This document summarizes a study that investigated the effects of chromium, cadmium, and lead on the growth, metal uptake, and antioxidant capacity of Typha angustifolia (narrow-leaved cattail). The study found that T. angustifolia showed high tolerance to 1 mM concentrations of Cr, Cd, and Pb with no visible toxicity symptoms. Exposure to Cd and Pb increased plant height and biomass, especially in Pb treatment. Cr, Cd, and Pb uptake increased along with increased uptake of nutrients like Ca and Fe. Antioxidant enzyme activities like SOD, POD, CAT, APX, and GPX increased in response to Cr, Cd, and Pb stress

1. Effects of Cadmium, Chromium and Lead
on Growth, Metal Uptake and Antioxidative
Capacity in Typha angustifolia
Alieu Mohamed Bah & Huaxin Dai & Jing Zhao &
Hongyan Sun & Fangbin Cao & Guoping Zhang &
Feibo Wu
Received: 1 February 2010 /Accepted: 1 June 2010 /
Published online: 16 June 2010
# Springer Science+Business Media, LLC 2010
Abstract This study investigates the modulation of antioxidant defence system of Typha
angustifolia after 30 days exposure of 1 mM chromium (Cr), cadmium (Cd), or lead (Pb).
T. angustifolia showed high tolerance to heavy metal toxicity with no visual toxic symptom
when exposed to metal stress, and Cd/Pb addition also increased plant height and biomass
especially in Pb treatment. Along with increased Cr, Cd, and Pb uptake in metal treatments,
there was enhanced uptake of plant nutrients including Ca and Fe, and Zn in Pb treatment.
A significant increase in malondialdehyde (MDA) content and superoxide dismutase (SOD)
and peroxidase (POD) activities were recorded in plants subjected to Cr, Cd, or Pb stress.
Furthermore, Pb stress also improved catalase (CAT), ascorbate peroxidase (APX), and
glutathione peroxidase (GPX) activities; whereas Cr stress depressed APX and GPX. The
results indicate that enzymatic antioxidants and Ca/Fe uptake were important for heavy
metal detoxification in T. angustifolia, stimulated antioxidative enzymes, and Ca, Fe, and
Zn uptake could partially explain its hyper-Pb tolerance.
Keywords Antioxidative enzyme . Heavy metal . Nutrition . Tolerance .
Typha angustifolia L
Introduction
Soil heavy metal contamination has become an increasing problem worldwide [1–3].
Among the heavy metals, cadmium (Cd), lead (Pb), and chromium (Cr), in particular,
causes increasingly international concern. For example, in the United States, 1,200 sites are
on the National Priority List (NPL) for the treatment of polluted soils, and about 63% of the
NPL sites are from toxic heavy metals including Pb, Cr, and Cd of 15%, 11%, and 8%,
respectively, indicating the extensiveness of this problem [4]. Cadmium is one of the most
known mobile elements, and it can be readily taken up by plants and transferred to aerial
Biol Trace Elem Res (2011) 142:77–92
DOI 10.1007/s12011-010-8746-6
A. M. Bah :H. Dai :J. Zhao :H. Sun :F. Cao :G. Zhang :F. Wu (*)
Department of Agronomy, College of Agriculture and Biotechnology, Huajiachi Campus,
Zhejiang University, Hangzhou 310029, People’s Republic of China
e-mail: wufeibo@zju.edu.cn

2. parts where it can accumulate to high levels. Consequently, it can enter the food chain and
become detrimental to human and animal health [1, 5]. Lead has many known toxic effects
on human health and its potential hazards to flora and fauna are of specific concern due to
its relative abundance at contaminated sites [6]. Chromium (VI) is hazardous heavy metal
which causes membrane damage, ultrastructural changes in the organelles, disrupted
metabolic activities, growth inhibition as well as oxidative damage to lipids, proteins, and
nucleic acids [7]. It was estimated that in India alone about 2,000 to 3,200 t of elemental Cr
escapes from the tanning industries into the environment annually [8]. Excessive metal
concentrations in the contaminated soils can result in soil quality degradation, crop yield
reduction, and poor quality of agricultural products. Furthermore, unlike organic pollutants,
heavy metals cannot be degraded through biological processes [9], and given the
widespread distribution of Pb, Cr, and Cd in soil due to human activities and the potential
human and ecological risks posed by these metals, it is crucially important to develop cost-
effective remediation strategies for these metals. One of such emerging technology is
phytoremediation through the use of green plants to extract, sequester, and detoxify the
pollutants. Consequently, selection and screening of plant species which are tolerant to
toxic levels of toxic heavy metals has grabbed attention in the treatment of metal polluted
soils. Typha angustifolia (narrow-leaved cattail), a perennial macrophyte, is characterized
by its fast growth, high productivity, and remarkable resistance to high levels of heavy
metals in the soil [10, 11]. However, little research has been carried out on the
physiochemical responses of T. angustifolia under high levels of Cr, Cd, and Pb stress,
and the mechanisms responsible for its hypertolerance to heavy metal stress and
detoxification remain unknown.
The tolerance capacity of plants to heavy metals depends on an interrelated network of
physiological and molecular mechanisms. One of the mechanisms that make a plant species
hypertolerant to heavy metal stress is the presence of strong antioxidant defence system [12,
13]. Some authors reported that heavy metal, such as Pb, Cd, and Cr, stress induces
oxidative stress by generating free radicals and toxic reactive oxygen species (ROS) [14]
that may damage plant major cell macromolecules (proteins, lipids, and DNA) [15].
Correspondingly plants developed several antioxidative defense systems, which is mainly
composed of metabolites and scavenging enzymes of active oxygen, to scavenge toxic free
radicals to protect themselves from the oxidant stress induced by heavy metals [16].
Concerning enzymatic antioxidants, different or even controversial patterns of heavy metal
toxicity on the activity of antioxidant enzymes scavenging ROS, including superoxide
dismutase (SOD; EC1.15.1.1), peroxidase (POD; EC 1.11.1.7), catalase (CAT; EC
1.11.1.6), glutathione peroxidases (GPXs; EC 1.11.1.7), and ascorbate peroxidase (APX;
EC 1.11.1.11), were found [17–19] in different plant species, tissues analyzed,
concentration, and duration of metal exposure. For example, it has been demonstrated that
Cd activated CAT [20, 21], SOD and POD [19, 22], and APX [17]. Conversely, a decrease
in SOD [23], CAT [22], APX and GR [24] activities were also reported under Cd exposure.
Therefore, a precise knowledge would be useful about the change in Cr-, Cd-, and Pb-
induced oxidant stress and enzymatic antioxidant system in T. Angustifolia.
This paper reports the biochemical analyses from a greenhouse experiment on T.
Angustifolia subjected to high level of Cr, Cd, and Pb. The objectives of this study were
to: (1) investigate the effects of Cr, Cd, and Pb on growth and nutrient uptake in T.
Angustifolia; and (2) evaluate the effects of Cr, Cd, and Pb on the activities of enzymatic
antioxidants (SOD, POD, CAT, APX, and GPX), Glutathione (GSH), and phytochelatin (PC)
to understand the biochemical detoxification strategies adopted by this plant. The results
should be helpful in the elucidation of heavy metal detoxification mechanisms of this plant.
78 Alieu et al.

3. And this knowledge may constitute a basis for molecular breeding and genetic engineering of
Cr-, Cd-, or Pb-tolerant crops that can be used for phytoremediation purposes.
Materials and Methods
Plant Materials and Treatments
The pot experiment was carried out in May to November 2008. Agricultural soil was
collected from the experimental farm (depth 0–15 cm) in Huajiachi Campus of Zhejiang
University, Hangzhou, China. The soil was air-dried and mixed daily until 8% water
content was reached. Air-dried soil of 4 kg was weighed and loaded into a plastic pot (5 L,
20-cm height). Pots were kept in a greenhouse under natural light condition during 60 days
after sowing. The soil used in this investigation had a pH of 6.8, with the available heavy
metal concentrations [ethylenediaminetetraacetic acid (EDTA)-soluble] of Cr, Cd, and Pb
(1.67, 0.15, and 9.63 mg kg-1
, respectively). The textural analysis showed the following
composition: sand 65.0%, silt 28.8%, and clay 6.2%, which indicated that this soil could be
classified as silt loam.
Seeds of T. angustifolia were scattered in the above-mentioned pots and irrigated with
tap water to keep humid. At 60 days, seedlings were thinned to leave 15 uniform, healthy
seedlings per pot, and then pots were transferred to a growth incubator with light intensity
of 300 μm m-2
s-1
and day/night temperature of 25±0.5°C/22±0.5°C with 14 h of day time.
There were two application dates for heavy metal treatments: D90 and D130, in which
seedlings were allowed to grow for another 30 and 70 days (i.e., 90 and 130 days after
sowing), respectively, before Cd, Cr(VI), and Pb application. During this period (i.e., days
61–90 and 61–130 after sowing), soils in the pots were kept humid (90–100% water
holding capacity) for the first 20 and 60 days (days 61–80 and 61–120 after sowing),
respectively, and then irrigation was stopped to reach the water holding capacity at about
50%. When the soil became droughty (90 and 130 days after sowing for D90 and D130,
respectively), 500 ml of distilled water (control, no addition of heavy metal), 1 mM
K2Cr2O7, 1 mM CdCl2, and 1 mM Pb(NO3)2 solution were added to each pot, and 10 days
later, the corresponding solution was added again to form four treatments of control, Cr, Cd,
and Pb for the two different growth period plants of 90 and 130 days after sowing (denoted
as Control-D90, Cr-D90, Cd-D90, Pb-D90 and Control-D130, Cr-D130, Cd-D130, Pb-
D130, respectively). The experiment was laid in a randomized block design and two plants
from each pot were marked for final harvest with four replicates. The soil was kept humid
thereafter. All reagents were analytical grade and all stock solutions were made with
deionized water.
After 30 days of the first heavy metal application (the two different growth period plants
of 120 = 60 + 30 + 30 and 160 = 60 + 70 + 30 days after sowing), plants were sampled for
the determination of the following traits.
Measurements of Plant Height, Dry Weight, and Metal Concentration
Previously tagged plants (two plants from each pot) were gently removed from soil,
separated into shoots and roots (including undeveloped rhizomes), washed with tap water.
and then rinsed in distilled water. Plant height were simultaneously measured, and then
dried at 80°C and weighed. Dried shoots and roots were powdered and weighed, then ashed
at 550°C for 12 h. The ash was digested with 5 ml 30% HNO3 and then diluted using
Effects of Cadmium, Chromium and Lead on Growth 79

4. deionized water [25]. Cadmium and Cr, Pb, Ca, Fe, Mn,, Cu and Zn concentrations were
determined using SHIMADZU AA-6300 flame atomic absorption spectrometry [26].
Assay of MDA Content and Antioxidative Enzyme Activities
Shoots were cut 0.5 cm above surface of soil, washed thoroughly with deionized water,
then immediately frozen in liquid nitrogen, and stored frozen at -80°C for the determination
of malondialdehyde (MDA) contents and antioxidative enzyme activities.
The level of lipid peroxidation was quantified as MDA content and was determined
as 2-thiobarbituric acid (TBA) reactive metabolites [19]. Fresh shoot tissues were
homogenized and extracted in 10 ml of 0.5% thiobarbituric acid made in 5%
trichloroacetic acid. Extract was heated at 95°C for 30 min and then quickly cooled
with ice. After centrifugation at 10 000×g for 10 min, the absorbance of the supernatant
was measured at 532 nm. Correction of nonspecific turbidity was made by subtracting the
absorbance value taken at 600 nm. The level of lipid peroxidation was calculated using an
extinction coefficient of 155 mM cm-1
.
For the determination of enzyme activities, frozen plant tissue was homogenized in 8 ml
50 mM sodium phosphate buffer (PBS, pH 7.8) using a prechilled mortar and pestle, then
centrifuged at 10000×g for 15 min at 4°C. The supernatant was used for enzyme activity
assay. SOD (EC 1.15.1.1), POD (EC 1.11.1.7), and CAT (EC 1.11.1.6) activities were
determined [27]. For the analysis of APX (EC 1.11.1.11), ascorbate (AsA) was used as the
substrate, and the decrease in ascorbate concentration followed as a decline in optical density at
290 nm, and the activity was calculated using the extinction coefficient 2.8 (mM cm-1
) for
ascorbate [28]. GPX activity was assayed accordingly [29].
Determination of GSH, Cysteine, and PC Contents
Shoot samples were rinsed by tap water and fully rinsed by deionized water, then frozen in
liquid N2, and stored at -70°C until the analysis of PC and other low molecular weight -SH
rich peptides. The monobromobimane (mBBr) was prepared daily and stored at 4°C. Milli-
Q water (18.3 MΩ) was used. The tissues were ground in liquid N2 and the powdered
samples (about 0.2 g) were homogenized in 2 mL 0.1% (w/v) CF3CO2H (Triflouroacetic
Acid (TFA), Sigma) with 6.3 mM diethylenetriamine-pentaccetic acid (DTPA, Sigma). The
homogenate was centrifuged at 10,000×g for 10 min at 4°C. Supernatant (250 μL) was
mixed with 450 μL 200 mM N-[2-hydroxyethyl]piperazine-N′-[3-propane sulfonic acid]
(HEPPS, Sigma) buffer containing 6.3 mM DTPA (pH 8.2,) and 10 μL 25 mM mBBr
(Sigma, dissolved in acetonitrile (ACN)). Derivatization was then carried out for 30 min in
the dark at room temperature. The reaction was terminated by adding 300 μL of 1 M
methanesulfonic acid (MSA, Sigma). Then the samples were stored in the dark at 4°C for
high performance liquid chromatography (HPLC) analysis within the next 2 or 3 days.
Reagent blanks were used to identify the reagent peaks.
By using a binary gradient of mobile phase A (0.1% TFA) and B (100% ACN) at
room temperature, the samples were analyzed on a Agilent 1100 HPLC system with a
fluorescence detector at 380 nm/470 nm (excitation/emission). The C18 column
(Agilent XDB-C18, 5 μm, 4.6×250 mm) was adopted, and the flow rate was set at
1 ml min-1
. Derivatives (20 μL) were run with a linear gradient (12–25% B for 15 min,
then 25–35% B for 14 min, and subsequently 35–50% B for 21 min). Before injecting a
new sample, the column was cleaned (5 min, 100% B) and equilibrated (10 min, 12% B).
The post-time was 5 min, resulting in a total analysis time of 70 min. All solvents were
80 Alieu et al.

5. degassed before use. Retention time and concentrations of Cys, GSH, and PC were
checked with different levels of Cys, GSH, PC2, PC3, and PC4 mixed standard. PC2, PC3,
and PC4 standard were obtained from Shanghai Science Peptide Biological Technology.
The standards were run after every six samples to monitor the slight shift of PC peaks in
retention time. All reagents were of reagent grade.
Statistic Analysis
All data presented are the mean values. The measurement was done with three replicates on
metal concentrations and four replicates on all enzyme activities and MDA content.
Statistical analyses were performed with data processing system (DPS) statistical software
package [30] using ANOVA followed by the least significant difference (LSD) test to
evaluate significant treatment effects at significance level of P≤0.05.
Results
Effects of Cr, Cd, and Pb on Plant Height and Biomass of T. angustifolia
The effects of Cr, Cd, and Pb application on plant growth traits of T. angustifolia after
30 days exposure are shown in Fig. 1. Only shoots and roots were examined for growth
parameters, for the plants were at the vegetative growth stage without inflorescence
development. There were no visual symptoms of metal toxicity such as chlorosis or
necrosis on T. angustifolia shoots 30 days after heavy metal application for both application
date (D90 and D130, heavy metal was applied 90 and 130 days after sowing, respectively).
However, a significant reduction in plant height, shoot and root dry weight by 3.3%, 5.7%,
and 54.5% in the Cr-D90 and by 17.1%, 30.0%, and 43.7% in Cr-D130 treatments (1 mM
K2Cr2O7 with the first application date of 90 and 130 days after sowing) were observed, as
compared with that of the controls. In contrast, Cd and Pb application induced slight or
even significant increase in plant height and biomass. For example, plant height, shoot and
root dry weight increased by 22.5%, 47.2%, and 6.1% in Cd-D90 treatment compared with
control-D90; shoot and root dry weight also increased by 25.7% and 29.6% in Pb-D130
over control-D130.
Effect of Cr, Cd and Pb on Tissue Metal Concentration of T. angustifolia
Shoot metal concentration in T. angustifolia is summarized in Table 1. Compared with
the control, the addition of Cr, Cd, and Pb markedly increased shoot Cr, Cd, and Pb
concentrations correspondingly by 6,681%, 5,766%, and 3,976%, respectively, on
average of the two application dates (D90 and D130). In addition, Cr application of Cr-
D90 and Cr-D130 significantly increased Cd (cf. 26% and 38%), Mn (113% and
783%), and Cu (10% and 125%) concentrations; increased Fe by 64% in Cr-D90 than
that of controls; markedly decreased Pb concentration in Cr-D90; and did not
significantly effect Zn and Ca concentrations. Cd application of Cd-D90 and Cd-
D130 increased Ca (47% and 65%) and Mn (61% and 368%) concentrations than that
of controls, but it markedly decreased Cr (30%, 50%) and Zn (42%, 25%)
concentrations and had no significant effect on Fe concentrations. Pb application,
especially in Pb-D90 treatment, induced significant reduction in Mn concentration and
increase in Zn concentration.
Effects of Cadmium, Chromium and Lead on Growth 81

6. Root metal concentration in T. angustifolia is summarized in Table 2. Similar to the
revelations in shoot, exposure to Cr, Cd, and Pb significantly increased root Cr, Cd, and
Pb concentrations correspondingly by 24,933%, 12,314% and 4,923% on average of the
two application dates compared with the controls. Furthermore, the treatments of Cr-D90
and Cr-D130 had synergistic effects on Cd, Pb, Ca, Fe, and Zn concentrations and 211%,
151%, 45%, 285%, and 32% and 148%, 253%, 111%, 60%, and 9% higher over the
controls, respectively. Meanwhile, Cr-D130 application significantly increased Mn by
194%, but has no effect on Cu compared to the control. Cd application of Cd-D90 and
Cd-D130 significantly increased Ca, Fe, and Zn concentrations by 95%, 17%, and 22%
and 116%, 41%, and 4%, respectively, compared to the controls; while it significantly
0
10
20
30
40
50
60
70
80
90
Cont.-D130 Cr-D130 Cd-D130 Pb-D130
Plantheight(cm)
0
0.05
0.1
0.15
0.2
0.25
0.3
Cont.-D130 Cr-D130 Cd-D130 Pb-D130
Treatment
RootDW(gperplant)
0
10
20
30
40
50
60
70
80
90
Cont.-D90 Cr-D90 Cd-D90 Pb-D90
Plantheight(cm)
0
0.05
0.1
0.15
0.2
Cont.-D90 Cr-D90 Cd-D90 Pb-D90
Treatment
RootDW(gperplant)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Cont.-D130 Cr-D130 Cd-D130 Pb-D130
ShootDW(gperplant)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Cont.-D90 Cr-D90 Cd-D90 Pb-D90
ShootDW(gperplant)
Fig. 1 Plant height, root length, and dry weight of T. angustifolia after 30 days exposure to Cr, Cd, and Pb.
Cont., Cr, Cd, and Pb correspond to distilled water (no addition of heavy metal), 1 mM K2Cr2O7, 1 mM
CdCl2, and 1 mM Pb(NO3)2, respectively; D90 and D130 refer to the first application date of heavy metals:
90 and 130 days after sowing. Error bars represent SD values
82 Alieu et al.

7. Table1EffectofCr,Cd,andPbAdditiononMetalConcentration(mgkg-1
DW)inShootsofT.angustifoliaPlantsfortheTwoApplicationDates
TreatmentCrCdPbCaFeMnCuZn
Control-D900.37±0.03c0.19±0.01b0.45±0.03b6861.1±1.48ab21.2±2.44b133.7±1.32b89.8±1.48b55.8±1.36bc
Cr-D9029.1±0.14a0.24±0.03b0.16±0.03b7183.2±0.42ab34.7±0.51a285.0±0.55a98.7±0.49a38.3±1.92c
Cd-D900.26±0.01d9.05±0.21a0.44±0.08b10074.0±1.56a19.9±0.76b214.6±2.64ab48.2±1.78d32.2±1.39c
Pb-D900.53±0.01b0.44±0.04b8.54±0.44a4109.9±0.35b19.5±0.44b32.2±0.64c64.5±0.62c74.9±0.95a
Control-D1300.48±0.01b0.16±0.07d0.11±0.47b4765.5±0.99b25.9±0.46a21.1±1.15c23.3±0.07b36.9±3.79ab
Cr-D13027.35±0.21a0.22±0.04c0.18±0.03b6392.7±2.90ab24.2±0.22a186.3±3.51a52.3±0.90a42.4±1.76a
Cd-D1300.24±0.01c11.15±0.49a0.13±0.01b7862.4±3.89a15.7±1.66a98.7±0.20b38.9±0.70ab27.8±0.06c
Pb-D1300.21±0d0.26±0.11b6.88±0.16a3475.9±1.34b17.7±0.4a20.4±0.11c21.8±0.70b30.8±2.61bc
Dataweremeansofthreeindependentreplications(means±SD).abc,differentlettersindicatesignificantdifferences(P<0.05)amongthefourtreatmentsandrefertoeach
subsetofdata.Control,Cr,Cd,andPbcorrespondtodistilledwater(noadditionofheavymetal),1mMK2Cr2O7,1mMCdCl2,and1mMPb(NO3)2,respectively,andapplied
for2times;D90andD130referredtothefirstapplicationdateofheavymetals,i.e.,90and130daysaftersowing
Effects of Cadmium, Chromium and Lead on Growth 83

8. Table2EffectofCr,CdandPbAdditiononMetalConcentration(mgkg−1
DW)inRootsofT.angustifoliaPlantsfortheTwoApplicationDates
TreatmentCrCdPbCaFeMnCuZn
Control-D901.15±0.24c0.79±0.11d0.90±0.17c6088.0±2.83d8.4±0.79c19.1±1.64ab8.7±0.08b59.0±2.86b
Cr-D90449.80±1.41a2.46±0.14b2.26±0.40b8803.8±1.20c32.3±0.39a14.6±0.67b9.3±0.33b77.7±2.49b
Cd-D901.34±0.11d108.0±0.28a0.81±0.03d11850.0±1.98b9.8±0.10c8.1±0.16c7.0±0.02b71.8±0.33b
Pb-D901.26±0.14b1.11±0.31c16.17±0.11a18216.2±2.97a21.0±0.75b20.5±0.91a21.2±0.30a240.8±2.30a
Control-D1302.12±0.14b0.48±0.16d0.34±0.04d786.8±3.11b71.4±0.64b24.8±0.05bc25.4±0.54b61.1±0.69b
Cr-D130232.20±0.49a1.19±0.07c1.20±0.01b1662.2±1.06a114.4±0.03a72.9±0.57a22.4±0.11b66.9±0.52ab
Cd-D1301.49±0.06c53.55±1.06a1.15±0.04c1697.2±0.42a100.4±0.01ab26.8±1.55b66.1±1.10a63.5±1.21ab
Pb-D1301.25±0.03d1.44±0.52b28.05±0.35a1594.9±2.26a115.1±1.77a22.8±1.39c34.8±1.26b71.3±1.84a
Dataaremeansofthreeindependentreplications(Means±SD).Differentletters(abc)indicatesignificantdifferences(P<0.05)amongthefourtreatmentsandrefertoeach
subsetofdata.Control,Cr,CdandPbcorrespondtodistilledwater(noadditionofheavymetal),1mMK2Cr2O7,1mMCdCl2and1mMPb(NO3)2,respectively,andapplied
fortwotimes;D90andD130refertothefirstapplicationdateofheavymetals,i.e.,90and130daysaftersowing
84 Alieu et al.

9. decreased Pb, Mn, and Cu concentrations by ca. 10%, 58%, and 20% in Cd-D90 and
Cr by 30% in Cr-D130, respectively. Pb application on the two dates (D90 and D130)
showed synergistic effects as all the parameters measured were significantly increased
except that in Pb-D130, Cr was decreased markedly by 41% compared to the control.
Correlations Among the Eight Elements
The relationship among the eight elements was analyzed for their shoot and root
concentrations (Table 3). In the roots a significantly positive correlation with Ca was only
detected in Pb. The significant correlation also occurred between Cu and Cr, Mn. In
addition, Zn was significantly correlated with Pb, Ca. The significantly positive correlation
was observed between Fe and Cd or Mn and between Zn and Pb. However, there was
strong negative correlation between Zn and Ca. Meanwhile, in the shoot a significant
positive correlation occurred between Cu and Cr, between Ca and Mn, and between Cr and
Fe. Furthermore, Ca was significantly correlated with Cd. The results also showed that Pb
was strongly negatively correlated with Ca and Cu, respectively.
Effect of Cr, Cd, and Pb Stress on Lipid Peroxidation
The level of MDA in shoots of T. angustifolia is shown in Fig. 2. Exposure to Cr, Cd, and
Pb resulted in a significant increase in MDA content especially in Cd treatment compared
to the control. Further analysis revealed that the level of MDA content increased by 37.2%,
89.9%, and 41.9% in Cr, Cd, and Pb treatments (average of the two application dates of
D90 and D130), respectively, compared with controls.
Effect of Cr, Cd, and Pb Stress on Antioxidant Enzyme Activities
Plants treated with Cr, Cd, and Pb showed significant increase (P<0.05) in the activities of
SOD and POD relative to control (Fig. 3). The increase was in the range of 2.3–37.4% and
17–855%, respectively, compared with the controls. Furthermore, Cd treatment induced the
highest SOD activity, but the least increase in POD activity over the control; while Pb
treatment induced the highest POD activity, but the least increase in SOD activity.
Figure 4 shows the activities of APX, GPX, and CAT and as affected by Cr, Cd, and Pb.
Ascobate peroxidase activity in the shoots of Cd- and Pb-treated plants increased compared
with controls, reading 20.0%, 4.1% higher for the first sampling time (D90), and 4.0%,
25.4% for D130, respectively. However, Cr treatment resulted in decreased APX activity
(cf. 15.7% and 14.5%) relative to the control.
Glutathione peroxidase activity in the shoots of Pb-treated plants increased significantly
compared with controls, being 10.7% and 20.4% higher for D90 and D130 sampling times,
respectively. No significant difference was found between Cd treatment and control. Whereas,
Cr-D130 induced 25.7% significant reduction in GPX activity though no difference in D90.
Catalase activity in the shoots of Pb-treated plants was significantly higher (P<0.05)
than in the control (14.2% and 90.1% for Pb-D90 and Pb-D130). However, there was no
significant difference between Cr- or Cd-treated plants and controls (Fig. 4).
Effect of Cr, Cd, and Pb Stress on Free Cysteine, GSH, and PC Contents
Fluorescence HPLC analysis revealed that no PC was produced in shoots of T. angustifolia
under Cd, Cr, or Pb stress and control, indicating that PC does not contribute to Cd, Cr, or
Effects of Cadmium, Chromium and Lead on Growth 85

10. Pb tolerance in this plant. That is in agreement with Wójcik et al. [31] who reported that PC
production was shown to be not responsible for the primary mechanism of Cd tolerance in
the Zn/Cd hyperaccumulator Thlaspi caerulescens.
The levels of free cysteine and GSH in T. angustifolia shoots are shown in Fig. 5.
Exposure to Cr, Cd, and Pb resulted in a significant decrease in GSH content, on average
0
2.5
5
7.5
10
12.5
15
0
2.5
5
7.5
10
12.5
15
Cont-D90 Cr-D90 Cd-D90 Pb-D90
Treatment Treatment
Cont-D130 Cr-D130 Cd-D130 Pb-D130
MDAcontent(µmolgFW)-1
MDAcontent(µmolgFW)-1
Fig. 2 Effect of Cr, Cd, and Pb on MDA content in shoots of the T. angustifolia plants. Cont., Cr, Cd, and
Pb correspond to distilled water (no addition of heavy metal), 1 mM K2Cr2O7, 1 mM CdCl2, and 1 mM Pb
(NO3)2, respectively; D90 and D130 refer to the first application date of heavy metals: 90 and 130 days after
sowing. Error bars represent SD values
Table 3 Correlations Among the Eight-element Concentrations in Plants of T. angustifolia
Cr Cd Pb Ca Fe Mn Cu Zn
Root concentration Root concentration
Cr 1
Cd −0.28 1
Pb −0.25 −0.3 1
Ca −0.21 −0.12 0.55* 1
Fe −0.18 0.64** 0.03 −0.47 1
Mn 0.33 0.14 −0.04 −0.75** 0.68** 1
Cu 0.78** −0.15 −0.1 0.45 0.2 0.64** 1
Zn 0.03 −0.29 0.67** 0.79** −0.23 −0.41 −0.24 1
Shoot concentration Shoot concentration
Cr 1
Cd −0.25 1
Pb −0.24 0.21 1
Ca −0.01 0.65** −0.55* 1
Fe 0.25 −0.13 −0.32 0.37 1
Mn 0.52* −0.2 −0.32 0.09 0.53* 1
Cu 0.74** 0.34 −0.48* 0.58* 0.44 0.41 1
Zn −0.08 −0.34 −0.02 −0.34 0.38 0.02 −0.2 1
Correlation is significant at the 0.05 (*) and 0.01 (**) levels, respectively
86 Alieu et al.

11. of the two application dates, being 15.6%, 19.5%, and 36.7%, respectively, lower than the
control. The content of cysteine of Cr and Cd treatments was 22.3%, 39.1% lower than
the control on average of the two application dates, respectively. However, Pb treatment
showed 52.1% marked increase in free cysteine compared to the control.
Discussion
Recently, heavy metal accumulation in biotic systems, as a consequence of human
activities, is becoming a major environmental issue worldwide, particularly, in agricultural
ecosystems, where it might endanger crop productivity and quality [23, 32, 33]. The
remediation of metal contaminated soils is urgently imperative because metals will persist
almost indefinitely in the environment due to its being not biodegradable [11, 33–36].
T. angustifolia, characterized by its remarkable resistance to high levels of heavy
metals in the soil, is one of the reasonable candidates for the induced phytoextraction
0
50
100
150
200
250
300
350
Cont.-D130 Cr-D130 Cd-D130 Pb-D130
0
50
100
150
200
250
300
350
Cont.-D90 Cr-D90 Cd-D90 Pb-D90
Cont-D130 Cr-D130 Cd-D130 Pb-D130Cont-D90 Cr-D90 Cd-D90 Pb-D90
SODactivity(unitg-1
FW)
SODactivity(unitg-1
FW)
0
0.5
1
1.5
2
2.5
3
3.5
0
1
2
3
4
5
6
7
Treatment Treatment
PODactivity(OD470g-1
FWmin-1
)
PODactivity(OD470g-1
FWmin-1
)
Fig. 3 Effect of Cr, Cd, and Pb on SOD and POD activities in shoots of the T. angustifolia plants. Cont., Cr,
Cd, and Pb correspond to distilled water (no addition of heavy metal), 1 mM K2Cr2O7, 1 mM CdCl2, and
1 mM Pb(NO3)2, respectively; D90 and D130 refer to the first application date of heavy metals: 90 and
130 days after sowing. Error bars represent SD values
Effects of Cadmium, Chromium and Lead on Growth 87

12. 0
0.2
0.4
0.6
0.8
1
1.2
Cont.D90 Cr-D90 Cd-D90 Pb-D90
APX(mmolg-1
FWmin-1
)
0
0.2
0.4
0.6
0.8
1
1.2
Cont.D130 Cr-D130 Cd-D130 Pb-D130
Cont.D90 Cr-D90 Cd-D90 Pb-D90 Cont.D130 Cr-D130 Cd-D130 Pb-D130
Cont.D90 Cr-D90 Cd-D90 Pb-D90 Cont.-D130 Cr-D130 Cd-D130 Pb-D130
0.0
0.2
0.4
0.6
0.8
1.0
1.2
GPX(mmolg-1
FWmin-1
)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Treatment Treatment
CATactivity(unitg-1FWmin-1)
APX(mmolg-1
FWmin-1
)GPX(mmolg-1
FWmin-1
)CATactivity(unitg-1FWmin-1)
0
0.2
0.4
0.6
0.8
1
1.2
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Fig. 4 Effect of Cr, Cd, and Pb on APX, GPX, and CAT activities in shoots of the T. angustifolia plants.
Cont., Cr, Cd, and Pb correspond to distilled water (no addition of heavy metal), 1 mM K2Cr2O7, 1 mM
CdCl2, and 1 mM Pb(NO3)2, respectively; D90 and D130 refer to the first application date of heavy metals:
90 and 130 days after sowing. Error bars represent SD values
88 Alieu et al.

13. of metal polluted soils [9, 10, 37]. However, little information is available about the
physiological responses of this plant to heavy metal toxicity. The present study
reported the effects of Cr, Cd, and Pb stress on plant growth, element accumulation in
T. angustifolia, and portrayed a general picture of antioxidant metabolism in response to
Cr, Cd, and Pb stress in order to understand the biochemical detoxification strategies
adopted by this plant against oxidative stress induced by heavy metal stress. No visual
symptoms of metal toxicity of chlorosis and necrosis were observed on the shoots of this
plant grown in 1 mM Cr, Cd, or Pb artificially polluted soil, which added evidence for its
high tolerance to heavy metal toxicity in agreement with previous reports [9, 11]. Plants
have mechanisms that allow them to tolerate relatively high concentration of Pb in
their environments without suffering any of these toxic effects. [38]. This was evident
in our research as 1 mM Cd or Pb induced slight or even significant increase in plant
height and root/shoot dry weight especially in Pb treatment (Fig. 1), demonstrating its
hypertolerance to Cd and Pb stress. Cr treatment, however, caused significant reduction in
the plant height and dry weights of both the plant parts. This reduction could possibly be
related to high Cr concentration in plant tissues, since the plant may have to use energy to
cope with the high Cr concentration in the tissues [38]. Nevertheless, no toxicity
0
10
20
30
40
50
60
Cont.-D130 Cr-D130 Cd-D130 Pb-D130
Cysteinecontent(µmolSHg
-1
FW)
Cysteinecontent(µmolSHg-1
FW)
0
10
20
30
40
50
60
Cont.-D90 Cr-D90 Cd-D90 Pb-D90
Cont.-D130 Cr-D130 Cd-D130 Pb-D130Cont.-D90 Cr-D90 Cd-D90 Pb-D90
0
50
100
150
200
250
300
350
400
0
50
100
150
200
Trearment Trearment
GSHcontent(µmolSHg-1
FW)
GSHcontent(µmolSHg-1
FW)
Fig. 5 Effect of Cr, Cd, and Pb on cysteine and GSH contents in shoots of the T. angustifolia plants.
Control, Cr, Cd, and Pb correspond to distilled water (no addition of heavy metal), 1 mM K2Cr2O7, 1 mM
CdCl2, and 1 mM Pb(NO3)2, respectively; D90 and D130 refer to the first application date of heavy metals:
90 and 130 days after sowing. Error bars represent SD values
Effects of Cadmium, Chromium and Lead on Growth 89

14. symptoms were observed for the plants, implying that this T. angustifolia tolerated Cr at
1 mM in this soil.
In the present study, Cr, Cd, and Pb uptake was significantly enhanced with the
application of Cr, Cd, and Pb in the soil. Regardless of the application date, Cr, Cd, and Pb
concentrations were higher in roots than in the shoots (Tables 1 and 2), implying that a
considerable amount of Cr, Cd, and Pb was retained in roots, and our results was
corroborated with other researchers who reported higher metal accumulation in roots than
shoots [39, 40]. Along with Cr, Cd, and Pb uptake in T. angustifolia, there was enhanced
uptake of plant nutrients including Ca and Fe. In addition, Pb treatment also markedly
improved Zn uptake (Tables 2). The fact that Cr, Cd, and Pb addition enhanced plant Ca
and Fe uptake as well as promoted greater Zn in the Pb treatments suggests that Ca and Fe
may play a role in Cr, Cd, and Pb detoxification by T. angustifolia, and that increased Zn
uptake may also contribute to its hyper-Pb tolerance as recorded in the increased biomass
over the control.
As lipid peroxidation is ascribed to oxidative damage, measurement of MDA level is
routinely used as a sensitive index of oxidative stress under stress conditions [41]. In the
present study, MDA content increased significantly when plants were subjected to Cr, Cd,
or Pb, compared with controls. In addition, there was a noticeable difference in the
alternation of MDA content caused by heavy metals exposure among the three metals. Cd
stress had more increase in MDA content than Cr and Pb. Heavy metals caused molecular
damage to plant cells either directly or indirectly through the formation of AOS [42, 43].
Enhancement of O2
*-
can produce the hydroperoxyl radical (*
OH, H2O2), which in turn
convert fatty acids to toxic lipid peroxides, destroying biological membranes. Increased
MDA in Cr, Cd, and Pb treatments, accordingly, suggests that Cr, Cd, and Pb stimulate
lipid peroxidation, resulting in oxidative stress.
To protect against oxidative stress, plants evolutionally developed enzymatic and
nonenzymatic ROS scavenging systems. These systems play a crucial role in protecting the
structure and function of membrane systems and maintaining cellular redox state [44]. Our
results demonstrated that in T. angustifolia plants subjected to 1 mM Cr, Cd, and Pb,
antioxidant defence mechanisms were activated, and different responses among the three
metals were also observed. Cd stress induced increase in SOD, POD, and APX activities,
but with no effect on GPX and CAT; Cr stress stimulated SOD and POD activities, but
depressed APX and GPX activities. Interestingly, the activities of SOD, POD, CAT, APX,
and GPX were significantly improved in the plants exposed to Pb stress (Figs. 3 and 4),
which may play a key role in Pb detoxification of T. angustifolia. The data obtained in this
study appear to support that enzymatic antioxidants were important for heavy metal
detoxification in T. angustifolia, and that Pb-induced increase in SOD, POD, CAT, APX,
and GPX could partially explain its higher tolerance to Pb stress. Our results was in
agreement with others, as when plants are in stress condition, the free radical species (forms
of active oxygen) may be increased, which will enhance the activities of these detoxifying
enzymes. The activities of SOD, CAT, and POD are induced in plants species by heavy
metals [18, 43, 45, 46]. In addition, improved Ca, Fe uptake may play a role in Cr, Cd, and
Pb detoxification by T. angustifolia, and increased Zn uptake may also contribute to its
hyper-Pb tolerance.
Acknowledgements This study was supported by the National Natural Science Foundation of China
(30671256). We appreciate Mr. Fei Chen from Agronomy Department of Zhejiang University, for his helpful
assistance during the experimental work.
90 Alieu et al.

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