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www.elsevier.com/locate/plantsci

Plant Science xxx (2004) xxx–xxx

F

Different effects of calcium and lanthanum on the expression

of phytochelatin synthase gene and cadmium

absorption in Lactuca sativa

Zhenyan He, Jiangchuan Li, Haiyan Zhang, Mi Ma*

Key Laboratory of Photosynthesis and Molecular Environmental Physiology, Institute of Botany,

Chinese Academy of Sciences, Beijing, Xiangshan 100093, PR China

Received 3 December 2003; received in revised form 23 June 2004; accepted 1 July 2004

Abstract

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OPhytochelatins (PCs) have an important role in metal detoxification in plants, however the relationship of the expression of Lactuca sativa

phytochelatin synthase gene, its catalyzing products—PCs and the absorption of cadmium (Cd) was still not clear. To investigate this

relationship, a full-length cDNA of a phytochelatin synthase gene (LsPCS1) was cloned from L. sativa and the physiological changes were

observed. When L. sativa (cabbage lettuce) plants were exposed to toxic cadmium, growth was retarded. Though L. sativa plants cadmium

tolerance was enhanced when synchronously treated with lanthanum (La3+) or calcium (Ca2+), the effect on the expression of phytochelatin

synthase gene and cadmium absorption was different. The transcript level of LsPCS1 and the PCs content in the plants grown under normal

conditions were very low, and the Cd absorption was minimal. In comparison with these plants, the expression level of LsPCS1 and the

absorption of Cd were enhanced both in leaf and root under Cd2+, Cd2+ + Ca2+ and Cd2+ + La3+ treatments. In comparison with Cd2+ treatment,

the accumulated mRNA level of LsPCS1, PCs content and the absorption of Cd were enhanced with Cd2+ + Ca2+ treatment. Though Cd2+ +

La3+ treatment showed a similar expression profile with Cd2+ + Ca2+ treatment, the absorption of Cd of Cd2+ + La3+ treatment was less than that

of Cd2+ treatment. Ca2+ alone has little effect on the expression of LsPCS1, whereas La3+ itself enhances the mRNA and PCs accumulation. It is

suggested that Ca2+ increases the expression of PC synthase gene under Cd2+ stress, which in turn enhances the plant tolerance and Cd

accumulation. Thus, fertilization with Ca2+ appears to be a promising strategy for increasing phytoremediation capacity. While La3+ could

decrease the accumulation of Cd, La3+ may play an important role on reducing the accumulation of Cd of plants grown in contaminated soil.

# 2004 Published by Elsevier Ireland Ltd.

Keywords: Cadmium; Calcium; Lanthanum; Phytochelatin synthase; Gene expression

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Toxic heavy metal pollution has become an increasing

worldwide environmental problem since the onset of the

industry revolution. Cadmium (Cd) used as a ‘model’ non-

essential metal of significant environmental relevance in

many studies is one of the most serious pollutants. About

38,900 tons of cadmium is discharged annually to the

environment [1]. Chronic cadmium poisoning may lead to

the damage of the skeletal system and kidney and induce

cancer. Recently it is reported that cadmium can act as an

estrogen mimic in the whole animal, inducing conditions

ranging from uterine hyperplasia to early onset of puberty, at

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* Corresponding author. Tel.: +86 10 8259 9422; fax: +86 10 8259 9422.

E-mail address: mami@ibcas.ac.cn (M. Ma).

0168-9452/$ – see front matter # 2004 Published by Elsevier Ireland Ltd.

doi:10.1016/j.plantsci.2004.07.001

doses as low as 5–10 mg/kg (single intraperitoneal injection)

[2]. Due to the exhaust emission, waste water and garbage

draining from industry, sewage irrigation, and the utilization

of pesticide, chemical combination and herbicide, the heavy

metals content of the plants growing in some contaminated

soil has exceeded the content of food standard. The plants

include two types that have tremendous effects on human

health. One is directly edible by human such as the

vegetables and the crops, another is indirectly utilized by

animals such as the clover that is an economically most

important forage legume worldwide [3]. Because of the non-

degradable nature of the cadmium, they can be passed to

various organisms through the food chains and lead to

various diseases in humans.

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It is estimated that cleanup of hazardous wastes using

conventional technologies will cost at least $200 billion in

the US alone [4]. Phytoremediation is a cost-effective and

environment-friendly technology [5–7], but there are many

problems to be solved, such as the scarcity of hyperaccu-

mulator, the complexity of the mechanisms of heavy metal

tolerance in plants, the low-efficiency of absorption for

heavy metal and so on.

Plants can produce Cys-rich peptides such as glutathione

(GSH), phytochelatins (PCs), or metallothioneins (MTs) for

detoxification or homeostasis of heavy metals [8–12]. PCs

are a kind of polypeptides with the structure (g-Glu-Cys)n-

Gly (where n = 2–11) and identified in a wide variety of plant

species and in some microorganisms [13,14]. It has been

demonstrated that PCs play an important role in metal

detoxification by transporting the metal Cd2+ to vacuole

[13], and PC synthase genes were isolated from Arabidopsis,

wheat and S. cerevisiae (AtPCS/CAD1, TaPCS and SpPCS)

in 1999 by three independent laboratories [15–17]. It is

reported that calcium (Ca) or lanthanum (La) can alleviate

cadmium toxicity in plant by anatomical and enzymological

methods [18,19]. Choi et al. [18] found that tobacco plants

actively excluded toxic Cd by forming and excreting Cd/Ca-

containing crystals through the head cells of trichomes.

Another study suggested the increased antioxidant enzymes

by La(NO3)3 enhance the antioxidant potential of the wheat

seedlings to reduce lead stress [19]. However, there are no

reports about what effects Ca and La have on the expression

of PC synthase gene, which has affinity with heavy metal

tolerance. We selected Lactuca sativa, a kind of common

vegetable, as the material to study how calcium and

lanthanum affect L. sativa cadmium tolerance and what

effects Ca and La have on the cadmium absorption in plants

and the expression of PC synthase gene, to find a way to

decrease the heavy metal accumulation in plants.

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1. Materials and methods

1.1. Plant material and treatments

1.1.1. Experiment I (seedlings in plastic Petri dishes)

L. sativa seeds were sterilized by rinsing in 70% ethanol

for 30 s, then in 0.1% HgCl2 for 10 min, and subsequently in

sterile, deionized water for 30 min, all on rocking platform.

Fifty sterilized seeds were sown in each plastic Petri dish in

hydroponic conditions containing 0.05 mM CdCl2, 4 mM

CaCl2 and 0.04 mg/L La(NO3)3. After differently treated for

5 d at 25 8C under continuous dark, the seedlings were

harvested, washed, and weighed, and the length of the root

was measured.

1.1.2. Experiment II (plants on sand medium)

L. sativa seeds were grown on sand conditions with to 1/2

MS (Murashige and Skoog 1962, CaCl2 free) medium for

8 h light and 16 h dark. After 20 d at 25 8C, the sand medium

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were added 0.5 mM CdCl2, 4 mM CaCl2 and 0.04 mg/L

La(NO3)3. Leaf and root were collected from all treatments

after plants were treated 1, 2, 6, 12, 24 and 48 h. Some

materials were frozen in liquid nitrogen and stored in

�80 8C used for RT-PCR analysis and phytochelatins

determination. Others were washed in deionized water for

2 min, dried at 60 8C for Cd elemental analysis. The plants

of 48 h treatment were weighed and the length of the root

was measured only.

1.2. Elemental analysis

Leaf and root were harvested, washed in deionized water

for 2 min, dried at 60 8C and then macerated in HNO3. After

acid digestion, the concentrations of Cd in the clear extracts

were measured with graphite atomic absorption spectrometer.

1.3. Amplification of the full LsPCS1 cDNA

Total RNA from the plant of L. sativa was extracted using

Trizol Reagent (Invitrogen, Karlsruhe, Germany) followed

by chloroform extraction and isopropanol precipitation.

Polyadenylated mRNAs were obtained using Oligotex

mRNA Spin-Column (clontech). Following reverse tran-

scription from mRNA, the first-strand cDNA was used as the

template in following reaction.

PC synthase genes are very conservative in the N-

terminal of all kinds of organisms. Two degenerate primers

were designed according to the conservative domains of the

PCS cDNA from the dicotyledon: Arabidopsis, Brassica

juncea and Glycine max. The sequences were: 50-ATGGC(T/

G)ATGGCG(A/G)GTTT((A/G)TATCG-30 (up) and 50-TGCCGTCGCAG(A/G)TGCA(G/A)(T/A)ACCT-30 (down).

PCR was performed with denaturing for 30 s at 95 8C,

annealing for 45 s at 60 8C and extending for 1 min at 72 8C.

The cycle was repeated 30 times using cDNA of L. sativa as

a template. The products of PCR were ligated into pGEM-T

Easy Vector (Promega). The products were transformed into

TOP 10-E. coli cells. Plasmid DNA was isolated and

sequenced. A 50 fragment of 800 bp was firstly obtained

from L. sativa cDNA. Sequence analysis was performed

using DNAman software.

The 30 fragment was obtained using SMARTTM RACE

cDNA Amplification Kit (clontech). The gene specific

primers (GSPs) for the 30-RACE reactions is as follows:

GSP2: 50-GCTTGACTGTTGCGAGCCTTTG-30, NGSP2:

50-TGCTCGCTTCAAGTACCCTCCTC-30.

1.4. RT-PCR analysis

Leaf and root were homogenized with a mortar and pestle

in liquid nitrogen. RNA was extracted using Trizol Reagent.

RT-PCR was manipulated using mRNA Selective PCR Kit

(Takara). PCR reactions using equal amounts of RNA

samples were performed. Cycle numbers were optimized

assure that the amplification reaction was tested in the

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Fig. 1. The effects of Ca and La on seeds germination of L. sativa treated

with Cd. Seedlings germination of L. sativa with different treatments (A),

fresh weight of seedlings with different treatments (B), the length of root

with different treatments (C). After treated for 5 d under continuous dark,

the seedlings were weighed and the length of the root was measured.

Untreated (CK), treated with 4 mM CaCl2 (Ca), treated with 0.04 mg/L

La(NO3)3 (La), treated with 0.05 mM CdCl2 (Cd), treated with 0.05 mM

CdCl2 and 4 mM CaCl2 (Cd + Ca) and treated with 0.05 mM CdCl2 and

0.04 mg/L La(NO3)3 (Cd + La). Values correspond to mean � S.E. of five

plants. The data among different treatments are significantly different at the

level of P � 0.01.

exponential phase. The following primers were designed for

the gene-specific transcript amplification: 50-GCTTGA-

CTGTTGCGAGCCTTTG-30 (up) and 50-ATCAATGC-

CATGTCCCTTCCAGCA-30 (down). A single band about

300 bp was detected. The relative level of each band of each

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Fig. 2. The effects of Ca and La on the growth of L. sativa plants treated

with Cd. Plants of 20-days-old were treated for 48 h. Fresh weight of

seedlings with different treatments (A), the length of root with different

treatments (B). Untreated (CK), treated with 0.5 mM CdCl2 (Cd), treated

with 0.5 mM CdCl2 and 4 mM CaCl2 (Cd + Ca) and treated with 0.5 mM

CdCl2 and 0.04 mg/L La(NO3)3 (Cd + La). Values correspond to mean �S.E. of five plants. The data among different treatments are significantly

different at the level of P � 0.01.

sample was quantified using Spot Densitometry (Alphai-

mager) and the data were calibrated against the quantities of

actin of L. sativa. The primers of Ls actin used in this

experiment are 50-TTTGCTGGAGATGATGCTCC-30 (up)

and 50-GTGGTACGACCACTGGCATA-30 (down).

1.5. GSH and phytochelatin determination

Phytochelatins are the major thiol-rich compounds

induced in metal-exposed although there are other thiol-

rich compounds such as MT [20]. In this experiment, the

amount of non-protein sulfhydryl compounds other than

glutathione was taken as a measure of phtochelatins. It can

be approximately estimated the production of PCs. PC-SH

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Fig. 3. The effects of Ca and La on the Cd accumulation of L. sativa plants

treated with Cd in leaf (A) and root (B) for a time frame 1–48 h. Plants

grown for 20 d were added to different treatments. Untreated (CK), treated

with 0.5 mM CdCl2 (Cd), treated with 0.5 mM CdCl2 and 4 mM CaCl2 (Cd

+ Ca) and treated with 0.5 mM CdCl2 and 0.04 mg/L La(NO3)3 (Cd + La).

Values correspond to mean � S.E. of three samples. The data among

different treatments are significantly different at the level of P � 0.05. And

the data of Cd + Ca and Cd + La are significantly different at the level of

P � 0.01.

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concentrations have been assessed by subtracting the GSH

concentration from the total acid-soluble thiol concentration

[21–26] in a number of previous studies.

Leaves and roots were homogenized with a mortar and

pestle in liquid nitrogen. Two milliliters 5% sulfosalicylic

acid and 6.3 mM diethylenetriaminepentaacetic acid were

then added. These homogenates were centrifuged for 10 min

at 12,000 � g at 4 8C, and supernatant was used for

spectrophotometric measurement of GSH and NPT. The

determination of GSH was manipulated as the total

glutathione quantification kit (Dojin East Company)

described. Phytochelatins were determined as non-protein

thiols (NPT) as described [21,22].

2. Results

2.1. Cadmium tolerance of L. sativa plants was enhanced

when synchronously treated with calcium (Ca) or

lanthanum (La)

The effects of Ca2+ and La3+ on the seeds germination

and plants growth of L. sativa treated with Cd2+ can be seen

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Fig. 4. (A) The alignment of the deduced amino acid sequence of LsPCS1 and

AY618896). AtPCS1: PC synthase gene of Arabidopsis thaliana (Accession No. N

are the conserved domain of phytochelatin synthase. (B) The result of NCBI co

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in Figs. 1 and 2. In comparison with untreated seeds, toxic

effects of Cd2+ were most obvious as evidenced by the

retardation of root growth. However, it was root growth that

Ca2+ and La3+ promoted in the presence of Cd2+ (Fig. 1C).

Ca2+ had a stronger effect than La3+. Moreover, fresh weight

was improved in the seedlings treated with Ca2+ and La3+ in

the presence of Cd treatments (Fig. 1B).

Under Cd2+ stress conditions, the most obvious toxic

syndrome of plants were drooping and losing freshness. To

some degree, Ca2+ and La3+ can detoxify Cd2+ in plants and

the plants had no obvious signs of wilting. Fresh weight of

plants (Fig. 2A) and the length of root (Fig. 2B) were

reduced by Cd2+. It is shown that Ca2+ and La3+ reduce the

effect of Cd2+, and the plants seedling seemed like that of

control plants (CK).

2.2. The effects of Ca and La on the Cd accumulation

of L. sativa plants treated with Cd

To understand the absorption of cadmium in L. sativa, the

cadmium concentration of leaf and root of all treatments

were measured by atomic absorption spectrometry. As

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AtPCS1. LsPCS: Putative PC synthase gene of L. sativa (Accession No.

M123774.2). The amino acid residues are numbered on the right. The blocks

nserved domain search.

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Fig. 5. The effects of Ca and La on the mRNA accumulation of L. sativa plants treated with Cd in leaf and root. LsPCS1 RT-PCR of different treatments for

different hours (A), the schematics of LsPCS1 mRNA accumulation in leaf (B and D) and root (C and E) under different treatments. Plants grown for 20 d were

added to different treatments. Untreated (CK), treated with 4 mM CaCl2 (Ca), treated with 0.04 mg/L La(NO3)3 (La), treated with 0.5 mM CdCl2 (Cd), treated

with 0.5 mM CdCl2 and 4 mM CaCl2 (Cd + Ca) and treated with 0.5 mM CdCl2 and 0.04 mg/L La(NO3)3 (Cd + La). The gel bands (A) were quantified using

Spot Densitometry (Alphaimager) and the products were normalized by comparison to quantities of actin of L. sativa.

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shown in Fig. 3, the Cd accumulation of the same treatment

both in root (Fig. 3B) and in leaf (Fig. 3B) had the similar

tendency and root accumulated more Cd than leaf did. The

Cd accumulation was nearly zero in CK plants. When Cd2+

was added to medium, the Cd concentration of the plants

apparently increased. Although the Cd concentration

increased with the increasing of treating time in both root

and leaf, the accumulation of Cd was more in Cd + Ca

treatment, but less in Cd + La treatment comparing with that

of Cd treatment alone. And the statistical analysis showed

the accumulation of Cd in Cd + Ca and Cd + La treatment

were significantly different (P � 0.01). After treated 48 h

with Cd, the Cd concentration of root and leaf in Cd

treatment was 513.9 and 416.8 mg/g DW, respectively,

whereas the concentration of Cd + Ca treatment increased to

732.4 mg/g DW in the root and 599.3 mg/g DW in the leaf,

respectively, which is equally 142.5% and 143.8% of that in

Cd treatment. While Cd concentration of Cd + La treatment

was only 400.2 mg/g DW in the root and 255.6 mg/g DW in

the leaf, which is 77.9% and 61.3% of the accumulation of

Cd treatment.

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Fig. 6. The effects of Ca and La on GSH contents of L. sativa plants treated with C

GSH contents of different treatments for different hours. Leaf (A) and root (B) GS

were added to different treatments. Untreated (CK), treated with 4 mM CaCl2 (Ca

treated with 0.5 mM CdCl2 and 4 mM CaCl2 (Cd + Ca) and treated with 0.5 mM C

of three samples. The data among different treatments are significantly different

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2.3. Amplification of the full length cDNA of L. sativa

phytochelatin synthase gene

To test the transcript level of phytochelatin synthase gene

of L. sativa with and without Cd, the full-length cDNA of L.

sativa phytochelatin synthase gene was cloned. Sequence

and homology analysis showed that the amplified cDNA

encode a putative phytochelatin synthase. The active site

region is located in the more conserved N-terminal portion

of PCS [27]. As the alignment shown in Fig. 4A, the deduced

N-terminal amino acids sequence shared over 90%

homology with the Arabidopsis PC synthase gene (AtPCS1),

which had been demonstrated the activity in catalyzing the

synthesis of phytochelatins [15–17]. The result of NCBI

conserved domain search is shown in Fig. 4B: the deduced

amino acids of the amplified cDNA have the functional

domain of phytochelatin synthase, which is the enzyme

responsible for the synthesis of heavy-metal-binding

peptides (phytochelatins). So the amplified cDNA from L.

sativa was named LsPCS1 (GenBank Accession No.

AY618896).

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d in leaf and root for a time frame 1–48 h. Leaf (A and C) and root (B and D)

H contents of different treatments for different hours. Plants grown for 20 d

), treated with 0.04 mg/L La(NO3)3 (La), treated with 0.5 mM CdCl2 (Cd),

dCl2 and 0.04 mg/L La(NO3)3 (Cd + La). Values correspond to mean � S.E.

at the level of P � 0.05.

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2.4. The effects of Ca and La on the transcript level of

LsPCS1 with and without Cd

To understand the molecular mechanisms of roles of La

and Ca on absorption of heavy metal in L. sativa, the

transcript expression changes of PC synthase gene were

tested. Only one gel band of 300 bp (Fig. 5A) were detected.

The relative level of LsPCS1 in uncompetitive RT-PCR

analysis under time frame of 1–48 h in leaf (B and D) and

root (C and E) was shown in Fig. 5, respectively. LsPCS1 of

CK leaf and root has a little transcript level. The transcript

levels of LsPCS1 leaf and root were all much higher under

Cd, Cd + Ca and Cd + La treatments than that of control.

Comparing the expression of LsPCS1 at leaf and root it can

be found that the root display a much higher expression level

than leaf. With increasing time, the accumulated mRNA level

of LsPCS1 of leaf and root present a similar tendency. Once

the accumulated mRNA of LsPCS1 of leaf and root enhance

to a higher level, it remained relatively stable. However, the

time was different for the transcript levels of LsPCSI to reach

peak level. It needed 12 h for the leaf in the Cd treatment, and

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Fig. 7. The effects of Ca and La on NPT contents of L. sativa plants treated with Cd

treatments for different hours. Plants grown for 20 d were added to different treatm

La(NO3)3 (La), treated with 0.5 mM CdCl2 (Cd), treated with 0.5 mM CdCl2 an

La(NO3)3 (Cd + La). Values correspond to mean � S.E. of three samples. The data a

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6 h for the Cd + Ca and Cd + La treatment, but 2 h for the root

in all treatments. Though Cd + Ca and Cd + La treatments

showed a similar expression profile, their transcript levels

differed slightly. The transcript level of Cd + Ca leaf was

higher than that of Cd + La leaf, while the expression of Cd +

Ca root was lower than that of Cd + Ca root.

2.5. The effects of Ca and La on PCs content with and

without Cd

Total contents of GSH (Fig. 6) and NPT (Fig. 7) were

analyzed in different treatments. PC-SH concentrations were

assessed by subtracting the GSH concentration from the

total acid-soluble thiol (NTP) concentration and the data

were shown in Fig. 8. Under control conditions (0 mM Cd),

GSH contents in Ca (Fig. 6A and B) and La (Fig. 6C and D)

treatments were not significantly different from those in CK

plants. When plants were subjected to 0.5 mM CdCl2treatment for different time, a slight increase in GSH content

was observed in Cd, Cd + Ca and Cd + La treatments

compared with untreated plants. The PCs content increased

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in leaf and root. Leaf (A and C) and root (B and D) NPT contents of different

ents. Untreated (CK), treated with 4 mM CaCl2 (Ca), treated with 0.04 mg/L

d 4 mM CaCl2 (Cd + Ca) and treated with 0.5 mM CdCl2 and 0.04 mg/L

mong different treatments are significantly different at the level of P � 0.05.

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Fig. 8. The effects of Ca and La on PCs contents of L. sativa plants treated with Cd in leaf and root. Leaf (A and C) and root (B and D) PCs contents of different

treatments for different hours. Plants grown for 20 d were added to different treatments. Untreated (CK), treated with 4 mM CaCl2 (Ca), treated with 0.04 mg/L

La(NO3)3 (La), treated with 0.5 mM CdCl2 (Cd), treated with 0.5 mM CdCl2 and 4 mM CaCl2 (Cd + Ca) and treated with 0.5 mM CdCl2 and 0.04 mg/L

La(NO3)3 (Cd + La).Values correspond to mean � S.E. of three samples. The data among different treatments are significantly different at the level of P � 0.05.

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under Cd stress than without Cd. Moreover, the PCs levels in

Cd + Ca (Fig. 8A and C) and Cd + La (Fig. 8B and D)

treatments were higher than those detected in Cd treatments.

Ca2+ alone has no effect on the PCs content, whereas La3+

itself enhances the PCs content.

With increasing time, the PCs content of leaf and root

present a similar tendency. It also can be found that the root

displays a much higher PCs level than leaf.

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O3. Discussion

The concept of phytoremediation has evoked considerable

interest in plant metal accumulation [12]. The results of this

work can suggest that the enhancement of the Cd tolerance in

L. sativa by Ca and La could relate to the phytochelatins.

As Zn and Cd are chemically very similar, they can

compete for binding and absorption sites in the plants [28–

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31]. So the experiment was expected that Ca competed for

binding and absorption sites and decrease the Cd absorption

too. But the results were not same to what expected.

This current study demonstrated that calcium and

lanthanum enhanced L. sativa cadmium tolerance (Figs. 1

and 2). However, the data (Fig. 3) suggests that the

accumulation of Cd was more in Cd + Ca treatment, but

less in Cd + La treatment comparing with that of Cd treatment

alone. It is easy to understand that La decreased the Cd

absorption and the cadmium tolerance of plant was enhanced.

Ca increased the Cd accumulation in the plant, while the

cadmium tolerance of plant was enhanced in Cd + Ca

treatment. Why?

Phytochelatins can chelate heavy metal and have an

important role in detoxification. Whether phytochelatins have

relationship with the more Cd accumulation in Cd + Ca

treatment or not? Figs. 5 and 8 showed that Cd enhanced the

LsPCS1 mRNA accumulation and the PCs content, and Cd +

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Ca enhanced the expression level of LsPCS1 comparing with

that of Cd treatment alone, but not Ca. It is suggested that the

enhancement of LsPCS1 expression leads to the increase of

the cadmium absorption in plants. We guess that the increased

PCs under the Ca chelated the most cadmium, then transported

to the vacuole, so Ca can decrease the toxicity of Cd.

How does Ca promote the expression of LsPCS1 in the

stress of Cd? Calcium is an important nutrient for plant

growth and Ca2+ influx and Ca2+ release from organelles

play important roles in many signaling cascades of the plant

[32,33]. We presume that Ca accelerates the transmission to

the Cd stress signal and enhances the LsPCS1 expression,

which increases the Cd absorption in plants, although further

study is needed to find the direct linkage. Pharmacology and

physics analysis will provide useful tools to study the

mechanisms. Lombi et al. [34] offered physiological

evidence for a high-affinity specific cadmium transporter

highly expressed in a Thlaspi caerulescens ecotype in the

root cell plasma membrane. It is possible that Ca induced the

high expression of LsPCS1 and cadmium transporter and led

to the enhancement of Cd absorption.

It has been known that lanthanon can enhance the

adaptability to stress conditions or increase the tolerance to

adversity such as low temperature, high temperature,

drought and salt. It was reported that lanthanon lightened

the plant damage by lead [19]. We were interesting in the

effects of La on seed germination and growth of L. sativa

under Cd stress. The results proved L. sativa plants

cadmium tolerance was enhanced when synchronously

treated with La. La itself enhances the LsPCS1 mRNA

accumulation and PCs content both in leaf and root. The

LsPCS1 expression on Cd + La treatment was higher in leaf

and root than Cd, but the Cd absorption is less than Cd

treatment. From the above results the following conclusions

could be made: The concentration of La is so low that La

cannot lead to antagonistic effects to Cd. However, the exact

mechanisms of the enhancement of LSPCS1 expression by La

itself remain unclear. It has been demonstrated by recent

research that phytochelatin have other bio-function in plants

[35]. Perhaps because La induced other mechanisms about

cadmium tolerance, which led to the decrease of Cd

absorption, La enhanced the cadmium tolerance of L. sativa

plants.

In conclusion, it was suggested that calcium and

lanthanum had different effects in Cd absorption. Further-

more, fertilization with Ca2+ appears to be a promising

strategy for increasing phytoremediation. As La can

decrease the accumulation of Cd, it may play an important

role on reducing the accumulation of Cd of the crops

growing in contaminated soil.

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NCAcknowledgments

The work was supported by the National Science

Foundation Council of PRC (Grant no. 30170086,

U

30370127), the Special Project of Transgenic Research

(JY03A2001) and Hi-tech Research and Development

Program of China (2001AA645010-5).

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PR

OO

F

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