Planta
DOI 10.1007/s00425-006-0433-7

O R I G I N A L A RT I C L E

Organic acids enhance the uptake of lead by wheat roots
Huanhua Wang Æ Xiaoquan Shan Æ Tao Liu Æ
Yaning Xie Æ Bei Wen Æ Shuzhen Zhang Æ Fang Han Æ
Martinus Th. van Genuchten

Received: 25 July 2006 / Accepted: 23 October 2006
 Springer-Verlag 2006

Abstract The uptake and bioavailability of lead (Pb)in soil–plant systems remain poorly understood. This
study indicates that acetic and malic acids enhance
the uptake of Pb by wheat (Triticum aestivum L.)
roots under hydroponic conditions. The net concentration-dependent uptake influx of Pb in the presence
and absence of organic acids was characterized byMichaelis–Menten type nonsaturating kinetic curves
that could be resolved into linear and saturable
components. Fitted maximum uptake rates (Vmax) of
the Michaelis–Menton saturable component in thepresence of acetic and malic acids were, respectively,
2.45 and 1.63 times those of the control, while theMichaelis–Menten Km values of 5.5, 3.7 and 2.2 lM,
respectively, remained unchanged. Enhanced Pb
uptake by organic acids was partially mediated by
Ca2+ and K+ channels, and also depended upon thephysiological function of the plasma membrane
P-type ATPase. Uptake may have been further
enhanced by an effectively thinner unstirred layer of

H. Wang  X. Shan (&)  B. Wen  S. Zhang 
F. HanState Key Laboratory of Environmental Chemistry and
Ecotoxicology, Research Center for Eco-Environmental
Sciences, Chinese Academy of Sciences,
P. O. Box 2871, Beijing 100085, Chinae-mail: xiaoquan@rcees.ac.cn
T. Liu  Y. Xie
Beijing Synchrotron Radiation Laboratory,
Institute of High Energy Physics,
Chinese Academy of Sciences, Beijing 100049, China
M. Th.van Genuchten
U.S. Salinity Laboratory, USDA-ARS,
450 West Big Springs Road, Riverside, CA 92501, USA

Pb adjacent to the roots, leading to more rapid
diffusion towards roots. X-ray absorption spectroscopic studies provided evidence that the coordination
environment of Pb in wheat roots was similar to thatof Pb(CH3COO)23H2O in that one Pb atom wascoordinated to four oxygen atoms via the carboxylategroup.
Keywords Ca2+ and K+ channels  Organic acids P-type ATPase  Uptake of lead (Pb)  Wheat(Triticum aestivum L.)  X-ray absorption spectroscopy

Introduction
Being one of the most frequently encountered metal
contaminants in many agricultural and industrial areas,
the environmental fate of Pb and its uptake by plants
has been the subject of much research (Manceau et al.
1996; Wu et al. 1999). Because of its limited solubility
due to the formation of insoluble precipitates, bioavailable levels of Pb in soils are generally low compared to other heavy metals, thus making it difficult toapply phytoremediation techniques to Pb-contaminated soils. Recent studies demonstrated that theaddition of synthetic chelates such as EDTA, HEDTA,
and DTPA to contaminated soils can rapidly and dramatically increase Pb release into the soil solution, thus
promoting Pb uptake by plants (Blaylock et al. 1997;
Vassil et al. 1998).
Organic acids are important plant root exudates andmicrobial metabolites in terms of their ability to increase the dissolution of metals from highly insoluble
mineral phases in soil, thereby increasing metal
mobility in the vicinity of roots and enhancing their123

Plantaavailability to plants (Naidu and Harter 1998; LópezBucio et al. 2000; Clemens et al. 2002). Several studies
have shown the important role that organic acids playat the soil–plant interface. For example, organic acidsin pot experiments triggered uranium accumulation inIndian mustard and Chinese cabbage (Huang et al.
1998), and increased chromium accumulation in tomato (Srivastava et al. 1999). Under hydroponic conditions, organic acids furthermore increased the uptakeof cadmium by tomato and durum wheat (Senden et al.
1995; Berkelaar and Hale 2003), and of lanthanum bywheat and barley (Wang et al. 2004; Han et al. 2005).
Organic acids play an important physiological rolein conferring metal tolerance in that a number of
mechanisms involving metal translocation or accumulation are influenced by organic acids. For example,
several studies have shown the key role of organic
acids in affecting plant tolerance to aluminum (Pellet
et al. 1995; Ma et al. 2001). The role of organic acids inmediating the uptake of Fe3+ was ascribed to ferric
reductase activity and the presence of cation channels(Schmidt 1999). Compared to other metals, limited
information exists on the interactions between organic
acids and Pb at or near the soil-root interface, and theresultant processes of Pb uptake by plant roots.
Laurie et al. (1991) discussed two possible pathways
for metal uptake under the influence of complexes.
One pathway involves the dissociation of a metal
complex (MeL) in the diffusion layer (solution phase)after which the released free metal ion (Me) may betransported to the root cell across the plasmalemma.
Another possible pathway involves the absorption of
the metal complex by the root cell membrane; thecomplex then either undergoes dissociation in the cell
membrane, with the free metal ion being transported
to the cell while the ligand (L) goes back to the solution phase, or the metal is transported to root cells
across the plasmalemma in the form of a complex.
Chloride was found to increase Cd uptake by chard
from a resin-buffered solution culture in which activities of Cd2+ were held constant (Smolders andMcLaughlin 1996). They suggested that either CdCl2–n2
species were transported across the plasma membrane,or that Cl1– enhanced the diffusion of Cd2+ through theunstirred liquid layer adjacent to the root surface or
through the apoplast to sites of Cd uptake within theroot itself. However, solution culture studies in thepresence and absence of EDTA indicate that CdEDTA may not be available for plant uptake (Checkai
et al. 1987). Berkelaar and Hale (2003) showed that thefree ion model (FIM) alone was likely insufficient topredict plant accumulation of metals. Hence, little
agreement exists on whether free metal ions or organic

123

ligand-complexes can be more easily taken up by
plants.
Recent biophysical studies on the interactions between lead and recombinant proteins and peptides
have provided considerable insight into the biologicalchemistry and molecular toxicology of lead (Godwin
2001). Because lead is non-essential and toxic to plants,
it is generally thought that plants are unlikely to havetransporters specific for lead. The first example of aplant transporter possibly mediating Pb2+ uptake has
been described by Arazi et al. (1999). They revealed aplasma membrane protein NtCBP4 that conferred Pb2+
hypersensitivity and correlated this with enhanced Pb2+
accumulation. Lead and calcium compete for the same
binding sites on proteins that belong to a large familyof ion binding compounds. Members of this family
include calmodulin, S-100, calretinin, calbindin, andparvalbumin. Lead substitutes for calcium in the activation of calmodulin-dependent phosphodiesterase.
CadA and ZntA have been discovered as two Pb(II)translocating P-type ATPase (Rensing et al. 1998). Stilllargely unanswered in the question how lead is taken
up into plant cells under the influence of organic acids,
and what the exact speciation of lead is within plant
cells.
The objective of this study was to gain insight into
the effects of organic acids on the uptake of Pb by
wheat roots under hydroponic conditions. Relevantmechanisms were investigated by using time- andconcentration-dependent Pb uptake experiments, andvarious cation channel blockers and the H+-ATPase
inhibitor vanadate. Speciation calculations of Pb in theabsence and presence of organic acids and diffuse
double layer model were further used to verify theenhanced diffusion of Pb in the nutrient solution towards wheat roots. We additionally employed such
structural sensitive characterization techniques as
X-ray absorption near edge spectroscopy (XANES) andextended X-ray absorption fine structure (EXAFS) to
investigate the coordination environment of Pb in
wheat roots.

Materials and methods
Plant cultivation
Wheat (Triticum aestivum L. cv. JM9158) seeds wereobtained from the Chinese Academy of Agricultural
Sciences (Beijing, China). Seeds were first surfacesterilized in 3% (v/v) H2O2 for 30 min, and thenthoroughly rinsed with deionized water. After soaking
the seeds in 2.8 mM Ca(NO3)2 for 4 h, they were al-Plantalowed to germinate on moisture filter paper for 4 daysin the dark and subsequently transferred to hydroponicsolutions containing major nutrients [one-thirdstrength of 1.5 mM KNO3, 1 mM Ca(NO3)2, 0.5 mM
MgSO4, 0.25 mM (NH4)H2PO4] and micronutrients[full-strength of 11.9 lM ethylenediaminetetraacetic
acid–iron (EDTA–Fe), 11.5 lM H3BO3, 1.25 lM
MnSO4, 0.2 lM ZnSO4, 0.075 lM CuSO4 and0.025 lM (NH4)6Mo7O24]. The solutions were buffered
to pH 5.8 with 2 mM Mes–Tris. Plants were grown in aplant growth chamber subject to continuous aeration
and with day and night temperature of 22C (16 h) and15C (8 h), respectively. A relative humidity between
50 and 60% and a photon flux intensity of 300 lmol m–
2 –1
s were maintained during the entire experimental
period. Fresh nutrient solutions were renewed every
3 days. The Pb treatment solutions contained the same
nutrients, but without NH4H2PO4 and EDTA–Fe,
while MgSO4 was applied at one-tenth strength in order to avoid precipitation.
Effect of molar concentration ratio of organic acids
to Pb on Pb accumulation
Uniform 22-day-old wheat seedlings were removed
from the nutrient solutions, rinsed with deionized water for 2 min, and placed in groups of four in themodified uptake solutions. As control we used an uptake solution containing 20 lM Pb as Pb(NO3)2 without the organic acids. Six different molar concentration
ratios (2:1, 5:1, 10:1, 20:1, 50:1, 100:1) of acetic or malic
acid to Pb were employed. Acetic and malic acids werechosen as representative of organic acids since they areubiquitous in many plant exudates (Cieśliński et al.
1998; Jones 1998). After exposure to amended solutions for 4 h, the seedlings were removed and thoroughly rinsed with deionized water and desorbed. The
desorbed roots were next gently blotted, weighed anddigested for final determination of Pb. The desorption
procedures were conducted for 15 min in ice-cold
5 mM CaCl2 solutions containing 5 mM Mes–Tris (pH6.0) to remove apoplastic Pb (Hart et al. 1998). Theseand all subsequent experiments with wheat seedlings
were performed in quadruplicate.
Short-term uptake and long-term accumulationof Pb as affected by organic acids
To study the time-dependency of Pb uptake, uniform
22-day-old wheat seedlings were transferred from thehydroponic solutions to uptake solutions containing
20 lM Pb with or without 200 lM of either acetic ormalic acids. The wheat seedlings were analyzed for Pb
content after uptake time periods of 5, 10, 20, 40, 60, 90
and 120 min, and 2, 4, 8, 12, 24 and 48 h. For the shortterm (0–2 h) uptake experiments, subsamples of theroots were collected followed by desorption for 15 minin 5 mM ice-cold CaCl2 (5 mM Mes–Tris, pH 6.0), or
without desorption prior to Pb determination. The
incubation solutions for the long-term (>12 h) accumulation experiments were replaced every 12 h. All
wheat roots were desorbed and digested for Pb determination.
Concentration-dependent kinetics of Pb uptake
as affected by organic acids
Three parallel experiments were performed simultaneously to test the effect of external Pb concentrations
on Pb uptake. Uniform 22-day-old wheat seedlings
were for this purpose incubated with fresh nutrient
solutions containing various concentrations of Pb (0, 5,
10, 20, 40, 60, 80, 100 lM). Acetic and malic acids wereadded separately in two of the experiments at molar
concentration ratio of 10:1 (organic acid:Pb). Afterincubation for 20 min, the seedlings were harvested
and the roots desorbed. Pb concentrations of the wheatroots were subsequently determined following aciddigestion.
Membrane permeability
Wheat seedlings were incubated in different treatment
solutions for 1–2 days. During this period the incubation solution was replaced every 12 h with a fresh
solution. Treatment solutions (including the control
nutrient and uptake solutions) were amended with
20 lM Pb with or without 200 lM acetic acid. At theend of the experiments, roots were thoroughly washedtwice with 5 mM ice-cold CaCl2 (5 mM Mes–Tris, pH
6.0) to remove nutrient solution from the apoplast andthen blotted on filter paper. Each sample consisting offour seedlings was subsequently introduced into a
25 ml of fresh 0.2 mM CaCl2 solution and incubatedfor 4 h in a water bath at 25C with gentle shaking(Andreu et al. 2000). Aliquots of this solution weretaken at 1 h intervals and the K+ efflux to the external
solution was measured using inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Optima
2000DV, Perkin Elmer Co., Wellesley, MA, USA).
Effects of cation channel blockers on Pb influx
To better understand the transport pathway of theincreased Pb influx, we next examined the physiologi-

123Planta

cal functions of the Ca2+ channel inhibitor of La3+ and
the K+ channel inhibitor of Cs+. The experiments werecarried out again with uniform 22-day-old wheat
seedlings, now incubated in nutrient solutions amended with 0.2 mM La3+ or Cs+ as La(NO3)3 or CsCl,
respectively. An unamended nutrient solution wasused as the control. La(NO3)3 and CsCl have previously been identified as cation channel blockers by
White (1997) and by Essah et al. (2003), who also
reported appropriate application concentrations. The
Pb concentrations in our experiments were maintained
at 20 lM. Acetic and malic acids were applied at molar
concentration ratio of 10:1 (organic acid:Pb). The
wheat seedlings were harvested after 4 h of incubation.
Roots were desorbed and acid digested prior to final
determination of Pb. As before, all experiments were
performed in quadruplicate.
Influence of vanadate on Pb uptake
The effect of vanadate on Pb uptake was analyzed by
pre-treating 22-day-old wheat seedlings with 0.1 mM
vanadate for 15 min prior to exposure to 20 lM Pb
solution with or without the organic acids. As before,
the molar concentration ratio of organic acids to
Pb was again maintained at 10:1. After 4 h of incubation, wheat roots were harvested, desorbed and aciddigested prior to Pb determination.
Speciation calculations
The chemical speciation of Pb in the incubation solutions in the presence and absence of organic acids waspredicted using the computer program Visual MINTEQ, version 2.50 (Gustafsson 2006). Full consideration was given in the calculations to mole balances,
relevant thermodynamic equilibrium constants andionic strengths.

of 0.15 cm) for the XAS experiments. Instrumental
conditions for measurement of the XAS spectra at
the Pb L3-edge are described in detail elsewhere (Qinet al. 2006).
Data processing and analyses were performed
with WinXAS 2.1 following standard procedures. Theenergy E0 (13,035 eV) was defined as the inflection
point of the absorption edge for the transform fromenergy to k space. The pre-edge absorption background
was fitted and subtracted by using Victoreen functions.
The post-edge absorption backgrounds were fitted witha spline function and subtracted from the absorption
spectra. The EXAFS functions were normalized using
the absorption edge jump, and next Fourier transformed into R-space with k2-weighting over the range
from 2.2 to 10.0 Å. We chose k = 2 weighting for Pb
since a higher k value decreased the signal-to-noise
ratio. The fits were performed in R-space with thecoordination number (N) fixed, while the interatomic
distance (R), the energy shift (E0), and the Debye–
Waller factor (r2) were allowed to float. The amplitude and phase shift parameters were obtained fromtheoretical calculations with Pb(CH3COO)23H2O asreference using the FEFF 8.0 code (Rehr et al. 1992).
Determination of Pb
The Pb contents of the various plant samples wereanalyzed by digesting them with 3-ml concentrated
HNO3–HClO4 (2:1, v:v) solutions under high pressure
conditions (Zhang and Shan 1997). Pb concentrations in the samples were determined using ICP-AES(Optima 2000 DV, Perkin Elmer Co.). Certified
reference materials (peach leaves, GBW 08501, PR
China) were used to ensure the quality of analyses.
Good agreement was obtained between the dataobtained with our method and the certified values.
Statistical analysis of data

X-ray absorption spectroscopic studies
We additionally performed X-ray absorption spectroscopic (XAS) studies to obtain direct information on
the coordination chemistry of Pb in the 22-day-old
wheat seedlings, which had been transferred into
20 lM Pb culture solutions containing 200 lM acetic
acid. X-ray absorption spectroscopy is an elementspecific and bulk-sensitive method. The seedlings forthis study were harvested after 2 days of uptake. The
fresh wheat roots (desorbed and undesorbed) wereground in an agate mortar and pestle under the protection of liquid N2. The fine root powders were nextpressed into pellets (diameter of 0.84 cm and thickness

123

The data were analyzed using the Microcal Origin 7.0(Microcal Software, Northampton, MA, USA) andSPSS 12.0 for windows (SPSS, Chicago, IL, USA)software packages.

Results
Effect of molar concentration ratio of organic acids
to Pb on the accumulation of Pb
Figure 1 shows the effects of acetic and malic acid on
the accumulation of Pb by the wheat roots as a function

1.5

1.6
1.2
0.8

Pb in roots-1(µ mol g FW)

Pb in roots (µmol g-1 FW)

Planta

0.4

a

1.2
0.9
0.6
0.3
0.0

0.0

0

20

40 60 80 100 120
Exposure time (min)20

40 60 80 100 120
Exposure time (min)

0
20
40
60
80 100Concentration ratio of organic acids to Pb
1.0of the molar concentration ratio of organic acid to Pb.
While acetic and malic acids qualitatively showedsimilar trends in terms of enhancing Pb accumulation,acetic acid had a much greater effect than malic acid.
The maximum concentrations of Pb in the roots occurred at a concentration ratio of 10:1. The Pb content
of the desorbed wheat roots at that ratio, after 4 h of
exposure, increased by 116 and 75% for acetic andmalic acid, respectively, relative to the control(P < 0.05). The Pb contents of the roots decreased witha further increase in the organic acid to Pb ratio. For
this reason we used a molar concentration ratio of 10:1
(organic acid:Pb) in most or all subsequent experiments.
Short-term uptake and long-term accumulationof Pb as affected by organic acids
Compared to seedlings grown in the control solution,
Pb concentrations of wheat roots grown in nutrientssolutions with the organic acids were found to beconsistently greater for both the short- and long-term
experiments. Figure 2a, b show the short-term (0–120 min) Pb uptake by wheat roots without and with
CaCl2 desorption, respectively. The uptake patterns
were similar for the control and the organic acidtreatment solutions. Acetic and malic acid bothnoticeably enhanced the short-term uptake of Pb. For
wheat roots without CaCl2 desorption, Pb uptake wasbiphasic in that a relatively fast initial phase was followed by a much slower and more linear second phase(Fig. 2a). The initial rapid phase (i.e., during the first
20 min) was presumably due to diffusion of Pb into
free space and binding to root cell walls. The slowerb

Pb in roots-1(µ mol g FW)

0.8
0.6
0.4
0.2
0.0

0

5

c

4
Pb in roots-1(µ mol g FW)

Fig. 1 Effect of molar concentration ratio (organic acids:Pb) for
acetic acid (filled square) and malic acid (filled circle) on the
uptake of Pb by wheat roots (Triticum aestivum L.). Values aremeans ± 1 SE of four replicates. The data at a concentration
ratio of 0 are for the controls (i.e., for nutrient solutions
containing Pb but no organic acids)

3
2
1
0

0

10

20
30
40
Exposure time (h)50Fig. 2 Time-dependent uptake of Pb by wheat roots (Triticum
aestivum L.) from solutions containing Pb but no organic acids(filled triangle), Pb and malic acid (filled circle), and Pb andacetic acid (filled square). Acetic and malic acids were applied ata molar concentration ratio of 10:1 (organic acid:Pb). Values aremeans ± 1 SE of four replicates. Results are for the short-time(£2 h) uptake experiments without a and with b desorption, andfor the long-time (up to 48 h) accumulation experiments withdesorption (c)linear phase of Pb uptake between 20 and 120 min may
correspond to transport across the plasma membrane.
The second phase could also be due to slower diffusion
into the apoplast and external binding to cells in thecortex.
Our results for the three uptake experiments with
CaCl2 desorption resemble previous findings by Lasatet al. (1996). The linear nature of Pb uptake by de-123PlantaConcentration-dependent Pb uptake as affectedby acetic and malic acids
Data from the relatively short uptake period (<20 min)were used to investigate uptake while minimizing thepossibility of Pb efflux across the plasma membraneback into the external solution. Figure 3a shows thatthe concentration-dependent Pb uptake data for thewheat seedlings are characterized by smooth, nonsaturating curves that become linear at Pb concentration
greater than about 20 lM. The organic acids increased
Pb uptake over the entire range of applied Pb in the
uptake solutions. The uptake kinetics of Pb in thepresence and absence of acetic and malic acids were
biphasic and could be described well with a modified
Michaelis–Menten type equation involving linear andsaturable components as follows (see also Fig. 3b):V ¼ aC þ

Vmax C;Km þ Cð1Þ

where V is the uptake rate, C is the Pb concentration in
the nutrient solution, a is parameter characterizing thelinear part of the uptake rate, and Vmax and Km are themaximum uptake rate and Michaelis–Menten rate
constant of the saturable (second) component of (1).
The r2 values for the acetic and malic acid treatments
and for the control were 0.993, 0.996 and 0.998,
respectively. The linear component presumably represents cell-wall-bound Pb remaining after desorption,

123a

Pb influx in roots(µmol g-1 FW h-1)

0.5
0.4
0.3
0.2
0.1
0.0

0

0.3
Pb influx in roots(µmol g-1 FW h-1)

sorbed wheat roots suggests that unidirectional Pb influx into the root symplast occurred for at least 120 min(Fig. 2b). The curves intercepted the axis slightly
above the origin, thus indicating the presence of relatively small amounts of rapidly bound Pb that were notremoved during the 15-min desorption process. This
undesorbed fraction probably consisted of Pb bound toreactive sites within the apoplast. After 2 h of exposure, Pb concentrations in the desorbed wheat roots of
the acetic and malic acid treatments were, respectively,
2.1 and 1.5 times greater than that of the control(P < 0.05).
Figure 2c shows the long-term Pb accumulation by
wheat roots. After about 4 h of rapid uptake, Pb continued to be accumulated for at least 48 h, but at a
slower rate. The accumulation did not reach a final
value after 48 h since the wheat seedlings were still
growing. Compared with Pb accumulation from thecontrol solution, the Pb content of desorbed wheatroots after 48 h of exposure was increased by 93 and
40% for acetic and malic acid, respectively (Fig. 2c,
P < 0.05).

20

40
60
80
Pb in solution (µM)

100

20

40
60
80
Pb in solution (µM)

100

b

0.2

0.1

0.0

0

Fig. 3 Concentration-dependent uptake kinetics of Pb by wheatroots (Triticum aestivum L.) from solutions containing Pb but noorganic acids (filled triangle), Pb and malic acid (filled circle), andPb and acetic acid (filled square). Acetic and malic acids wereapplied at a molar concentration ratio of 10:1 (organic acid:Pb). a
Measured overall kinetic curves for Pb uptake influx; values aremeans ± 1 SE of four replicates; b Deconvolution of the overall
kinetic curves into linear and saturable components according to
Eq. 1. The uptake curves are for Pb (dotted line), Pb and malic
acid (solid line), and Pb and acetic acid (dashed line)

while the saturable component represents true uptake
across the plasma membrane. Values of Vmax for theacetic and malic acid treatments were found to be 179.2
and 120.5 nmol g–1 fw h–1, respectively, which were2.45 and 1.63 times that of the control, respectively.
The corresponding Km values were within the sameorder of magnitude (5.5, 3.7 and 2.2 lM, respectively).
These results indicate that acetic and malic acids significantly enhanced Pb influx into the root symplasm.
Membrane integrity
In order to test whether organic acids at high concentrations and Pb exert toxic effect on the membrane
integrity of wheat roots, the membrane integrity was
examined by measuring the K+ efflux to the external
solution. Results indicate that the K+ concentration of
the external solution remained almost the same when
the plants were exposed to the nutrient solutions alone,PlantaFigure 5 shows the effects of the Ca2+ channel inhibitorof La3+, the K+ channel inhibitor of Cs+, as well as
vanadate on Pb uptake. Results indicate that La3+ andCs+ significantly inhibited the Pb influx (P < 0.05). If
we designate the Pb contents of wheat roots with noorganic acid, and with acetic and malic acids, as thecontrol values, La3+ decreased the Pb contents of thewheat roots by 74, 72 and 71%, respectively. Analogously, Cs+ decreased the Pb contents by 62, 59 and57%, respectively. Pre-treatment of wheat roots byvanadate, a H+-ATPase inhibitor, for 15 min, caused
the Pb contents of wheat roots to decrease by 55, 37,
and 51%, respectively (Fig. 5, P < 0.05).
We note here that, except for the ion channels(White 1997), two other possible mechanisms involvedin the passage of ions through the plasma membranesof cortical and xylem parenchyma are ion pumping andthe presence of carriers.

1.6

NN+acetic acid
N+Pb
N+Pb+acetic acid

0.8
0.4
0.0

1d

Vanadate

0.9
0.6
0.3

+

+
Pb

Pb

m
al
ic
ac
id

ac
et
ic
ac
id

0.0

Fig. 5 Effects of cation channel inhibitors and vanadate on theinflux of Pb in wheat (Triticum aestivum L.) roots as influenced
by organic acids. Values are means ± 1 SE of four replicatesSpeciation of Pb in the absence and presenceof organic acids
The speciation of Pb in the absence and presence ofacetic and malic acids as computed with the Visual
MINTEQ program is shown in Table 1. The results
indicate that free Pb2+ dominated the nutrient anduptake solutions, and that organic acids decreased thefree Pb2+ concentration as compared to the control.
Malic acid decreased free Pb2+ concentration morethan acetic acid. Moreover, as the molar concentration
ratio of organic acids to Pb increased from 2:1 to 100:1,
the percentage of free Pb2+ decreased from 94.6 to61.7% for acetic acid and from 91.5 to 31.7% for malic
acid. The Pb-acetate and Pb-malate contents were verylow (only 5.19 and 18.5%, respectively) when the molar
concentration ratio of acetic and malic acids to Pb wasset at 10:1. However, the Pb-acetate and Pb-malate
contents increased when the ratio of organic acids to
Pb increased from 2:1 to 100:1.
Effect of acetic and malic acids on the thicknessof unstirred layer of Pb adjacent to roots

1.2

+

-1

K in solution (µmol g FW)

2.0

1.2

Control
CsCl
La(NO3)3Pb

Effects of cation channels blockers and vanadateon Pb uptake rate

1.5

Pb in roots (µmol g-1 FW)or to the nutrient solutions with 200 lM acetic acid orwith 20 lM Pb or with 200 lM acetic acid and 20 lM
Pb together, over culture time intervals of 1–2 days on
the base of fresh wheat root weight (Fig. 4). There
were no significant differences between the K+ contents of the external solution for the different plant
culture conditions. These results suggest that plasma
membrane integrity was not adversely affected by theapplication of 200 lM acetic acid or 20 lM Pb. We
hence conclude that the observed Pb uptake
enhancement in wheat roots in the presence of organic
acids was not due to toxic action of either Pb or theorganic acids.

2d

Fig. 4 Potassium concentration of the external solution after
treatment for 1 and 2 d with and without the presence of 200 lM
acetic acid and 20 lM Pb. N represents the nutrient solution.
Values are means ± 1 SE of four replicates

Plant roots generally have a negatively charged surface
due to the presence of O– and COO– groups, whichleads to the formation of an unstirred layer adjacent to
the root surface. The unstirred layer of a nutrient
solution contains an excess of ions called counterions(e.g., i+ in Fig. 6) that carry charge equal in magnitude
and opposite in sign to that exhibited by the root surface. The thickness of the unstirred layer varies inversely with the ionic strength of the nutrient solution.
The characteristic thickness is given by 0.28 · I–0.5 nm,

123Planta
Table 1 Pb chemical speciation (% of total Pb) of nutrient solutions in the presence and absence of organic acids as calculated with
Visual MINTEQ using our initial solution composition
Acetic acid:Pba

Pb

Pb2+
PbOH+
PbSO4(aq)PbNO3+
Pb-Ac+Pb(Ac)2(aq)Pb-(Mal)22–
Pb-Mal(aq)

95.7
1.33
1.55
1.46

Malic acid:Pba

2:1

5:1

10:1

20:1

50:1

100:1

2:1

5:1

10:1

20:1

50:1

100:1

94.6
1.31
1.53
1.44
1.09

93.1
1.29
1.51
1.41
2.67

90.7
1.25
1.46
1.37
5.19
0.02

86.2
1.19
1.38
1.30
9.83
0.09

75.1
1.02
1.17
1.12
21.2
0.48

61.7
0.83
0.93
0.91
34.1
1.53

91.5
1.27
1.49
1.35

85.9
1.18
1.38
1.26

78.0
1.07
1.24
1.17

66.0
0.90
1.03
0.96

46.0
0.61
0.67
0.64

31.7
0.40
0.42
0.42

4.39

0.01
10.3

0.04
18.5

0.13
31.0

0.57
51.5

1.50
65.6

Results are for a pH of 5.8 and a temperature of 25C. Pb was maintained at 20 lM
Ac acetic acid, Mal malic acidaDifferent molar concentration ratio of organic acids to Pb

unstirred layer bulk solution
plant
root

COO-

i+

i+

i+

Coordination environment of Pb in wheat roots

i+

O-

i+

i+

COOi+

O-

inorganic ion
in solution
unstirred layer counterion(enriched in species ofchargedopposite charge to that of surface)surface group

Fig. 6 Schematic showing how a negatively charged root surface
attracts cations, including Pb2+(e.g., i+), in the unstirred layer of anutrient solution

where I is the nutrient solution’s ionic strength inmolar units (e.g., Schwarzenbach et al. 2003). The
thickness of the unstirred layer was 6.56 nm for thenutrient solution without Pb and without organic acids.
When the molar concentration ratio of acetic or malic
acid to Pb increased from 2:1 to 10:1, the thickness of
the unstirred layer decreased from 6.42 to 6.30 nm for
acetic acid and from 6.16 to 5.92 nm for malic acid.
These results suggest that the unstirred layer wasthinner in the presence of organic acids, thus creating asituation in which the counterions (including Pb2+) are
more readily absorbed or taken up by wheat roots. Thisfinding is consistent with the observed enhanced uptake of Pb by wheat (Fig. 1). However, when the molar
concentration ratio of organic acids to Pb further increased and the thickness of the unstirred layer further
decreased, the uptake of Pb by wheat roots eventually
decreased. We postulate that this is due to the adverse

123

effect of higher organic acid concentrations on theplasma membrane.

Normalized XANES spectra of Pb for the root samples
and model compounds are shown in Fig. 7. A sharp
peak occurred in Pb L3-XANES, which was caused
primarily by 2p to 5d electronic transition of Pb. Better
spectral agreement was obtained between root samples(inflection at 13049.3 eV) and Pb(CH3COO)23H2O(inflection at 13048.5 eV). For Pb(NO3)2 the peakshifted to a lower energy at 13042.3 eV and showed
greater intensity. The XANES spectra of the root
samples also exhibited a clear maximum at 13085.3 eV.
Similar maxima were observed for Pb(CH3COO)2
3H2O, but to a lesser extent, while the Pb(NO3)2and b-PbO spectra did not show such features. These
distinct differences in the XANES spectra arise fromdifferent local geometries and occupancies of the
final electronic states. The similarities in the XANES
structure for root samples and Pb(CH3COO)23H2O
demonstrate that their local atomic configurations
surrounding the central Pb atoms are similar. In
Pb(CH3COO)23H2O, Pb is bound with carboxyl
functional groups, which indicates that Pb in roots arecoordinated via similar chemical environments. Similar
results were obtained by Sarret et al. (1998).
The k2-weighted EXAFS spectra and Fouriertransforms for the wheat root samples and referencecompounds are presented in Fig. 8a, b. Compared tothe undesorbed roots, the k2-weighted EXAFS spectrum for the desorbed roots showed more noise above
6.2 Å–1 due to lower Pb contents in the desorbed roots.
The first two oscillations for the root samples at 3.6
and 5.5 Å–1 showed a fingerprint feature, and arePlantato the second-shell of Pb–N. The similarities between
the spectra of the root samples and Pb(CH3COO)2
3H2O further support the binding of lead(II) to wheatroots through a similar mechanism, i.e., Pb was most
likely coordinated to oxygen atoms via carboxylic
groups.
The fitted results are listed in Table 2 for the firstshell. The data indicate that the nearest-neighbor distance of the Pb–O bond in wheat roots is similar to
those for Pb(CH3COO)23H2O but deviates from thoseof Pb(NO3)2. No XAS study was carried out for wheatroots treated with malic acid since we were unable toobtain lead malate reference compounds.

Pb(NO3)2
β-PbO
Pb(CH3COO)2·3H2Oµ (E)Undesorbed root
Desorbed root

Discussion
13.1

13.2

Energy (kev)Organic acids may influence metal solubility anduptake through their indirect effects on microbialactivity, rhizosphere physico-chemical properties, androot growth dynamics, as well as more directly throughacidification, complexation, precipitation and oxidation-reduction reactions in the rhizosphere (Cieśliński
et al. 1998). Still, relatively little information exists on
the effects of organic acids on the uptake of Pb byplants. The results of our experiments clearly demonstrate that organic acids enhance Pb uptake by wheatroots (Figs. 1, 2). The significant greater Vmax values in
the presence of acetic and malic acids reflect theenhanced net influx of Pb into symplasm.
In the uptake experiments, binding to the cell wall
could have confounded the estimation of Pb transport
into the cytosol. In principle, extracellular absorption must not be considered true uptake by plants.

Fig. 7 Normalized Pb L3-edge XANES for desorbed andundesorbed wheat (Triticum aestivum L.) roots and modelcompounds

similar to those for Pb(CH3COO)23H2O. The Fouriertransforms reflect a relative radial distribution functionof lead(II) with neighboring atoms located in the local
coordination shells. The dominant peak at 1.62 Åarose from the first shell of Pb–O. However, this peakfor model compound Pb(NO3)2 was shifted 0.3 Å
higher than the others. The second weak peak around
3.3 Å for the root samples probably arose from thesecond-shell due to metal–metal bonding. For
Pb(CH3COO)23H2O the second peak may be due tothe effect of Pb–Pb and/or Pb–C bonds. In addition, asecond weak peak around 3.7 Å for Pb(NO3)2 was duebaPb(NO3)2

β-PbO

k2 (k)

Fig. 8 Pb L3-edge EXAFS
data (a) and Fouriertransforms of the EXAFS
data (b) along with fits to thedata. The solid line represents
the data, while the dotted line
was fitted to the data. Resultsof the data analysis arepresented in Table 2

Pb(NO3)2

F(R)

13.0

β-PbO

Pb(CH3COO)2·3H2O

Pb(CH3COO)2·3H2O
Undesorbed root

Undesorbed root
Desorbed root

Desorbed root

2

4

6

k(Å-1)

8

10

0

2

4

6

8

R(Å)

123

Planta
Table 2 Results of fits to the
EXAFS data

Errors of approximately 20%
in the coordination number
and 0.01 Å in the bonddistance

Samples

Undesorbed roots
Desorbed roots
Pb(CH3COO)23H2O
Pb(NO3)2First Pb–O shellN

R (Å)r (Å )N

R (Å)

r2 (Å2)

4.13
4.02
4
6

2.36
2.26
2.30
2.63

0.008
0.011
0.006
0.002

4
6

2.39
2.72

0.007
0.008

Therefore, removal of Pb from the root surface isessential for accurate measurement of uptake of metals
by plants. Desorbed Pb could have originated from thecell walls and/or from the cytosol via efflux across theplasma membrane back into the external solution. In
our experiments, desorption was performed in an icecold water bath. Under such conditions the active Pb
efflux from cytoplasm is presumably inhibited. Basedon our experimental results we conclude that most of
the desorbed Pb originated from the root cell walls.
Pb has been found to significantly inhibit voltagegated Ca2+ channel activity in the plasma membrane ofwheat roots (Huang and Cunningham 1996). The
inhibition of Ca-channel activity by Pb2+ could result
either from competitive transport of Pb2+ through Cachannel or from its blockage by Pb. While monitoring
Pb entry into isolated cells, Tomsig and Suszkiw (1991)
observed permeation of Pb through Ca-channels. In
maize and wheat plasma membrane vesicles the Ca2+
channel is very sensitive to La3+ (Tyerman and Skerrett 1999). Bregante et al. (1997) reported that 1 mM
Cs+ was able to block in a very fast and voltagedependent manner up to 96% of the potassium current
in protoplasts of maize roots. Our results suggest that
La3+ and Cs+ significantly inhibit the uptake of Pb by
wheat roots (Fig. 5). It is generally recognized that Cs+
is a K+ channel blocker, while not affecting any Ca2+
channels. La3+ is a broad range Ca2+ channel blocker,
but also blocks non-selective cation channels, anion
channels and the general metabolism. Interestingly,
Pb2+ can bind to Ca2+-binding sites in regulatory proteins such as calmodulin. Recently, the tobacco plasma
membrane protein NtCBP4 and the Arabidopsis gene
CNGC1 were reported to be components of a transport
pathway responsible for Pb2+ entry into plant cells(Sunkar et al. 2000). The involvement of CNGC1 andCNGC2 in the transport of potassium has been reported also (Leng et al. 1999). These results support
our evidence that the uptake of Pb2+ was mediated atleast in part by K+ and Ca2+ channels.
Uptake of cationic solutes is likely to be driven
largely also by the negative membrane potential acrossthe plasma membrane, which is generated in part bymetabolically dependent processes such as proton
extrusion via the plasma membrane H+-ATPase

123

Second Pb–O shell
2

2(Kochian 1991). The pmf generated by plasma membrane H+-pumping ATPase powers transport through avariety of carriers; it also influences ion channel
activity through its impact on Vmax. The plasma
membrane H+-ATPase is a P-type ATPase. All P-type
ATPase membranes are inhibited by orthovanadate(H2VO–4). Pre-treatment of wheat roots by vanadatesignificantly inhibited Pb uptake in our study (Fig. 5),thus suggesting that the enhanced Pb uptake into thecytosol of wheat was dependent upon physiological
functions of the plasma membrane P-type ATPase.
The above discussion on the mechanisms of enhanced uptake of Pb in the presence of organic acids
focuses on mostly plant-physiological aspects of theuptake problem. Interestingly, the mechanisms canalso be discussed from a solution chemistry perspective. Table 1 shows the speciation of Pb in the nutrientand uptake solutions as estimated with Visual MINTEQ, version 2.50. Our results suggest that free Pb2+
was the dominant species, even in the presence of organic acids. In view of the size and polarity of intact
Pb(II)–organic ligand complexes, absorption of thesecomplexes is very unlikely. Also, synthetic chelatescannot slip through the plasma membrane since they
are too large and too polar to move through the plasmalemma lipid bilayer (Berne and Levy 1998). However, it seems possible that diffusion of Pb-acetate or
Pb-malate complexes was more rapid than free Pb2+
because of the high charge density of Pb2+, thus facilitating these complexes to move more rapidly towards
the roots. Once these chelates reach the root surface,
the surfaces will promote their dissociation into freePb2+, which are then more readily absorbed or taken
up by the roots.
The reason for acetic and malic acids exert different
enhancement effects on the uptake of Pb may be
explained by their different chelating capabilities. The
stability constants of complexes formed between Pb
and acetic acid are less than those between Pb andmalic acid, which is contrary to the order of theenhancement effects obtained in our experiments. It isapparent that the lower the stability constants of thePb(II)–organic ligand complexes, the greater theenhancement effect. This means that Pb(II)–organic
ligand complexes are more easily dissociated by wheat

Plantaroots, leading to more uptake of dissociated Pb ions by
the wheat roots.
Negatively charged plant root surface promote thedevelopment of an adjacent unstirred layer, and hence
relatively high Pb2+ concentration gradient near theroots. According to Kochian et al. (1989), diffusive
gradients in the unstirred layer around the roots or in
the apoplast can occur and influence the uptake rate ofelements, even in well-stirred solutions. Our results
suggest that the unstirred layer was effectively thinner
in the presence of organic acids, thus allowing Pb2+ tobe more readily absorbed or taken up by roots. Thisfinding supports the enhanced uptake of Pb as shown in
Fig. 1.
Lead can be tetravalent or divalent. Divalent leadpossesses a 6s2 outer shell electronic configuration. The
two lone pair electrons are often stereochemically
active and induce a strong deformation of divalent leadpolyhedron. Another source of complexity of thecoordination chemistry of lead comes from the highvariability of its coordination number. All possible
coordination values (between 3 and 12) have beenreported in the literature. Our EXAFS data indicatethat one Pb(II) atom was coordinated to four oxygen
atoms and that the coordination environment of intracellar Pb in wheat roots is similar to that ofPb(CH3COO)23H2O. It is worth noting that thecoordinated oxygen atom is not from Pb(NO3)2 insolution but from carboxylate functional groups. The
coordination number for root samples was particularly
low compared to that of Pb(CH3COO)23H2O becauseof the high structure disorder of the Pb coordination
shells in root samples. Similar observations for Pb–O
coordination have been reported for organic macromolecules such as humic acids, fulvic acids or salicylate
in soils (e.g., Manceau et al. 1996), for lichen cells exudates such as oxalate and parietinic acid (Sarret et al.
1998), and for lignocellulosic biomaterial (Dupont
et al. 2002).
The major conclusions from this study are that (1)the maximum Michaelis–Menten influx rate (Vmax) for
Pb uptake was enhanced by the presence of organic
acids; (2) the enhanced influx of Pb into wheat roots
was partially mediated by Ca2+ and K+ channels, anddepended upon the plasma membrane P-type ATPase;(3) the enhanced uptake of Pb was attributed to theenhanced diffusion of Pb towards roots by organic
acids; and (4) one Pb(II) atom was coordinated to fouroxygen atoms in wheat roots such that the coordination
environment of Pb in wheat roots was similar to that ofPb(CH3COO)23H2O. This last conclusion supports
earlier findings that the binding of Pb(II) in wheatroots occurs via carboxylic groups. However, weemphasize that our findings were the result of experiments performed with solution cultures and therefore
may not be directly applicable to actual field situations.
Acknowledgments This study was supported by the National
Natural Science Foundation of China (Grant No. 20237010 and20177030).

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