Plant Soil (2011) 339:377389

DOI 10.1007/s11104-010-0589-6

REGULAR ARTICLE

Lead bioaccumulation in Acacia farnesiana and its effect

on lipid peroxidation and glutathione production
Amalia Maldonado-Magaa & Ernesto Favela-Torres &
Fernando Rivera-Cabrera & Tania L. Volke-Sepulveda

Received: 22 July 2010 / Accepted: 21 September 2010 / Published online: 2 October 2010
# Springer Science+Business Media B.V. 2010

Abstract Phytoremediation offers a cheap, efficient bioconcentration (>8.5) and low translocation (0.03)
and environmentally friendly option for cleaning sites factors. These results indicate the suitability of A.
contaminated with toxic elements. However, there is a farnesiana for lead-phytostabilization purposes. Lead
need to find new plant species for phytoremediation and concentrations below 500 mgL1 had no significant
to understand the mechanisms involved in processes effect on lipid peroxidation and enhanced the glutathi-
such as tolerance, accumulation, exclusion and metab- one content, suggesting that the ability of A. farnesiana
olism of toxic metals in plants. Thereby, in this study, to withstand the Pb-induced oxidative stress could be
the ability of Acacia farnesiana (L.) Willd to tolerate related to glutathione metabolism.
and accumulate lead was analyzed. Seedlings grown in
vitro with 250 and 500 mg Pb2+ L1 showed an Keywords Acacia farnesiana . Lead
increase in their growth, achieving tolerance indexes phytoremediation . Glutathione . Lipid peroxidation .
close to 100%. In seedlings exposed to 1,000 mg Pb2+ Oxidative stress
L1, growth was strongly inhibited, finding an effective
concentration 50% (EC50) from 720 to 766 mg Pb2+ Abbreviations
L1. A. farnesiana accumulated 80% of the Pb2+ in ROS Reactive oxygen species
roots (up to 51,928 mgkg1 of air-dried tissue) in MS Murashige & Skoog
seedlings exposed to 1,000 mg Pb2+ L1, with high EDTA Ethylene-diamine-tetraacetic acid
EU Experimental unit
FW Fresh weight
Responsible Editor: Juan Barcelo. ADW Air-dried weight
A. Maldonado-Magaa : E. Favela-Torres :
TI Tolerance index
T. L. Volke-Sepulveda (*) EC50 Effective concentration 50%
Departamento de Biotecnologa, BCF Bioconcentration factor
Universidad Autnoma Metropolitana-Iztapalapa, TF Translocation factor
San Rafael Atlixco 186, Col. Vicentina, Iztapalapa,
GR Glutathione reductase
09340 Mxico, D.F., Mexico
e-mail: tvs@xanum.uam.mx MDA Malondialdehyde
DTNB 5,5-dithiobis-2-nitrobenzoic acid
F. Rivera-Cabrera TNB 5-thio-2-nitrobenzoic acid
Departamento de Ciencias de la Salud,
GSHT Total glutathione
Universidad Autnoma Metropolitana-Iztapalapa,
San Rafael Atlixco 186, Col. Vicentina, Iztapalapa, GSH Reduced glutathione
09340 Mxico, D.F., Mexico GSSG Glutathione disulfide (oxidized glutathione)
378
Introduction
Mining has been one of the major economic activities
during centuries; however, it has generated large
amounts of wastes (tailings and melting scum) that
were improperly disposed in the environment for
many years, impacting vast areas of soils with toxic
elements. In Mexico, lead and cadmium are two of
the main toxic metals included in tailings (Volke
Sepulveda et al. 2005). Due to their high toxicity for
humans, they are considered important environmental
pollutants. According to Singh et al. (2003), the
global discharge of Cd2+ and Pb2+ over the past five
decades reached 22,000 t and 783,000 t, respectively.
Because of this situation, there is a need to develop
strategies to remediate ecosystems that have been
injured by metal pollution. Phytoremediation offers a
cheap, efficient and environmentally friendly option
for cleaning contaminated sites, allowing revegetation
of degraded lands (Vangronsveld et al. 2009). This
technique involves the use of plants and their
associated microorganisms to remediate contaminated
matrices through the extraction, transformation, deg-
radation and/or stabilization of organic and inorganic
pollutants (Meagher 2000; Mench et al. 2009).
Nevertheless, many phytoremediation techniques are
still under development, which creates the need for
conducting research to find new plant species with
phytoremediating potential and to generate a deeper
understanding about processes involved in tolerance,
accumulation, exclusion and metabolism of toxic
metals in plants. The use of trees as a vegetation
cover for phytoremediation of metal-polluted soils
could have a significant potential, as demonstrated for
species such as poplar, spruce and pine (Brunner et al.
2008). However, studies performed up to date about
this topic are relatively insufficient. Acacia is an
important genus of the family Fabaceae that includes
~1,200 species. In particular, Acacia farnesiana (L.)
Willd. is a shrub or small-tree distributed in arid and
semiarid areas in Mexico, where most of the metal
polluted sites are located. This legume species has
great ecological value because it can help to control
soil erosion and improve soil fertility through symbi-
osis with Rhizobium and mycorrhizal fungi (Caedo-
Ortiz et al. 2000). Moreover, A. farnesiana is
extremely tolerant to drought and easily adaptable to
extreme temperatures and pH levels, and therefore,
can be grown in a wide range of soils, including sands
Plant Soil (2011) 339:377389
and clays, as well as disturbed, nutrient depleted, and
saline soils or very rocky sites (Francis 2002).
Additionally, legume crops are reported to be tolerant
to several heavy metals (Reddy et al. 2005).
Since toxic metals cannot be degraded by physi-
cochemical or biological processes once released into
the soil, their remediation can be achieved basically
through changes in the redox state, favoring mecha-
nisms that modify the solubility, mobility and/or
toxicity of metals making possible the remediation
process through their separation or dissolution (Gadd
2000; Barkay and Schaefer 2001). Thereby, phytor-
emediation is one of the few technically and econom-
ically feasible alternatives for the remediation of
metal-polluted soils by phytoextraction and/or phy-
tostabilization mechanisms (Schnoor et al. 1995;
Meagher 2000; Kuiper et al. 2004). The plants
capabilities to efficiently phytoremediate a metal-
contaminated soil depend on their resistance to a
particular pollutant. This resistance can be attributed
to mechanisms of exclusion or tolerance (Klassen et
al. 2000). The molecular mechanisms proposed to
explain the tolerance of some plants to toxic metals
include binding to cell walls, compartmentalization in
vacuoles and adaptive mechanisms of metabolic and
enzymatic nature, such as the activation of enzymatic
and non-enzymatic systems to eliminate reactive
oxygen species (ROS) (Schtzendbel and Polle
2002).
ROS are partially reduced forms of atmospheric
oxygen, which level maintenance in cells under
normal conditions is a well regulated process. Metal
toxicity enhances the production of ROS up to 30-
fold reaching concentrations between 5 and 15 M
H2O2, causing an increment in the oxidative stress as
a result of an imbalance between ROS production
and the antioxidant defense mechanisms (Mittler
2002). The lack of control on the oxidative stress
represents a hazard to the cells, since ROS can react
with lipids, proteins and nucleic acids, causing lipid
peroxidation and, eventually, the loss of membrane
integrity, protein denaturation and enzymes inactivation
(Schtzendbel and Polle 2002; Blokhina et al. 2003;
Imlay 2008; Wang et al. 2008). Many higher plants
possess antioxidant mechanisms including soluble and
membrane-bound compounds, particularly in mito-
chondria and chloroplasts (Schtzendbel and Polle
2002; Collin et al. 2008). Geebelena et al. (2002)
demonstrated that even low concentrations of intracel-
Plant Soil (2011) 339:377389
lular Pb2+ in Phaseolus vulgaris, substantially in-
creased the ROS formation leading to the oxidation
of thiol groups in enzymes and peroxidation of
unsaturated fatty acids in membranes, leading eventu-
ally to cell death. Pb2+ can also replace essential metals
or cofactors in enzyme active sites, triggering an
imbalance of trace metals or the depletion of glutathi-
one, causing the accumulation of ROS (Schtzendbel
and Polle 2002; Wang et al. 2008). Independently of
the production pathway, ROS are highly cytotoxic and
their levels in plant cells are controlled by antioxidant
enzymatic and non-enzymatic systems. Modulation of
the levels of antioxidant systems in plant cells is an
important adaptive response to resist adverse condi-
tions produced by exposure to toxic metals. Thus, to
avoid the toxicity by metals, plants have developed a
series of protective mechanisms that remove ROS
before they can injure sensitive parts of the cellular
components (Sharma et al. 2005). The elimination of
ROS is controlled by antioxidant systems that include
low molecular weight molecules with antioxidant
capacity, enzymes that regenerate the reduced forms
of these antioxidant molecules, and enzymes that
interact with ROS (Blokhina et al. 2003).
In this context, the aim of the present study was to
determine the Pb2+ phytoremediating potential of
Acacia farnesiana, a legume species widely distrib-
uted in arid and semi-arid regions in Mexico, as well
as to determine the Pb2+ effect on plant growth and
antioxidant capacity of this species, using an in vitro
system.
Materials and methods
Plant material and culture conditions
A. farnesiana seedlings were grown under in vitro
conditions from seeds collected from a mature wild
tree grown surrounded by mining wastes. Seeds were
surface-sterilized with 2% commercial detergent
(30 min), 70% ethanol (30 s) and 1.8% sodium
hyphochloride (30 min) followed by repeated rinses
with sterile deionized water.
Seeds were mechanically scarified under aseptic
conditions and sown in Magenta boxes (Sigma)
containing Murashige & Skoog medium (MS, Sigma)
added with sucrose (30 gL1, Sigma), Phytagel (2 g
L1, Sigma), Na2EDTA (0.5mM, Baker) and different
379
concentrations of Pb2+ (0, 250, 500 and 1,000 mgL1)
supplied as Pb(NO3)2; different NO31 concentrations
added to media as Pb(NO3)2 were compensated by the
addition of NaNO3. All media were adjusted to pH 5.7
with 0.5N NaOH before autoclaving (121C, 15 min).
Seedlings were maintained at 251C at 50 mmolm2
s1 light intensity for 16 h photoperiod during 60 days.
For each Pb concentration, four replicates were
evaluated, where one experimental unit (EU) was the
biomass obtained from four seedlings. Sixteen EU
were used at each sampling time (15, 30, 45 and
60 days) for fresh weight determinations. After 60 days,
4 EU for each Pb concentration were used for Pb
analysis in air-dried plant tissues and 4 for determi-
nations of total glutathione (GSHT) and malondialde-
hyde (MDA) content, and glutathione reductase (GR)
activity in fresh tissues. At each sampling time,
measurements were performed by destructive analyses.
Determination of growth parameters and Pb2+
tolerance
Morphological observations and growth measure-
ments were made in all seedlings 15, 30, 45 and
60 days after planting. For this purpose, biomass and
length of the roots and the shoots (the distance from
culture medium surface to upper end of the longest
leaf) were measured for each treatment. The biomass
was measured on fresh (FW) and air-dried weight
(ADW) basis after drying at 60C up to constant
weight. Growth measurements were used to estimate
the lead-tolerance index (TI) and the effective
concentration 50% (EC50) for A. farnesiana in the
presence of lead. TI was calculated as follows (Deng
et al. 2006):
Root elongation with Pb
TI%  100
Root elongation without Pb
EC50 was estimated from a doseresponse curve,
as the lead concentration in which the root length and
the total fresh weight decrease 50% regarding to the
maximum values achieved (Naumann et al. 2007).
Determination of the lead content
For lead analysis, roots were washed with 10mM
EDTA to remove adsorbed Pb2+ (Gthberg et al.
2004). Then, roots and shoots were oven-dried (60C,
2448 h) and about 100 mg of air-dried tissue were
380
completely digested after 10 min in a microwave
digestion system (CEM, MARSXpress) with 5 mL of
69% HNO3 (J.T. Baker ACS Reagent Grade) and
4 mL of deionized water (18 Mcm1, Milli-Q
Millipore). All samples were filtered (0.45 m, GN-
6 Metricell) and the final sample volume was adjusted
to 10 mL with deionized water. Lead concentration in
extracts was analyzed by flame atomic-absorption
spectrometry (Shimadzu, AA-6300). Standard curves
were prepared with a Pb 2+ standard solution
(1,000 gmL1 J.T. Baker Instra-analyzed). All the
laboratory ware was prepared by soaking overnight in
a 10% HNO3 solution.
Lead concentration measurements were used to
estimate the bioconcentration factor (BCF) and the
translocation factor (TF). BCF was defined as the
ratio of lead concentration in plant tissues ([Pb2+]plant)
to lead concentration in medium ([Pb2+]medium). TF
was defined as the ratio of lead concentration in
shoots ([Pb2+]shoots) to lead concentration in roots
([Pb2+]roots) (Audet and Charest 2007):
Pb2 plant Pb2 shoots
BCF TF
Pb2 medium Pb2 roots
Lipid peroxidation, glutathione and GR activity
assays
Preparation of crude extracts
For determination of lipid peroxidation, GR activity
and GSHT content, fresh shoots and roots samples
(100300 mgFW) of seedlings were ground with
liquid nitrogen in a mortar and homogenized with
1.5 mL of the respective extraction buffer. Ground
tissues were homogenized with phosphate buffer (100
mM, pH 7.5) for lipid peroxidation and GR assays, or
with 5% metaphosphoric acid for glutathione quanti-
fication. All homogenates were centrifuged at
18,500g for 15 min at 4C, and the resultant
supernatants were used for determinations.
Lipid peroxidation assay
Crude extracts were used for determination of lipid
peroxidation using a commercial assay kit (Biomol,
BML-AK170). The assay is based on the quantifica-
tion of MDA, the main product of membrane
Plant Soil (2011) 339:377389
polyunsaturated fatty acid peroxidation (Esterbauer
and Cheeseman 1990). One molecule of MDA reacts
with two molecules of the chromogenic agent N-
methyl-2- phenylindole, when the reaction mixture is
catalyzed by HCl at 45C. The reaction yields a stable
chromogen with maximum absorbance at 586 nm.
Lipid peroxidation was expressed as the MDA
content in nmolgFW1.
Glutathione determination
Glutathione content was determined spectrophotomet-
rically at 412 nm using 5,5-dithiobis-2-nitrobenzoic
acid (DTNB), as described by Akerboom and Sies
(1981), using a commercial kit assay (Sigma-Aldrich).
This assay measures the content of both total
glutathione (GSHT) and oxidized glutathione (gluta-
thione disulfide, GSSG) using a kinetic assay in
which catalytic amounts of glutathione cause a
continuous reduction of DTNB to TNB (5-thio-2-
nitrobenzoic acid). Then, the GSSG formed is
recycled by GR and NADPH, and the product
(TNB) is measured at 412 nm. The rate of TNB
production is directly proportional to this recycling
reaction, which is in turn directly proportional to the
concentration of GSH in the sample.
GR
NADPH H GSSG ! NADPH 2 GSH
GSH DTNB ! GSTNB TNB
The reaction mixture contained 150 L of a
working mixture, 50 L of 0.16 mgmL1 NADPH,
10 L of 5% metaphosphoric acid and 10 L of the
enzyme extract. The working mixture contained
1 mL of 100 mM potassium phosphate buffer
(pH 7.0 with 1mM EDTA), 28.5 L of 1.5 mg
mL1 DTNB solution and 28.5 L of 6 units mL1
GR solution. The contents of reduced glutathione
(GSH), GSSG, and GSH T (GSH + GSSG) are
expressed in nmolgFW1.
Glutathione reductase (E.C. 1.6.4.2) assay
GR activity was determined in roots or shoots enzyme
crude extracts following the methodology proposed
by Smith et al. (1988) using a commercial kit assay
(Sigma-Aldrich). The assay is based on the increase in
absorbance at 412 nm when DTNB and GSH react to
generate TNB. The reaction mixture (200 L)
contained 100 L of 2mM oxidized glutathione,
Plant Soil (2011) 339:377389
30 L of 100 mM potassium phosphate buffer
(pH 7.5, with 1mM EDTA), 50 L of 3mM DTNB,
10 L of 2mM NADPH and 10 L of the enzyme
extract. GSSG was replaced by water in the reaction
mixture used as blank. Absorbance was monitored
using a spectrophotometer (Perkin-Elmer UVvis,
Lambda 2) and enzyme activity was calculated from
the initial rate of the reaction using an extinction
coefficient () of 14.15mM1 cm1 for TNB. One unit
of GR was defined as the amount of enzyme that
causes the reduction of 1 mol of DTNB to TNB at
25C and pH 7.5.
Statistical analysis
Each treatment was applied in a randomized design
with four replicates, where one EU was the biomass
obtained from four seedlings. One-way ANOVA and
a Duncan test with a significance level of P0.05 was
used to compare the means of the growth parameters,
MDA and GSH contents, and the GR activity of A.
farnesiana seedlings exposed to the different Pb2+
concentrations. All results are expressed as means,
with their corresponding standard errors and letters
that indicated statistical differences between the
means. Statistical analyses were performed using the
SAS software.
Fig. 1 Effect of lead con-
centration on the growth
and morphology of A. far-
nesiana seedlings, after
60 days of culture. Arrows
show chlorosis signs in
seedlings grown at
1,000 mg Pb2+ L1
0 mg PbL-1
381
Results
Lead effect on plant growth and tolerance index
Lead concentration had an important effect on the
growth of A. farnesiana seedlings (Fig. 1). At
concentrations of 250 and 500 mg Pb2+ L1, the
growth of seedlings increased as compared to the
controls without the metal, showing thicker and
longer roots and higher number of secondary roots.
On the contrary, seedlings exposed to a lead concen-
tration of 1,000 mgL1 showed signs of chlorosis and
a marked growth inhibition, manifested in a smaller
size, fewer leaves and shorter roots as compared to
the control. These morphological features were
quantitatively confirmed by measuring the fresh
weight (FW) and the length of roots and shoots
(Fig. 2). Particularly, in seedlings exposed to 250 mg
Pb2+ L1, growth was stimulated, reaching a total FW
between 11% and 39% higher than the controls. Root
length was between 23% and 35% higher than that
observed in the control seedlings. At 1,000 mg Pb2+
L1, the total FW of seedlings decreased from 30% to
74% as compared to the FW of controls. In addition,
root growth was strongly inhibited (being 9093%
shorter with respect to seedlings without Pb2+) during
the entire duration of the experiment.
250 mg PbL-1 500 mg PbL-1 1000 mg PbL-1
382 Plant Soil (2011) 339:377389

0.8 20
0 mg Pb L-1 a b *
250 mg Pb L-1
* * *
Total fresh weight (g)

0.6 500 mg Pb L-1 15

Root lenght (cm)

1000 mg Pb L-1 * *
* *
0.4 * 10

*
0.2 * 5
* *
* * * *
0 0
0 15 30 45 60 0 15 30 45 60
Time (days) Time (days)

Fig. 2 Effect of lead concentration and time of exposure on conditions. Significant differences (P0.05) regarding to the
total biomass production (a), and root length (b) in seedlings of control (seedlings grown without lead) are marked with an
A. farnesiana during 60 days of growth under in vitro asterisk

Lead concentrations from 250 to 500 mgL1
significantly stimulated the growth of A. farnesiana
in comparison with seedlings cultivated without the
metal. This response was associated with high TI
values, 37 and 26 units above the control (100%), for
250 and 500 mgL1, respectively (Fig. 3). This
behavior suggests the operation of a tolerance
mechanism against adverse effects of lead at concen-
trations of 250 and 500 mg Pb2+ L1. In contrast,
when Pb2+ concentration in the medium was in-
creased up to 1,000 mgL1, the TI showed an
important decrease to values as low as 8% to 10%,
independently of the harvest time. Thus, EC50 values
of 765 mg Pb L1 and 719 mg Pb L1 were obtained
150
a seedlings exposed to 1,000 mg Pb2+ L1. The observed
125
a high BCF values should be attributed to the high
Tolerance index (%)
for seedlings of A. farnesiana exposed to lead during
60 days using root length and total FW as response
variables, respectively.
Lead uptake and accumulation
Pb2+ uptake and accumulation in biomass of A.
farnesiana increased with increasing Pb2+ concentra-
tion in the medium (Table 1), reaching up to 979 and
51,928 mg Pb2+ per kg of air-dried shoots and roots,
respectively, in seedlings exposed to 1,000 mg Pb2+
L1 during 60 days. The capability of A. farnesiana
seedlings to accumulate high Pb2+ concentrations was
associated to the BCF, which values were found
between 8.5 and 10.8 for concentrations of lead below
500 mgL1, and reached values up to 53.58.0 in

100
amount of metal accumulated in the roots (Table 1),
b being 30-fold higher than the levels in the shoots. In
75 seedlings exposed to 250 and 500 mg Pb2+ L1, an
average of 93% from the Pb2+ accumulated in the
50 entire plant tissues was retained in the roots, while in
seedlings grown at 1,000 mgL1 the lead content in the
25 765 mg L-1 c roots reached 8011% from the accumulated. These
0 data were associated with very low values (below
0 250 500 750 1000 0.032) of the TF in all the Pb2+ concentrations tested.
Pb concentration (mg L-1)
2+
Effect of Pb2+ on MDA concentration
Fig. 3 Effect of Pb concentration on the lead-tolerance index
(TI) of A. farnesiana. Dotted line shows the data considered for
the estimation of EC50. Values with the same letter are not MDA content in the shoots of A. farnesiana seedlings
significantly different (P0.05) grown 60 days under different Pb2+concentrations
Plant Soil (2011) 339:377389 383

Table 1 Lead uptake and accumulation in shoots and roots of A. farnesiana after 60 days of exposure to different initial Pb2+
concentration. Values of bioconcentration (BCF) and translocation (TF) factors are also shown

Pb2+ (mgL1) Pb2+ in plant tissues (mgkg ADW1) BCF TF

Shoots Roots

0
B B B A
250 8313 2,615214 10.80.8 0.0320.003
B B B A
500 13613 4,135998 8.52.1 0.0330.008
A A A A
1,000 979512 51,9287,000 53.58.0 0.0280.016

Data with the same letter by column are not significantly different (P0.05)

was around two times higher than MDA level in the
roots (Table 2). In shoots, Pb2+concentration did not
have a significant effect on the MDA content
compared to the control seedlings. On the other hand,
in roots of seedlings exposed to concentrations of 250
and 500 mg Pb2+ L1, the MDA content was
maintained around to 13 nmolg FW1 showing no
significant difference regarding to controls, however,
in roots exposed to 1,000 mg Pb2+ L1, the MDA
level increased about two times, reaching up to
24 nmolg FW1.
tration was increased the GSH/GSSG ratio decreased
to values 1.4 and 2.2 times below the control, for
treatments with 500 and 1,000 Pb2+ L1, respectively
(Table 2). In roots, the GSHT content ranged from
25.5 to 54.2 nmol g FW1 and it was slightly
stimulated as lead concentration increased. From the
total concentration of glutathione, more than 50% was
found in its reduced form (GSH) yielding GSH/GSSG
ratios from 1.5 to 2.9 (Table 2).
GR activity

Effect of Pb2+ on the glutathione content
Total concentration of glutathione (GSHT) in shoots
of seedlings treated with lead concentrations between
0 and 500 mgL1 was higher (4.58.5 times) than in
roots (Fig. 4). The production of GSHT was signifi-
cantly stimulated (46%) in shoots of seedlings
exposed to 250 mg Pb2+ L1 compared to the control,
but its content decreased (up to 73%) with the
increase in Pb2+ concentration. These results were
associated with a significant increase (3.5-fold) in the
GSH/GSSG ratio in plants treated with 250 mg Pb2+
L1 regarding to the control, while as Pb2+ concen-
GR activity in shoots was 2.36.1-fold higher in
tissues of seedlings exposed to lead for 30 days than
in those exposed for 60 days (Fig. 5). At 30 days, the
GR activity was stimulated with increasing lead
concentration in the media, attaining from 1.2 to 1.6
times more activity regarding the control seedlings. In
roots, GR activity was significantly stimulated only in
seedlings grown at 1,000 Pb2+ L1 (Fig. 5b). At
60 days, lead concentration did not have a significant
effect on the GR activity in shoots compared to the
activity in the controls, with values ranging from 179
to 239 molg FW1 min1. Enzyme activity in roots
was slightly enhanced with increasing lead concen-

Table 2 Malondialdehyde
(MDA) content and GSH/ Plant tissue Pb2+ (mgL1) MDA (nmolg FW1) GSH/GSSG ratio
GSSG ratio in shoots and
A B
roots of A. farnesiana Shoots 0 28.42.3 3.260.61
exposed to different initial A A
250 29.84.7 6.731.36
Pb2+ concentration during 500 28.70.7 A
1.830.66 C
60 days A C
1,000 27.53.5 1.020.51
Data with the same letter by Roots 0 13.93.0 b
2.100.87 a
column are not significantly b a
different (P0.05). Upper- 250 12.31.6 1.520.48
b a
case and lower-case indicate 500 13.01.9 1.530.92
independent testing for each 1,000 24.13.8 a
2.861.87 a

plant organ
384 Plant Soil (2011) 339:377389

350 100
a
a GSH-T b

Glutathione (nmol.g FW -1)

Glutathione (nmol.g FW -1)

280 80 GSSG
GSH
A
210 b 60 a
c
B b
140 40 bc
c
BC
C A
70 d 20 BC
D C a
a ab
bc c b b b
0 0
0 250 500 750 1000 0 250 500 750 1000

Pb concentration (mg L-1) Pb concentration (mg L-1)

Fig. 4 Effect of lead concentration on the total concentration of exposed to Pb2+ during 60 days. Data with the same letter are
glutathione (GSHT), oxidized glutathione (GSSG) and reduced not significantly different (P0.05); upper-case and lower-case
glutathione (GSH) in shoots (a) and roots (b) of A. farnesiana indicate independent testing for each variable

trations, achieving a maximum of 15917 molg A. farnesiana, a lead concentration of 720 and
FW1 min1 in roots exposed to 500 Pb2+ L1. 766 mgL1 decreased plant growth by 50%, quanti-
fied as the total FW and the root length as response
variables, respectively. At a high lead concentration
Discussion (1,000 mg Pb L1), this negative effect in growth was
also accompanied with notable physiological changes
Lead accumulation and its effect on the plant growth which can be considered toxicity symptoms (Fig. 1).
This adverse effect in growth could be attributed to
Total plant biomass, growth rate, root length and the accumulation of high lead concentrations in the
shoot length are variables commonly used as indica- roots (Table 1), inhibiting its development (Diwan et
tors of growth performance of plant species exposed al. 2010). In contrast, seedlings exposed to 250 mg Pb
to toxic elements, such as lead. In fact, some metal L1 showed a marked increase in all the evaluated
ions in the plant environment can act as a stress factor growth parameters, presenting also TI values higher
causing physiological changes or, in extreme cases, a than 100% (Fig. 3). This effect might be attributed to
complete inhibition of plant growth (Baker 1987). For a phenomenon known as hormesis, an adaptive
1000 1000
a a b
GR shoots ( mol.g FW-1.min-1)

30 d 60 d
GR roots ( mol.g FW -1.min-1)

800 b 800
b
a
600 c 600

400 400 b b b

A A
200
A A 200 A A
AB
B
0 0
0 250 500 1000 0 250 500 1000
-1
Pb concentration (mg L-1) Pb concentration (mg L )

Fig. 5 Glutathione reductase (GR) activity in shoots (a) and not significantly different (P0.05), where upper-case and
roots (b) of A. farnesiana exposed to different Pb2+ concen- lower-case indicate independent testing for each time
trations during 30 and 60 days. Values with the same letter are
Plant Soil (2011) 339:377389
response characterized by increasing and reducing
responses at low and high concentrations, respective-
ly, of a given pollutant (Calabrese et al. 2007). Thus,
based on the above considerations, it is possible that a
lead concentration of 250 mgL1 could induce low
levels of stress, activating cellular and molecular
mechanisms that enhance the ability of this species
to withstand more severe stresses.
According to Baker (1987), a plant species can
resist the heavy metal stress by either of two
strategies: (i) avoidance or exclusion, which refers to
the external protection from the influence of stress,
and (ii) tolerance, which is conferred by specific
physiological mechanisms that enable the plant to
function normally even in the presence of high
concentrations of toxic metals. Our results suggest
that A. farnesiana have both mechanisms of resis-
tance, since the plant accumulated more than 80% in
roots, which could be considered as an exclusion
mechanism (Brunner et al. 2008). The plant was also
able to grow normally at concentrations up to 500 mg
Pb L1, even when lead was accumulated at concen-
trations of 13613 mgkg ADW1 in the shoots,
which implies its translocation to aerial tissues
(tolerance). According to Deng et al. (2006), plants
grown in sites contaminated with toxic metals might
have some degree of tolerance to those particular
pollutants. In dry-land plants, such as A. farnesiana,
metal tolerance is usually a trait evolved among
populations growing in contaminated sites. Then, the
Pb tolerance of this species could be attributed to
special features developed among the populations
growing at the sampling site, probably associated to
the ability to avoid the Pb uptake into their aerial
biomass (Brunner et al. 2008). In fact, Deng et al.
(2006) found that species with a poor capacity for
root exclusion were more sensitive to the presence of
Pb, while species which possess the mechanisms for
metal exclusion showed a very high Pb tolerance,
attributed to their ability to exclude Pb from the
shoots.
BCF has been used as a measure of the metal
accumulation efficiency. In accordance to Audet and
Charest (2007), BCF values higher than 1 are
indicative of potential hyperaccumulator species.
However, this ratio is a measure of the metal
concentration in the whole plant (roots and shoots)
and, therefore, a high value indicates the capability of
a plant to accumulate metals in both roots and shoots.
385
In the case of A. farnesiana, the high BCF values
found should be attributed mainly to the high amount
of the metal accumulated in the roots (Table 1). The
very low lead translocation from roots-to-shoots (TF
0.03) suggests that A. farnesiana could be a good
candidate for phytostabilization of Pb2+ (Rizzi et al.
2004). Actually, it has been demonstrated that a
common form of metal detoxification by some forest
trees (such as spruce, pine, and poplar) is the uptake
prevention by mechanisms such as the root exudation
of organic acids and/or the metal accumulation in
roots by binding to the cell wall, to specific proteins
such as metallothioneins, and/or to biomolecules such
as phytochelatins or glutathione (Brunner et al. 2008).
Pb2+ effect on lipid peroxidation
A suitable criterion to select plants for metal
phytoremediation purposes could be the presence of
biochemical mechanisms related to heavy metal
tolerance. Important biological mechanisms of metal
detoxification involve the induction of metal-binding
ligands and antioxidant defense mechanisms against
the excessive ROS formation (Mittler 2002; Diwan et
al. 2010). Membrane lipids are the major target of free
radical attack, since the protonation of superoxide
radical (O2) can produce hydroperoxyl radical
(HO2), which can convert fatty acids to toxic lipid
peroxides. Therefore, measurement of the products of
lipid peroxidation, such as MDA, has been commonly
used to assess oxidative stress/injury (Del Rio et al.
2005; Diwan et al. 2010).
Although we found that lead was mainly accu-
mulated in the roots, MDA levels in the shoots
were about 2 times higher than in roots. However,
no significant differences in the MDA concentration
between shoots of seedlings exposed to Pb2+ and
the control were found, indicating that lead exposure
did not increase the oxidative stress in the above-
ground tissues, even at the highest concentration
tested. Liu et al. (2009) also found no significant
differences in the MDA content when garlic seed-
lings were exposed during 20 days to Pb concen-
trations between 2.1 and 21 mgL1; but, when the Pb
concentration was increased to 207 mgL1, the MDA
levels were significantly enhanced (about 2 times).
In contrast, Verma and Dubey (2003) and Dey et al.
(2007) found that increasing Pb concentrations (from
41 to 414 mgL1) significantly enhanced the lipid
386
peroxidation in shoots of species such as rice and
wheat. We found an enhanced lipid peroxidation in
roots grown at 1,000 mg Pb L1, which might
indicate an induction of oxidative stress attributable
to the accumulation of high lead concentrations in
roots (51,9287,000 mgkg DW1) that drastically
inhibited the plant development.
It is important to point out that in A. farnesiana
grown at a lead concentration as high as 500 mgL1,
neither lipid peroxidative products nor oxidative
stress were enhanced. This result supports that A.
farnesiana is a species that can resist the heavy metal-
induced stress.
Effect of Pb2+ on glutathione content
Glutathione is the most abundant low molecular weight
thiol in plant cells, with estimated concentrations in
chloroplasts from 1 to 4.5 mM (Rouhier et al. 2008). In
response to stress conditions, plants increase the
glutathione concentration and the activity of enzymes
that synthesize it. In fact, it has been found a strong
correlation between elevated glutathione concentra-
tions and the ability of plants to withstand the metal-
induced oxidative stress (Freeman et al. 2004; Singh et
al. 2006). Besides its role in maintenance of the redox
balance, glutathione acts as a powerful detoxifier of
toxic metals and xenobiotics through conjugation
reactions and it can serve as a precursor of phytoche-
latins (Blokhina et al. 2003; Grato et al. 2005;
Rouhier et al. 2008).
In the present work, it was found a higher total
glutathione content (GSHT) in shoots than in roots,
which could be attributable to the occurrence of
photosynthesis in green tissues, one of the main
pathways of ROS production in the aerobic metabo-
lism (Mittler 2002; Apel and Hirt 2004). This result
could be also related to the higher MDA concentra-
tion in shoots, where an increase in ROS production
could be inducing the synthesis of antioxidants such
as glutathione. GSH is oxidized by ROS to produce
GSSG, which is further reduced to GSH in a reaction
catalyzed by the GR (Grato et al. 2005). As GSSG
dissociates upon reduction into two GSH units, its
influence on the cellular redox status depends on both
the total glutathione concentration and the GSH/
GSSG ratio (Rouhier et al. 2008). Consequently, the
balance between GSH and GSSG is a suitable
indicator of oxidative stress in living cells, being a
Plant Soil (2011) 339:377389
high GSH/GSSG ratio essential for the effective
elimination of ROS in plant cells (Apel and Hirt
2004; Meyer 2008).
The significant increase in both the GSHT produc-
tion and the GSH/GSSG ratio in shoots of seedlings
exposed to 250 mg Pb2+ L1 compared to the control,
suggests that A. farnesiana could withstand the lead-
induced oxidative stress through mechanisms related
to glutathione metabolism, which could be also
related to the hormesis effect in these seedlings. This
result also indicate that the production of the reduced
form of glutathione was about 6-fold higher than the
corresponding to the stoichiometric GSH/GSSG mass
ratio (1.003), which suggests that A. farnesiana
exposed to 250 mg Pb2+ L1 had an increased
reducing power. A high GSH/GSSG ratio helps plant
species in several physiological functions, which
include activation and inactivation of redox-
dependent enzyme systems and regeneration of
ascorbic acid (a powerful cellular antioxidant) under
oxidative stress conditions (Singh et al. 2006). Thus,
our results suggest that although the GSH/GSSG ratio
decreased with increasing Pb concentrations, the
antioxidant mechanism related to the GSH metabo-
lism in A. farnesiana was efficient, since at concen-
trations up to 500 mg Pb2+ L1 this ratio was above 1
(Table 2).
Similar to our results, Sun et al. (2005) observed
that increasing lead concentrations (from 10 to 93 mg
L1) significantly stimulated the GSH production (up
to 4-fold) in stems, leaves and roots of mine plants of
Sedum alfredii. Singh et al. (2006) found an increase
(~33%) in the GSH content in plants of Pteris vittata
exposed to arsenic (10 mg L1) during 5 days,
achieving a GSH/GSSG ratio of 1.68. However, as
the arsenic concentration increased, the GSSG content
was enhanced, yielding a reduction in the GSH/GSSG
ratio. These authors also found that the increase in the
GSH/GSSG ratio was related to a higher arsenic
tolerance.
Effect of Pb2+ on GR activity
The maintenance of a proper GSH/GSSG ratio in the
cells depends on GR, a ubiquitous enzyme that
catalyzes the NADPH-dependent reduction of GSSG
to GSH. Thus, GR activity is essential for the
glutathione redox cycle, since it maintains adequate
levels of the reduced cellular glutathione, which
Plant Soil (2011) 339:377389
serves as an antioxidant reacting with free radicals
and organic peroxides (Smith et al. 1988). After
30 days of exposure, GR activity in shoots of A.
farnesiana increased as lead concentration was
increased, attaining 1.2- to 1.6-fold more activity than
in the control seedlings. In roots, GR activity was
only significantly stimulated in seedlings grown at
1,000 Pb2+ L1 compared to the control. However,
after 60 days of exposure GR activity in tissues of A.
farnesiana was markedly reduced. There have been
reports that lead also cause an increase in GR activity
in maize calli and Oryza sativa (Verma and Dubey
2003). Reddy et al. (2005) observed that GR activity
was increased in two legume species (Macrotyloma
uniflorum and Cicer arietinum) treated with lead
(200800 mgkg1) during 30 days, finding higher
activities in roots than in shoots. Diwan et al. (2010)
also found an enhanced GR activity in shoots of
Vigna radiata in response to increasing concentrations
of Cr (2.610.4 mgL1) during a short period of
exposure. However, similar to what we observed, as
the time of exposure increased, the enzyme activity
decreased.
The increase on the GR activity in A. farnesiana
at 30 days of treatment may be attributed to an
increase of its substrate GSSG (Nouairi et al. 2009).
Thus, as the GR activity increases catalyzing the
reduction of GSSG to GSH, it confers a higher
tolerance to the plant. On the other hand, the marked
decrease registered in the GR activity after 60 days
of lead exposure, can be explained by the reaction of
Pb with sulfhydril groups and thereby the inactiva-
tion of thiol-containing enzymes such as GR (Reddy
et al. 2005).
Conclusions
The present study demonstrates that A. farnesiana can
be potentially used for phytoremediation of metal-
polluted sites; particularly, for lead phytostabilization
because of its tolerance to relatively high amounts of
this metal and its ability to accumulate high concen-
trations in roots, limiting the metal translocation to
shoots. Seedlings grown with 250 and 500 mg Pb2+
L1 showed a marked increase in their growth,
leading to TI>100%. This effect indicates an adaptive
response occurring at certain concentrations of a
pollutant, known as hormesis, which induces low
387
levels of stress and activates existing mechanisms that
enhance the ability of A. farnesiana to withstand more
severe stresses. An EC50 between 720 and 766 mg
Pb2+ L1 was found.
This study demonstrates that A. farnesiana is able
to resist lead stress by mechanisms of exclusion and
tolerance. A. farnesiana accumulated 80% of Pb2+ in
roots (exclusion) and grew healthy at 500 mg Pb L1
(tolerance), even when lead was translocated to the
aerial tissues. These results corroborate the suitability
of A. farnesiana for Pb2+-phytostabilization purposes,
since relatively high BCFs (>8.5) and very low TFs
(0.03) were attained. Plants with this feature can be
used for revegetation of mine tailings, hence reducing
the dispersion of pollutants and minimizing the
transfer of metals into the food chain. To our
knowledge, this is the first study reporting the
potential capability of Acacia farnesiana for lead-
phytostabilization purposes.
Lead exposure did not have a significant effect
on lipid peroxidation in shoots of A. farnesiana,
suggesting a resistance mechanism against the heavy
metal stress. Signs of oxidative stress were detected
in roots exposed to 1,000 mg Pb L1, which could be
related to the high lead accumulation observed. The
enhanced level of GSH and the high GSH/GSSG
ratio, especially in shoots of seedlings grown at
250 mg Pb2+ L1 suggest that a mechanism to
withstand the Pb-induced oxidative stress in A.
farnesiana could be related to glutathione metabo-
lism. It seems that GSH has an active role in the
ROS detoxification, both directly (non-enzymatic)
and indirectly, through the action of GR.
Acknowledgments We would like to acknowledge to Con-
sejo Nacional de Ciencia y Tecnologia (CONACyT) for its
financial support in part of this research. A. Maldonado-
Magaa also thanks CONACyT for the financial support
(scholarship 211555).
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