Differential responses of three sweetpotato metallothionein genesto abiotic stress and heavy metals

Sun Ha Kim • Jae Cheol Jeong • Young Ock Ahn •

Haeng-Soon Lee • Sang-Soo Kwak

Received: 20 April 2014 / Accepted: 1 July 2014 / Published online: 17 July 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Metallothioneins (MTs) are cysteine-rich, low

molecular weight, metal-binding proteins that are widely

distributed in living organisms. Plants produce metal-che-

lating proteins such as MTs to overcome the toxic effects

of heavy metals. We cloned three MT genes from sweet-

potato leaves [Ipomoea batatas (L.) Lam.]. The three IbMT

genes were classified according to their cysteine residue

alignment into type 1 (IbMT1), type 2 (IbMT2), and type 3

(IbMT3). IbMT1 was the most abundantly transcribed MT.

It was predominantly expressed in leaves, roots, and callus.

IbMT2 transcript was detected only in stems and fibrous

roots, whereas IbMT3 was strongly expressed in leaves and

stems. The IbMT expression profiles were investigated in

plants exposed to heavy metals and abiotic stresses. The

levels of IbMT1 expression were strongly elevated in

response to Cd and Fe, and moderately higher in response

to Cu. The IbMT3 expression pattern in response to heavy

metals was similar to that of IbMT1. Exposure to abiotic

stresses such as methyl viologen (MV; paraquat), NaCl,

polyethylene glycol (PEG), and H2O2 up-regulated IbMT

expression; IbMT1 responded strongly to MV and NaCl,

whereas IbMT3 was induced by low temperature and PEG.

Transgenic Escherichia coli overexpressing IbMT1 protein

exhibited results suggest that IbMT could be a useful tool

for engineering plants with enhanced tolerance to envi-

ronmental stresses and heavy metals.

Keywords Sweetpotato � Metallothionein � Reactive

oxygen species � Heavy metal � Phytoremediation

Abbreviations

MTs Metallothioneins

MV Methyl viologen

PCs Phytochelatins

PEG Polyethylene glycol

ROS Reactive oxygen species

H2O2 Hydrogen peroxide

Introduction

Heavy metal contamination of water and soil degrades the

environment, causes direct health hazards to animals and

human beings, and decreases crop productivity [1]. A

variety of technologies have been investigated for reme-

diation of sites contaminated with heavy metals. One cost-

effective approach is phytoremediation, in which plants

that actively sequester heavy metals are sown on contam-

inated site. Plants that are successfully used for phyto-

remediation have short lifetimes with high biomass, and

hyperaccumulate heavy metals. Heavy metal ions play

essential roles in several biological process in plants, but

excess amounts inhibit plant growth due to root damage,

decreased chlorophyll content, and leaf chlorosis [2, 3].

Plants produce metal-chelating peptides and proteins

such as phytochelatins (PCs) and metallothioneins (MTs)

to overcome heavy-metal stress [4]. MTs are cysteine-rich,

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-014-3582-y) contains supplementarymaterial, which is available to authorized users.

S. H. Kim � J. C. Jeong � Y. O. Ahn � H.-S. Lee (&) �S.-S. Kwak (&)

Plant Systems Engineering Research Center, Korea Research

Institute of Bioscience and Biotechnology (KRIBB), 125

Gwahangno, Daejeon 305-806, Korea

e-mail: hslee@kribb.re.kr

S.-S. Kwak

e-mail: sskwak@kribb.re.kr

123

Mol Biol Rep (2014) 41:6957–6966

DOI 10.1007/s11033-014-3582-y

metal-binding proteins with low molecular weight that are

widely distributed in living organisms. MTs are involved in

the detoxification of heavy metals and in intracellular metal

ion homeostasis [5], and are implicated in scavenging

reactive oxygen species (ROS) [6–10] and stress responses

[11]. MTs are grouped into two classes (I and II) on the

basis of the cysteine (Cys) residue alignment [4, 5, 12, 13].

The MT cysteines are responsible for metal binding via

mercaptide bonds [13]. Plant MTs belong to class II, and

constitute a separate family that is subclassified into four

types on the basis of the cysteine residue distribution [4,

12]. Expression patterns of these four types can be corre-

lated with different tissues: type 1 is predominantly

expressed in roots, type 2 in shoot, type 3 in ripening fruits

or leaves, and type 4 is restricted to developing seeds [12].

Metallothionein gene expression is regulated by abiotic

stress, including heavy-metal stress, and plays an important

role in metal detoxification and homeostasis [4, 12–15].

Because MT proteins were first isolated as cadmium-

binding peptides in horse kidney [16], earlier studies of

plant MTs focused on their role in maintaining intracellular

metal homeostasis and mediating responses to metal tox-

icity [12]. However, more recent studies report additional

roles for plant MTs in development, fruit ripening, senes-

cence, and defense against oxidative stress. A recent study

showed that expression of the Brassica juncea MT2 in

Arabidopsis enhanced tolerance to copper and cadmium,

but inhibited root elongation [17]. Two MT-like genes were

up-regulated during natural leaf senescence in sweetpotato

[18]. Cotton MT3 scavenged ROS and enhanced plant

tolerance to oxidative stresses caused by NaCl, polyethyl-

ene glycol (PEG), and low temperature [6].

Bioremediation and phytoremediation using bacteria or

plants are very attractive strategies compared with physi-

cochemical methods for removal of heavy metals from

contaminated sites. They have lower costs and higher

efficiency for cleaning wastes, sediments, and soils con-

taminated with low levels of toxic heavy metals [19–21].

Although it is likely that both PCs and MTs participate in

heavy-metal detoxification in plants, distinctive roles have

not been clearly demonstrated. Plant MTs are thought to be

primarily involved in cellular ion homeostasis. Several

plant MT genes have been characterized, but the in vivo

function of each MT type has not been elucidated. The

mechanisms mediating heavy-metal tolerance remain

unclear in Ipomoea. Sweetpotato (Ipomoea batatas) is a

globally important root crop that is relatively tolerant to

unfavorable growth conditions and environmental stresses.

The molecular basis of heavy-metal stress tolerance in

sweetpotato has not been reported. In this study, we

investigate the responses of sweetpotato MTs to heavy

metals and abiotic stresses, and assess the effects of IbMT

overexpression in Escherichia coli.

Materials and methods

Plant material and growth conditions

Ten-day-old, white-flesh sweetpotato [I. batatas (L.) Lam.

cv. Yulmi, (Ym)] plantlets were cultivated in growth

chambers with 12-h day/12-h night cycles at 25 �C/18 �C.

Calli were induced from shoot meristems cultured on

Murashige-Skoog (MS) medium supplemented with 1 mg/l

2,4-dichlorophenoxyacetic acid (2,4-D), 3 % sucrose, and

0.4 % gelrite (MS1D). Calli were propagated on MS1D

medium with subculture at 14-d intervals, and maintained

at 25 �C in the dark.

Cloning IbMTs and sequence analysis

To clone IbMT1, IbMT2, and IbMT3 from sweetpotato,

IbMT primers were designed based on sequence obtained

from the NCBI database (http://ncbi.nlm.nih.gov). IbMT1

was amplified from the cDNA of sweetpotato (Ym) storage

root by RT-PCR using the advantage 2 pfu DNA poly-

merase mix (Clontech, Japan), and the IbMT gene-specific

primers listed in Table 1. The PCR products were cloned

into pGEM-TEasy vector (Promega, USA) and sequenced.

Multiple sequence alignments with other plant MTs were

computed using the CLUSTAL W program.

Stress treatments

Ten-day-old sweetpotato plantlets and 7-day-old calli (Ym)

were used for assessing plant responses to stress treat-

ments. To evaluate responses to hormones, plantlets and

calli were treated with 200 lM salicylic acid (SA), 10 lM

abscisic acid (ABA), 50 lM methyl jasmonate (MeJA),

and 0.3 % ethephon (ET). To evaluate responses to heavy

metal toxicity, plantlets were treated for 48 h to 300 lM

liquid of CaCl2, CuSO4, FeSO4, ZnSO4, CdSO4, Pb(NO)3,

MgSO4, and MnSO4, respectively. To evaluate responses

to abiotic stress, plantlets were exposed to 100 lM methyl

Table 1 Primer sequences for semi-quantitative RT-PCR

Primer name Primer sequence

lbMT1-F 50-ATGTCTTCCGGTTGCAAG-30

lbMT1-R 50-GCTGTCCCCACAGCTACAGT-30

lbMT2-F 50-ATGTCTTGCTGCGGAGGA-30

lbMT2-R 50-TCAACATTTGCAAGGGTCG-30

lbMT3-F 50-ATGTCGGACAAGTGCGGA-30

lbMT3-R 50-TTAGTGGCCACAGGTGCG-30

a-Tubulin-F 50-CAACTACCAGCCACCAACTGT-30

a-Tubulin-R 50-CAAGATCCTCACGAGCTTCAC-30

Note F forward, R reverse

6958 Mol Biol Rep (2014) 41:6957–6966

123

viologen (MV), 10 mM hydrogen peroxide (H2O2),

300 mM NaCl, or 25 % PEG. Cold tolerance was assessed

by exposing plantlets to 4 �C. Tissue samples of plantlets

were collected at 3, 9, 18, and 48 h after treatment with

hormones, heavy metals, or abiotic stress, frozen in liquid

nitrogen, and stored at -80 �C until analysis.

RNA isolation and RT-PCR analysis

Frozen plants tissues were ground in liquid nitrogen with a

mortar and pestle, and total RNA was extracted with the Easy-

Spin total RNA extraction kit (iNtRON, Korea) according to

the manufacturer’s instructions. RNA samples were treated

with DNase I for 60 min before further analysis to digest

contaminating genomic DNA. First-strand cDNA synthesis

was performed in a 20 ll reaction mixture containing 1 lg

RNA, 0.5 lg oligo dT, 40 lM each of the four dNTPs, 2 ll

109 buffer, 200 units of M-MLV reverse transcriptase, and 20

units of RNase inhibitor (MBI-Fermentas, Germany). The

reaction mixture was incubated at 42 �C for 60 min, and

stopped by heating at 65 �C for 10 min.

To determine gene expression patterns, semi-quantitative

PCR using gene-specific primers was performed using the

a-tubulin (accession no. DV037573) transcript level as

internal standard. The IbMT gene-specific primers used are

listed in Table 1. PCR reaction mixtures included the sin-

gle-stranded cDNA template along with sense and antisense

primers. The thermal cycling conditions were 94 �C for

30 s, 56–58 �C for 30 s, 72 �C for 30 s, with a total of 25

cycles. PCR products were separated by 2 % agarose gel

electrophoresis, and detected under ultraviolet illumination.

Construction of expression plasmids pET28a(?)-IbMTs

For construction of E. coli expression vector pET28a(?)

(Novagen, Germany), the following IbMT primers were

designed from IbMT sequences, with restriction enzyme sites

BamHI and SalI underlined: IbMT1-BamHI, 50-GC

GGATCCATGTCTTCCGGTTGCAAG-30; IbMT1-SalI, 50-CTGTCGACACAGTTGCAAGGGTC-30; IbMT2-BamHI,

50-GCGGATCCATGTCTTGCTGCGGAGGA-30; IbMT2-

SalI, 50-CTGTCGACAATTTGAAGGGTCG-30; IbMT3-

BamHI, 50-GCGGATCCATGTCGGACAAGTGCGGA-30;and IbMT3-SalI, 50-CTGTCGACGTGGCCACAGGTGCG-

30. PCR was performed in 20 ll reactions containing 1 lg of

pGEM-IbMT template and 0.1 lM of IbMT primers (con-

taining the BamHI and SalI restriction sites) under the fol-

lowing conditions: initial denaturation step at 95 �C for

2 min, followed by 30 cycles of denaturation at 95 �C for

20 s, annealing at 58 �C for 30 s, polymerization at 72 �C for

30 s, and a final extension at 72 �C for 10 min. PCR products

were digested with BamHI and SalI to release the IbMT

fragment. The IbMT bands were recovered from the gel and

sub-cloned into the expression vector pET28a(?) using the

DNA ligation kit (TaKaRa, Japan) to create pET28a(?)-

IbMT. Competent E. coli DH5a cells were transformed with

pET28a(?)-IbMT plasmids, positive clones were selected by

blue/white screening, and confirmed by restriction enzyme

analysis with BamHI and SalI.

Measurement of growth and heavy metal accumulation

in E. coli overexpressing IbMT

Purified pET28a(?)-IbMT plasmids (0.5 ll) were used to

transform 100 ll of E. coli BL21 (Stratagene, USA),

which were grown on LB plates containing 50 lg/ml

kanamycin at 37 �C overnight to select cells harboring

pET28a(?)-IbMT. A single colony was inoculated into

3 ml LB liquid medium, grown at 37 �C overnight,

100 ll of the overnight culture was added to a glass tube

containing 50 ml LB plus kanamycin, and then cultured at

37 �C. When cells grew to OD600 = 0.6, 1 mM isopro-

pyl-a-D-thiogalactopyranoside (IPTG) was added to

induce the lac promoter.

To measure the accumulation of heavy metals, E. coli

cells overexpressing IbMT, [pET28a(?)-IbMT] were grown

in liquid medium containing 1 mM CdCl2 or 500 lM ZnSO4

at 37 �C. When cells grew to OD600 = 0.6, 1 mM IPTG

was added and then incubated for a further 24 h at 30 �C.

Cells were harvesting, pellets were dried inverted in a fume

hood, and digested with 100 % nitric acid for 4 h at 95 �C.

After cooling at room temperature, the digests were diluted

with deionized water to a final volume of 50 ml, and ana-

lyzed for metals using inductively coupled plasma atomic

emission spectroscopy (ICP-AES; OPTIMA 7300DV; Per-

kin Elmer, USA).

Results

Multiple alignment and phylogenetic analysis of I.

batatas metallothionein genes

We cloned three MT genes from sweetpotato leaves. Each

MT was classified as type 1, type 2, or type 3 according to

the cysteine alignment, and designated as IbMT1, IbMT2,

or IbMT3, respectively. The full IbMT1 cDNA sequence

consisted of 201 nucleotides that encoded a 67 amino acid

polypeptide of approximately 4.6 kDa. IbMT2 cDNA

consisted of 249 nucleotides that encoded an 83 amino acid

polypeptide of approximately 4.2 kDa. IbMT3 cDNA

consisted of 195 nucleotides that encoded a 65 amino acid

polypeptide of approximately 4.8 kDa. The predicted pro-

teins contained two Cys-rich domains in the N- and

C-terminal regions. The deduced IbMT1 peptide contained

four Cys in the N-terminal region and five Cys in the

Mol Biol Rep (2014) 41:6957–6966 6959

123

C-terminal region, which were separated by a central, Cys-

free, 35 amino acid spacer. The deduced IbMT2 peptide

contained conserved sequences, MSCCGGN at the N-ter-

minal. The deduced IbMT3 peptide contained four Cys in

the N-terminal region and five Cys in the C-terminal

region, which were separated by a central, Cys-free, 32

amino acid spacer (Fig. 1a).

The genetic similarity of IbMTs with other plant MTs

was determined using blastx (https://blast.ncbi.nlm.nih.gov)

and CLUSTAL W (http://www.ualberta.ca/*stothard/

javascript/color_align_cons.html/). IbMT1 had the highest

homology with the putative MT of I. batatas L. cv. Kokei

(AB193246.1). IbMT2 and IbMT3 had the highest homology

with AF177760.1 and AF242374.1 of I. batatas L. cv. Tai-

nong57, respectively. Multiple alignments indicated that

IbMTs shared high homology with many MTs from other

species. To assess the evolutionary history of IbMTs, a

phylogenetic tree was constructed based on the deduced

amino acid sequence similarities of plant MT genes

(Fig. 1b).

A

B

Fig. 1 a Multiple alignment of three IbMT genes and other plant

MTs. Conserved cysteine residues are indicated by stars; conserved

amino acids are shaded. b Phylogenetic analysis of three IbMTs with

other plant MTs. Evolutionary history was inferred using the

neighbor-joining method; the optimal tree with the sum of branch

length = 4.13877083 is shown [33]. Evolutionary distances were

computed using the Poisson correction method [34], in units of the

number of amino acid substitutions per site. The analysis involved 16

amino acid sequences. All positions containing gaps and missing data

were eliminated. There were a total of 24 positions in the final dataset.

Evolutionary analyses were conducted in MEGA6 [35]

6960 Mol Biol Rep (2014) 41:6957–6966

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Tissue-specific expression of IbMTs in sweetpotato

plants

To examine the tissue-specific expression patterns of

IbMTs in sweetpotato, RT-PCR analysis was performed

with total RNAs extracted from different tissues, including

leaf, stem, fibrous roots, storage roots, and cultured cells

(Figs. 2, 3). IbMT1 and IbMT3 had relatively higher basal

expression levels compared to that of IbMT2 under normal

conditions. IbMT1 was predominantly expressed in leaves

and fibrous roots, whereas IbMT2 was weakly expressed

only in stem and fibrous roots. IbMT3 was expressed in all

tissues examined, with strong expression in leaves and

stems. IbMT1 was strongly expressed only in cultured cells.

Differential expression of IbMT1 and IbMT3

in response to abiotic stress and heavy metals

The expression patterns of IbMTs in response to abiotic

stress and heavy metal were investigated in 10-day-old

sweetpotato plantlets. IbMT1 and IbMT3 expression dis-

played similar patterns; however, IbMT2 expression was

undetectable during these experiments because of the low

transcript levels. Therefore, we analyzed IbMT1 and

IbMT3, which had high expression levels that enabled

further study.

RT-PCR was used to evaluate the effects of abiotic

stress, including MV, NaCl, H2O2, PEG, and low tem-

perature (Fig. 4). Treatment with 100 lM MV induced

strong IbMT1 expression at 3 h after treatment, and

expression remained at a higher level thereafter. Treat-

ment with 100 lM MV induced IbMT3 expression at 3 h

after treatment; subsequently, transcript levels declined

gradually to the basal level. IbMT1 expression was

strongly enhanced at 9–18 h after treatment with

300 mM NaCl. IbMT3 expression patterns were similar,

showing an elevation at 9 h, followed by a decrease at

18 h, to a level that remained constant. Other stresses

such as cold (4 �C), 100 mM H2O2, and 25 % PEG did

not significantly affect IbMT1 and IbMT3 expression

levels. The IbMT1 and IbMT3 expression patterns show

similarities in their responses to H2O2 and PEG treat-

ments. Both MT genes were slightly up-regulated by

H2O2 at 18–48 h, and by PEG at 9 h after treatment.

IbMT1 expression was not appreciably induced by cold

treatment, whereas IbMT3 expression was apparent at

18 h after cold treatment.

We investigated the response to heavy metal stress using

CaCl2, FeSO4, CdSO4, CuSO4, MgSO4, ZnSO4, MnSO4,

and Pb(NO)3 (Fig. 5). IbMT1 and IbMT3 expression

showed the same patterns in response to CaCl2 treatment;

they were slightly down-regulated at 3–18 h after treat-

ment, and recovered to the basal expression level at 48 h.

IbMT1 and IbMT3 expression was weakly up-regulated at 3

and 18 h in response to FeSO4 treatment. IbMT1 expres-

sion was strongly induced by CdSO4 at 9–48 h after

treatment, and IbMT3 expression was clearly induced at

9 h. IbMT1 expression was up-regulated at 3–9 h after

treatment with CuSO4, but we did not detect significant

changes in IbMT3 transcription levels at 18–48 h. IbMT

expression displayed different responses to MgSO4; IbMT3

expression was completely suppressed by 9–18 h after

treatment, whereas IbMT1 expression did not change.

IbMT1 and IbMT3 expression decreased at 3 h after treat-

ment with ZnSO4; subsequently, it gradually increased up

to 48 h after treatment. We could not detect significant

changes in MT transcript levels after MnSO4 treatment.

However, IbMT1 and IbMT3 expression was strongly

induced by Pb(NO)3 at 48 h after treatment.

Transgenic culture cells expressing IbMT1 respond

to hormones, heavy metals, and abiotic stress

IbMT1 was specifically expressed in callus, whereas IbMT2

and IbMT3 were not detected (Fig. 3). To determine the

expression of IbMT1 in cultured cells, sweetpotato calli at

10 days after subculture were treated with four heavy

metals, four plant hormones, four abiotic stresses, and were

assessed at 3 and 18 h after treatment (Fig. 6). IbMT1

transcript levels in calli were markedly elevated in

response to ABA, MeJA, and ET at 3 h after treatment.

MeJA-induced IbMT1 expression continued at higher lev-

els until 18 h after treatment. SA-induced IbMT1 expres-

sion was higher at 18 h after treatment. In heavy metal

treatment, IbMT1 transcript levels were higher at 3 h after

Fe and Mn treatment, whereas Cu induced IbMT1

αα-tubulin

IbMT3

IbMT2

IbMT1

Leaf StemFibrous roots

Storage roots

Fig. 2 Semi-quantitative RT-PCR analysis of IbMTs expression

patterns in plant tissues of I. batatas cv. Yulmi

αα-tubulin IbMT1 IbMT2 IbMT3 callus

Fig. 3 Semi-quantitative RT-PCR analysis of IbMTs expression

patterns in cultured cells of I. batatas cv. Yulmi

Mol Biol Rep (2014) 41:6957–6966 6961

123

expression after 18 h. IbMT1 expression did not respond to

Zn. In abiotic stress studies, MV and NaCl slightly down-

regulated IbMT1 expression, whereas H2O2 markedly

down-regulated IbMT1 expression after 3 h. IbMT1

expression was clearly up-regulated by NaCl and low

temperature after 18 h (Fig. 6c).

Cold

MV

H2O2

tubulin

IbMT1

IbMT3

0 3 9 18 48 h

0 3 9 18 48 h

0 3 9 18 48 h

0 3 9 18 48 h

0 3 9 18 48 h

NaCl

PEG

tubulin

IbMT1

IbMT3

tubulin

IbMT1

IbMT3

Fig. 4 Semi-quantitative RT-

PCR analysis of IbMT1 and

IbMT3 expression in

sweetpotato plantlets in

response to abiotic stresses.

Total RNA was extracted at 0,

3, 9, 18, and 48 h after exposure

to 100 lM MV, 10 mM H2O2,

300 mM NaCl, or 25 % PEG.

Cold tolerance was assessed by

exposure to 4 �C

MgSO4

MnSO4

ZnSO4

tubulin

IbMT1

IbMT3

tubulin

IbMT1

IbMT3

Pb(NO)3

CuSO4

FeSO4

CdSO4

CaCl2

0 3 9 18 48 h

0 3 9 18 48 h

0 3 9 18 48 h

0 3 9 18 48 h

0 3 9 18 48 h

0 3 9 18 48 h

0 3 9 18 48 h

0 3 9 18 48 h

tubulin

IbMT1

IbMT3

tubulin

IbMT1

IbMT3

Fig. 5 Semi-quantitative RT-

PCR analysis of IbMT1 and

IbMT3 expression in

sweetpotato plantlets in

response to heavy metal. Total

RNA was extracted at 0, 3, 9,

18, and 48 h after exposure to

heavy metals including 300 lM

each CaCl2, CuSO4, FeSO4,

ZnSO4, CdSO4, Pb(NO)3,

MgSO4, and MnSO4

6962 Mol Biol Rep (2014) 41:6957–6966

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IbMTs confer heavy metal tolerance to transgenic

E. coli

To determine whether IbMTs conferred heavy metal tol-

erance, three E. coli expression vectors were constructed

harboring IbMT1, IbMT2 and IbMT3 in pET28a (pET28a-

IbMT1, pET28a-IbMT2, and pET28a-IbMT3). For inde-

pendent proof of function, each IbMT was expressed as a

recombinant protein in E. coli. Soluble IbMT proteins were

extracted from bacterial lysates and affinity-purified on a

Ni-agarose matrix. Purified recombinant IbMT proteins

were analyzed by SDS-PAGE (Supplemental Fig. S1).

IbMT1 protein was detected after purification on the Ni-

column, but not in crude extract. IbMT2 partitioned into the

insoluble crude extract. IbMT3 was expressed in the solu-

ble crude extract.

The growth of untransformed E. coli cells (BL21, NT)

was compared with that of cells harboring the empty vector

(pET28a) or the IbMT expression vectors (Fig. 7). In the

absence of heavy metals, all bacterial strains exhibited

identical growth (data not shown). However, exposure to

media containing CdCl2 or ZnSO4 differentially affected

cell growth. In CdCl2 medium, cells expressing IbMT1 and

IbMT3 grew better than those expressing empty vector and

untransformed NT cells. The growth of pET28a-IbMT1

cells increased for 8 h and then decreased. After 8 h culture

in CdCl2 medium, the growth of IbMT1 cells was three

times higher than that of cells harboring empty vector and

untransformed NT cells. The growth of pET28a-IbMT3

cells was inhibited with culture time in CdCl2 medium.

However, after 8 h culture in CdCl2 medium, the growth of

IbMT3 cells was approximately 1.31 and 1.81 times higher

than that of empty vector and NT cells, even though all

cells showed a similar inhibition growth curve in CdCl2medium (Fig. 7a). In ZnSO4 medium, pET28a-IbMT1 cells

Fig. 6 Semi-quantitative RT-PCR analysis of IbMT1 expression in

sweetpotato culture cells in response to stress-related hormones,

heavy metals, and abiotic stresses. Total RNA was extracted at 3 and

18 h after treatments. To evaluate responses to hormone treatment,

calli were treated with 200 lM salicylic acid (SA), 10 lM abscisic

acid (ABA), 50 lM methyl jasmonate (MeJA), or 0.3 % ethephon

(ET). Sweetpotato culture cells were exposed to 100 lM MV, 10 mM

H2O2, 300 mM NaCl, or 25 % PEG. Cold tolerance was assessed by

exposure to 4 �C. Calli were exposed to 300 lM CuSO4, FeSO4,

ZnSO4, or MnSO4. For abiotic stress assessment, cultured cells were

exposed to 100 lM MV, 10 mM H2O2, 300 mM NaCl, or 25 % PEG

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Gro

wth

(O

D a

t 60

0 n

m)

NT pET28apET28a-IbMT1 pET28a-IbMT3

CdCl2

ZnSO4

A

B

C

0

20

40

60

80

100

Co

nte

nts

(p

pm

/g F

W)

NTpET28apET28a-IbMT1pET28a-IbMT3

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0hr 2hr 4hr 6hr 8hr 12hrs 24hr

CdCl2 ZnSO4

0hr 2hr 4hr 6hr 8hr 12hrs 24hr

Gro

wth

(O

D a

t 60

0nm

)

NT pET28apET28a-IbMT1 pET28a-IbMT3

Fig. 7 Metallothioneins confer heavy metal tolerance to E. coli

expressing IbMT1 and IbMT3. Growth rates of MT-expressing trans-

formants, empty vector control, untransformed control cells, and E. coli

expressing IbMT1 or IbMT3 were measured in the presence of 500 lM

CdCl2 (a) and 300 lM ZnSO4 (b). OD600 was continuously monitored.

Data represent mean ± SD of three independent replicates. c CdCl2 and

ZnSO4 accumulation in NT, IbMT1, and IbMT3 E. coli (BL21). Cells

were grown in LB medium containing 1 mM CdCl2 or 500 lM ZnSO4

for 1 day before sampling. Data represent mean ± SD of three

independent experiments

Mol Biol Rep (2014) 41:6957–6966 6963

123

grew better than that of cells harboring empty vector and

untransformed NT cells, but growth decreased after 8 h.

ZnSO4 inhibited the growth of pET28a-IbMT3 cells, but

growth was higher than that of cell harboring empty vector

and untransformed NT cells after 12 h (Fig. 7b). All cells

showed the same growth rate in CuSO4 medium, indicating

that CuSO4 does not affect cell growth (data not shown).

We confirmed that IbMTs conferred tolerance to heavy

metals (CdCl2 and ZnSO4) in E. coli. To examine whether

this tolerance is mediated by accumulation of heavy met-

als, E. coli cells overexpressing IbMT1 and IbMT3 were

cultured in medium with 1 mM CdCl2 and 500 lM ZnSO4,

respectively, and their metal ion contents were measured

(Fig. 7c). In CdCl2 medium, IbMT1-overexpressing cells

contained lower Cd2 ? levels compared to those of empty

vector and untransformed controls. In ZnSO4 medium,

IbMT3-overexpressing cells accumulated Zn to levels 2.5

times higher than those in empty vector and untransformed

controls.

Discussion

Research on plant MTs has focused primarily on eluci-

dating protein function. Shared common functions of plant

MTs include metal chelation, regulation of ion homeosta-

sis, and ROS quenching under different stress conditions.

The physiological roles of each MT gene in sweetpotato

plants have not been clearly defined. In this study, we

cloned three MT genes from sweetpotato leaves, charac-

terized their protein classification, and investigated their

expression patterns in response to hormones, heavy metals,

and abiotic stress using RT-PCR analysis. Each MT was

classified as type 1, type 2, or type 3 according to the

cysteine alignment (Fig. 1b). We tested potential in vivo

functions of recombinant IbMTs in heavy metal stress using

the E. coli expression system. In a previous report, two MT-

like genes, Y459 and G14 [18], were isolated from sweet-

potato cv. Tainong 57. Y459 was a type 2 MT that was

strongly expressed in senescent leaves, whereas G14 was a

type 4 MT that was likely a house-keeping gene.

Several reports indicate that MT expression is spatially

and temporally controlled in plants, and plant MTs display

tissue-specific expression. Plant type 1 MTs are more

abundantly expressed in roots than leaves, whereas type 2

MTs are expressed primarily in leaves and at a lower level

in roots [10, 12, 14, 18, 22, 23]. Type 3 MTs are expressed

in leaves or in ripening fruit [24], whereas type 4 MTs

appear to be restricted to developing seeds [25]. However,

the current study found that IbMT1 was predominantly

expressed in leaves and fibrous roots, and only IbMT1 was

specifically expressed in cultured cells (Fig. 2). IbMT2 was

detected only in the stem, and IbMT3 was predominantly

expressed in leaves and stems (Fig. 2). IbMT1 showed

relatively higher expression levels compared to those of

IbMT2 and IbMT3 under normal conditions in different

plant tissues, suggesting that IbMT1 may have diverse roles

or functions.

In previous studies, MT expression was induced by

heavy metals and abiotic stresses in different plants [15, 17,

26, 27]. Rice OsMT expression was induced by several

abiotic stresses including salts (NaCl and NaHCO3),

drought (PEG), and metal ions (CuCl2, ZnCl2, and CdCl2)

[28]. In the current study, IbMT1 and IbMT3 exhibited

similar expression patterns in response to abiotic stresses

and heavy metals (Figs. 3, 4). However, the IbMT1

expression level was higher than that of IbMT2. Our results

showed that IbMT1 was strongly induced by treatment with

MV, NaCl, CdCl2, and ZnSO4. In culture cells, IbMT1

differentially responded to abiotic stresses, stress-related

hormones, and heavy metals (Fig. 5). These results indicate

that expression of sweetpotato MTs is differentially regu-

lated in response to exogenous factors.

Previous reports discussed expressing plant MTs in

E. coli to assess if they confer tolerance to heavy metals.

We examined in vivo functions of recombinant IbMTs

using E. coli expressing pET28a-IbMTs vectors, and

examining cell growth in the presence of heavy metals.

Cell growth was affected by toxic concentrations of heavy

metals, and IbMT overexpression significantly improved

growth kinetics. E. coli expressing IbMT1 and IbMT3 grew

better in media containing high concentrations of CdCl2and ZnSO4, respectively, than those without IbMTs.

IbMT1-expressing cells had better growth rate compared

with that of IbMT3-expressing cells (Fig. 6). Both IbMT1-

and IbMT3-expressing cells accumulated metal ions,

except in CdCl2 medium, in which cells showed no accu-

mulation of Cd. By contrast, IbMT3-expressing cells

accumulated high levels of Zn, and grew relatively well. It

appears that IbMT1 may function to export cadmium ions

to the extracellular space. IbMT3 may function in metal

chelation in the vacuole for detoxification (Fig. 7c). These

results are consistent with other reports that Cd tolerance

and accumulation in sugarcane is mediated by other

mechanisms [29]. OsMT1b has greater affinity for Cd and

Ni ions compared to Zn and Cu ions, suggesting that

OsMT1b may protect rice cells against toxic metals, or play

a role in detoxification [23, 30]. The over-expression of

sheep MTII and maltose binding protein in the cytoplasm

of E. coli allowed the bioaccumulation of the heavy metals

such as Zn2?, Cu2? and Cd2?. This result suggested that

combination of metal export system and chelate system

like MT or PCs may also play an important role in bioac-

cumulation of heavy metal ion [31]. Arabidopsis MT3

enhances Cd tolerance when expressed in Vicia faba guard

cells [26]. Thlaspi caerulecens MT3 confers Cd, Cu, or Zn

6964 Mol Biol Rep (2014) 41:6957–6966

123

tolerance [27, 32]. Tamarix hispida MT-like ThMT3

enhances tolerance to Cd, Zn, Cu, and NaCl stress when

expressed in Saccharomyces cerevisiae [9]. It is anticipated

that IbMT1 and IbMT3 will play important roles in release

of accumulated metal ions and metal ion chelation,

respectively. Analysis of tolerance to heavy metal stress

conditions in plants expressing IbMTs remains to be

investigated in future studies. We suggest that three MT

genes from I. batatas will be useful tools for engineering

heavy metal tolerance and hyperaccumulation of metal ions

for new phytoremediation plants.

In conclusion, we cloned three MT genes from sweet-

potato leaves, which were classified as type 1, type 2, and

type 3. IbMT1 had relatively higher levels of basal

expression compared to those of IbMT2 and IbMT3 under

normal conditions. IbMT1 was predominantly expressed in

leaves, roots and callus, whereas IbMT2 was detected only

in the stem. IbMT3 was specifically expressed in stems and

leaves. In sweetpotato plantlets, IbMT1 and IbMT3

expression was strongly induced by Zn, Cd, Pb, MV, and

NaCl. In calli, IbMT1 was enhanced by ABA, MeJA, eth-

ephon, NaCl, and low temperature. Recombinant IbMT

proteins were successfully expressed in E. coli, and IbMT1

and IbMT3 conferred tolerance to heavy metal stress. The

results suggest that IbMTs are differentially regulated by

different exogenous factors. Thus, our results demonstrate

that the three MT genes from I. batatas may be useful tools

to develop transgenic plants and microorganisms with

enhanced tolerance to heavy metals and abiotic stresses,

which can be used for phytoremediation or tolerance to

environmental stresses.

Acknowledgments This work was carried out with the support of

‘‘Cooperative Research Program for Agriculture Science & Tech-

nology Development (Project No. PJ009506 and PJ008097)’’ Rural

Development Administration, Republic of Korea, and the KRIBB

Initiative Program.

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