ORIGINAL PAPER

Phytochelatin synthase of Thlaspi caerulescens enhanced toleranceand accumulation of heavy metals when expressed in yeastand tobacco

Ge-Yu Liu • Yu-Xiu Zhang • Tuan-Yao Chai

Received: 9 September 2010 / Revised: 1 January 2011 / Accepted: 11 January 2011 / Published online: 16 February 2011

� Springer-Verlag 2011

Abstract Phytochelatin synthase (PCS) is key enzyme

for heavy metal detoxification and accumulation in plant.

In this study, we isolated the PCS gene TcPCS1 from the

hyperaccumulator Thlaspi caerulescens. Overexpression of

TcPCS1 enhanced PC production in tobacco. Cd accumu-

lation in the roots and shoots of TcPCS1 transgenic seed-

lings was increased compared to the wild type (WT), while

Cd translocation from roots to shoots was not affected

under Cd treatment. The root length of the TcPCS1 trans-

genic tobacco seedlings was significantly longer than that

of the WT under Cd stress. These data indicate that

TcPCS1 expression might increase Cd accumulation and

tolerance in transgenic tobacco. In addition, the malondi-

aldehyde content in TcPCS1 plants was below that of the

wild type. However, the antioxidant enzyme activities of

superoxide dismutase, peroxidase and catalase were found

to be significantly higher than those of the WT when the

transgenic plant was exposed to Cd stress. This suggests

that the increase in PC production might enhance the Cd

accumulation and thus increase the oxidative stress induced

by the cadmium. The production of PCs could cause a

transient decrease in the cytosolic glutathione (GSH) pool,

and Cd and lower GSH concentration caused an increase in

the oxidative response. We also determined TcPCS1 in

Thlaspi caerulescens was regulated after exposure to var-

ious concentrations of CdCl2 over different treatment

times. Expression of TcPCS1 leading to increased Cd

accumulation and enhanced metal tolerance, but the Cd

contents were restrained by adding zinc in Saccharomyces

cerevisiae transformants.

Keywords Phytochelatin synthase � Thlaspi caerulescens �Heavy metal � Tolerance � Accumulation � Antioxidative

enzyme

Abbreviations

CAT Catalase

MDA Malondialdehyde

PEG Polyethylene glycol

POD Peroxidase

SOD Superoxide dismutase

WT Wild type

PCs Phytochelatins

Introduction

Heavy metal pollution is widespread and caused by various

anthropogenic activities. The high concentrations of dif-

ferent heavy metals in the environment of plants can affect

transpiration (Sengar et al. 2008), photosynthesis (Kupper

et al. 2007) and synthesis of RNA (DalCorso et al. 2008),

thereby strongly inhibiting plant growth and development

(Street et al. 2007) and even causing plant death (Kozlov

2004). Cadmium (Cd) is the most toxic of the heavy

metals. The toxicity of Cd may result from its binding to

the sulfhydryl groups of proteins, leading to an inhibition

Communicated by K. Chong.

G.-Y. Liu � T.-Y. Chai (&)

College of Life Science, Graduate University of Chinese

Academy of Sciences, Yuquan Rd 19, Beijing 100049, China

e-mail: tychai@gucas.ac.cn

Y.-X. Zhang

Department of Biological Engineering,

School of Chemical and Environmental Engineering,

China University of Mining and Technology (Beijing),

Xueyuan Rd 11, Beijing 100083, China

123

Plant Cell Rep (2011) 30:1067–1076

DOI 10.1007/s00299-011-1013-2

of activity due to disruption of the protein structure.

Alternatively, perturbations in the calcium level in the cell

(Perfus-Barbeoch et al. 2002), disruption of cellular redox

control (Schutzendubel and Polle 2002), and/or induction

of the production of reactive oxygen species (Romero-

Puertas et al. 2004) may also be mechanisms of Cd toxicity

in plants.

It is well known that one of the major mechanisms that

plants use to cope with toxic Cd levels is the production of

heavy metal-binding peptides called phytochelatins (PCs)

(Grill et al. 1985). PCs are cysteine-rich polypeptides with

the general structure of (c-Glu-Cys)nGly (n = 2–11) that

are widely present in plants, fungi, and some other

organisms (Grill et al. 1985; Piechalak et al. 2003). They

are heavy metal-binding thiolate peptides that are enzy-

matically synthesized from glutathione (GSH) by phyto-

chelatin synthase (PCS). PC synthase activity was first

found in cultured plant cells by Grill et al., followed by the

identification and characterization of the PC synthase genes

in higher plants and fission yeast. PCS genes have since

been found in different kinds of plants, bacteria, and even

in animals (Clemens et al. 2001; Tsuji et al. 2004).

Due to the important role of PCs in Cd detoxification,

several attempts were made to enhance their synthesis in

plants. Expression of Arabidopsis PCS genomic DNA and

cDNA in Indian mustard was recently found to enhance the

plant’s tolerance of Cd, arsenic (As), and Zn (Gasic and

Korban 2007a, b). Expression of a PCS gene of Cynodon

dactylon L. in tobacco plants enhanced PC production by

3.88-fold and subsequently enhanced Cd accumulation by

3.21-fold in transgenic tobacco. However, Lee et al. found

that a few transgenic Arabidopsis lines were more Cd-

sensitive than the wild type (Lee et al. 2003a). Recently,

Wojas et al. also reported that overexpression of the At-

PCS1 gene in tobacco led to Cd hypersensitivity. Thus,

there have been contradictory results on the effect of PCS

overexpression in plants, ranging from an increased Cd

tolerance to no effect on Cd tolerance, or, paradoxically,

even Cd hypersensitivity.

Thlaspi caerulescens is a well-known heavy metal

hyperaccumulator that has been studied extensively for

its abilities to hyperaccumulate several heavy metals,

mainly Cd, zinc (Zn) and nickel (Ni). Certain ecotypes of

T. caerulescens can accumulate as much as 30,000 ppm of Zn

and approximately 10,000 ppm of Cd in the shoot biomass

without any signs of toxicity. Because it is slow-growing

and has a low shoot biomass, T. caerulescens has been used

as a model system for the investigation and identification of

the underlying molecular and physiological mechanisms of

hyperaccumulation, with the ultimate goal of transferring

these mechanisms to higher-biomass plant species. PCS

function has been proven in many species to produce PCs

for binding heavy metals and transporting them as a PC-

heavy metal complex. Interestingly, Ebbs et al. found that

PCs do not appear to be involved in metal tolerance in the

hyperaccumulator Gange ecotype. While leaf and root PC

levels for metal tolerance in T. caerulescens and the related

Prayon ecotype showed a similar positive correlation with

tissue Cd, the total PC levels in the Gange ecotype were

generally lower, despite correspondingly higher metal

concentrations (Ebbs et al. 2002). Many genes involved in

the metal accumulation mechanism of T. caerulescens have

shown significant function in transgenic plants (Wei et al.

2009; Gendre et al. 2007; Bernard et al. 2004). The PCS

gene of T. caerulescens may play a unique function in

the transformants. In this investigation, we expressed the

T. caerulescens phytochelatin synthase (TcPCS1) gene in

wild-type tobacco and measured the tolerance and accu-

mulation of Cd and the levels of ROS, comparing the wild-

type and transgenic plants.

Materials and methods

Plant materials and stress treatments

The T. caerulescens seeds were kindly provided by Dr.

Mark Aarts at the Laboratory of Genetics, Plant Science

Department, Wageningen University, Netherlands. To

obtain the seedlings, seeds were sown under sterile con-

ditions in Petri dishes containing equal parts Murashige

and Skoog medium that was solidified with 0.8% (w/v)

agar. Cultures were maintained at 22 ± 3�C over a 16 h

photoperiod with a photosynthetic photon flux density of

45 lmol m-2 s-1. After germination, the seedlings were

transferred to Hoagland solution for plant propagation

under the same culture conditions. Fourteen-day-old

seedlings were treated with 100, 200 or 500 lM of CdCl2for either 4 or 8 h to analyze the PCS expression pattern.

cDNA cloning, plasmid construction, and semi-

quantitative RT-PCR analysis

The full-length TcPCS1 cDNA for yeast vector construc-

tion was amplified using the primer sets pY-TcPCS-F

(50-CGGATCCGATGGCTATGGCGAGTTTG-30) and

pY-TcPCS-R (50-GGAATTCCAGTGTGAATTTGGGTA

G-30), which were designed based on the cDNA sequence

of TcPCS1 and the restriction sets of yeast vector pYES2.

The PCR was done in a reaction volume of 25 lL and

amplified for 30 cycles at 94�C for 30 s, 53�C for 30 s, and

72�C for 1.5 min using standard PCR conditions (100 ng

of DNA, 2.5 lL of 109 PCR buffer with 15 mM MgCl2,

200 lM dNTPs, 10 lmol of each primer and 0.2 U Taq

DNA polymerase) in an ABI 9700 thermocycler.

The amplified products were purified using the standard

1068 Plant Cell Rep (2011) 30:1067–1076

123

freeze–thaw method for cloning. After digestion with

BamHI and EcoRI (sites underlined above), the fragment

was cloned into the yeast vector pYES2.

Total RNA was isolated from treated seedlings using

TRIZOL Reagent (Invitrogen, USA). For semi-quantitative

RT-PCR, the concentration of RNA was accurately quan-

tified by spectrophotometric measurement. cDNAs were

synthesized from DNase-treated total RNA with the reverse

transcription system kit (Promega,USA). Reverse tran-

scription (RT) PCR conditions were 94�C for 2 min fol-

lowed by 30 cycles of 94�C for 30 s, 55�C for 30 s, and

72�C for 1 min. RT-PCR was performed using the primer

sets TcP-F (50-TTGCCCGTTTCAAGTATCCTCCTC-30)and TcP-N (50-ATGCACCACAACTCCAGTCACCA-30).Actin was used as an internal control (Guan et al. 2008).

All RT-PCR analyses were done in triplicate.

Yeast culture, transformation and complementation

assays

Saccharomyces cerevisiae strain YK44 (ura3-52 his3-200,

DZRC, DCot1, mating type a) was kindly provided by Prof.

Dietrich H. Nies of the University of Martin-Luther. This

strain is sensitive to Zn and Cd. Yeast transformations were

performed as described previously in Ito et al. (1983).

The yeast cells harboring pYES2 or pYES2-TcPCS1

were cultured in SD-ura medium at 30�C overnight, after

which 400 lL of the culture (OD600 = 0.5) was col-

lected by centrifuge. The pellet was resuspended in

deionized water and then inoculated into 40 ml of YP-

GAL (yeast extract peptone galactose) supplemented with

20 lM CdCl2 and 50 lM ZnCl2 for single metal treat-

ments. Combined treatments were 10 lM or 20 lM Cd2?

(final concentration) with 50 lM ZnCl2, or 25 or 50 lM

Zn2? with 20 lM CdCl2. All of these treatments were for

24 h.

Transformant cells were grown on SD-ura medium. The

cultures were diluted to ODs of 10-1–10-3 and spotted

onto YPGAL plates containing 100 lM CdCl2 or 1 mM

ZnCl2. All experiments were done in triplicate.

Plant transformation

The TcPCS1 cDNA expression cassette was inserted into

the BamHI and SmaI sites of the plant expression vector

pBI121 to construct the expression vector pBI121-TcPCS1.

The recombinant plasmid pBI121-TcPCS1 was introduced

into the Agrobacterium tumefaciens EHA105; this strain

was then used to transform Nicotiana tabacum NC89 by

the Agrobacterium-mediated transformation method. Total

RNA was isolated from both the WT and TcPCS1 trans-

genic plants. Northern blots were performed to verify

whether the introduced genes were expressed in the plants.

Root length measurement and sampling

Wild-type and transgenic plants pcs6 and pcs47 were

germinated and grown on 1/2 MS medium in 90-mm-

diameter plates. Two-day-old seedlings were transferred to

a plate with 1/2 MS medium containing differing concen-

trations of Cd (supplied as CdCl2). The plates were main-

tained vertically in the culture room for 14 days to compare

the root length of wild-type and transgenic plants. At least

ten seedlings of each line were then measured in triplicate

experiments.

Elemental analysis of transgenic plants and yeast

The assessment of metal tolerance was performed on the

separate tissues of the plants. Cells were harvested by

centrifugation. All samples were washed twice with

50 mM EDTA in cold distilled ultra-pure water, dried at

65�C for 24 h and then the metal concentration was

determined using an inductively coupled plasma mass

spectrometry (ICP-MS) device (Varian). All of the stan-

dard solutions were supplied by Merck.

Biochemical assays

The T1 generation of the pcs6 15-day-old seedlings and WT

plants were cultivated in Hoagland’s nutrient solution for

7 days. Rooted tillers of approximately the same size were

selected and held in Hoagland’s nutrient solution containing

100 lM CdCl2 for 2–48 h. Fresh leaves were collected, and

POD and SOD activities were measured as described by

Scebba et al. (2006). MDA was determined by measuring

the concentration of thiobarbituric acid-reacting substances

(Buege and Aust 1978). PC content was analyzed by GSH

subtracted from total non-protein sulphydryl groups. Four

plants were analyzed from each line. All of the above

experiments were carried out four times.

Statistical analysis

In all statistical tests, P \ 0.05 was interpreted as statis-

tically different. Results were analyzed using the SAS

statistical package (Version5.1, SAS Institute Inc., Cary,

NC, USA)

Results

Tissue expression pattern of TcPCS1 and heavy metal

stress in T. caerulescens

The endogenous expression of TcPCS1 in response

to cadmium stress was analyzed by semi-quantitative

Plant Cell Rep (2011) 30:1067–1076 1069

123

RT-PCR. TcPCS1 mRNA showed no differences between

the roots, leaves, and stems of T. caerulescens without

treatment (Fig. 1a). However, TcPCS1 expression

increased gradually in the leaves when given a treatment of

100, 200, or 500 lmol CdCl2 for 4 or 8 h.

Functional analysis of the TcPCS1 gene in yeast

The complete open reading frame (ORF) of TcPCS1 was

inserted between the inducible GAL1 promoter and the

CYC1 terminator of the pYES2 shuttle vector. YK44 cells

were transformed with this plasmid. Northern blot analysis

showed that the TcPCS1 gene was expressed when the

S. cerevisiae were placed into a galactose-induction med-

ium. No signal was detected when RNA was analyzed from

transformants harboring the empty vector pYES2 under

inducing conditions (Fig. 2a), indicating that there was no

cross-hybridization with endogenously expressed yeast

genes.

We tested the effects of PC formation in different Cd2?

or Zn2? treatments in cells normally synthesizing PCs by

expressing TcPCS1 under the control of the inducible

pGAL1 promoter in S. cerevisiae strain YK44. When cells

were grown in the presence of 100 lM Cd2? or 1 mM

Zn2? in the galactose medium, an increased tolerance of

Cd or Zn was observed for the TcPCS1 transformed cells,

while TcPCS1 and empty vector cells grew equally well in

glucose medium under control conditions (Fig. 2b).

An additional assay was performed to assess the func-

tion of TcPCS1 in combined heavy metal stress. Cells were

transformed into YPGAL medium with combined metal

treatments. When various concentrations of Cd and Zn

were given, a reciprocal synergistic effect was observed

between PC production and metal accumulation. The

TcPCS1-overexpressing yeast cells showed differences in

the ability of the metals to accumulate between the single

metal (Cd) and combined metals (Cd/Zn) stress conditions.

The heavy metal accumulation capacity of TcPCS1-trans-

formants was usually three times, but always at least two

times, more than that of the control cells when given

20 lM Cd. Interestingly, heavy metal accumulation dif-

fered when the combined metals treatment was given. No

significant changes in Cd accumulation were observed

when the Zn content was 25 lM, while Cd accumulation

was slightly decreased in the 20 lM Cd and 50 lM Zn

treatment group. The PC concentration of cells containing

TcPCS1 showed no difference when Zn was added. This

suggests that the decline in Cd concentration was not

accompanied by a decrease in PC production during

combined metal stress (Fig. 3a, b).

Transgenic line identification and functional analysis

of TcPCS1 expression in tobacco

After transformation and regeneration, three lines of

tobacco transformed with the TcPCS1 construct were

named pcs3, pcs6, and pcs47 and were confirmed by

Northern blot. Seeds of the plants with the highest RNA

expression of the TcPCS1 gene (pcs6 and pcs47) were

collected and used for further tests (Fig. 4).

Both plant growth and root development were found to

be sensitive to heavy-metal stress. To validate whether the

positive lines had better cadmium tolerance and accumu-

lation ability, the transgenic lines and wild-type tobacco

Fig. 1 RT-PCR analysis of the expression of the TcPCS1 gene in T.caerulescens tissues and seedlings in the presence of CdCl2.

a TcPCS1 expression in the root stem and leaf without treatment

(CK). b TcPCS1 gene expression pattern with different CdCl2treatment for CK (lane 1) without any treatment; 100, 200 or 500 lM

for 4 h (lanes 2, 3, and 5); and 200 or 500 lM for 8 h (lanes 4 and 6)

Fig. 2 Northern blot (a) and tolerance analysis of transgenic yeast

cultured in a galactose-induced medium (b). A Northern blot analysis

on RNA isolated from yeast that were transformed with TcPCS1 or

the empty vector. b The growth of S. cerevisiae YK44 (ura3-52 his3-

200, DZRC DCot1, mating type a) cells expressing TcPCS1 or the

pYES2 empty-vector control cells grown in YPGAL plates containing

100 lM CdCl2 or 1 mM ZnCl2 for 72 h

1070 Plant Cell Rep (2011) 30:1067–1076

123

plants were both evaluated for the effect of cadmium stress

on root growth in two separate experiments. For Cd tol-

erance, transgenic lines and wild-type seeds were germi-

nated and grown on medium supplemented with various Cd

concentrations (0, 10, 20, 50 lM CdCl2) for 14 days, and

the root length was then measured. As shown in Fig. 5b,

the wild-type and transgenic plants showed different tol-

erances to cadmium, as various root lengths were observed

under various treatments. In the absence of Cd2?, the root

growth of lines pcs6 and pcs47 were comparable to that of

the wild type, while under treatments of 10 lM and 20 lM

CdCl2, the root length of transgenic lines were longer

(Fig. 5a) and significantly, more than that of the wild type

as indicated by SAS analysis (SAS used in all experiments

below; Fig. 5b). The growth of both transgenic lines and

WT roots was almost completely inhibited when given the

50 lM CdCl2 treatment (Fig. 5a, b).

To assess whether the increase in the tolerance and

accumulation of Cd2? was caused by overexpression of

TcPCS1, the Cd2? content in the roots and shoots of pcs6

and pcs47 seedlings were compared with that in the same

organs of WT seedlings. As was done previously, the

seedlings were transferred into liquid medium containing

100 lM CdCl2 and the content of Cd2? in the roots and

shoots was measured after 7 days of treatment. As reported

in Fig. 6, the root Cd2? content of the pcs6 and pcs47

plants was 1.47 and 1.36 times greater than that of WT,

respectively. In the shoots, the Cd concentration of the pcs6

and pcs47 seedlings was 1.36 and 1.24 times higher than in

the WT plant, respectively. Thus, the overexpression of

TcPCS1 led to a statistically significant increase in Cd2?

accumulation in the pcs seedlings. Accordingly, the Cd2?

concentration was proportionately higher in the organs of

the pcs than in WT seedlings: 1.42-fold in roots and 1.3-

fold in shoots. The pcs plants showed a significant increase

in Cd2? content in the roots and in the shoots compared to

WT plants.

PC GSH and MDA content and ROS activity analysis

To determine the difference in the production levels of

PCs, we compared PC levels in the transgenic and WT

plants when the plants were grown hydroponically in the

presence of 20 lM CdCl2. As shown in Fig. 7a, b, the PC

content of pcs6 and pcs47 plants were about 1.8 and 1.7

times higher than the wild type, respectively, when given

the 20 lM CdCl2 treatment. Overexpression of PC syn-

thase and a high concentration of Cd ions led to an increase

in the production of PCs and decrease in GSH content. In

the transgenic plants, PC content increased by about 1.1-

fold compared with wild-type plants. GSH contents result

revealed that the transgenic plants GSH contents were little

lower than wild type with no treatment, however, the

decreases of GSH contents of pcs6 and pcs47 transgenic

plants were 45 and 36% compared with control.

Quantification of the cytotoxic product malondialdehyde

(MDA) is used as a measure of lipid peroxidation and is

therefore diagnostic of the severity of the oxidative stress

induced by the presence of heavy metals (Ohkawa et al.

1979). The MDA content of the wild-type and transgenic

lines following the imposition of 100 lM Cd2? stress is

illustrated in Fig. 8a. The MDA content increased in a

time-dependent manner in the leaves of both the transgenic

and WT plants under Cd stress. However, the MDA content

in the WT plants was higher than that in the transgenic

plants after Cd exposure, indicating that the membrane

damage imposed by Cd in the WT was more serious than

that in the transgenic plants.

CAT, SOD and POD, which are also involved in anti-

oxidative protection, were analyzed as well. In this inves-

tigation, several significant differences were found between

the transgenic and WT roots with regard to antioxidative

enzyme activities and responses to Cd. Without Cd2?, the

0

1

2

3

20/0 20/25 20/50

Cd/Zn concentration (µM)C

d co

nten

ts (

µm

ol/g

DW

)

BA

0

2

4

6

8

20/0 20/25 20/50Cd/Zn concentration (µM)

PCs

cont

ents

( µm

ol/m

g pr

otei

n)

pYES-TcPCS1

pYES

pYES-TcPCS1

pYES

Fig. 3 Heavy metal

accumulation and PC content of

transformants harboring pYES2

or pYES2-TcPCS1 in the

presence of different heavy

metal concentrations. The

transformants were grown in

YPGAL media having different

Cd and Zn concentrations for

24 h. Cells were harvested and

the heavy metal content was

checked by ICP-MS.

Mean ± standard error of the

mean (SEM) shown (n = 3)

Fig. 4 RNA expression of TcPCS1 in independent plant lines was

tested by Northern blot

Plant Cell Rep (2011) 30:1067–1076 1071

123

endogenous SOD, CAT and POD activity in pcs (Fig. 8a)

was the same as that in the WT plant (Fig. 8). The activity

of CAT was found to decline after Cd exposure. The CAT

activity of transgenic lines was higher than that in WT

under 100 lM CdCl2 stress, although both SOD and POD

activities showed increasing trends after exposure to

100 lM Cd in both WT and transgenic tobacco plants. The

SOD and POD activities in the transgenic tobacco plants

sharply increased, while a smooth ascending curve was

found for WT. These results suggest that the overexpres-

sion of TcPCS1 led to an increase in the antioxidative

activity and a decrease in the oxidative damage induced by

Cd toxicity.

Discussion

PCS or PCS-like genes are widespread in many distantly

related organisms, including cyanobacteria, algae, ferns,

fungi, and nematodes (Cobbett and Goldsbrough 2002).

Expression of the PCS genes can improve Cd tolerance and

accumulation in the transformants in species of plants,

fungi, or even bacteria. The evidence presented here sug-

gests that by expressing TcPCS1 in yeast and plants, both

the sequestration and the tolerance of Cd can be increased.

In higher plants, PC synthase is regarded as an enzyme

that is constitutively expressed, independent of heavy metal

exposure (Grill et al. 1989; Chen et al. 1997). If PC

B

A

50 µM Cd

10 µM Cd

20 µM Cd

WT WT pcs6 pcs47

*

*

0

1

2

3

0 10 20 50Cd concentration ( M)

root

leng

th (

cm)

TcPCSWT

Fig. 5 Root length comparison of transgenic lines exposed to

different Cd concentrations. a Tobacco seedlings, either WT or

transgenic, were grown on agar medium containing either 0.01, 0.02

or 0.05 mM CdCl2. WT wild-type tobacco, pcs6 pcs47 transgenic line

having the TcPCS1 cDNA. b Root lengths were measured after 7

days. TcPCS average root lengths of transgenic lines, WT average root

length of WT. Values shown is the mean ± standard error of the

mean. *Significant differences (P \ 0.05) between the means when

compared to WT plants grown in media having the same CdCl2concentration

1072 Plant Cell Rep (2011) 30:1067–1076

123

synthase can be transcriptionally regulated by metals, then

the PCS expression pattern should be different due to dif-

ferent species or after treatment with heavy metals (Amaro

et al. 2009; Dong 2005; Gonzalez-Mendoza et al. 2007).

We used semi-quantitative RT-PCR to assess the pattern of

TcPCS1 gene expression in T. caerulescens plants in dif-

ferent tissues or after different treatments. The data showed

that TcPCS1 expression was the same between the root,

stem or leaf in control plants (Fig. 1a), while TcPCS1

cDNA was upregulated in plants exposed to 100, 200, and

500 lM CdCl2 for 4 and 8 h treatments in the leaf

(Fig. 1b). T. caerulescens is a hyperaccumulator, so the

expression of TcPCS1 resulted in a response that was dif-

ferent from that seen in normal plant species after heavy

metal stress: the majority of Cd2? was translocated from

the root to the leaf following CdCl2 treatment. The increase

of Cd concentration there must have induced PCS gene

expression rapidly to produce the level of PCs needed to

chelate the Cd2? after Cd stress in T. caerulescens. A

similar result was reported earlier with regard to PCS

activity in the hyperaccumulators Avicennia germinans

(Gonzalez-Mendoza et al. 2007)and Brassica juncea (Heiss

et al. 2003). When the concentration and the treatment time

of the heavy metals are increased, toxicity to the organelles

is also increased, so the PCS expression is enhanced to

increase the production of PCs (Tennstedt et al. 2009).

It has been noted that the expression of PC synthase

genes can result in the tolerance and accumulation of heavy

metals or metalloids mediated by the production of PC

peptides in yeast and plants (Wawrzynska et al. 2005;

Singh et al. 2008). However, the heavy metal tolerance or

accumulation properties of the different PC genes isolated

*

*

*

*

0

100

200

300

400

rootshoot

Cd

cont

ents

(µ

mol

g-1

DW

)WTpcs6pcs47

Fig. 6 Cadmium accumulation in the tissues of pcs6, pcs47 and wild

type (WT) plants in 100 lM CdCl2 liquid medium for 7 days. Values

shown are the mean ± the standard error of the mean. *Significant

differences (P \ 0.01) between WT and transgenic lines when

subjected to the same treatment

Control

*

*

*

*

0

4

8

12

rootshoot

WTpcs6pcs47

Cd 20µM*

*

*

*

0

4

8

12

rootshoot

Tota

l PC

con

tent

(µm

ol-1

g FW

)

Tota

l PC

con

tent

(µm

ol-1

g FW

) WTpcs6pcs47

** **

0

2

4

6

rootshoot

WTpcs6pcs47

**

**

0

2

4

6

shoot rootGSH

con

tent

(µm

ol-1

g pr

otei

n) WTpcs6pcs47

A B

C D

GSH

con

tent

(µm

ol-1

g pr

otei

n)

Fig. 7 PCs (a, b) and GSH (c, d) contents in the shoot and root

tissues of WT and TcPCS1 tobacco plants. a, c Control; b, d 20 lM

CdCl2. WT wild-type tobacco, pcs6 pcs47 TcPCS1 transgenic lines

with TcPCS1 cDNA, values shown are the mean ± the standard error

of the mean. *Significant differences (P \ 0.05) between the WT and

transgenic lines when subjected to the same treatment

Plant Cell Rep (2011) 30:1067–1076 1073

123

from various species were dramatically different in the

transformants. We found that the heterologous overex-

pression of TcPCS1 enhanced the accumulation of Cd in

YK44 transformants. During the past decades, S. cerevisiae

has been used to develop genetically engineered cells to

increase their metal chelation ability for use during biore-

mediation. Through the overexpression of metal-chelating

peptides such as metallothioneins or synthetic phytochel-

atins, there have been significant improvements in heavy

metal accumulation in these organisms (Valls et al. 1998).

In this work, we found that the Cd2? content in the TcPCS1

yeast transformants was higher than in the empty vector

transformants after 24 h of Cd2? stress and that the trans-

formants grew faster than the control strain under 100 lM

Cd and 1 mM Zn heavy metal stress (Fig. 3). This might be

due to a higher expression level of TcPCS1 and thus to

higher PC content.

Pollution from multiple heavy metals is a commonly

seen phenomenon at mining sites (Lei et al. 2009). Most

previous studies of bioengineered cells having the PCS

gene investigated monometal exposure and did not inves-

tigate the interaction of different metals with respect to PC

production (Kang et al. 2007; Singh et al. 2008). The

synergetic effect of Zn and Cd on PC production and metal

accumulation was investigated in this study. TcPCS1 gene

expression was found to enhance the ability of transfor-

mants to accumulate Cd under Cd stress compared with the

control. As Zn was added, the accumulation of Cd gradu-

ally decreased. The PC production was not affected by

addition of Zn (Fig. 3a, b). Nakazawa and Takenaga (1998)

suggested that in the presence of Cd at levels that allowed

the metal-binding site of PC synthase to be unsaturated, the

effect of the interaction between Cd and the other metals

activating the enzyme may be additive or synergistic

(Takamura et al. 1998). Hart et al. (2002) pointed out that

the uptake of Cd and Zn was driven by a combination of

linear binding to the cell wall and a saturable, Michaelis–

Menten influx across the plasma membrane (Hart et al.

2002). A decrease in intracellular Cd concentration would

occur if the transporter of Cd2? was occupied by Zn2?, but

the decrease of Cd concentration should not be impacted by

the production of PCs.

To date, descriptions of the overexpression of AtPCS1,

TaPCS1 and SpPCS1 in N. tabacum have been published.

Cd hypersensitivity was reported to result from AtPCS1

overexpression (Li et al. 2004), but this result was not seen

in all studies (Pomponi et al. 2006; Gasic and Korban

2007a; Lee et al. 2003a, b). It has been suggested that

A B

C D

0

10

20

30

40

SOD

Uni

ts m

g-1

prot

ein

0

POD

Uni

ts m

g-1

prot

ein

0

10

20

30

40

0 4 8 12 24 48h 0 4 8 12 24 48h

0 4 8 12 24 48h

0 4 8 12 24 48h

MD

A (µ

mol

g-1

FW)

WT pcs6 pcs47 WT pcs6 pcs47

WT pcs6 pcs47

WT pcs6 pcs47

0

20

40

60

CA

T U

nits

mg-

1 Pr

otei

n

10

20

30

Fig. 8 a Malondialdehyde (MDA) content (mmol g-1 of fresh

weight). b The specific catalase (CAT) activity (mmol min-1

mg-1 protein), c peroxidase (POD) activity (mmol min-1 mg-1

protein) and d superoxide dismutase (SOD) activity (Unit mg-1 pro-

tein) found in the leaves of the transgenic lines and wild-type plants

grown in 100 lM CdCl2 for 0, 4, 8, 12, 24, and 48 h. pcs6, pcs47transgenic lines, WT wild-type control line. Values shown are the

mean ± the standard error of the mean. Three plants were measured

and averaged for each line

1074 Plant Cell Rep (2011) 30:1067–1076

123

different PCS expression levels and genetic differences

between target plant species may contribute to the diverse

phenotypes of PCS transformants. The work presented here

demonstrated that when TcPCS1 isolated from a hyperac-

cumulator was expressed in tobacco plants, it increased the

tolerance of the transgenic plants to Cd, which was

accompanied by PC production (Fig. 7). The transgenic

plants showed more tolerance than WT tobacco to Cd

stress. Root length elongation results showed that the root

growth in the transgenic plants was significantly different

after 10 or 20 lM Cd treatment compared to the WT.

When given 50 lM Cd treatments, the root lengths were

not significantly different between the WT and pcs plants.

This suggests that TcPCS1 expression can increase the

tolerance of transgenic plants at low, but not high, con-

centrations of Cd. Both WT and transgenic plant growth

were completely inhibited at 50 lM CdCl2.

An increased root-to-shoot ratio of Cd accumulation in

transgenic plants indicated that the metal was more

strongly retained in the roots of these plants than in the

wild type. Overexpression of TcPCS1 led to a statistically

significant twofold increase in Cd2? accumulation in both

the PC and wild-type seedlings. However, the ratio of Cd2?

in shoots versus roots was not different for pcs6 and WT

plants. The ratio of Cd2? in shoots versus roots indicated

that TcPCS1 overexpression caused a more abundant

accumulation of Cd2? and that it might not increase Cd

translocation to the shoots.

The variations in Cd2? tolerance and accumulation in

the transgenic plants were also in agreement with the MDA

content and the activities of the antioxidant enzymes CAT,

SOD, and POD (Fig. 8). MDA content is diagnostic for the

occurrence of oxidative stress; therefore, the increase in the

MDA content seen in the transgenic lines suggests that they

were better able to deal with Cd2? stress than the WT or

other transgenic lines (Fig. 8a). The activities of the anti-

oxidant enzymes CAT, SOD, and POD were greatly

affected by Cd2? stress. Both wild-type and transgenic

lines had lower activity of these enzymes after the appli-

cation of Cd2? stress, and all of the transgenic lines had a

higher level of SOD and POD activity than the wild type

during Cd stress from 2 to 48 h. It is well known that GSH

plays an important role in controlling the antioxidative

defense system and in redox homeostasis of plant cells

(Sethi et al. 2008; Mendoza-Cozatl and Moreno-Sanchez

2006). GSH is a substrate for PC synthesis, and activation

of PC synthase results in rapid formation of phytochelatins,

which causes a transient decrease in the cytosolic GSH

pool (Nocito et al. 2006). The increased PC production

improved the chelation of Cd, while amount of Cd was

accumulated in transgenic plants. The low content of GSH

and additional Cd2? caused oxidation resistance in the

transgenic leaves. The improvement in antioxidant enzyme

activity was needed to reduce the oxidative response and

toxicity to the tissues and organs of the transgenic plant.

In conclusion, TcPCS1 expression can be induced in

T. caerulescens by cadmium stress. Heavy metal accumu-

lation and tolerance in yeast transformants can be improved

by TcPCS1 expression under either single or combined

metal stress. TcPCS1 cDNA expression may increase the

antioxidative activity and reduce the toxicity of Cd and

oxidative stress.

Acknowledgments The research was supported by the National

Major Special Project on New Varieties Cultivation for Transgenic

Organisms (Grant nos. 2009ZX08009-130B), the National High

Technology Planning Program of China (Grant nos. 2009AA06Z320)

and China National Natural Sciences Foundation (Grant nos.

50874112).

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