Journal of Experimental Botany, Vol. 56, No. 420, pp. 2765–2775, October 2005
doi:10.1093/jxb/eri270 Advance Access publication 30 August, 2005
RESEARCH PAPER
Accumulation and fractionation of rare earth elements
(REEs) in wheat: controlled by phosphate precipitation,
cell wall absorption and solution complexation
ShiMing Ding1,2, Tao Liang2,*, ChaoSheng Zhang3, JunCai Yan4 and ZiLi Zhang4
1
Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, PR China
Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, No. 11A,
Datun Road, Chaoyang District, Beijing 100101, PR China
2
3
4
Department of Geography, National University of Ireland, Galway, Ireland
Department of Soil and Agrichemistry, Anhui Agricultural University, Hefei 230036, PR China
Received 22 March 2005; Accepted 18 July 2005
Abstract
Previous studies on rare earth element (REE) bioaccumulation have focused on their accumulation rate and
fractionation, but the processes involved remain unclear. In this study, the accumulation and fractionation
of REEs in wheat (Triticum aestivum L.) were investigated using solution culture with exogenous mixed
REEs. A decrease in REE contents was observed from
the roots to the tops of wheat. Significant fractionations of REEs were found in wheat organs as compared to the exogenous mixed REEs. Middle REE
(MREE, the elements from Sm to Gd) enrichment and
an M-type tetrad effect (an effect that can cause a split
of REE patterns into four consecutive convex segments) were observed in the roots, which were probably caused by phosphate precipitation of REEs in/on
the roots and absorption of REEs to root cell walls.
Light REE (LREE, the elements from La to Eu) and
heavy REE (HREE, the elements from Gd to Lu) enrichments were observed in the stems and leaves, respectively, accompanied by conspicuous W-type tetrad
effects (an opposite effect to the M-type tetrad effect)
in the REE patterns. HREE enrichment decreased from
the older to the younger leaves and increased upwards
within a single leaf. It is suggested that the solution
complexation that occurred in the xylem vessels plays
an important role in REE fractionations in the aboveground parts of wheat.
Key words: Accumulation, fractionation, rare earth elements
(REEs), the tetrad effect, Triticum aestivum L., wheat.
Introduction
The rare earth elements (REEs) comprise a group of 17
trivalent metallic elements with similar chemical properties.
They vary in relative atomic mass from 139 (lanthanum) to
175 (lutetium). Also commonly included in the group are
scandium (45) and yttrium (89). Lanthanum, the most well
known, has the useful property of being electron opaque
compared with other Group III metal cations (e.g. Al3+,
Ga3+, Sc3+) (Robards and Robb, 1974). Laboratory experiments have dealt with the potential for REEs to replace and
compete with Ca for binding sites on proteins, and with
effects on membrane stability (Brown et al., 1990). Previous studies have also identified some notable similarities
in the early reactions by primary roots to REE3+ and to Al3+
(Bennet and Breen, 1992; Ishikawa et al., 1996; Kataoka
et al., 2002). This enables REEs potentially to be ultrastructural tracers for Al toxicity to plants.
There is an increasing interest in the bioaccumulation
processes of REEs due to the wide application of REEs in
a variety of non-nuclear industries and agriculture, resulting in possible environmental contamination (Choppin
et al., 1986; Wang et al., 2001). Meanwhile, due to their
unique chemical structures as a group of elements, they
may be used to trace the sources of inorganic elements in
plants (Fu et al., 1998, 2001; Fu and Tasuku, 2000) and the
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2766 Ding et al.
behaviour of actinides for risk assessment of radioactive
waste disposal (Brookins, 1989; Miekeley et al., 1994).
Investigations into the bioaccumulation characteristics of
REEs have been carried out in recent years as sensitive
techniques such as inductively coupled plasma-mass spectrometry (ICP-MS) and neutron activation analysis (NAA)
have became available (Ichihashi et al., 1992; Wyttenbach
et al., 1994, 1998; Fu et al., 1998, 2001; Fu and Tasuku,
2000; Liang et al., 2001; Wei et al., 2001). Concentrations
of REEs in plants are extremely variable, with about
700 ng g1 La reported in one species of fern (Matteuccia)
(Fu et al., 1998), but it can be less than 10 ng g1 La in the
needles of Norway spruce (Wyttenbach et al., 1994).
Possible reasons for the differences in REE concentrations
in plants include differences of REE concentrations in soils
and differences of plant species (Miekeley et al., 1994; Fu
et al., 2001). Like other trace elements, REE concentrations
are elevated in roots and they decrease in the order of
roots>leaves>seeds (Wyttenbach et al., 1998). However,
the reasons for this kind of variation are not clear, and more
investigation into the accumulation of REEs in plants is
needed.
For decades, geologists have recognized that REEs
are very useful tracers in studies of geochemistry because
of their generally coherent and predictable behaviour
(Henderson, 1984). The property of REEs as tracers can
also be applied to explain the mechanisms of element intake
by plants (Fu et al., 1998). For example, based on REE
patterns, Fu et al. (2001) suggested that silicate particles in
soils might be a significant source of inorganic elements for
plants. However, this may be complicated by the fractionation among individual REEs.
Despite extensive studies on REE uptake and transport in
plants, little attention has been paid to the REE fractionation in bioaccumulation processes. The results of no or no
obvious fractionation were often claimed, but they were
most probably due to analytical difficulties or uncritical use
of the term ‘no fractionation’ (Wyttenbach et al., 1998).
Fractionations of REEs in biogeochemical processes have
been observed, for example, fractionation between light
REEs (LREEs, La–Eu) and heavy REEs (HREEs, Gd–Lu)
(Wyttenbach et al., 1998; Wei et al., 2001), non-redoxsensitive REE anomaly (such as Gd, Yb and Lu)
(Wyttenbach et al., 1998; Xu et al., 2002), and redoxsensitive REE anomaly (Ce and Eu) (Wyttenbach et al.,
1998; Fu et al., 2001; Wei et al., 2001; Ding et al., 2003).
Most of the natural chemical fractionations can be attributed to the differences in their ionic radii (with increasing contraction of the 5s and 5p electrons with the increase
of atomic number) and variations in valences (Ce3+ or
Ce4+, Eu2+ or Eu3+).
However, a radius-independent variation in the chondritenormalized REE patterns (the REE abundances are normalized to those in chondrite, and then the pattern is
achieved by plotting the ratios on a logarithmic scale
against the atomic number: the REEs in chondrite are
assumed to be no fractionation) called the tetrad effect was
also found in plants (Fu et al., 1998, 2001; Fu and Tasuku,
2000). This effect can cause a split of REE patterns into
four segments called tetrads [first tetrad, La–Ce–Pr–Nd;
second tetrad, (Pm)–Sm–Eu–Gd; third tetrad, Gd–Tb–Dy–
Ho; and the fourth tetrad, Er–Tm–Yb–Lu] with ‘breaks’ at
Gd, between Nd and Pm, and between Ho and Er,
corresponding to the half-, quarter-, and three-quartersfilled 4f subshell, respectively (Mclennan, 1994). Fidelis
and Siekierski (1966) and Peppard et al. (1969) initially
observed the tetrad effect in patterns of liquid–liquid REE
distribution coefficients. Since then, the tetrad effect has
been recognized in chemistry as affecting REE complexing
behaviour, which is assumed to be influenced by variations
in the exchange interactions of unpaired 4f-electrons, spinorbit coupling, or crystal field stabilization (Irber, 1999).
There are two types of variations of the tetrad effect, and the
overall shapes of the tetrads are either convex or concave
and form M-shaped distribution in solid and W-shaped
distribution in solution, respectively (Masuda et al., 1987).
A W-type tetrad effect was observed in fern (Matteuccia),
implying that the REEs must once have been in a dissolved
state (Fu et al., 1998).
Few studies have been carried out on the REE fractionation in the soil–plant system via laboratory experiments.
Field investigation may not be suitable for studies on the
mechanisms of REE fractionation due to the extremely low
concentration ratios of plant/soil, contamination of plant
samples with soil and dust particles and the uncertainty of
the normalization procedure (Wyttenbach et al., 1994;
Mclennan, 1994). In this study, the water culture of wheat
with the application of exogenous mixed REEs was
adopted. Accumulation and fractionation of REEs among
wheat organs or tissues were investigated, and the absorption, distribution, and fractionation mechanisms of REEs
were discussed.
Materials and methods
Stock solution of mixed REEs
Stock solution of 2.1 mM mixed REEs used in this study was
composed of 14 lanthanides (La to Lu except Pm), with an identical
concentration of each element ([Ln3+]=0.15 mM, Ln represents any
lanthanide). Each REE stock solution of about 10 mM was prepared
by dissolving oxide with 1 M HCl, except that Ce was made from
CeCl3. Then the solution of each element was calibrated by an ICPMS and prepared for the mixed-REE solution. All solutions used in
the present study were made using deionized water of 18-mega ohm
quality or better.
Plant materials and growth conditions
Seeds of wheat (Triticum aestivum L. cv. Jin-Dong 8) were sterilized
for 15 min in a 1% solution of sodium hypochlorite, rinsed with tap
water, and soaked in deionized water for 24 h. The seeds were then
placed on a nylon net, which was fixed on an aerated solution
containing 0.2 mM CaCl2 in a 7.0 l plastic container. After being kept
REEs in wheat
in the dark for 5 d at 25 8C, the seedlings were transplanted to 3.0 l
plastic pots (20 seedlings per pot) containing aerated one-fifth
strength Hoagland solution. This nutrient solution contained the
following macronutrients (mM): KNO3 (1.0), Ca(NO3)2 (1.0),
MgSO4 (0.4), and KH2PO4 (0.2); and the micronutrients (lM): FeEDTA (4), H3BO3 (3.0), MnCl2 (0.5), CuSO4 (0.2), ZnSO4 (0.4), and
(NH4)6Mo7O24 (1.0). The solution was adjusted to pH 5.5 with dilute
HCl or NaOH and renewed every other day. Plants were grown under
controlled-environment conditions with a 14/10 h 25/20 8C day/night
scheme and light intensity of 40 W m2 until the appearance of the
fifth leaf (about 5 weeks). A part of them were then used for the
accumulation and fractionation experiments. The rest of the seedlings
continued to grow in half-strength Hoagland solution for another
5 weeks, and were then used for the collection of the xylem sap.
Accumulation and fractionation of REEs in wheat
The seedlings with five leaves were exposed to 1 mM CaCl2 solution
(pH 5.5) for 30 min to reduce PO3
4 from adhering to the root surfaces
(Ca2+ was used to maintain the stability of root tissues) (Marschner,
1995), then to 1 mM CaCl2 solution (pH 5.5) containing 5 lM mixed
REEs for 1 d, and the nutrient solution (one-fifth strength Hoagland
solution) for another day. The reason for this intermittent treatment
was to avoid the interactions between REEs and other nutrients such
as PO3
4 : This process was repeated three times, and on the seventh
day after treatment, the seedlings were further cultured in 1 mM
CaCl2 solution (pH 5.5) for 30 min to reduce the REEs adhering to
root surfaces. Half of the seedlings were then harvested, while the
remaining half were transferred to half-strength Hoagland nutrient
solution without mixed REEs. When three new leaves (sixth, seventh,
and eighth) appeared, the plants were sampled. The plant samples
were separated into roots, stems, and leaves and the leaves were
separated into the first to the fifth leaf at the fifth-leaf stage and the
first to the eighth leaf at the eighth-leaf stage (numbered from the
oldest to the youngest). The third leaf at the fifth-leaf stage was cut
into three equal parts according to its length, and named as ‘base’,
‘centre’, and ‘top’ from the bottom to the top.
Collection of the xylem sap
Prior to collection of the xylem sap, the 10-week-old seedlings were
cultivated overnight in a solution containing 1 mM CaCl2 (pH 5.5),
then exposed to 50 lM mixed REEs in 1 mM CaCl2 (pH 5.5)
solution. After 24 h of culture, the stem was severed 2 cm above the
root and xylem sap was collected for 8 h with a micropipette during
the daytime. The sap in the micropipette was removed into a beaker
with 0.1 M HCl and then evaporated to near dryness. The residue
was dissolved with 0.44 M HNO3. Concentrations of REEs were
determined as described below.
Cell-wall-bound REEs in wheat roots
Seedlings of wheat precultured in 0.2 mM CaCl2 were transplanted to
the above nutrient solution and grown for two more weeks. The plants
with three leaves were then exposed to 1 mM CaCl2 solution (pH 5.5)
containing 5 lM mixed REEs for 48 h. The roots were sampled and
parts of them were used to extract cell walls. Cell wall extraction was
conducted according to the methods described by Hu and Brown
(1994) and Ma et al. (1999) with minor modification. The root
samples (about 5 g fresh weight) were homogenized with 10 ml icecold distilled water using a glass grinder. The homogenate was
centrifuged at 1000 g for 10 min, and the precipitate was resuspended
in ice-cold water and centrifuged again. The precipitate was then
washed three times with 10 vols of 80% ethanol and one with 10 vols
of methanol:chloroform mixture (1:1, v/v), followed by 10 vols of
acetone. In every step of washing, the supernatant was collected by
centrifugation. Finally, the precipitate was oven-dried under 75 8C
and used for detection of REEs.
2767
REE-phosphate/citrate interactions in vitro
200 ml of 0.01 M NaNO3 solution containing 50 lM mixed REEs
was mixed with an equal volume of 0.01 M NaNO3 containing
200 lM NaH2PO4 or 200 lM NaH2PO4+50 lM citrate. The initial
solutions were sufficiently acidic (pH 3.0) to keep REEs from
precipitating. After sampling for the initial concentrations of REEs,
the pH was slowly raised by adding dilute NaHCO3 and was finally
stable at 3.760.1 while stirring vigorously. Thereafter, solutions were
sampled at regular time intervals. The samples were filtered through
a 0.22 lM membrane filter and the filtrates were prepared for determination of REEs.
REE detection and quality control
The wheat samples were washed with tap water and with deionized
water successively, then oven-dried at 75 8C until the dry weight
reached a constant value. The dry samples were ground to fine
powder. Each sample with an amount of 0.05 g of root, 0.1 g of stem
or leaf, 0.025 g of cell walls was digested with the mixture of 5 ml
HNO3 and 0.5 ml HClO4 in a 50 ml beaker. The digested solution was
evaporated to near dryness. The residue was dissolved with 0.44 M
HNO3 and transferred into a 10 ml flask, then diluted to the specific
volume with deionized water. REEs in solution were determined by
ICP-MS (PQ II Turbo, VG). Prior to detection, 0.2 ml of a 1 lg ml1
indium (In) solution was added in the solution to be used as an
internal standard to compensate for matrix suppression and signal
drifting. The wheat seeds used in this study were also determined and
the REE contents were found to be extremely low (about 0.05 mg
kg1 DW).
Quality control was achieved with a certified reference sample
GBW07603 of shrub leaves from the National Research Center for
Certified Reference Materials, Beijing, China. Seven duplicates were
made. The results for the reference samples were in line with the
reference values, with the standard deviations less than 5%, except
for Sm (6.7%) and Lu (5.4%).
Quantification of the tetrad effect
The proposed method was adopted to quantify the degree of the tetrad
effect according to Irber (1999) and Monecke et al. (2002). The
method was designed for REE patterns that exhibit a split into four
tetrads. To illustrate the four segments of an REE pattern showing the
tetrad effect, the segments are consecutively numbered as i=1, 2, 3,
and 4. The size of the tetrad effect in each segment can be given as
rffiffiffiffiffiffiffiffiffiffiffiffiffi
XBi XCi
ti =
ð1Þ
XAi XDi
where XAi, XBi, XCi, and XDi, are the concentration of the first to the
fourth element in the tetrad, respectively. The second tetrad (Pm to
Gd) cannot be calculated because of the missing Pm in nature.
The values of ti determined from three segments can be averaged to
produce an overall value of T:
Size of the tetrad effect = T = ðt1 3t3 3t4 Þ
1=3
ð2Þ
The tetrad effect can be simply characterized by an M-shape or
a W-shape for the T values being larger than one or smaller than one,
respectively.
Results
After short-term exposure to 1 mM CaCl2 solution (pH 5.5)
containing 5 lM mixed REEs, considerable amounts of
REEs were absorbed by wheat and distributed to every
organ of the seedling (Fig. 1A). More than 99% of the total
2768 Ding et al.
Fig. 1. Contents and patterns of REEs in wheat at the fifth-leaf stage. (A) Contents of REEs in each organ of wheat (lg). (B) REE patterns in different
leaves. (C) REE patterns in the stems. (D) REE patterns in the roots. The seedlings at the fifth-leaf stage were exposed to 5 lM mixed REE solution
and nutrient solution on alternate days for 6 d. Values are means 6SD of three replicates.
REEs was concentrated in the roots and only a small
amount had been transported to the above-ground parts.
After further culture in the solution without mixed REEs,
REE contents of the roots decreased from 55.662 lg to
50.519 lg, and the contents of the above-ground parts
increased from 0.591 lg to 3.404 lg. However, the total
REEs in the whole plant did not change (Fig. 2A). REE
contents in the leaves varied with leaf position at both
growth stages. A decrease in REE contents was observed
from the oldest to the youngest leaf. The three youngest
leaves at the eighth-leaf stage (the sixth to the eighth leaf),
which appeared after the cessation of mixed REEs treatment, contained extremely low REE contents. A decrease in
REE contents was also found from the base to the top of the
third leaf sampled at the fifth-leaf stage (Table 1). The total
contents of LREEs decreased about 50% from the base to
the top, compared with a slight decrease of HREEs.
As mentioned earlier, the plants were cultivated in a
5 lM mixed REE solution, which is composed of 14
lanthanides with identical concentrations of each element.
After intake of REEs into the wheat seedlings, REE patterns
should present flat lines if there were no fractionations.
However, significant fractionations of REEs occurred in
wheat (Figs 1B–D, 2B–D). The roots strongly enriched
middle REEs (MREEs, e.g. Sm, Eu, and Gd), and the stems
exhibited a slight enrichment of LREEs. Unlike the roots
and stems, a significant enrichment of HREEs was found in
the leaves. The enrichment of HREEs decreased upwards,
associated with the reduction of the total concentrations of
REEs (Fig. 3). An inverse feature was found within the
third leaf sampled at the fifth-leaf stage, where the total
concentrations of REEs slightly decreased from the base to
the top, but the HREE enrichment significantly increased
upwards (Table 1; Fig. 4).
Besides the radius-dependent fractionation of REEs in
wheat, the tetrad effect was also observed in the REE
patterns (Figs 1, 2). The size of the tetrad effect in different
organs was calculated according to equations (1) and (2).
The M-type and W-type tetrad-effect-like patterns appeared
in the roots and above-ground parts, respectively (Fig. 5).
After 24 h treatment with 50 lM mixed REEs, REE
pattern in the xylem sap was similar to that in the leaves,
with obvious HREE enrichment and W-type tetrad effect
(T=0.81) (Fig. 6). After 48 h exposure in 5 lM mixed REE
solution, the total concentration of REEs in the cell walls
of roots was 5.9260.63 lmol g1 DW; about 72% of the
total (8.1760.54 lmol g1 DW) was in the roots. A slight
enrichment of MREEs was observed in the cell walls, with
REEs in wheat
2769
Fig. 2. Contents and patterns of REEs in wheat at the eighth-leaf stage. (A) Contents of REEs in each organ of wheat (lg). (B) REE patterns in different
leaves. (C) REE patterns in the stems. (D) REE patterns in the roots. The seedlings after exposure to 5 lM mixed REE solution and nutrient solution on
alternate days for 6 d at the fifth-leaf stage were further grown in nutrient solution until three new leaves (sixth, seventh, and eighth) appeared. Values are
means6SD of three replicates.
Table 1. Accumulation and fractionation features of REEs in
different parts of the third leaf at the fifth-leaf stage
The seedlings at the fifth-leaf stage were exposed to 5 lM mixed REEs
solution and nutrient solution on alternate days for 6 d. At harvest, the
third leaf was separated into three equal parts, and named as ‘base’,
‘centre’ and ‘top’ from the bottom to the top. Values are means 6SD of
three replicates.
Features
Content (lg)
Concentration
(lmol g1 DW)
HREE/LREE
LREE
HREE
REE
REE
Base
Centre
Top
0.01560.000
0.02460.001
0.03960.002
0.08560.004
0.01260.000
0.02560.001
0.03760.003
0.08260.005
0.00160.000
0.02160.001
0.03060.002
0.06360.004
1.6060.07
2.0360.16
2.4660.25
a slight depletion of HREEs compared with the LREEs.
However, the M-type tetrad effect, originally present in
the roots (T=1.09), disappeared in the cell walls (T=0.98)
(Fig. 7).
After the reaction of REE ions with phosphate in solution
for 10 h at pH 3.7, significant depletions of MREEs, with
obvious W-type tetrad effects were observed in the dissolved REEs (Fig. 8A). When citrate was added to the
solution, the MREE depletions in the patterns gradually
changed to HREE enrichments (Fig. 8B), and the REE
patterns were finally close to those observed in wheat
leaves (Figs 1B, 2B, 4).
Discussion
Accumulation and mobility of REEs in wheat
The accumulation of metals from external solution into the
shoot (mainly leaves) can be separated into two processes:
a non-metabolic, passive movement from the external
solution to the roots and a transpiration-dependent translocation from the roots to the shoots via the xylem
(Marschner, 1995; Salt et al., 1995). Uptake and translocation may vary considerably depending on plant species
and metal types. For example, it is well known that Ca
concentrations are higher in old leaves than in young
2770 Ding et al.
Fig. 3. Variations of the total concentration of REEs (RREE) and
concentration ratio of HREE/LREE in the first to the fifth leaf at the fifthleaf stage (the curves formed by five data points) and the first to the eighth
leaf at the eighth-leaf stage (the curves formed by eight data points). The
seedlings at the fifth-leaf stage were exposed to 5 lM mixed REE
solution and nutrient solution on alternate days for 6 d. Half of them were
further grown in nutrient solution until three new leaves appeared. LREEs
refer to La-Eu. HREEs refer to Gd-Lu. Values are means 6SD of three
replicates.
Fig. 5. The tetrad effect (T) in different organs of wheat at the fifth-leaf
stage and eighth-leaf stage, respectively. The seedlings at the fifth-leaf
stage were exposed to 5 lM mixed REE solution and nutrient solution
on alternate days for 6 d. Half of them were further grown in nutrient
solution until three new leaves appeared. The T values were calculated
according to equations (1) and (2). Values are means 6SD of three
replicates.
Fig. 6. REE pattern in the xylem sap of wheat. 10-week-old seedlings
were exposed to 50 lM mixed REE solution for 24 h, the stem was then
severed 2 cm above the root and the xylem sap was collected for 8 h with
a micropipette. Values are means 6SD of three replicates.
Fig. 4. REE patterns in different parts of the third leaf sampled at the
fifth-leaf stage. The seedlings at the fifth-leaf stage were exposed to 5 lM
mixed REE solution and nutrient solution on alternate days for 6 d. The
third leaf was separated into three equal parts, as named as ‘base’, ‘centre’
and ‘top’ from the bottom to the top. Values are means 6SD of three
replicates.
leaves, and this is related to transpiration rates, phloem
mobility, age of leaves, and the low mobility of Ca (Behling
et al., 1989). An acropetal decrease in Al concentrations
was found in buckwheat (Fagopyrum esculentum Moench),
caused by the low mobility of Al (Shen and Ma, 2001).
Fig. 7. REE patterns in the roots and cell walls of the roots. 3-week-old
seedlings were exposed to 5 lM mixed REE solution for 48 h, the roots
were then sampled and parts of them were used for extraction of the cell
walls. Values are means 6SD of three replicates.
REEs in wheat
2771
Fig. 8. REE patterns in solution during a 10 h precipitation of phosphate with mixed REEs in the absence (A) and presence (B) of citrate. 200 ml of 0.01
M NaNO3 solution containing 50 lM mixed REEs was mixed with an equal volume of 0.01 M NaNO3 containing 200 lM NaH2PO4 or 200 lM
NaH2PO4+50 lM citrate. The initial solution pH was increased from 3.0 to 3.7 to increase the rate and extent of precipitation. The solutions were
sampled at 0, 1, 2, 4, and 10 h, respectively. REE patterns are depicted as Ct/C0, where C0 is the initially identical concentration of mixed REEs and Ct
is the later concentration in solution.
A similar distribution of REEs was found in wheat (Figs
1A, 2A). It means that the accumulation of REEs in wheat
is dominated by their mobility, and the acropetal decrease is
caused by the low mobility of REEs. This is mainly
attributed to the high valencies of REE ions, leading to
high affinity of REEs with plant cell walls, as the cell walls
contained 72% of the total REEs in the roots. X-ray
absorption spectroscopic techniques verified the binding of
La(III) in barley roots via carboxylate groups and hydration
of La(III) (Han et al., 2005). Phosphate precipitation of
REEs occurring in/on the roots or the transport path is
another important factor, which will be explained later.
Redistribution of an element from old to young organs
mainly takes place via the phloem (Marschner, 1995). The
redistribution of REEs in wheat showed that REE contents
were extremely low in the young leaves (Fig. 2A). This
means that it is probably hard for REEs to transport in
the phloem. The phloem consists of living cells containing
substances on which metal ions are easy to bind (Greger,
1999) and thus may prevent REE translocation in it. The
accumulation of REEs continued to increase in the older
leaves and stems (Fig. 2A), but it seems that they originated
from the REEs remaining in the roots due to the constant
total contents of REEs in wheat. It is deduced that the
acropetal transport via the xylem continues to play an important role in the accumulation of REEs due to the high
concentration ratio of root/leaf.
REE fractionation in the roots
Conspicuous fractionation of REEs was observed in the
roots, characterized by a relative MREE enrichment and
a slight M-type tetrad-effect-like feature (Figs 1D, 2D).
Laboratory experiments verified that the precipitation of
phosphates in the solution system caused the formation of
MREE-enriched precipitates, leading to the relative depletions of MREEs and the W-type tetrad effects in the solution (Fig. 8A). Similar results were reported by Byrne et al.
(1996). When the REE patterns in the solution (Fig. 8A)
were compared with those in the roots (Figs 1D, 2D),
inverse fractionation features were observed between
them. Based on the fact that, at pH >4.0, phosphate precipitation of REEs can easily take place (Diatloff et al.,
1993), it is suggested that the fractionation features of REEs
observed in the roots of wheat are partly caused by REE
phosphate (LnPO4) precipitation in the roots or on the root
surfaces. Ishikawa et al. (1996) reported that the localization of La3+ and Yb3+ in root-tip portions of rice and pea
treated with La3+ and Yb3+ was fully consistent with that of
phosphate and the phosphate concentration increased both
in the apoplast and symplast, suggesting that the distribution of these ions might result from the precipitation of the
metal ions with the phosphate ions released in the apoplast.
Quiquampoix et al. (1990) confirmed the presence of deposits containing Gd and phosphorus in the extracellular
space of root tissue of maize using electron microscopy and
X-ray microanalysis. It is concluded that phosphate precipitation is an important factor to control the accumulation
and especially the fractionation of REEs in wheat roots.
Selective absorption to cell walls may also play a certain
role on REE fractionation in the roots, as the REE pattern in
the cell walls exhibited slight MREE enrichment (Fig. 7).
However, no tetrad effect was observed in the pattern of cell
walls. It means that the tetrad-effect fractionation of REEs
in the roots result exclusively from the precipitation of
phosphate. Release of REE ions originally absorbed to the
cell walls might occur during the extraction process of
the cell walls. Due to the decrease of ionic potential of the
2772 Ding et al.
dehydrated REE ions from La to Lu, heavier REEs can
form stronger covalent bonds with protoplasmic substances
(Ishikawa et al., 1996), and the release of these elements
might be more serious. It may explain the slight MREE
enrichment and the depletion of HREEs in the cell walls
compared with those in the roots (Fig. 7). In fact, a more
significant enrichment of MREEs was observed when cell
walls were extracted from the roots of wheat grown in an
REE-free medium followed by short-term absorption in
mixed REE solution (data not shown). The mechanisms
involved in this remain unclear.
Tetrad effect in the above-ground parts
The existence of the W-type tetrad effect in the aboveground parts of wheat suggests that REEs must be in
a dissolved state before they are transported to the aboveground parts, as the W-type tetrad effect exists in fluids and
the M-type tetrad effect is observed in the corresponding
solid materials that react with these fluids (Masuda et al.,
1987). The roots, as mentioned above, showed the M-type
tetrad effect derived from precipitation of phosphates with
REEs (Figs 1D, 2D). As a result, REE patterns in the root
solution should exhibit the W-type tetrad effect and REE
patterns in the above-ground parts should also show the
same feature, as the dissolved REEs in the roots are
exclusively the source of REEs in the above-ground parts.
This hypothesis is consistent with the results in this study
(Figs 1, 2). REE pattern in the xylem sap also showed a
W-type tetrad effect (Fig. 7).
Origin of HREE-enrichment in the leaves
As mentioned above, the roots exhibited a MREEenrichment feature due to the phosphate precipitation and
cell wall absorption (Figs 1D, 2D), which should result in
a relative MREE-depletion feature in REE patterns in the
xylem solution, and further lead to the same feature in the
above-ground parts after the transfer of REEs into these
organs. However, the results observed in this study are far
from being the case (Figs 1B, C, 2B, C). Obviously there
are other processes different from phosphate precipitation
and cell wall absorption causing the changes.
Difference of speciation among individual REEs in the
xylem solution might be an important factor to cause the
REEs to fractionate from each other. Therefore, REE
speciation in simulated xylem solution was calculated initially using an ionic speciation model. The results showed
that EDTA complexes were the major species for the dissolved REEs and HREE enrichment existed in the complexes (data not shown). Based on the simulation, it was
deduced that the enrichment of HREEs in wheat leaves was
caused by EDTA complexation, where more HREEs
were complexed by EDTA due to the increase of stability
constants with the increase of atomic number across
lanthanides (Byrne and Li, 1995), leading to more HREEs
moving to the above-ground parts and less was adsorbed to
cell walls or precipitated by phosphate. In order to verify
the hypothesis, an additional experiment was carried out.
The wheat was grown in an EDTA- and REE-free nutrient
solution followed by exposure to mixed REE solutions
containing different levels of EDTA. The results showed
that REE accumulation and HREE enrichment in wheat
leaves generally decreased with the increase of EDTA
levels (data not shown). Other inorganic ligands (e.g. SO2
4
data not shown) and organic ligands (e.g. citrate; Ding
et al., 2005) have similar effects. Like EDTA, stability
constants of these ligands with REEs increase with the
increase of atomic number across the lanthanides. However, when acetic acid was used as an exogenous ligand, of
which the stability constants generally remain constant
among individual REEs, the HREE enrichment in wheat
leaves remained unchanged (data not shown). The results
of the above studies indicated REEs should be absorbed
by wheat roots mostly in the form of free ions, and the
enrichment of HREEs in wheat leaves do not originate from
the complexation of exogenous ligands such as EDTA.
This is consistent with researches on other metal ions. It is
general accepted that the total dissolved metal concentration of a metal is not a good predictor of its bioavailability
for terrestrial higher plants. Accumulation and/or toxicity
of dissolved metals correlates best with activities of free,
uncomplexed metal ions in solution (Campbell, 1995;
Parker et al., 1995, 2001), although a few exceptions to the
findings have been reported (White, 2001; Parker et al.,
2001).
Exudates from plant transport tissues contain many
natural complexing compounds such as small (Cl, carboxylic acids, amino acids) and macromolecular substances
(proteins, DNA, polysaccharides) (White et al., 1981a).
Most transition metal ions and some main group metal
ions form stable complexes with these ligands present in the
xylem of plants, and transport, at least to some extent,
in complexed form (Lobinski and Potin-Gautier, 1998;
Rauser, 1999). There is also in vitro electrophoretic
evidence for stable metal complex formation and quantitative estimates of the equilibrium distribution of metals,
ligands, metal complexes, and other solutes in the xylem
fluid (White et al., 1981b, c; Mullins et al., 1986). Due to
the high ionic potential of the dehydrated REE ions, the
REEs in the xylem vessels should mainly be translocated in
complexed forms. Such complexations occurring in xylem
fluid can prevent the phosphate precipitation, as well as cell
wall absorption, of the REEs in solution and, moreover,
lead to the formation of a HREE-enriched pattern in solution because of the increase of stability constants of REE
complexes with the increase of atomic number of REEs for
the most ligands (e.g. citrate, as shown in Fig. 8B) (Wood,
1990; Millero, 1992; Byrne and Li, 1995). As a result,
the HREEs become more mobile than the LREEs and
cause the HREEs to be enriched in wheat leaves relative to
REEs in wheat
the LREEs. The HREE enrichment in leaves, therefore, is
assumed to be the result of the combined effects of solution
complexation of ligands produced by plants, cell wall
adsorption, and phosphate precipitation. The REE complexes have been separated from the xylem solution using
a nanometer-sized titanium dioxide (Li et al., 2004), and it
is suggested that REEs are associated with organic substances, with an HREE-enriched pattern in the complexes
(data not shown).
Unceasing absorption to plant cell walls and precipitation by phosphate along the transport path can explain the
acropetal decrease of REE content within a single leaf
(Table 1) and among individual leaves (Figs 1A, 2A).
Then, a question arises as to why the HREE enrichment
increased from the base to the top of a single leaf (Fig. 4),
but decreased from newer leaves to older leaves (Figs 1B,
2B). As discussed before, the REEs translocate to aboveground parts of wheat mainly in the form of complexes, and
certainly the complexed REEs are more stable and more
mobile than the free ions of REEs. That is to say, the
HREEs would be preferentially transported to the end of
the transpiration stream (i.e. top of a single leaf) and to the
older leaves via the transpiration stream since more HREEs
translocate in complexed forms compared with the LREEs.
Therefore, a ligand- and transpiration-mediated transfer of
REEs in the xylem may account for the above phenomena.
Slight enrichment of LREEs in the stems
The slight enrichment of LREEs in the stems (Figs 1B, 2B)
possibly suggests that there is strong complexing environment present in the transport path before the transfer of
REEs into the leaves. It is enough for the environment to
keep the HREEs from chemical precipitation or absorption
to cell walls in the stems while it is less effective on the
LREEs. Fu et al. (2001) pointed out that the degree of
fractionation between the LREEs and HREEs might be
a reflection of the amount of ligands in the xylem solution.
Implications of this study
It is suggested that soil-normalized REE fractionation
patterns might be controlled via a complexation of REEs
by root exudates in the rhizosphere (Wyttenbach et al.,
1998). This is consistent with the results of this study that
solution complexation is a major factor affecting REE
accumulation and fractionation in plants. However, exogenous and intrinsic ligands may play different (i.e. nearly
opposite) roles on them. The fact that high concentrations
of low-molecular organic ligands, such as citrate, malate,
and oxalate, in rhizosphere solutions (Jones, 1998) may be
the reason why LREE enrichment is widely observed in
plants when normalized to the host soils (Wyttenbach et al.,
1998; Fu et al., 1998, 2001; Wei et al., 2001), while strong
complexations of intrinsic chelators in the xylem may be
responsible for the HREE enrichment (Ding et al., 2003).
2773
It is well known that absorption onto negatively-charged
sites in cell walls is the major reason for the root-concentrated
feature of heavy metals in plants (Hardiman and Jacoby,
1984; Marschner, 1995), and it is also the same for REEs.
However, as adsorption onto the cell walls is of limited
capacity and has a limited effect on heavy metal activity
(Hall, 2002), phosphate precipitation might be another
important factor to influence REE accumulation and fractionation in the bioaccumulation processes due to extremely
low KspLnPO4 [e.g. K0sp ðGdPO4 Þ ffi 1024 ] (Byrne et al.,
1996). Under natural conditions, concentrations of inorganic
phosphates in soil solutions are extremely low (0.6–5 lM)
(Baber, 1984), and they are close to that of the total
concentrations of REEs in soil solutions (average of 9 lM
in 33 types of soils in China) (Xing and Xu, 2003).
However, phosphates in plants and xylem solutions can
be raised by several hundred times via active absorption
by plants (Hocking, 1980; Marschner, 1995), which would
largely facilitate precipitation of REEs in plants, and further
contribute to the acropetal decrease of REE accumulation
and the occurrence of the tetrad effect (Fu et al., 1998;
Fu and Tasuku, 2000). The results in this study give
valuable information on exploring the mechanisms of
REE accumulation and fractionation in bioaccumulation
processes.
Acknowledgement
This work was sponsored by the Key Project of National Natural
Science Foundation of China (No. 40232022).
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