexes
at molar ratios (Pb:EDTA) of 1 : 0.5, 1 : 1, 1 : 2 and 1 : 4
for 24 h. These complexes were made by mixing 0.5, 1.0,
Soil was taken from the experimental farm of Kagawa
University as described previously (Ueno et al., 2004).
The pH of the soil was 6.0 and the concentration of Pb in
the soil solution was 0.01 mg L21. The soil was sieved
(2 mm mesh) and air-dried. Lead as 2PbCO3.Pb(OH)2
was added at a rate of 2500 mg Pb kg21 soil. Six-day-old
seedlings were prepared as described above and were
planted in a 1.5-L plastic pot (two seedlings per pot)
filled with Pb-enhanced soil. The plants were grown in a
greenhouse for 6 weeks. Soil moisture was kept at field
capacity with daily addition of tap water. A solution of
Na2-EDTA-2H2O or HNO3 was added at a rate of
0.3 mmol kg21 soil at day 8 before harvest.
Soil solution was collected nondestructively with a
looped hollow fibre placed in the pots (Ueno et al.,
2004). Briefly, the soil solution sampler system, which consisted of a looped hollow fibre, a silicon tube and a
Xu et al. — Uptake of EDTA – Pb by Sorghum
disposable syringe, was set up in a pot. The soil solution
was collected in the morning for 2 h once a week before
application of chemicals and at 1, 2, 3, 5 and 7 d after
addition of chemicals. Approximately 5 mL of solution
were collected each time. After collection, the pH and concentrations of Pb were measured immediately as described
below.
Determination of Pb concentration
The plant samples were digested in glass tubes containing 5 mL of concentrated HNO3 in a heater (TAITECH,
Tokyo, Japan). The temperature started from 70 8C and
then increased to 130 8C with intermittent shaking until
the samples were completely oxidized. When the digested
solution became clear, the sample volume in each tube
was reduced to about 1 mL by evaporating the extra
HNO3 at 145 8C, and then the tubes were filled with
about 20 mL of distilled water. The concentration of Pb
in the solution was determined by an atomic absorption
spectrometer (Hitachi Z-5000, Tokyo, Japan).
R E S U LT S
The concentration of Pb in the sorghum shoots was compared between plants exposed to different forms of Pb.
The concentration of 1 mM used was commonly found in
the soil solution of experiments with EDTA-assisted phytoextraction (e.g. Huang et al., 1997). At this concentration,
plant growth did not differ significantly between different
treatments due to short exposure. The plants exposed to
ionic Pb and EDTA – Pb at 1 : 0.5 (Pb:EDTA) ratio for 2 d
accumulated five times more Pb than those exposed to
EDTA– Pb at 1 : 1 and 1 : 2 ratios (Fig. 1). When there
was excess EDTA (Pb:EDTA, 1 : 4), the concentration of
shoot Pb doubled in comparison with the EDTA– Pb at
1 : 1 ratio (Fig. 1). The accumulation of Pb in the roots
(data not shown) was much higher than that in the shoot,
but showed a similar trend to that observed for the shoots.
F I G . 1. Effect of different Pb forms on the concentration of Pb in the
shoot of sorghum. Twenty-day-old seedlings were exposed for 48 h to a
0.5 mM CaCl2 solution (pH 5.0) containing 0, 1 mM Pb(NO3)2 or
EDTA–Pb complexes at a molar ratio of 1 : 0.5, 1 : 1, 1 : 2 and 1 : 4
(Pb:EDTA). Data shown are means + s.d. (n ¼ 3).
871
The time-course experiment showed that the shoot Pb
concentration was very low during the first 6 h in the
plants exposed to either ionic Pb or the EDTA– Pb
complex at a ratio of 1 : 1 (Fig. 2A). However, the shoot
Pb concentration increased thereafter in the plants
exposed to ionic Pb, but not in the plants exposed to
EDTA– Pb complex (Fig. 2A). The concentration of Pb in
the roots was much higher than that in the shoot
(Fig. 2B). Furthermore, the root Pb concentration increased
rapidly with exposure time and saturated at 9 h. The Pb concentration in the external treatment solution was also monitored. Twenty-four hours after plants were treated with the
EDTA– Pb complex, the concentration of total soluble Pb in
the external solution increased compared with that of the
initial solution (data not shown).
The concentration of Pb in the xylem sap was measured
in the plants exposed to 1 mM ionic Pb or EDTA– Pb (1 : 1).
Xylem sap was not available for the plants exposed to ionic
Pb because of plant toxicity (see below). The Pb concentration in the xylem sap increased with the exposure time
to EDTA – Pb, but the concentration was only 0.04 mM at
8 h, which was about 1/25 000 of the concentration in the
external solution (Fig. 3).
The effect of transpiration on Pb accumulation was examined by adding ABA to the treatment solution. ABA is known
to suppress transpiration. In the presence of 0.1 mM ABA,
transpiration during 12 h decreased by 41.5 % and 37.5 %,
respectively, in the plants exposed to ionic Pb and
F I G . 2. Time-dependent concentration of Pb in the shoot (A) and the root
(B) of sorghum. Twenty-one-day-old seedlings were exposed to a 0.5 mM
CaCl2 solution (pH 5.0) containing 1 mM Pb(NO3)2 or EDTA –Pb (1 : 1)
complex. Plants were harvested at different times. Vertical bars represent
means + s.d. (n ¼ 3).
872
Xu et al. — Uptake of EDTA – Pb by Sorghum
F I G . 3. Concentration of Pb in the xylem sap of sorghum. Seedlings (21 d
old) were exposed to a 0.5 mM CaCl2 solution ( pH 5.0) containing 1 mM
EDTA –Pb (1 : 1) complex. At different times, the stem was severed and
the xylem sap was collected for 30 min. Vertical bars represent means +
s.d (n ¼ 3).
EDTA – Pb (1 : 1) (Fig. 4A). The Pb concentration in the
shoots also decreased corresponding with the decreased transpiration in plants exposed to either Pb form (Fig. 4B).
The toxicity of ionic or EDTA-complexed Pb was
assessed using root elongation. Root elongation was inhibited with 1 mM ionic Pb by 90 % (Fig. 5). In contrast, Pb
complexed with EDTA at 1 : 1 and 1 : 2 molar ratios did
not inhibit root elongation (Fig. 5). At 1 : 4 ratio of Pb to
EDTA, root elongation was also severely inhibited. Root
elongation of sorghum exposed to EDTA – Pb at the mole
F I G . 5. Effect of different forms of Pb on the root elongation of sorghum.
Six-day-old seedlings were exposed to a 0.5 mM CaCl2 solution (pH 5.0)
containing 0, 1 mM Pb(NO3)2 or EDTA– Pb complexes at a molar ratio
of 1 : 0.5, 1 : 1, 1 : 2 or 1 : 4 for 24 h. Vertical bars represent means + s.d.
(n ¼ 10).
ratio of 1 : 0.5 (Pb:EDTA) was also inhibited, suggesting
that the concentration of unchelated Pb in the solution
was high enough to be toxic. The effect of EDTA alone
on root elongation in sorghum was also investigated. Root
elongation was inhibited by 38 % and 82 % by 2 mM and
4 mM EDTA, respectively, suggesting that EDTA was also
toxic to plant roots at high concentrations (data not shown).
The Pb content in the different root segments was determined. Over all, the Pb content in the roots exposed to ionic
Pb was much higher than that in the roots exposed to
EDTA – Pb complex at a molar ratio of Pb:EDTA higher
than 1 (Table 1). For example, the root apices (0 – 1 cm)
exposed to ionic Pb contained 41.7 nmol root segment21,
while those exposed to EDTA – Pb (1 : 1) contained only
22.9 pmol root segment21, which was almost 1/2000 of
that exposed to ionic Pb (Table 1). In the roots exposed
to ionic Pb, more Pb accumulated in the root tips of
0 –1 cm than in the mature regions (.2 cm), suggesting
that the root apex is the first target of Pb toxicity.
The roots were stained with the Fluostain I dye, which
shows a pale blue fluorescence after binding to the cellulose
of the cell walls (Hughes and McCully, 1975). Therefore,
TA B L E 1. Pb content extracted from different root segments
Pb : EDTA
F I G . 4. Effect of transpiration on the accumulation of Pb by sorghum.
Seedlings (15 d old) were allowed to take up Pb in a nutrient solution containing 1 mM ionic Pb or EDTA –Pb in the presence or absence of 0.1 mM
ABA for 12 h. (A) transpiration; (B) shoot Pb concentration. Vertical bars
represent means + s.d. (n ¼ 3).
0 –1 cm
1 –2 cm
2 –3 cm
Pb content (nmol root segment21)
0:0
n.d.
1:0
41.70 + 1.40
1 : 0.5
37.62 + 1.75
n.d.
12.11 + 0.03
7.97 + 0.65
n.d.
10.30 + 0.35
5.69 + 0.38
Pb content (pmol root segment21)
1:1
22.88 + 23.75
1:2
1.24 + 1.13
1:4
0.15 + 0.26
13.03 + 8.05
1.15 + 0.58
8.19 + 3.37
13.36 + 2.75
6.05 + 0.54
8.10 + 0.83
Six-day-old seedlings were exposed for 24 h to a 0.5 mM CaCl2
solution (pH 5.0) containing 1 mM Pb(NO3)2 or EDTA– Pb complexes at
a molar ratio of 1 : 0.5, 1 : 1, 1 : 2 or 1 : 4.
Data shown represent means + s.d. (n ¼ 3).
n.d., Not detected.
Xu et al. — Uptake of EDTA – Pb by Sorghum
this dye can be used as a tracer of apoplastic solute flow in
the roots. The presence of this dye at 0.01 % and 0.001 %
has no effect on root elongation (data not shown).
Fluorescence was only observed at the surface of the epidermal cells in the roots in the control treatment (no Pb or
EDTA– Pb) or in those exposed to EDTA– Pb (1 : 1)
(Fig. 6), suggesting that the dye was stopped at the root
surface and cannot enter the stele. In contrast, in the roots
exposed to ionic Pb, fluorescence was observed not only
in the epidermal cells but also in the whole cortex and
central stele as early as 6 h after exposure (Fig. 6). At
24 h, the roots exposed to ionic Pb became too soft for
sectioning due to loss of vigour (Fig. 6).
When plants exposed to either ionic Pb or EDTA – Pb
(1 : 1) for 1 d were transferred to a solution containing
EDTA– Pb (1 : 1), an enhanced accumulation of Pb the
next day was observed in the plants pre-treated with ionic
Pb only (Fig. 7). This result suggests that ionic Pb
damaged the physiological barrier in the root and facilitated
the transport of EDTA – Pb into the stele.
Pb accumulation from different forms was further
examined in a Pb-contaminated soil. Application of
EDTA or HNO3 to the soil 8 d before harvest resulted in
different Pb concentrations in the soil solution and in
shoot accumulation. The concentration of Pb in the
soil solution was much higher after addition of EDTA
than HNO3 (Fig. 8A). However, the accumulation of Pb
was much lower in plants grown on soil with added
EDTA than those with added HNO3 (Fig. 8B). These
results indicate that EDTA was very effective in solubilizing
Pb in soil, but only a small fraction of the solubilized Pb
was taken up.
873
F I G . 7. Effect of pretreatment with ionic Pb on the concentration (A) and
accumulation (B) of EDTA –Pb in the shoot of sorghum. The seedlings,
pre-treated with 1 mM ionic Pb or EDTA– Pb (1 : 1) for 1 d, were transferred to a solution containing EDTA –Pb (1 : 1). After 1 d of exposure
to the EDTA–Pb solution, the seedlings were harvested. Data of shoot
Pb accumulation show the Pb accumulation during the second day.
Vertical bars represent means + s.d (n ¼ 3).
DISCUSSION
F I G . 6. Sorghum roots stained with Fluostain I. The plants were exposed
to a 0.5 mM CaCl2 solution ( pH 5.0) containing 0, 1 mM Pb(NO3)2 or
EDTA–Pb (1 : 1) complexes in the presence of Fluostain I. At 6 h and
24 h, hand cross-sections were prepared 1 mm back from the root tip.
Scale bar ¼ 100 mm.
Because of a physiological barrier in the roots, metals
cannot reach the stele freely. For a metal to be accumulated
in the shoots from the soil environment, at least three steps
are required. The first one is the transport of a metal from
soil to the surface of root cells via diffusion or mass flow.
The second step is the transport of a metal from the
outside (rhizosphere) to the root cells and the third one is
the release of a metal into the xylem (xylem loading)
(Mitani and Ma, 2005). Transport proteins are involved in
the latter two processes. The present study investigated
whether a transport system exists for EDTA– Pb in
sorghum. The present results show that plant roots do not
possess a transporter for the EDTA – Pb complex based on
the following evidence. First, the short-term uptake experiment showed that the uptake of Pb was greatly reduced
when EDTA was present (Figs 1 and 2). Two kinds of transporters for cations, ionic and chelated forms, have been
reported depending on metals and plant species. Most
cations such as Zn, Fe and Cu are transported in the ionic
forms (Kochian, 2000), and it is generally believed
that the chelated form of metals are less available
for uptake as compared with the ionic ones. However, in
gramineous plants, Fe is transported in a chelated form
(Fe– phytosiderophore complex) (Murata et al., 2006).
874
Xu et al. — Uptake of EDTA – Pb by Sorghum
F I G . 8. Effect of EDTA or HNO3 on the Pb solubility in soil (A) and
accumulation in sorghum (B). Plants were grown in a Pb-enhanced soil
(2500 mg Pb kg21) for 6 weeks. Eight days before harvest, EDTA and
HNO3 were added as a solution to the soil at the rate of 0.3 mmol kg21.
Soil solution was collected after application. Vertical bars represent
means + s.d. (n ¼ 3).
Low uptake from EDTA– Pb suggests that a transporter is
not involved. Secondly, the Pb concentration in the xylem
sap of plants exposed to EDTA –Pb was only 1/25 000 of
the external solution (Fig. 3). This result is in agreement
with the recent finding by Schaider et al. (2006). They
found that under normal conditions, xylem sap solute concentrations from EDTA– metal complexes are relatively low
(i.e. ,0.5 % of concentration in solution) in Brassica
juncea. If a transporter is involved, the concentration in
the xylem sap is usually higher than that in the external
solution. For example, Al in the xylem sap reached
4-fold higher concentration than that in the external solution
as early as 2 h after exposure in buckwheat (Ma and
Hiradate, 2000). Thirdly, the concentration of total
soluble Pb in the external solution of the EDTA– Pb treatment increased with time, suggesting that uptake of
EDTA – Pb, if any, was slower than uptake of water. This
means that there is a lack of active uptake of EDTA– Pb
by the roots. Usually, a depletion of a metal in the external
solution is observed if its uptake is mediated by a transporter. Fourthly, the Pb uptake from EDTA –Pb depended on
transpiration (Fig. 4), which suggests that the EDTA– Pb
complex is transported passively and that transpiration is
the driving force for Pb translocation.
The higher accumulation from ionic Pb can be attributed
to root damage. Ionic Pb inhibited root elongation (Fig. 5).
Root staining showed that there is a physiological barrier on
the epidermal cells of the root tips, which may prevent
solutes from passing freely through the cortex into the
stele (Fig. 6, controlled roots), although the barrier has
not been identified. Exposure to ionic Pb caused damage
of the physiological barrier (Fig. 6), resulting in a free
flow of Pb to the shoot. This is supported by the findings
that Pb accumulation is controlled by transpiration
(Fig. 4) and that Pb accumulation in the shoot dramatically
increased after 6 h in the plants exposed to ionic Pb
(Fig. 2A). It is also supported by the finding that pretreatment with ionic Pb enhances the subsequent accumulation from EDTA –Pb (Fig. 7). In rice, it was also reported
that the root structure was damaged by excessive Na, resulting in enhanced flow of Na into the xylem vessels and
increased accumulation of Na in the shoot (Ochiai and
Matoh, 2002).
In contrast to ionic Pb, EDTA – Pb was not toxic to
sorghum roots at the level investigated (Fig. 5) and the
physiological barrier was also not damaged in the roots
(Fig. 6). This result is consistent with a previous study
with Indian mustard (Vassil et al., 1998). In their study,
phytotoxicity of Pb was observed when the concentration
of EDTA was lower than that of Pb in the treatment solution. The toxicity of Pb is suggested to be caused by the
binding of Pb to cellular components (Geebelen et al.,
2002). Chelation of Pb by EDTA prevents this binding,
therefore detoxifying Pb. This is supported by the finding
that a lower concentration of Pb was detected in the roots
exposed to EDTA– Pb than those exposed to ionic Pb
(Table 1).
Previous studies reported that the accumulation of Pb was
greatly enhanced by application of EDTA in soil (e.g.
Blaylock et al., 1997; Huang et al., 1997). In an ultrastructural study of chelated Pb transport in Chamaecytisus proliferus spp. Proliferus ‘palmensis’ grown in a solution,
the Pb transported from roots to shoots increased in the
EDTA – Pb treatment (Jarvis and Leung, 2001). Indian
mustard plants exposed to Pb and EDTA in nutrient solution were able to accumulate up to 55 mmol kg21 Pb in
dry shoot tissue [1.1 % (w/w)] (Vassil et al., 1998). They
suggested that the soluble EDTA– Pb complex is transported through the plant, via the xylem, and accumulates
in the leaves. However, this high Pb accumulation may be
attributed to the excess use of EDTA. Excessive EDTA
causes toxicity to the root and shoot growth (Fig. 5 and
Vassil et al., 1998). It may also disrupt the physiological
barriers including the Casparian strip (Nowack et al.,
2006). In fact, there is a threshold of EDTA concentration
to ‘induce’ the accumulation of high Pb (Vassil et al.,
1998). At a ratio of EDTA – Pb lower than 1, no EDTA
was detected in the shoot. When the ratio of EDTA to Pb
was higher than 2, an increase in EDTA was observed in
Indian mustard with a concomitant decrease in shoot
growth. These aforementioned observations support the
interpretation that the apparent accumulation of
Pb, observed by Vassil et al. (1998), was caused by the toxicity of excessive EDTA. Excessive EDTA may remove Zn
and Ca ions in the plasma membranes, which are essential
for stabilizing the membranes, thereby destroying the
Xu et al. — Uptake of EDTA – Pb by Sorghum
physiological barriers of the roots. EDTA can also chelate
various essential divalent cations such as Fe, Zn and Cu.
Therefore, excessive presence of EDTA may disrupt the
biochemistry of the leaf cells and ultimately cause cell
death (Vassil et al., 1998).
Two major limiting factors in Pb phytoextraction from
contaminated soils are the low bioavailability of Pb in the
soil and the low Pb translocation from roots to shoots.
Although EDTA is an efficient chelator of Pb, the present
results show that plant roots do not possess a system for
transporting EDTA – Pb. Although excessive use of EDTA
can enhance the accumulation of Pb to some extent, there
is an environmental risk of EDTA due to its low biodegradability. Therefore, for an efficient phytoextraction of Pb,
approaches which facilitate the uptake of the EDTA– Pb
complex are required. From the results of the soil experiment reported here, it seems that lowering soil pH is
more effective for Pb accumulation in the plant than applying EDTA to the soil (Fig. 8).
AC KN OW L E DG E M E N T S
This study was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (No. 13556010 to
J. F. Ma and by CAS International Partnership Project
(CXTD-Z2005-4)). We thank Lawrence Datnoff and
Fangjie Zhao for their critical reading of this paper.
L IT E RAT URE C IT E D
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