Chemosphere 60 (2005) 22–31

www.elsevier.com/locate/chemosphere

Effects of exogenous organic chelators on

phytochelatins production and its relationship with
cadmium toxicity in wheat (Triticum aestivum L.)
under cadmium stress
Q. Sun a, X.R. Wang a,*
, S.M. Ding b, X.F. Yuan a

a
State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Nanjing University,
22 Hankou Road, Nanjing 210093, PR China
b
Institute of Geographical Sciences and Natural Resources Research, CAS, Beijing 100101, PR China

Received 20 July 2004; received in revised form 7 October 2004; accepted 26 October 2004
Available online 29 January 2005

Abstract

Phytochelatins (PCs) have been proposed as a potential biomarker for metal toxicity. In this study, cadmium (Cd)
toxicity, PCs production and their relationship in wheat under Cd stress were examined using various exogenous
organic chelator-buffered nutrient solutions. Single Cd stress produced strong toxic effects, as indicated by decreases
of growth parameters, high level of lipid peroxidation in leaf and overproduction of PCs in root. Exogenous organic
chelators with proper dose more or less reduced Cd toxicity by increasing growth parameters and decreasing lipid per-
oxidation in leaves. Of organic chelators (EDTA, DTPA, citric acid, malic acid and oxalic acid), EDTA was the most
effective in decreasing Cd toxicity in plants, followed by DTPA and citric acid. Simultaneously, the concentrations of
Cd-induced PCs in roots decreased, and the greatest decrease was caused by application of EDTA and DTPA. Linearly
positive relationships were observed between Cd toxicity and root PCs concentrations under the influences of organic
chelators, particularly EDTA, DTPA and citric acid. Furthermore, present results provide stronger evidence that PCs
synthesis in plant cells was related to free Cd ion concentrations, not total Cd, and demonstrate that the levels of PCs
production in plants correlated well with toxic effects caused by the bioavailable Cd levels.

2005 Elsevier Ltd. All rights reserved.

Keywords: Organic chelator; Phytochelatins; Cadmium toxicity; Biomarker; Wheat (Triticum aestivum L.)

1. Introduction

Increasing environmental pollution caused by heavy

metals, due to industrial and agricultural activities, is
*
Corresponding author. Tel.: +86 25 83595222; fax: +86 25 becoming a significant problem in the modern world.
83707304. Of heavy metals, cadmium (Cd) is a potentially
E-mail addresses: sunqinhf@sohu.com (Q. Sun), ekxr@ important environmental contaminant, particularly
nju.edu.cn (X.R. Wang). where there is high anthropogenic pressure. According

0045-6535/$ - see front matter
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2004.10.068
 Q. Sun et al. / Chemosphere 60 (2005) 22–31
to statistics, the farmland polluted by Cd in China has
reached 20 · 104 hm2 and produced 14.6 · 108 kg agri-
cultural products every year (Li et al., 2003). The bioac-
cumulation of Cd in food chain and biological
circulation can be highly dangerous to all organisms.
It is thus of greater concern to decrease Cd uptake
and accumulation in higher plants, especially in cereals
aimed for food production. For the purpose of the safe
crop production, it is not enough to predict yield reduc-
tion or quality decline due to excess heavy metals in soil
only based on chemical soil analysis. The use of bio-
marker, such as phytochelatins, specially induced in
plants upon exposure to heavy metals, may be an addi-
tional tool or diagnostic criterion in metal research and
in practice (Keltjens and van Beusichem, 1998b).
Phytochelatins (PCs) are a family of non-protein
thiol-rich peptides including classic PCs with a general
structure of (c-Glu-Cys)n-Gly (n = 2–11) and iso-PCs,
in which C-terminal Gly is absent or replaced by other
amino acid such as Ser, Glu, b-Ala, Gln (Rauser, 1995;
Kubota et al., 2000). Despite the structural differences
in individual PCs species, they perform identical physio-
logical functions in plants, owing to common nature of
increasing repetition of c-Glu-Cys dipeptide. Inducible
PCs are catalyzed by a specific enzyme, named as PCs
synthase, which is activated by the presence of heavy
metals in the cytoplasm (Grill et al., 1989; Loeffler
et al., 1989), and use glutathione (GSH) or PCn 1 as a
substrate. It seems that PCs synthesis originates from
heavy metal uptake. In fact, since Grill et al. (1985,
1987) revealed that the levels of PCs in plants increased
under heavy metal stress, considerable interest has been
attracted to PCs as a potential biomarker for metal
toxicity due to its unique responsive properties. For this
purpose, numerous researchers have examined the direct
relationships between metal exposure and PCs synthesis
(Schat and Kalff, 1992; De Knecht et al., 1995; Keltjens
and van Beusichem, 1998a,b; Sneller et al., 1999), which
largely strengthens the potentiality of PCs as a bio-
marker. However, the definitive role of PCs in assessing
metal toxicity has not been demonstrated, and there
has been considerable debate concerning the reliability
of PCs as an acceptable biomarker. For example, it
was reported that no linear relationship occurs between
PCs levels and the concentrations of Cd, Cu, Mn and
Zn in some phytoplankton from natural lake (Knauer
et al., 1998). Ernst et al. (2000) found that when plants
are grown on soils rich in Cu, the amount of PCs produc-
tion in the early vegetative phase can not be related to
vegetative and seed biomass at the reproductive stage.
Therefore, the possibility of PCs as a biomarker is wor-
thy of further investigation.
Biological effects of metals in plant tissues depend on
the hydrated metal ion or inorganic complexes rather
than on the total metal concentrations (Welch and
Norvell, 1999). This is also true of PCs production in
23
algae cell (Ahner et al., 1994). However, most previous
studies on metal toxicity and PCs production in plants
were conducted in metal-defined medium without differ-
entiating possible changes of metal speciation in this
complex system. Such studies are not reliable for assess-
ing effects and biochemical responses, given that the toxi-
city mechanisms of an element are strongly dependent
on its chemical forms.
Here, we investigated Cd toxicity, PCs and GSH con-
centrations in wheat exposed to Cd under the conditions
of exogenous organic chelators, which can change Cd
bioavailability in solution. Furthermore, the relation-
ship between PCs production and degree of Cd toxicity
was examined. To elucidate the effects of exogenous or-
ganic chelators on Cd bioavailability, VMINTEQ pro-
gram was used to calculate Cd and other essential
element species in nutrient solutions. In this experiment,
wheat was used as test material because of its critical
importance in agricultural field, which is one of the most
frequently consumed cereals in the world.
2. Materials and methods
2.1. Plant culture and experimental design
Wheat seeds (Triticum aestivum L., cv. Yangmai 10)
were purchased commercially. The seeds were surface-
sterilized with 75% ethanol for 10 min, washed thor-
oughly with tap water and distilled water, then placed
on moist filter paper. The filter paper was placed in an
illuminated incubator at 25 ± 1 C for germination.
After being kept in the dark for 2 d, the germinated
seedlings were transferred into nylon net and cultivated
with distilled water for 2 d, and then with half-strength
aerated Hoagland and Arnon nutrient solution
(pH = 6) for another 2 d. Afterwards, the uniform seed-
lings were transplanted into individual plastic pot (9
seedlings per pot) containing 1 l complete aerated Hoa-
gland and Arnon nutrient solution (pH = 6). Black plas-
tic pots were used to shade the light to minimize algal
growth in the nutrition medium. Plants were grown in
a growth chamber under controlled-environment condi-
tions with a 14 h/25 C day and 10 h/20 C night scheme,
and photo flux intensity of 700–800 lM m 2 s 1.
After 3-d preculture in individual pot, the seedlings
were exposed to 20 lM Cd (added as CdCl2) combined
with a range of organic chelators (abbreviated as
OCs). Detailed treatments were as follows: control (ex-
pressed as ck), 20 lM Cd + 0 lM OCs (T0), 20 lM
Cd + 10 lM OCs (T1), 20 lM Cd + 50 lM OCs (T2),
20 lM Cd + 100 lM OCs (T3) and 20 lM Cd + 500 lM
OCs (T4). Each treatment was triplicate. Organic chela-
tors examined included EDTA and DTPA and organic
acids (malic acid, citric acid and oxalic acid), all of
which were added as pure acids, except for EDTA as
24 Q. Sun et al. / Chemosphere 60 (2005) 22–31
Table 1
Free Cd ion concentrations (expressed as pMe) in various organic chelator-buffered Cd nutrient solutions
Concentrations of organic chelators (lM) EDTA
0 5.72
10 6.63
50 8.06
100 8.85
500 0 0
pM is equivalent to log[Me2+]. 0 indicates the nearly all Cd chelated by EDTA and DTPA.
Na2EDTA. In order to compare effects of organic chela-
tors on Cd toxicity and PCs production, the same levels
were used. The free Cd ion concentrations after exoge-
nous organic chelators were calculated using VMIN-
TEQ program (Visual MINTEQ, 2.15a based on
MINTEQA2; Brown and Allison, 1990) and are listed
in Table 1 (expressed as pMe = log[Me2+]). Notice that
Cd ion concentrations significantly decreased as addi-
tion of EDTA and DTPA increased, and to a lesser ex-
tent by organic acids. The treatment solutions (pH = 6)
were refreshed daily to maintain constant Cd and vari-
ous organic chelator concentrations, and also aerated
continuously. After 8 d, fresh leaves were sampled to
determine melondialodehyde (MDA) contents. One
day later the roots of all seedlings were immerged in cold
10 mM CaCl2 for 10 min to remove the adhering Cd in
root surfaces. After washing with distilled water, the
plant samples were cut into roots, stems and leaves,
and each tissue was subdivided into two parts. One
was immediately weighed and frozen in liquid nitrogen
and kept at 50 C for analysis of total non-protein
SH compounds and glutathione, and the other was for
analysis of Cd concentrations. Length of the longest
roots of each seedling was also recorded at all
treatments.
2.2. Determination of Cd toxic effects
The effects of Cd on wheat growth were evaluated by
determining root length, fresh weights of shoots and
roots, as well as levels of lipid peroxidation products
in leaves. The levels of lipid peroxidation in leaves were
reflected by melondialodehyde (MDA) contents assayed
according to the method described by He et al. (2003).
2.3. Determination of total non-protein SH compounds
Total non-protein SH compounds (TNP-SH) was ex-
tracted and assayed according to the method suggested
by De Vos et al. (1992). In short, TNP-SH was extracted
by homogenizing 0.5 g frozen plant materials with 2 ml
of a 5% 5-sulphosalicyclic acid (SSA) with 6.3 mM
diethylenetriaminepentaacetic (DTPA) at 0 C (using
mortar, pestle and quartz sand). The homogenate was
centrifuged at 12 000g at 4 C for 10 min. Clear superna-
DTPA Citric acid Malic acid Oxalic acid
5.72 5.72 5.72 5.72
6.81 5.72 5.73 5.73
0 5.73 5.73 5.74
0 5.74 5.74 5.75
5.81 5.78 5.81
tants were collected and immediately used for the assay
of TNP-SH.
The concentrations of TNP-SH were determined
using EllmanÕs reagent (DTNB). 300 ll of supernatant
was mixed with 630 ll of 0.5 M K2HPO4, final pH 7.5,
and the absorbance was measured at 412 nm (30 C).
After addition of 25 ll of DTNB solution (6.3 mM
DTNB in 0.143 M K2HPO4 and 6.3 mM DTPA, pH
7.5), the A412 was measured again after 2 min (€DTNB =
13 600 mol l 1 cm 1). The increase in absorbance was
corrected for the absorbance of DTNB. Cadmium did
not affect the increase in absorbance.
2.4. Determination of total glutathione
Total glutathione (GSH + GSSG) was extracted and
assayed according to the method reported by Hissin and
Hilf (1976) and Gupta et al. (1998). Frozen plant
materials were homogenized in 0.1 M sodium phos-
phate—0.005 M EDTA buffer (pH = 8.0) and 25%
metaphosphoric acid (used as protein precipitant). The
homogenate was centrifuged at 12 000g at 4 C for
15 min to obtain supernatant for total GSH determina-
tion. Total GSH was determined fluorometrically and
fluorescence intensity was recorded at 420 nm after
excitation at 350 nm on a Hitachi Fluorescence Spectro-
photometer (model no. 850).
2.5. Determination of Cd concentrations
In order to determine the total amount of Cd taken
up by plant tissues, 1 g of the fresh plant materials was
homogenized in a mortar and pestle and digested with
a mixture of HNO3/HClO4 (85/15, v/v). The concentra-
tions of Cd were determined by Atomic Absorption
Spectrophotometry (Hitachi Z-81001).
2.6. Data analysis
Index of Tolerance (IT) for the roots was calculated
following the protocol of Wilkins (1957): IT = (average
length of roots in tested solution/average length of roots
in control) · 100%. The relative inhibition rate (RIR) of
root elongation was defined as RIR = ((average length
of roots in control average length of roots in tested
 Q. Sun et al. / Chemosphere 60 (2005) 22–31 25

solution)/average length of roots in control) · 100% (i.e. 650

RIR = 1 IT). The levels of PCs production were calcu- 600
lated by subtracting the amount of GSH from the total

Root FW (mg plant-1)

550
amount of non-protein SH compounds. Because sub-
types of PCs peptides were not identified, PCs concen- 500
trations were expressed in SH units (i.e. PCs-SH) (De 450
Vos et al., 1992). All results were subjected to one-way 400
analysis of variance (ANOVA) in SPSS statistical pack- 350
age (version 10.0 for windows). Linear regression analy- 300 malic acid citric acid
sis was performed in Microsoft Excel. oxalic acid DTPA
250 EDTA
200
3. Results
400
malic acid citric acid
3.1. Effects of exogenous organic chelators on Cd 350 oxalic acid DTPA
toxicity in wheat EDTA

Root FW (mg plant-1)

300
3.1.1. Index of Tolerance (IT) of the roots 250
Root length of wheat exposed to Cd alone (T0) was
significantly inhibited by approximately 40%. However, 200
IT values of the roots increased to varying degree with 150
increasing levels of them after exogenous organic chela-
tors into Cd-treatment solutions (Fig. 1). These results 100
illustrated that the presence of organic chelators with 50
proper dose could decrease Cd toxicity. Of organic che- ck T0 T1 T2 T3 T4
lators tested, EDTA had the greatest effects, followed by Treatments of organic chelators
DTPA and citric acid in a concentration-dependent Fig. 2. Effects of exogenous organic chelators on fresh weights
manner, while malic acid and oxalic acid with the equiv- of shoots and roots of wheat exposed to 20 lM Cd for 9 d. Data
alent levels had slight effects (Fig. 1). represent means ± standard deviation (n = 3).

3.1.2. Shoot and root biomass

In order to further examine the condition of wheat
28% and 38%, respectively. However, they were raised
growth at all treatments, shoot and root biomasses were
more or less by proper application of organic chelators
investigated by measuring fresh weights. Compared with
(Fig. 2). Of organic chelators, EDTA was the most
the control plants, single Cd stress (T0) caused a signi-
effective.
ficant decrease in shoot and root fresh weights by about
3.1.3. Melondialodehyde (MDA) contents in leaves
Exposure to Cd alone (T0) resulted in a significant
110 MDA accumulation in leaf (Fig. 3), indicating that Cd
malic acid
citric acid induced strong oxidative damage in wheat. The levels
IT (Index of Tolerance, %)

100
oxalic acid of MDA in leaves showed a more or less decrease after
90 DTPA exogenous organic chelators into Cd-treatment solutions
EDTA (Fig. 3), implying that the presence of organic chelators
80 alleviates oxidative damage caused by Cd. The drastic
decreases of MDA contents in leaves were observed at
70 50 lM or higher levels of EDTA and DTPA applied,
whereas organic acids with the equivalent levels showed
60
weaker effects.
50
T0 T1 T2 T3 T4 3.2. Effects of exogenous organic chelators on Cd
Treatments of organic chelators uptake and accumulation in wheat
Fig. 1. Index of Tolerance (IT)—estimated by means of
WilkinsÕ test for wheat roots exposed to 20 lM Cd with Cadmium was mainly accumulated in roots of wheat
exogenous organic chelators for 9 d. Detailed treatments seen in seedlings, followed by stems and leaves (Fig. 4). Cad-
Section 2 (the same below). Data represent means ± standard mium concentrations in different tissues of wheat were
deviation (n = 3). significantly influenced by exogenous organic chelators
26 Q. Sun et al. / Chemosphere 60 (2005) 22–31

7.0 4B), suggesting that citric acid treatment could stimulate

MDA contents (nmol g-1 FW)
6.0
5.0
4.0
3.0
2.0
1.0
0.0
ck
Treatments of organic chelators
Fig. 3. Effects of exogenous organic chelators on MDA
contents in leaves of wheat exposed to 20 lM Cd for 8 d. Data
represent means ± standard deviation (n = 3).
(Fig. 4). Compared with Cd alone (T0), exogenous
DTPA and EDTA generated a sharp reduction of Cd
concentrations in leaves, stems and roots at 50 lM or
higher levels (Fig. 4D and E), while malic acid and oxa-
lic acid caused a significant decrease of Cd concentra-
tions in wheat tissues only at higher levels (Fig. 4A
and C), and such an effect was not as great as EDTA
and DTPA. However, a progressive increase of Cd con-
centrations was found in roots with increasing supply of
citric acid, whereas a decrease in stems and leaves (Fig.
Cd uptake from solution to root whilst inhibiting Cd
transport from root to shoot. These results indicated
that the presence of organic chelators in solution affects
Cd bioavailability with a high degree of variability and
ultimately Cd uptake by plants.
malic acid
citric acid
3.3. Effects of exogenous organic chelators on PCs-SH
oxalic acid and GSH concentrations in wheat
DTPA
EDTA Single Cd stress (T0) induced the overproduction of
T0 T1 T2 T3 T4 TNP-SH in root, which was 3-fold higher than that of
leaf. The increase of TNP-SH upon Cd exposure corre-
sponded to the production of PCs-SH, approximately
90% of which was accounted for by PCs-SH (Fig. 5).
Application of organic chelators markedly affected
PCs-SH concentrations in roots (Fig. 5F–J), but not in
leaves (Fig. 5A–E). PCs-SH concentrations in roots
drastically reduced to 0.676 and 0.879 lmol g 1 FW at
50 lM DTPA and EDTA respectively, which were close
to that of the control root, followed by little change at
higher levels. PCs-SH concentrations in roots also
showed obvious decreases at 100 lM or higher level of
citric acid, malic acid and oxalic acid applied, and a lar-
ger decrease was observed with citric acid treatment. Re-
sults further showed that the presence of organic
chelators reduced the bioavailable Cd levels in wheat,
which are responsible for Cd toxicity. Moreover, five or-

2.00 2.40 1.80

1.80 (A) 2.20 (B) 1.60 (C)
1.60 2.00
1.40
1.80
1.40 1.20
1.60
1.20 1.40 1.00
1.00 1.20
0.80
0.80 1.00
0.60 0.80 0.60
0.40 0.60 0.40
Cd (µmol g –1 FW)

0.40
0.20 0.20 0.20
0.00 0.00 0.00
ck T0 T1 T2 T3 T4 ck T0 T1 T2 T3 T4 ck T0 T1 T2 T3 T4

1.80 1.80
1.60 (D) 1.60 (E)
1.40 1.40
1.20 1.20
1.00 1.00
0.80 0.80
0.60 0.60
0.40 0.40
0.20 0.20
0.00 0.00
ck T0 T1 T2 T3 T4 ck T0 T1 T2 T3 T4
Treatments of organic chelators

Fig. 4. Effects of exogenous malic acid (A), citric acid (B), oxalic acid (C), DTPA (D) and EDTA (E) on Cd concentrations in leaves
(), stems (h) and roots (n) of wheat exposed to 20 lM Cd for 9 d. Data represent means ± standard deviation (n = 3).
 Q. Sun et al. / Chemosphere 60 (2005) 22–31 27

1.4 5
1.2 (A) (F)
4
1
0.8 3

0.6 2
0.4
1
0.2
0 0
1.4 3.5
(B) (G)
1.2 3
1 2.5
0.8 2
0.6 1.5
0.4 1
0.2 0.5
Non-protein SH compounds (µmol g–1 FW)

0 0
1.4 5
(H)
1.2 (C)
4
1
0.8 3

0.6 2
0.4
1
0.2
0 0
1.4 3.5
1.2 (D) 3 (I)

1 2.5
0.8 2
0.6 1.5
0.4 1
0.2 0.5
0 0
1.2 3.5
(E) 3 (J)
1
2.5
0.8
2
0.6
1.5
0.4 1
0.2 0.5
0 0
ck T0 T1 T2 T3 T4 ck T0 T1 T2 T3 T4
Treatments of organic chelators

Fig. 5. Effects of exogenous organic chelators on total non-protein SH compounds (TNP-SH, ), phytochelatins (PCs-SH, n) and
glutathione (GSH, h) in leaves (L) and roots (R) of wheat exposed to 20 lM Cd for 9 d. Panel A and F were supplied with malic acid,
panel B and G with citric acid, panel C and H with oxalic acid, panel D and I with DTPA, panel E and J with EDTA. Data represent
means standard deviation (n = 3).
28 Q. Sun et al. / Chemosphere 60 (2005) 22–31

ganic chelators tested displayed significant differences in 4. Discussion

their effects on Cd bioavailability and ultimately its toxi-
city, and EDTA and DTPA were more effective than
organic acids.
Single Cd stress (T0) also stimulated a larger produc-
tion of GSH in root than in leaf of wheat. After exoge-
nous organic chelators, changes of GSH in roots were
similar to that of PCs-SH. Interestingly, GSH concen-
trations in root and leaf at the concentration of
500 lM DTPA were higher than those of the other
DTPA concentrations. This may result from nutrient
deficiency or stress at the highest level of DTPA applied.
3.4. Relationships of PCs-SH concentrations and Cd
toxicity in wheat roots
Cadmium toxicity for all treated plants was quanti-
fied by the relative inhibition rate (RIR) of root elonga-
tion. Foliar symptom (chlorosis) observed at the highest
concentrations of DTPA in Cd-treatment solution and
drastic decreases of Cd uptake (Fig. 4D) in wheat sug-
gested that excess DTPA exerted an adverse effect on
plant growth. This datum point was excluded from the
regression. As shown in Fig. 6, linearly positive relation-
ships existed between root PCs-SH concentrations and
Cd toxicity in the presence of organic chelators. Nota-
bly, these relationships were more significant at a supply
of EDTA, DTPA and citric acid.
In a number of earlier studies PCs production has
been assessed by subtracting the amount of GSH from
the amount of total non-protein SH compounds (De
Knecht et al., 1992; De Vos et al., 1992; Schat and Kalff,
1992) and also adopted in recent years (Hartley-Whi-
taker et al., 2001a,b). Based on these experiments, the
amount of total non-protein SH compounds other than
GSH was taken as a measure of PCs production in this
study. Present results showed that most of non-protein
SH compounds induced by Cd in roots of wheat were
accounted for by PCs-SH (about 90%) (Fig. 5). This
was in good agreement with initial results by Grill
et al. (1985) and De Knecht et al. (1994). Furthermore,
it was found that PCs-SH was present in considerable
amounts in the control plants, as noted by Keltjens
and van Beusichem (1998b) and Maier et al. (2003). This
further supports hypothesis that aside from detoxifica-
tion, PCs play an important role in intracellular metal
homeostasis (Grill et al., 1985; Thumann et al., 1991).
It has been shown that PCs production is dependent
on the presence of heavy metals in the cell cytoplasm
(Grill et al., 1989; Loeffler et al., 1989). This can be
clearly demonstrated in this work. After addition of
EDTA, DTPA, malic acid and oxalic acid, the decreases
of Cd concentrations in wheat were accompanied by
those of PCs-SH concentrations in roots (Figs. 4 and
5). And these concomitant relationships were greatly

3.5 3 4
3 3.5
2.5
2.5 3
2 2.5
2
1.5 2
1.5 1.5
y = 0.1066x - 1.1817 1 y = 0.0849x - 0.5109 y = 0.2681x - 7.4041
1 1
PCs-SH (µmol g–1 FW)

R2 = 0.7927NS R2 = 0.9679** R2 = 0.6509NS

0.5 0.5 0.5
(A) (B) (C)
0 0 0
25 30 35 40 45 10 25 40 55 30 35 40
RIR (%)
3 3
y = 0.0667x - 0.1254
2.5 2.5
R2 = 0.9592**
2 2
1.5 1.5
1 1 y = 0.0431x + 0.9031
R2 = 0.9782**
0.5 0.5
(D) (E)
0 0
0 15 30 45 01 5 30 45
RIR (%) RIR (%)

Fig. 6. Relationships between Cd toxicity, quantified by the relative inhibition rate (RIR) of root elongation and PCs-SH
concentrations in roots of wheat exposed to 20 lM Cd with exogenous malic acid (A), citric acid (B), oxalic acid (C), DTPA (D) and
EDTA (E) for 9 d. Each point represents means of three replicates. In the regressive equation y and x indicate PCs-SH concentrations
and relative inhibition rate (RIR) of root elongation, respectively. R2 labeled by ‘‘**’’ indicate significant correlation at p = 0.01,
respectively. NS indicates no significant correlation.
 Q. Sun et al. / Chemosphere 60 (2005) 22–31
significant in the presence of EDTA and DTPA. It was
also reported that intracellular PCs concentrations in
the diatom are related to free Cd ion concentrations,
rather than its total concentrations (Ahner et al.,
1994). Present study provided convincing information
that this dependant relation was also present in plant
cells. Increasing supply of citric acid increased Cd up-
take by wheat roots (Fig. 4B), and yet led to a lower syn-
thesis of PCs-SH (Fig. 5G) and lower Cd toxicity (Figs.
1–3), indicating PCs synthesis in plant cells indeed de-
pends on free Cd levels, instead of total Cd contents.
It has been suggested that PCs synthesis will be termi-
nated once metals ion activating a PCs synthase are che-
lated either by the metal-free PC2 or PC7 or the addition
of a metal chelator such as EDTA (Loeffler et al., 1989).
According to the suggestion (Loeffler et al., 1989), it is
inferred that lower production of PCs with citric acid
treatments may be mainly due to the occurrence of
increasing Cd-citrate complexes in vivo, which causes a
decrease in cellular Cd ion concentrations and thus the
lowering of PCs production.
Greater effort has been made to correlate metal-con-
tents of soils and plants to toxic effects. However, in
most cases, the relationships were either poor or absent.
Our results clearly showed that the evaluation of Cd toxi-
city was inconsistent by measuring total Cd contents.
The presence of citric acid with proper dose in Cd-treat-
ment solutions significantly increased Cd concentrations
in wheat roots (Fig. 4B), but inversely alleviated Cd toxi-
city (Figs. 1–3). It has been demonstrated that most Cd
in tobacco leaves is located in the vacuole and com-
plexed by vacuolar organic acids (such as citrate, oxalate
and malate) (Krotz et al., 1989; Vögeli-Lange and Wag-
ner, 1990). Therefore, analyzing Cd by the total root
only reflects total Cd contents in roots, not the cytosolic
part of the Cd, which is involved in Cd toxic effects, so
that only metal analysis may lead to an overestimation
of metal toxicity.
Metal-induced PCs may function as a biomarker for
metal toxicity in plants, especially in crops (Keltjens and
van Beusichem, 1998a,b). For particular metals, e.g. Cd,
Cu and Pb, linear relationships between metal toxicity,
measured as growth inhibition, and expression of PCs
have been found in Silene vulgaris, Zea mays, Tristicum
aestivum L. and microalga Stichococcus bacillaris (Schat
and Kalff, 1992; De Knecht et al., 1995; Keltjens and
van Beusichem, 1998a,b; Sneller et al., 1999; Pawlik-
Skowrońska, 2002). Present results were further in favor
of a potential role for PCs related to metal toxicity.
Herewith, Cd toxicity was assessed by the relative inhi-
bition rate (RIR) of root elongation. Because plant roots
are the first point of contact for toxic metals, by measur-
ing processes occurring in roots we can clearly identify
how plants respond to toxic metals. Under various or-
ganic chelator-buffered conditions, the degree of Cd toxi-
city was positively correlated with Cd-induced PCs-SH
29
concentrations (Fig. 6). However, under the conditions
of exogenous malic acid and oxalic acid, these correla-
tions were not clear. Supplement of malic acid and oxa-
lic acid only slightly reduced Cd toxicity in wheat even at
the highest level (Figs. 1–3). Considering their lesser ef-
fects on Cd toxicity, the results were not surprising.
As a matter of fact, based on the laboratory data,
several studies in the field have been carried out to relate
metal toxicity to the level of PCs in plant. For example,
Grill et al. (1988) found that an elevated PCs concentra-
tion is observed in plants growing in areas of extreme
metal pollution near mine tailings. Later, Ahner et al.
(1994) found a positive relationship between PCs con-
centrations in marine phytoplankton and the distance
to Boston Harbour. Gawel et al. (2001) showed that
PCs are bioindicators of atmospheric metal exposure
via direct foliar uptake in three trees species growing
near Sudbury, Ontario and Canada. These practical re-
sults enhanced the potentiality of PCs as a biomarker to
assess metal toxicity.
Of organic chelators tested, apparent differences in
alleviating Cd toxicity were observed. Synthetic chela-
tors (EDTA and DTPA) had greater effects than organic
acids (Figs. 1–3). In particular, application of EDTA
with proper dose completely reduced Cd toxic effects.
The fundamental reason for these differences might be
derived from their different binding ability to Cd. As cal-
culated by the chemical equilibrium program VMIN-
TEQ, Cd ion concentrations in solutions drastically
decreased with increasing concentrations of EDTA and
DTPA (Table 1), and lesser by citric acid, malic acid
and oxalic acid. It is known that kinetically labile such
as the hydrated Cd2+ ion or inorganic complexes are
bioavailable to plant tissues (Welch and Norvell,
1999). It may be true that Cd and EDTA/DTPA in solu-
tion form Cd-EDTA/DTPA complexes, which are high
molecular weight with relative high stability with regard
to metal-exchange reactions and consequently reduce Cd
bioavailability. This can be further confirmed in this
work, as shown by drastic declines of Cd uptake and
PCs-SH concentrations in wheat exposed to relatively
higher EDTA/DTPA-Cd concentrations (Figs. 4D, E
and 5I, J). In contrast to our own, Maier et al. (2003) ob-
served greater Cd accumulation in roots and old leaves
of lettuce exposed to higher Cd-EDTA concentrations.
Moreover, in the research on lead accumulation in Bras-
sica juncea, it has been demonstrated that application of
synthetic chelators, such as EDTA, both in soil (Epstein
et al., 1999) and hydroponic culture conditions (Vassil
et al., 1998), increased its uptake to plants by forming
Pb-EDTA complexes. These discrepant results may be
caused by metal- and species-specific difference.
Hue (1986) reported that the detoxifying ability of
organic acid depends on the OH/COOH relative posi-
tion in the main carbon backbone, regarding citric acid
as a stronger detoxifying agent than malic acid, acetic
30 Q. Sun et al. / Chemosphere 60 (2005) 22–31
acid and lactic acid. Present results with wheat showed
that citric acid exhibited stronger effects on decreasing
Cd toxicity than malic acid and oxalic acid, accompa-
nied by enhanced uptake of Cd and suppressed trans-
port of Cd to above-ground parts. This is desirable,
not only because it alleviates Cd injury, but also it re-
duces the transportion of Cd into above-ground parts.
One possible explanation for this phenomenon is that
citric acid forms complexes with Cd, and facilitates Cd
uptake from solution to root, and then absorbed Cd-cit-
ric complexes stores in inert parts, such as vacuole,
resulting in lowing of the transportation of Cd from root
to shoot. This hypothesis can be further supported by
increasing decreases of PCs-SH concentrations in wheat
roots with citric acid treatments.
Together with earlier researches from the laboratory
experiments and field measurements, present results fur-
ther demonstrated that the levels of PCs-SH in plants
cells could be regarded as a good tool to assess ecotoxi-
city of environments contaminated by metals, especially
Cd. However, the specific metals to which plants are ex-
posed under field conditions cannot be tested from the
PCs production, which is also induced by many other
metals (Rauser, 1995). Therefore, the application of
PCs as a biomarker in more than one heavy metal-con-
taminated environment will be necessary to be con-
nected with other traditional chemical methods of
detecting heavy metals.
In the case of controlling Cd bioavailability in wheat,
our study showed that EDTA was a better ligand of Cd
than the other four organic chelators. Excess DTPA
drastically reduced Cd uptake by wheat, and even the ef-
fects were stronger than EDTA (Fig. 4). However, ex-
cess DTPA exerted adverse effects on plant growth and
physiology, as observed by significant decreases of
growth parameters (Figs. 1 and 2) and chlorophyll con-
tents of leaves (data not shown) at the highest level of
DTPA applied. This may be derived from stronger bind-
ing ability of DTPA to metals, especially Fe (data not
shown), resulting in Fe deficiency (chlorosis) in wheat
plants. Therefore in the application of synthetic chela-
tors to improve metal ecotoxicity, we should be careful
to select feasible organic chelators, pay more attention
to suitable dose of application, and be alert to their sec-
ondary influences on plant growth.
5. Conclusion
Cadmium toxicity stimulated the overproduction of
PCs-SH in roots, not leaves of wheat. The presence of
organic chelators in Cd-treatment solutions reduced
Cd toxicity to varying degree, concomitant with de-
creases of PCs-SH production in roots. Of organic che-
lators tested, EDTA, DTPA and citric acid had
relatively stronger effects on reducing Cd toxicity and
PCs production. Furthermore, PCs biosynthesis in plant
cells was found to be closely related to the presence of
Cd in plants and further dependent on free Cd ion con-
centrations, instead of total Cd. It is suggested that ana-
lysis of metal-induced PCs is an additional means for
studies on some metal bioavailability and subsequently
their toxicity in complex environments.
Acknowledgment
This work was supported by the National Natural
Science Foundation of China (No. 20237010) and Inno-
vation Foundation of Advanced UniversitiesÕ Graduate
of Jiangsu Province, China.
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